Abstract Collection from the 10th Annual Meeting of the European Section of the International Academy of Cardiovascular Sciences

Prevention of atherosclerotic plaque formation, reduction in plaque burden and stabilization of a vulnerable plaque are of therapeutic significance in decreasing the incidence of acute events. Initially, we found an imbalance between the inflammation and reparative process, leading to plaque instability. The findings supported the critical role of TLR4 and TREM-1 in underlying pathophysiology of chronic inflammation, atherosclerotic plaque formation and plaque instability. Thus, we examined the effect of the selective inhibitors of TREM-1 (LR-12) and TLR4 (TAK242) in a microswine model of carotid artery atherosclerosis. Hypercholesterolemic Yucatan microswine were treated with a control vehicle and TREM-1 or TLR4 inhibitor at the time of intimal injury and were sacrificed after 5–6 months. Optical coherence tomography (OCT) showed plaques with thick fibrous cap and occluded arteries in the controls. Radiological evaluation of carotid arteries revealed decreased neointimal hyperplasia and plaque formation with treatment using the TREM-1 inhibitor compared to the scrambled peptide group. There was no significant effect of TAK242 when dissolved in 30% ethanol. However, TAK242 dissolved in a clinically relevant vehicle significantly decreased the plaque size and enhanced plaque stabilization. Histomorphologically, there was neointimal hyperplasia with significantly increased inflammation and elastin degradation in the controls compared to the inhibitor groups on H&E and Movat pentachrome staining. The gene and protein expression for the markers of plaque vulnerability, including MMP-7, IL-6, IL-12/23, and CD36, were significantly decreased with treatment using the TREM and TLR4 inhibitors (dissolved in clinically relevant vehicle) compared to individual vehicle control. The findings of this study support the therapeutic efficacy of inhibiting either TLR4 or TREM-1 signalling alone or in combination in attenuating atherosclerotic plaque vulnerability, reducing plaque burden and thus preventing the occurrence of transient ischaemic attack and stroke.

carotid artery stenosis – stroke – unstable plaque – TREM-1 – TLR4

This work was supported by the research grant R01 HL144125 from the National Institutes of Health, USA.

Hydrogen has the potential to be a valuable agent in clinical medicine due to its antioxidant and anti-inflammatory properties. This gas easily passes through membranes and quickly reaches the tissues, where it acts by quenching the hydroxyl radical, irrespective of other reactive oxygen species. The current interest in this gas is reflected in studies of ischaemia-reperfusion injury, cardiovascular disease, transplantation surgery, haemorrhagic shock and post-cardiac arrest syndrome. After successful resuscitation of cardiac arrest by achieving the return of spontaneous circulation, the whole body is subject to an ischaemia-reperfusion injury that involves a massive ROS generation, which is believed to subsequently damage the endothelial cells and the endothelial glycocalyx (EG) layer. The latter is a delicate sugar-based gel-like structure covering the endoluminal aspect of endothelial cells and is ascribed to have a multitude of functions, including endothelium protection, regulation of vasomotion, antioxidation, anti-thrombosis and regulation of capillary permeability, hence preventing the free movement of water and electrolytes. Preserving the EG layer is considered a clinically relevant study objective, as damage to this layer results in the loss of its protective functions. Positive effects of hydrogen gas have previously been reported in cardiac arrest settings in animals and humans. Moreover, a preventive effect was observed on syndecan-1 release in a rat model of haemorrhagic shock. The lecture describes the author's experience in experimental research with hydrogen gas and the protection of EG in a rat model of cardiac arrest.

hydrogen – ischaemia-reperfusion – cardiopulmonary resuscitation – endothelial glycocalyx –

This work was supported by the University Hospital Hradec Kralove, Czech Republic: MH CZ-DRO (UHHK, 00179906).

The antiarrhythmic and cardiac electrophysiological effects of SZV-2649, a new amiodarone-like antiarrhythmic compound that lacks the benzofuran moiety, were studied in a mammalian cell line as well as in rat and dog cardiac preparations. SZV-2649 exerted antiarrhythmic effects against coronary artery occlusion/reperfusion-induced ventricular arrhythmias in rats and in acetylcholine- and burst stimulation–induced atrial fibrillation in dogs. SZV-2649 inhibited hERG and GIRK currents in HEK cells with IC50 values of 342 nM and 529 nM, respectively. In canine cardiac ventricular myocytes, SZV-2649 (10 μM) decreased the densities of IKr, Ito, INaL and ICaL currents. The compound (2.5–10 μM) elicited Vaughan Williams Class IB type V_max reducing and Class III type action potential duration prolonging effects in dog right ventricular papillary muscle. In canine atrial muscle, SZV-2629 (2.5–10 μM) moderately prolonged action potential duration, and this effect was greatly augmented in preparations pretreated with 1 μM carbachol. In conclusion, SZV-2649, similar to amiodarone, has antiarrhythmic effects based on its multiple ion channel blocking properties. However, since its chemical structure shows major differences compared to amiodarone, it can be expected that SZV-2649 would exhibit fewer serious adverse effects than amiodarone and may be a promising molecule for further development.

arrhythmias – mexiletine – amiodarone – atrial fibrillation

This work was supported by the National Research Development and Innovation Office (NKFIH-K147212).

Quercetin (QCT) is a natural polyphenolic substance that has been shown to exert several beneficial effects in the cardiovascular system, including antioxidant, anti-inflammatory and antihypertensive effects. In our experimental studies, we focused on potential the cardioprotective effects of QCT in myocardial (I/R) injury in different experimental settings, including the presence of selected comorbidities, co-medications and ageing.

The current presentation aims to summarize data obtained in our studies documenting the effects of QCT in several animal models of myocardial I/R injury and to uncover the role of age, metabolic comorbidities including diabetes and obesity, and co-medication with the anthracycline doxorubicin in its cardiac effects.

QCT in the dose of 20 mg/kg/day was administered orally for 4–6 weeks to rats of different age and rats with selected comorbidities/co-medications. After the end of treatment, hearts were isolated and ex vivo exposed to I/R (30-min global ischaemia/2-h reperfusion). Recovery of cardiac function and infarct size were assessed as the physiological outputs of the experiments. Molecular mechanisms of QCT action were evaluated as well.

The results showed that QCT exerts cardioprotective effects in I/R injury in healthy young and doxorubicin-treated rats but is inefficient in preventing I/R injury in rats with comorbidities (hypertensive/type 2 diabetic). Moreover, the cardioprotective efficiency of QCT is negatively influenced by increasing age. In conclusion, QCT might be potentially cardioprotective in preventing myocardial I/R injury; however, ageing and/or the presence of comorbidities may decrease or even abolish the anti-ischaemic effects of QCT.

ischaemia-reperfusion – quercetin – cardioprotection – ageing – comorbidities

Supported by VEGA 2/0104/20 and APVV-21-0194.

Perivascular adipose tissue (PVAT) plays an important role in the regulation of vascular homeostasis. Under physiological conditions, PVAT produces vasodilating and anti-inflammatory factors. However, in obesity/metabolic syndrome, PVAT phenotype changes to a pro-inflammatory one. Recently, it has been demonstrated that green tea polyphenols, such as (−)-epigallocatechin (EGC), oxidize the gasotransmitter hydrogen sulphide (H2S) to polysulphides (H2Sn), the important signalling molecules (Redox Biol 2020; 37:101731). We examined the effect of EGC on periaortic adipose tissue (PAT) phenotype in rats fed a high fat diet (HFD). Rats were fed a regular diet or HFD for 1 month and either treated with EGC or left untreated (10 mg/kg/day). The expression of pro- and anti-inflammatory factors was measured in PAT by RT-qPCR. H2S and H2Sn levels in PVAT were measured by an electrochemical sensor as well as the specific fluorescent probes.

Expression/secretion of leptin, resistin, TNF-alpha, IL-6 and MCP-1 was higher and adiponectin was lower in PAT of obese rats, which was accompanied by increased H2S production. The H2S and H2Sn donors, Na2S and Na₂S₄, had pro- and anti-inflammatory effects on PVAT, respectively. Administration of EGC in HFD rats reduced the expression of leptin, resistin, TNF-alpha, IL-6 and MCP-1 and increased the expression of adiponectin. In addition, EGC reduced the M1 macrophage marker, inducible NO synthase (iNOS), and increased the expression of M2 markers, IL-10 and arginase-1. The effects of EGC were mimicked by Na₂S₄. EGC decreased H2S and increased H2Sn in PAT of obese rats. Similar results were observed ex vivo in EGC-treated PVAT explants.

EGC has the anti-inflammatory effect on PVAT mediated by oxidation of H₂S to H₂S_n. This effect may contribute to the anti-atherosclerotic properties of green tea polyphenols and may explain, at least in part, the beneficial vascular effect of green tea in the metabolic syndrome.

hydrogen sulphide – polysulphides – perivascular adipose tissue – atherosclerosis – obesity

This study was supported by the Medical University of Lublin, Lublin, Poland, under the grant DS476.

Background: Cardioprotection against myocardial ischaemia/reperfusion injury (MIRI) is still challenging; however, recently, non-coding RNAs, including microRNAs, have opened up a new pharmacological multi-target approach to reduce myocardial damage. We have previously shown that cardiac microRNA-125b* (microRNA-125b-2-3p; miR-125b*) increased cell viability in isolated cardiac myocytes subjected to simulated IRI; therefore, it is termed a ProtectomiR. We aimed to characterize the pharmacokinetics and pharmacodynamics, as well as the effect of miR-125b* mimic on infarct size in an in vivo mouse model of acute myocardial infarction.

Materials and Methods: To characterize the pharmacokinetics of miR-125b*, a single intravenous (i.v.) bolus of 10 μg miR-125b* mimic or its scramble miRNA control or vehicle (a neutral lipid emulsion) was administered to male C57BL/6 mice, and 1, 2, 4, 8 and 24 h later, miR-125b* expression was determined from plasma and heart samples. The effect of miR-125b* on myocardial infarct size was assessed after administration of the same dose of miR-125b* mimic (and non-targeting miRNA or vehicle as negative controls) at the 10th minute of a 45-min coronary occlusion followed by 24-h reperfusion. To assess the molecular mechanism of cardioprotection, using bioinformatics, mRNA targets of miR-125b* were selected, and their ventricular expression were experimentally validated.

Results: Circulating and cardiac expression of miR-125b* were significantly increased at 1 h after miR-125b* mimic treatment. Infarct size was significantly reduced after i.v. administration of miR-125b* mimic as compared to the vehicle. The expression of Ccna2, Eef2k and CaCnb2 (miR-125b*'s target mRNAs) were significantly reduced 8 h after i.v. bolus injection of the miRNA mimic.

Conclusions: This is the first demonstration of the pharmacokinetics and explorative pharmacodynamics of miR-125b* mimic in mice. Furthermore, we have shown for the first time that miR-125b* mimic provides cardioprotection in a well-translatable mouse model of acute myocardial infarction when administered as a single i.v. bolus before recanalization.

microRNA-125b* – cardioprotection – acute myocardial infarction – pharmacokinetics – infarct size

This study was supported by the 2020-1.1.5-GYORSÍTÓSÁV call programme (2020-1.1.5-GYORSÍTÓSÁV-2021-00011; Project no. RRF-2.3.1-21-2022-00003 ‘National Heart Laboratory, Hungary’; Hungarian National Scientific Research Fund (OTKA-138223).

Taurine is a well-known non-essential amino acid that is very important for cell function. This circulating factor is produced mainly by the brain and the heart. Nutrients, particularly seafood, ensure its supplementation. Taurine's beneficial effects are numerous, particularly at the cardiovascular level. A prolonged diet rich in sodium salt can lead to salt-sensitive hypertension. Until recently, it was accepted that there are sexual differences between men and women in salt-sensitive hypertension. Indeed, knowledge of the process behind the development of female-specific salt sensitivity remains to be elucidated. Recently, in humans, it has been shown that sodium-sensitive hypertension is due in part to morphological remodelling and ion homeostasis of human contractile vascular smooth muscle cells (hcVSMCs) induced by glycocalyx destruction. Our study shows that chronic exposure to extracellular sodium is associated with greater remodelling of ionic and morphological homeostasis in male compared to female hcVSMCs. Long-term treatment with taurine prevents hypernatraemia-induced remodelling of hcVSMCs in a sex-independent manner.

taurine – salt-sensitive hypertension – sex dependency – human vascular smooth muscle

This work was supported by a grant from CIHR and NSERC.

The main aim of this study was to determine whether molecular hydrogen (H2) administration through the hydrogen-rich water (HRW) intake can affect cardiovascular system regulation and lactate response during and immediately after an experimental exercise protocol in well-trained soccer players. An experiment was designed as a randomized, double-blind, placebo-controlled crossover trial. The heart rate (HR) responses during and following a repeated sprint ability protocol (15 m ± 30 m with 20 s recovery between each sprint) were performed in 12 professional soccer players aged 18.8 ± 1.3 years. The HRW contained 0.9 ppm of dissolved H₂. HR was monitored using the HR monitor during the repeated spring protocol (maximal achieved heart rate – HR_max), average HR and at 1 (HRR1) and 3 min (HRR3) post-exercise HR. Post-exercise blood lactate concentration was measured after the last sprint. A statistically significant difference was found between HRW and placebo in HRR1 (HRW = 46.8 ± 10.6 bpm, placebo = 41.6 ± 12.6 bpm, P = 0.017) and HRR3 (HRW = 67.8 ± 9.0 bpm, placebo = 62.7 ± 8.2 bpm, P = 0.022). However, neither lactate concentrations nor exercise HR were significantly different between HRW and placebo. In conclusion, it seems that acute HRW administration can improve the HR recovery of team sport athletes performing maximal repeated sprints that may translate to improved performance during training and competition. Therefore, it appears that H₂ may be considered a promising dietary supplement in the future.

hydrogen-rich water – recovery – vagal reactivation – training – sprint

Muscular-enriched Lamin Interacting Protein (MLIP) is increasingly recognized as a pivotal component in maintaining cellular homeostasis across various tissues, particularly within muscle cells. MLIP interacts intimately with the nuclear lamina, influencing nuclear architecture, gene expression and mechanotransduction pathways. This protein's role extends to the regulation of cellular stress responses, differentiation and proliferation, highlighting its multifaceted involvement in cellular physiology. Recent studies underscore MLIP's significance in muscle development and function, where its dysregulation is linked to muscular dystrophies and other myopathies. Notably, MLIP modulates key signalling cascades such as the PI3K/AKT and MAPK pathways, which are critical for cell survival and growth. These insights suggest that MLIP not only contributes to the structural integrity and dynamic functions of the nucleus but also plays a crucial role in cellular adaptive mechanisms. Given its central role in cellular homeostasis, MLIP emerges as a promising therapeutic target. Strategies to modulate MLIP activity could offer novel interventions for muscle-related diseases and potentially other conditions characterized by nuclear architecture disruptions and impaired stress responses. This presentation aims to synthesize current knowledge on MLIP's biological functions and therapeutic prospects, proposing future research directions to harness its potential in clinical applications.

cellular homeostasis – cardiomyopathies – therapeutic potential – pi3k/akt pathway – nuclear lamina

This work was supported by the Qatar University (QUCG-CAS-23/24-214) and Qatar National Research Fund (UREP30-000-3-001).

Two decades and > 200 trials have shown that adult stem/progenitor cells are safe, and several Phase I/II studies suggest that they are efficacious in heart failure and refractory angina, although large Phase III trials are needed to establish efficacy conclusively. In contrast, embryonic stem cells have never been tested in a controlled trial in cardiovascular medicine and are plagued by major problems, including ethical concerns, arrhythmias, tumourigenicity, rejection requiring immunosuppression, genomic instability, prohibitive dose requirements, phenotypic heterogeneity and lack of long-term engraftment. The most reasonable interpretation of current data is that embryonic stem cell-based therapies are not likely to have clinical application for heart disease. At the preclinical level, there is overwhelming evidence that various stem/progenitor cells can improve cardiac function after myocardial infarction. The mechanism remains unclear, but it does not involve regeneration of new myocytes; rather, transplanted cells work via secretion of factors that act in a paracrine fashion. The use of repeated dosing is particularly important because mounting evidence shows that the efficacy of cell therapy is underestimated if only one dose is used. In the clinical arena, heart failure and refractory angina appear to be the main target populations. The use of the intravenous route for cell delivery may fundamentally transform the field of cell therapy. Regarding cardioprotection, this field has stalled, and no interventions have proven efficacious in ST-segment Elevation Myocardial Infarction despite myriad clinical trials. Gene therapy is a promising approach that has not yet been tested adequately in the clinical arena. This lecture critically examines the current state of clinical research on cell-based therapies, cardioprotection and gene therapy, highlighting the controversies in the field, improvements in clinical trial design and the application of new strategies.

heart disease – cardioprotection – gene therapy

The global rise in diabetes, obesity, cardiovascular diseases (CVDs), and healthcare costs is alarming. Consequently, there is an urgent need to identify cost-effective strategies for the prevention of CVDs, diabetes and obesity. Consuming healthful foods rich in flavonoids, polyphenols and carotenoids—such as fresh fruits and vegetables—and probiotics/prebiotics, along with reducing intake of salt, sugar-sweetened beverages and saturated fats; quitting smoking; reducing excessive drinking and engaging in moderate exercise for 30 min a day, are collectively recognized as effective in preventing CVDs. This comprehensive approach is likely the most cost-efficient for promoting health and preventing chronic conditions such as cancer, diabetes, obesity, CVDs and neurodegenerative diseases. Strong evidence supports that a Mediterranean-type diet, which includes whole grains, omega-3 fatty acids, poultry, fish, nuts, seeds, olive oil, dairy products, limited red meat and moderate red wine consumption, is associated with lower mortality and morbidity from CVDs. Additionally, recent studies indicate that dietary supplements containing flavonoids, carotenoids and antioxidants can influence gene and protein expression, altering metabolic pathways and maintaining homeostasis, thus reducing the risk of chronic diseases with complex causes. The positive effects of plant-derived bioactive compounds and their metabolites are due to their combined antioxidant and anti-inflammatory properties. Probiotics, prebiotics and synbiotics contribute to health by fostering healthy gut microbiota, enhancing gut endothelial integrity and strengthening the body's immune function.

cardiovascular diseases – healthful foods – Mediterranean diet – probiotics – gut microbes

Cardiac fibrosis is a clinically important contributor to heart failure and arrhythmias, leading to an increased risk of death, typically in response to stressors such as myocardial infarction or pressure overload. Despite many years of research, no therapies currently exist to directly attenuate cardiac fibrosis, and new approaches are required. For fibrosis to occur, resident cardiac fibroblasts undergo a phenotypic conversion to myofibroblasts, which secrete large quantities of extracellular matrix proteins and contribute to cardiac remodelling. Blocking fibroblast activation is thus a promising means to stop, and possibly reverse, cardiac fibrosis. We have shown that scleraxis is required for cardiac fibroblast activation and that, as a transcription factor, it works by transactivating a suite of pro-fibrotic genes that are responsible for the myofibroblast phenotype, including alpha-smooth muscle actin, periostin and collagen 1 alpha 2. In addition, scleraxis controls the expression of glutaminase 1, the rate-limiting enzyme of glutaminolysis, which appears to be a required energy-producing pathway for myofibroblasts. Our data have shown that blockade of scleraxis by knockout or knockdown prevents the activation of pro-fibrotic genes as well as glutaminase 1, interfering with fibroblast activation. Drug-mediated blockade of glutaminolysis has also shown promising impacts on fibroblast activation. In vivo, scleraxis knockout in cardiac fibroblasts is sufficient to both prevent and reverse cardiac fibrosis, resulting in significantly improved cardiac function, reduced remodelling and increased survival following pressure overload. Our evidence indicates that scleraxis loss both prevents fibrotic gene expression and blocks the energy-producing pathway required for myofibroblast activity. These results support targeting scleraxis to reduce the clinical burden of cardiac fibrosis and may yield the first anti-fibrotic therapeutic.

heart failure – gene expression – transcription factor – fibrosis – therapy

Supported by Canadian Institutes of Health Research.

Although different antiplatelet agents are used for the prevention of thrombosis and treatment of ischaemic heart disease, very little information regarding the therapeutic potential of these agents in heart failure is available. We have investigated the effects of some antiplatelet agents, such as sarpogrelate (SAR) and cilostazol (CIL) treatments, on cardiac dysfunction, cardiac remodelling and subcellular defects in heart failure due to myocardial infarction. Heart failure in rats was induced by occluding the coronary artery for 8 weeks, and the drug treatment was started 4 weeks after inducing myocardial infarction. Marked depression in cardiac output and ejection fraction and increases in heart rate, left ventricle (LV) thickness and LV volume in the infarcted animals were attenuated by SAR and CIL. Alterations in myofibril Ca²⁺-ATPase, as well as myosin isozyme contents and gene expression in the failing heart were reduced by SAR and CIL. Likewise, changes in sarcoplasmic reticular Ca²⁺ uptake and release activities, Ca²⁺ pump and Ca²⁺ release protein content as well as their mRNA levels were attenuated by both drug treatments. These results provide evidence that both SAR and CIL delay the progression of heart failure and improve cardiac function by attenuating cardiac remodelling, subcellular defects and abnormalities in cardiac gene expression. It is suggested that antiplatelet agents may prove to be a viable therapy for the treatment of heart failure. (Infrastructure support for this study was provided by the St. Boniface Hospital Foundation.)

heart failure – antiplatelet agents – cardiac remodelling

Allogeneic (unrelated donor) mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) are being tested in animal studies and clinical trials for cardiac regeneration and repair. The outcome of initial studies was very encouraging, and transplanted stem cells were safe after transplantation in the recipient heart. However, poor survival of transplanted stem cells in the infarcted heart has impaired the clinical translation of stem cell-based therapies. We have performed investigations to understand the mechanisms of poor survival of transplanted stem cells in the heart. We found that allogeneic stem cells after transplantation in the ischaemic heart turned immunogenic and were subsequently rejected by the host immune system. In our ongoing studies, we are developing biomaterial-based strategies to prevent rejection of transplanted stem cells in the heart. We synthesized and characterized titanium carbide (Ti₃C₂) MXene quantum dots (MQDs). The immunomodulatory MQDs selectively reduce activation of CD4+IFN-γ+ T-lymphocytes and promote the expansion of immunosuppressive CD4+CD25+FoxP3+ regulatory T-cells in an activated lymphocyte population. We also incorporated MQDs into a chitosan-based hydrogel to create a 3D platform for stem cell delivery to the heart. This composite immunomodulatory hydrogel-based platform improved survival of stem cells and mitigated allo-immune responses. We also found that MQDs have the potential to mitigate allograft vasculopathy and prevent rejection of transplanted organs. These studies highlight the potential of MXene-based next-generation biomaterials for cardiac regenerative medicine.

cardiac regeneration – immunomodulation – stem cells

Transverse (t)-tubules are important for the rapid and synchronous rise in calcium, which brings about the contraction of cardiac myocytes. In the atrial, t-tubules are almost completely lost at the point of heart failure. We set out to determine if it is possible to restore t-tubules to cardiac myocytes following their loss in heart failure and if their restoration can restore the calcium transient. Heart failure was induced by rapid ventricular pacing in sheep. Atrial t-tubules were almost entirely lost, and the calcium transient amplitude decreased. In a subset of sheep, rapid pacing was terminated, and the animals were allowed to recover for 5 weeks. Following recovery from heart failure, atrial t-tubule density was restored, but t-tubules were structurally disordered. Despite their disorder, we show recovered t-tubules were able to trigger calcium release. The recovery of atrial t-tubules restored the L-type calcium current, the calcium content of the sarcoplasmic reticulum and the calcium transient. Our data suggest that myotubularin, a lipid phosphatase, is important for t-tubule restoration.

t-tubule – calcium – heart failure recovery

Funded by British Heart Foundation.

Folic acid, a synthetic form of folate (vitamin B9), has been associated with several cardioprotective effects, primarily in reducing non-proteinogenic amino acid homocysteine level (epigenetic biomarker linked to an increased risk of cardiovascular, neurodegenerative disease and cancer when elevated) and metabolizing into other amino acids. Moreover, folate is involved in methylation reactions, which are crucial for regulating gene expression, involved in DNA synthesis and repair, and maintaining cardiovascular health. Folic acid effects have been studied in homocysteine-dependent (induced hyperhomocysteinaemia, isoprenaline-induced myocardial injury) and homocysteine-independent (monocrotaline-induced heart failure, streptozotocin-induced diabetes mellitus) rat models of cardiometabolic diseases for their potential cardioprotective effects on coronary flow, endothelial function, oxidative stress, inflammation and activities of cardiometabolic enzymes. Folic acid has been found to reduce homocysteine and glucose levels when elevated, exhibit vasodilator and antioxidant properties, improve endothelial dysfunction and affect cardiac, hemostatic and hepatorenal biomarkers; cardiac oxidative stress and cardiometabolic enzyme activities. Individual responses to folic acid may vary, and these effects also depend on the applied dose of folic acid, the method of application and the used experimental model of cardiometabolic disease. It is important to note that while folic acid supplementation may help lower homocysteine levels, its direct impact on reducing the risk of cardiovascular events like heart attack, and stroke is still a subject of ongoing research and debate. However, research on its direct cardioprotective effects is ongoing, and it is often recommended as part of a balanced diet rather than as a standalone treatment for heart disease.

folic acid – cardiometabolic diseases – cardioprotection

Supported by The Ministry of Science, Technological Development and Innovation of the Republic of Serbia.

The prevalence of hypertension has been steadily increasing over the past three decades, necessitating the constant development of novel treatments for this condition. Plants from the Mediterranean cuisine, such as oregano, have a history of traditional use in hypertension treatment. Carvacrol, a major constituent of essential oils from the Lamiaceae family, is known for its vasorelaxant properties and potential hypotensive effects. Therefore, this study aims to investigate the role of Transient Receptor Potential Ankyrin 1 (TRPA1) channels in carvacrol-induced vasodilation and to design novel compounds based on the structure of carvacrol with enhanced activity. An isolated tissue bath methodology was used to investigate the role of TRPA1 in carvacrol-induced vasodilation. Different in silico approaches were combined in the design and evaluation of novel TRPA1 agonists. The selective TRPA1 antagonist A967079 significantly reduced carvacrol-induced vasodilation of isolated human mesenteric arteries. A reliable 3D-QSAR model with good prediction accuracy (R² = 0.83; Q² = 0.59; R_pred^2 = 0.84) was created, and the obtained results were used to design novel TRPA1 agonists. Predicted pEC50 activities of these compounds were higher (4.996–5.235) compared to the experimental pEC50 of 4.77 for carvacrol. All created molecules had satisfying predicted Absorption, Distribution, Metabolism, Excretion, Toxicity (ADMET) properties, with six of them having better characteristics than carvacrol. Molecular docking studies demonstrated that the designed molecules bind to the same amino acid residues of the TRPA1 channel as carvacrol, with eight compounds showing lower binding energies. Molecular dynamics simulations revealed the good stability of carvacrol-TRPA1 and D27-TRPA1 complexes. Conclusion: Activation of TRPA1 channels is partly responsible for the vasorelaxant effect of human mesenteric arteries caused by carvacrol. Based on the results from various in silico studies, compound D27 exhibits the most promising predicted characteristics as a potential novel TRPA1 agonist regarding its activity, ADMET profile, interactions with residues and stability in complex with the TRPA1 channel.

TRPA1 – carvacrol – vasodilation – hypertension – 3D-QSAR

This work was supported by the Ministry of Scientific and Technological Development and Higher Education of Republic of Srpska: 19.032/961-28/23.

There is a circadian rhythm in the normal heart rhythm (in heart rate and PR, QRS and QT intervals) as well as in the incidence of bradyarrhythmias, atrial fibrillation, atrioventricular block, ventricular fibrillation and sudden cardiac death. This lecture summarizes our current understanding of the underlying mechanisms: The oldest explanation is that cardiac circadian rhythms are the result of an acute post-translational regulation of cardiac ion channels by the autonomic nervous system under the control of the suprachiasmatic nucleus, but, paradoxically, there is little or no evidence of this. Instead, there is a plausible suggestion that rhythms in the transcription of cardiac ion channels driven by local circadian clocks in the heart and circadian rhythms in plasma catecholamines and glucocorticoid (all ultimately under the control of the suprachiasmatic nucleus) are involved. Understanding these fundamental processes could potentially highlight new ways of treating cardiac arrhythmias and novel ‘chronotherapeutic’ strategies – for example, we have recently shown that a glucocorticoid receptor blocker can prevent the morning increase in ventricular arrhythmia susceptibility.

circadian rhythm – arrhythmia – ion channels

This work was support by the British Heart Foundation Intermediate Fellowship (FS/19/1/34035), British Heart Foundation Project grant (PG/22/10919), British Heart Foundation Programme Grant (RG/18/2/33392) and a Fondation Leducq TNE award (‘FANTASY’ 19CV03).

Despite nearly four decades of intensive research, drug treatment for myocardial infarction and subsequent post-infarction heart failure remains an unmet clinical need, possibly due to its complex molecular mechanism. Therefore, targeting a molecular network rather than a single molecular drug target may be a more effective strategy for cardioprotective therapy. An unbiased, comprehensive analysis of gene expression patterns in normal and protected ischaemic myocardium may lead to the exploration of transcriptomic and proteomic molecular networks to identify novel molecular targets for cardioprotection. The non-coding oligonucleotides microRNAs (miRNAs) targeting multiple mRNAs form a dynamic transcriptomic molecular network. Targeted perturbation of the molecular network by miRNA oligonucleotide compounds is a promising approach for the treatment of diseases of complex molecular mechanisms. By a systematic unbiased analysis of the transcriptomic network of healthy, infarcted, as well as protected by ischaemic conditioning heart samples, we have discovered endogenous cardioprotective miRNAs (e.g. miR125b-3p, miR-450a-3p) and validated them in vitro and in vivo and patented the nucleotide sequences of around these miRNAs. We termed these miRNAs protectomiRs. We are developing a miRNA discovery platform consisting of bioinformatic software packages (see miRNAtarget. com) to build transcriptomic and proteomic molecular networks and identify potential oligonucleotide drug candidates to treat myocardial infarction and other diseases with unmet clinical needs. We improve the performance of these algorithms using machine learning to create an effective drug discovery enabling platform. In conclusion, miRNA therapy may represent a breakthrough in the treatment of complex diseases such as myocardial infarction. Here, we have developed a miRNA discovery enabling platform and are currently developing our miRNA compound pipeline towards clinical phases.

myocardial infarction, heart failure, miRNA, transcriptomic

Intracellular cAMP levels produced upon β-AR stimulation are counterbalanced by the degradation of the cyclic nucleotide by enzymes called phosphodiesterases (PDEs). Our past studies showed that PDEs not only terminate the activation of cAMP effectors but also compartmentalize this second messenger in discrete subcellular compartments. Among the six PDE families expressed in cardiomyocytes to degrade cAMP, we recently focused our interest on PDE2 for several reasons. First, PDE2 hydrolytic activity is stimulated 10 to 30-fold by cGMP via binding to the regulatory N-terminal GAF-B domain, making PDE2 a unique integrator of cAMP/cGMP signals. Second, using selective PDE2 inhibitors, we showed that PDE2 inhibits cardiac L-type Ca2 channel activity in various species, including humans, and controls excitation-contraction coupling. Third, in contrast to PDE3 and PDE4, the two main enzymes degrading cAMP in the heart, whose expression and activities are generally decreased in pathological hypertrophy and heart failure (HF), PDE2 is increased in HF to blunt β-AR/cAMP signals, suggesting a protective mechanism against increased circulating catecholamines. Fourth, transgenic (TG) mice with constitutive cardiac overexpression of PDE2 exhibit improved cardiac function and are protected against catecholamine-induced arrhythmias after ischaemic injury.

In an attempt to further delineate the impact of PDE2 upregulation on cardiac function and its putative beneficial effects in HF, we performed gene therapy with adeno-associated virus serotype 9 (AAV9) allowing preferential cardiac overexpression of PDE2. This strategy allows expression from a definite time point rather than constitutive cardiac expression as previously employed. With this strategy, we demonstrate in vivo that PDE2 overexpression protects the heart upon chronic exposure to sympathomimetic amines by reducing remodelling and left ventricular dysfunction. Strikingly, increased cardiac PDE2 expression prevents ventricular arrhythmias upon stress, and this correlates with improved excitation-contraction coupling at the level of single cardiomyocytes.

Thus, our results demonstrate that increasing PDE2 activity is beneficial in HF. They suggest that a gene therapy with PDE2 in cardiac cells, or PDE2 activators yet to be discovered, could be of therapeutic value.

heart failure – cAMP-phosphodiesterase – excitation-contraction coupling – arrhythmia – gene therapy

This work was supported by the Leducq Foundation for Cardiovascular Research (19CVD02), ERA-CVD ‘PDE4HEART’, ANR-23-CE14-0051-01, Fédération Française de Cardiologie, and Fondation Lefoulon-Delalande.

Cardiovascular diseases are the leading cause of morbidity and mortality worldwide, imposing significant health and economic burdens on individuals, families and societies. Accordingly, a substantial effort is exerted to find novel, effective therapeutics for these diseases. Recently, inhibitors of the poly(ADP-ribose) polymerase (PARPi), originally developed for cancer therapy, have shown promise for repurposing in cardiovascular diseases. These drugs' cytoprotective, antioxidant and anti-inflammatory properties can be pivotal in treating heart conditions characterized by tissue damage and inflammation. Studies suggest that PARPi could protect cardiac cells from ischaemia-reperfusion injury and reduce adverse remodelling post-myocardial infarction. Additionally, their anti-inflammatory properties might mitigate atherosclerosis progression. Although prolonged administration of PARPi could lead to malignant transformation due to DNA damage accumulation, the short-term application of these drugs may be beneficial in post-infarct cardiac remodelling. We are presenting experimental evidence for the use of PARPi in the aforementioned conditions and providing mechanistic background for the protective effects of PARPi, including the effects of these drugs on protein kinase B/Akt and mitogen-activated protein kinase signalling.

PARP inhibitors – Akt – MAPK – repurposing – cardiovascular diseases

Environmental pollution is one of the biggest problems facing the modern world because it affects the health of virtually all living beings on the planet, increasing morbidity and mortality. In this chapter, we have described the effects of acute and chronic pollution on the lungs and heart, as well as three hypotheses that could explain how polluting particles reach different organs, especially the lungs and heart. Polluting particles reach the lungs through the respiration of living beings, where they activate pro-inflammatory cytokines, which reach the heart, producing damage to the mitochondria that increases the formation of free radicals and decreases the synthesis of ATP. This mechanism leads to a situation of myocardial ischaemia whose end point is myocardial infarction. This can occur with or without coronary obstruction; obviously, the presence of the latter aggravates the situation. Acute pollution produces cellular and molecular changes that lead to alterations in important cellular structures. Chronic pollution over time produces structural remodelling of the lungs and heart, leading to cardiac pathologies that, due to pressure and volume overload, modify the geometry of the organs. In summary, whether due to acute or chronic exposure to polluting particles, we are in the presence of an important risk factor for cardiovascular health that increases the incidence of myocardial infarction and heart failure in patients exposed to breathing severely polluted air. Although there is no specific treatment to reduce the effects of pollution, experimental evidence suggests that chronic exercise could be beneficial and improve the living conditions of these patients.

pollution – myocardial ischaemia – cytokines – lungs – myocardial infarction

This work was supported by the University of Buenos Aires - CONICET – ANPCYT.

Heart failure with reduced ejection fraction (HFrEF) remains a serious and highly relevant issue in cardiology. Due to advances in pharmacological and device-based treatments, a growing number of patients are experiencing favourable changes in left ventricular (LV) geometry, accompanied by improvements in LV ejection fraction (LVEF). This process is referred to as LV reverse remodelling (LVRR), and patients meeting certain criteria are classified as having heart failure with improved ejection fraction (HFimpEF). If the symptoms and signs of heart failure disappear, we may speak of heart failure remission. An important fact is that these favourable structural changes in the myocardium are associated with better clinical outcomes. In patients with HFimpEF, an improved quality of life, better exercise tolerance, lower risk of heart failure-related hospitalizations and a reduced risk of sudden cardiac death have been observed. Given these positive clinical indicators, LVRR has become a key therapeutic goal in patients with HFrEF. The occurrence of reverse remodelling has been observed in approximately 22% of HFrEF cases. The likelihood of LVRR depends on heart failure aetiology, clinical characteristics, laboratory tests and imaging findings. Generally, patients with shorter disease duration, non-ischaemic aetiology, smaller LV end-diastolic dimensions, a lower percentage of late gadolinium enhancement (LGE) on cardiac MRI, higher global longitudinal strain (GLS), and good tolerance to pharmacological therapy are more likely to experience LVRR. Personalized prediction offers an opportunity for individualized patient management, including decisions on the timing and implementation of device therapy. In conclusion, LVRR not only becomes one of the main therapeutic targets in patients with HFrEF, but investigating the mechanisms and predictors of LVRR also opens up possibilities for potential new treatment options for these patients.

heart failure – remodelling – recovery

Structural heart disease is widely recognized as the cause of most abnormalities in the electrical activity of the heart. However, persistent cardiac electrical abnormalities are frequently not the consequence but rather the ignored cause or an important additional etiological factor in the development of chronic heart failure (HF). Such electrical disturbances comprise persistent arrhythmias and/or left bundle branch block (LBBB). Therefore, electrically induced cardiomyopathy (EI-CMP) can be defined as a potentially reversible myocardial disease in which persistent arrhythmia and/or LBBB causes left ventricular systolic dysfunction (LVSD), which is phenotypically manifested as HF with reduced LV ejection fraction (HFrEF).

Experimental studies on animals exposed to rapid atrial or ventricular pacing were instrumental in gaining insight into the pathophysiological mechanisms of LVSD and, ultimately, HF. Tissue, myocyte, and electrical remodelling are observed in both animal models and human hearts with EI-CMPs. A decline in LV function is linked to impaired calcium handling dynamics and basal ATP activity. The remodelling process is characterized by a number of key features, including fibrosis, inflammation, apoptosis, expansion of the extracellular matrix, myocyte disarray, mitochondrial dysfunction and prolonged ventricular repolarization.

In humans, potential etiological factors for EI-CMP include:

Atrial fibrillation/flutter (irrespective of ventricular rate)

The majority of electrical abnormalities with the potential to induce or exacerbate heart failure with reduced ejection fraction (HFrEF) are treatable by catheter ablation or resynchronization therapy (CRT) utilizing biventricular pacing or pacing of the conduction system. When applied in a timely manner, these interventions can result in a complete or partial reversal of LVSD. Therefore, ensuring an accurate and timely diagnosis, followed by appropriate therapy, is crucial for the prognosis of patients with EI-CMP.

cardiac arrhythmias – left bundle branch block – heart failure – catheter ablation – cardiac resynchronization therapy

The clinical outcome of patients with an acute myocardial infarction who undergo reperfusion therapy by primary percutaneous coronary intervention is determined by the final infarct size. However, coronary microvascular injury is also increasingly appreciated as a decisive determinant of clinical outcome, beyond that of infarct size. In fact, a recent definition and grading of infarct severity encompasses not only cardiomyocyte death but also coronary microvascular injury. The mechanisms of coronary microvascular injury in the perfusion territory of a coronary artery with plaque rupture or plaque erosion are multifold: (a) microembolization of plaque debris with superimposed thrombotic material from the atherosclerotic lesion; (b) release of vasoconstrictor, pro-thrombotic and pro-inflammatory substances from the atherosclerotic lesion; (c) increased vascular permeability and oedema formation, which acts back by compression on the microcirculation; (d) platelet and platelet-leukocyte aggregates; (e) erythrocyte stasis; (f ) capillary constriction by pericytes; (g) endothelial shedding and capillary obstruction; (h) capillary rupture and intramyocardial haemorrhage. Many interventions that protect from myocardial ischaemia/reperfusion and reduce infarct size also attenuate coronary microvascular injury, including ischaemic conditioning protocols and cardioprotective drugs. The only intervention that appears to specifically protect the coronary microcirculation is angiopoietin-like 4.

myocardial ischaemia – reperfusion – cardioprotection – infarct size – coronary microvascular injury

This work was supported by the German Research Foundation (CRC 1116 B8, RTG 2989), EU Cost Action CARDIOPROTECTION and METAHEART.

Diabetes and diabetic cardiomyopathy are among the leading causes of morbidity worldwide, and their prevalence is still increasing. Diabetes is associated with tissue hypoxia and also results in the dysregulation of the hypoxia-inducible factor 1α (HIF-1α) signalling pathway, potentially contributing to the development of cardiomyopathy. The pathogenesis of diabetic cardiomyopathy involves changes in gene expression profiles, in which epigenetic modifications of RNA (epitranscriptomics) may play a role. In this study, we aimed to investigate the role of HIF-1α in the alteration of proteomic profiles in pancreatic islets and myocardial tissue, with a particular focus on epitranscriptomic regulators, following the induction of type 2 diabetes mellitus and the progression of diabetic cardiomyopathy. To achieve this, we utilized a Hif1a ± mouse model, where diabetic cardiomyopathy was induced through a combination of metabolic (high-fat diet; HFD) and hypertensive stress (L-NAME-induced inhibition of NO synthase) over 8 weeks. Diabetic animals developed cardiac hypertrophy and showed a reduction in cardiac output. Glucose tolerance tests, along with insulin assays, demonstrated the expected increases in glycaemia and insulin levels following HFD + L-NAME treatment in both wild-type and Hif1a ± mice. However, these levels were significantly higher in Hif1a ± mice compared to wild-type mice. Proteomic analysis revealed that the cardiac proteomic profile was influenced by both HFD + L-NAME treatment and HIF-1α deficiency, while pancreatic islets exhibited relatively homogeneous proteomic profiles across the experimental groups. Epitranscriptomic regulators were altered in a distinct pattern in both the heart and pancreatic islets in response to HFD + L-NAME treatment in both wild-type and Hif1a ± mice. This research contributes to the complex mosaic of diabetes research, offering new insights into the molecular basis of diabetic cardiomyopathy and highlighting the critical role of HIF-1α and epitranscriptomic regulation in the pathogenesis of this disease.

heart – diabetes – HIF-1a – epitranscriptomics

This work was supported by the National Institute for Research of Metabolic and Cardiovascular Diseases (Programme EXCELES, ID Project No. LX22NPO5104) – Funded by the European Union – Next Generation EU and the Grant Agency of the Czech Republic, grant No. 24-10497S.

Sodium-glucose co-transporter 2 inhibitors (SGLT2i), a novel class of glucose-lowering medications, have revolutionized the management of heart failure (HF) across the ejection fraction (EF) spectrum, regardless of the presence of diabetes. Extensive experimental and clinical evidence has unequivocally documented the cardiovascular benefits of SGLT2i, which extend far beyond glycaemia control and are supported by several ‘off-target’ effects. Oxidative stress is a central pathophysiological mechanism that disrupts cardiomyocyte homeostasis in the vast majority of cardiometabolic diseases, but the sources of reactive oxygen species (ROS) are far from being fully elucidated. Monoamine oxidase (MAO), with two isoforms MAO-A and B, contributes to cardiovascular oxidative stress by continuously producing ROS at the outer mitochondrial membrane. This study, performed in overweight, non-diabetic patients with chronic HF and the entire range of EF with indication of elective cardiac surgery, was aimed at assessing whether the SGLT2i included in HF guidelines, empagliflozin and dapagliflozin, decrease atrial MAO expression and alleviate oxidative stress elicited by exposure to angiotensin II (AII) and high glucose (HG). Right atrial appendages were incubated ex vivo with either empagliflozin or dapagliflozin (1, 10 μM, 12 h) in the presence or absence of AII (100 nM) and HG (400 mg/dL) and used for the assessment of MAO-A and B gene and protein expression (by qPCR and immunofluorescence) and oxidative stress (by FOX and dihydroethidium assays). Stimulation with AII and HG increased atrial expression of both MAOs and augmented ROS production. The effects were mitigated by both concentrations of the SGLT2i. Atrial oxidative stress is positively correlated with the echocardiographic parameters of heart cavities and negatively with left ventricular EF. In conclusion, in non-diabetic overweight patients with HF, MAO contributes to the cardiac oxidative stress in conditions that mimic the renin-angiotensin system activation and hyperglycaemia. MAO expression was mitigated with empagliflozin and dapagliflozin, as a novel ‘off-target’ class effect of the SGLT2i.

SGLT2i – MAO – atrial oxidative stress

Usnic acid (UA), a natural dibenzofuran derivative found in numerous lichens, has gained considerable research interest due to its broad spectrum of biological activities. It holds promise for therapeutic use, including antimicrobial, anticancer and antioxidant properties. Despite its long-standing use in traditional medicine and well-established benefits, there is a distinct lack of research on the cardioprotective effects of UA. This study aims to evaluate the effects of UA on doxorubicin-induced cardiotoxicity in a rat model.

UA was isolated from the acetonic extract of the lichen Xanthoparmelia stenophylla and confirmed by comparison with a standard reference sample. The study utilized 40 male Wistar albino rats, with UA being administered orally at a dose of 25 mg/kg per day for 28 consecutive days. After this treatment phase, doxorubicin was injected intraperitoneally at a cumulative dose of 15 mg/kg. Three days post-doxorubicin administration, the rats' hearts were excised and analysed ex vivo using a Langendorff apparatus. Blood samples and coronary venous effluent were collected for oxidative stress assessment, and heart tissues were preserved for histological and gene expression analyses. Administering UA at a dose of 25 mg/kg over 28 days resulted in notable improvements in cardiac function, particularly in the dp/dt max parameter, and a reduction in oxidative stress markers in both blood and coronary venous effluent in a doxorubicin-induced cardiotoxicity model. Histological analysis revealed a significant decrease in degenerative changes in the UA-treated group. Furthermore, qPCR results demonstrated a reduction in oxidative stress and activation of anti-apoptotic mechanisms in the groups receiving UA treatment. Our findings demonstrate that UA possesses significant cardioprotective antioxidant properties, suggesting its potential utility as a cardioprotective agent. Further investigation is required to elucidate the underlying mechanisms through which UA exerts its effects.

lichen – usnic acid – oxidative stress – doxorubicin – cardiotoxicity

Sudden cardiac death causes around 300,000 adult deaths each year in Europe. Ventricular tachycardia/fibrillation (VT/VF) is an important immediate cause of sudden cardiac death. Electrical tornados (reentrant waves, rotors) spinning at high frequency and generating turbulent electrical activity in the ventricles are the origin of VT/VF. When these tornados occur, the heart is unable to effectively pump blood; the only treatment is a powerful electrical shock applied to the ventricles. Cellular and molecular studies may offer an alternative by explaining how tornados form, how they generate electrical turbulence and how they can be annihilated. We have identified a groundbreaking membrane macromolecular complex known as a ‘channelosome’, which contains NaV1.5, the channel responsible for the sodium inward current (INa), and Kir2.1, the strong inward rectifier potassium channel responsible for (IK1). Through their channelosome, these critical cardiac ion channels reciprocally modulate each other's cell surface expression and function, which enables them to rigorously control electrical excitability and impulse conduction velocity in the ventricles. The consequences of Kir2.1-NaV1.5 channelosome dysfunction are manifold: the resting membrane potential is depolarized, excitability is reduced, action potential upstroke velocity is reduced, and action potential duration and QT interval are prolonged. These proarrhythmic changes promote the formation of electrical tornados and are key in their maintenance. Such new knowledge provides strong evidence to consider macromolecular complexes and ion channel interplay in the development of antiarrhythmic therapeutic strategies to prevent rotor formation and sudden cardiac death in patients with heart failure and other cardiac diseases. Sudden cardiac death poses significant medical and economic challenges to society. These exciting new concepts offer a novel paradigm for the understanding of cardiac electrical function. Most importantly, they may lead to advanced targeted therapy based on the rational design of new small molecules or peptides that promote channelosome location and function at the cell membrane as a more effective alternative for preventing life-threatening ventricular arrhythmias in many patients.

reentrant arrhythmias – Ion channels – channelosomes – excitability – conduction velocity

This work was funded by the La Caixa Banking Foundation (project code LCF/PR/HR19/52160013); grants PI20/01220 and PI23/01039 by Instituto de Salud Carlos III (ISCIII); MCIU grants BFU2016-75144-R and PID2020-116935RB-I00, co-funded by FEDER; Fundación La Marató de TV3 (736/C/2020); European Union's Horizon 2020 [GA-965286]. CNIC is ISCIII, the Ministerio de Ciencia e Innovación and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033).

This study examined the effect of combined valsartan/sacubitril administration on the browning promotion in healthy rats and rats with metabolic syndrome (MS). Using in silico studies, the signalling pathways by which the applied treatment induces browning were identified. The study included 24 Wistar Albino rats, grouped into healthy and MS-induced animals, further divided into two subgroups depending on the applied treatment. Animals from the experimental groups were treated orally with valsartan/sacubitril (Entresto) suspension (68 mg/kg) for 4 weeks. After the animals were sacrificed, biochemical parameters were determined, while three types of adipose tissue (retroperitoneal, epididymal and inguinal) were used for pathohistological and PCR analysis with adipocyte density and surface area assessment. Valsartan/sacubitril treatment had multiple positive effects on improving glycoregulation, insulin levels and lipid status. Besides antioxidant and anti-inflammatory effects, the applied treatment promotes browning by increasing UCP-1 expression as the main thermogenic biomolecule. The special contribution of this study is reflected through elucidation of the mechanisms by which valsartan/sacubitril induces browning and primarily refers to mTORC1 activation as a common target molecule through which this dual pharmacological therapy induces UCP-1 expression in adipose tissue of animals with MS.

valsartan – sacubitril – adipose tissue – browning – metabolic syndrome

Cardiovascular diseases, and in particular cardiac arrhythmias such as ventricular fibrillation, play a leading role in mortality statistics in developed countries. Accordingly, cardiac arrhythmias have become a major area of cardiovascular research. Drug therapy has traditionally been the main treatment for ventricular and supraventricular arrhythmias. It is therefore important to understand the mechanism of action of antiarrhythmic drugs at the organ, tissue, cellular and subcellular levels in order to develop new, more effective, less proarrhythmic agents. One such potential development direction is the development of a series of amiodarone-like antiarrhythmic agents in which repolarization-inducing (class III) activity is combined with other pharmacological properties (combined class III +I/B, II and/or IV activity) that are considered to be beneficial. Therefore, we investigated the antiarrhythmic and cardiac electrophysiological effects of a novel antiarrhythmic compound (SZV-2649), similar to amiodarone but lacking the benzofuran chemical structure, in rat and canine cardiac cell line cardiac preparations. SZV-2649 exerted antiarrhythmic effects on coronary occlusion reperfusion-induced ventricular fibrillation in rats and acetylcholine and burst stimulation-induced atrial fibrillation in dogs. SZV-2649 inhibited hERG/IKr and GIRK/IK,ACh currents in expressed HEK293 cell lines and in vitro in canine atrial muscle preparations. Based on these observations, it was concluded that SZV-2649 has antiarrhythmic effects similar to amiodarone, with multiple ion channel blocking properties. However, as its chemical structure is substantially different from that of amiodarone, it is expected to have less severe side effects than amiodarone, making it a promising target for future development.

SZV-2649 – antiarrhythmic drugs – proarrhythmic action – cardiac action potential – ion currents

This work was supported by the National Research Development and Innovation Office (NKFIH K 135464, K 142738, K 147212, FK 138223 and TKP2021-EGA-32, as well as GINOP-2.3.2.-15-2016-00006 and GINOP-2.3.2.-15-2016-00040), the Ministry of Human Capacities Hungary (EFOP-3.6.2-16-2017-00006), the Albert Szent-Györgyi Medical School institutional grant (SZTE AOK-KKA 2021, SZGYA 2021 and SZTE AOK-KKA 2022), Hungarian Research Network (HUN-REN TKI project to A.V., N.J. and I.B.) and the Hungarian Academy of Sciences (bo_481_21).

Cytokines such as TNFα have been implicated in cardiac dysfunction and toxicity associated with doxorubicin (DOX). While TNFα can elicit different cellular responses, including survival or death, the mechanisms underlying these divergent outcomes in the heart remain cryptic. The E3 ubiquitin ligase TRAF2 provides a critical signalling platform for K63-linked ubiquitination of RIPK1, crucial for NF-κB activation by TNFα. Whether alterations in TNFα-TRAF2 signalling underlie the cardiotoxic effects of DOX, remains poorly understood.

To investigate TRAF2 signalling in the pathogenesis of DOX cardiotoxicity.

Using a combination of in vivo (4 weekly injections of DOX (5 mg/kg/week) in cardiac myocyte-restricted expression of AAV9-GFP and AAV9-TRAF2 mice (C57/BL6J), and in vitro approaches (rat, mouse and human iPSCs derived cardiac myocytes), we monitored TNFα levels, LDH, cardiac ultrastructure and function, mitochondrial bioenergetics and cardiac cell viability.

In contrast to vehicle-treated mice, severe ultrastructural defects, including cytoplasmic swelling, mitochondrial perturbations and elevated TNFα levels, were observed in the hearts of mice treated with DOX. While investigating the involvement of TNFα in DOX cardiotoxicity, we discovered that in the absence of DOX, NF-κB was readily activated by TNFα. However, TNFα-mediated NF-κB activation was impaired in cardiac myocytes treated with DOX. This coincided with the loss of K63-linked polyubiquitination of RIPK1, attributed to the proteasomal degradation of TRAF2. Further, TRAF2 protein expression was markedly reduced in the hearts of cancer patients treated with DOX. Impaired TRAF2 signalling resulted in the activation of Bnip3 and mitochondrial perturbations, including disrupted bioenergetics, loss of membrane potential and permeability transition pore opening. We further established that the reciprocal actions of the ubiquitinating and deubiquitinating enzymes c-IAP1 and USP19, respectively, regulated the proteasomal degradation of TRAF2 in DOX-treated cardiac myocytes. Importantly, an E3 ligase mutant of c-IAP1 (c-IAP1 H588A) or gain of function of USP19 prevented proteasomal degradation of TRAF2 and DOX-induced cell death. Further, wild-type TRAF2, but not a RING finger mutant defective for K63 ubiquitination of RIPK1, restored NF-κB signalling and suppressed DOX-induced cardiac cell death. Finally, cardiomyocyte-restricted expression of TRAF2 (AAV9-TRAF2) in vivo protected against mitochondrial defects and cardiac dysfunction induced by DOX.

Conclusions: Our findings reveal a novel signalling axis that functionally connects the cardiotoxic effects of DOX to proteasomal degradation of TRAF2. Disruption of the critical TRAF2 survival pathway by DOX sensitized cardiac myocytes to TNFα-mediated necrotic cell death.

cell death – doxorubicin – innate immunity – heart failure

Supported by Canadian Institute for Health Research.

Cardioprotective strategies aim to preserve the myocardium from ischaemia/reperfusion injury and reduce infarct size and its consequences. Various stimuli delivered at sites remote from the heart (remote conditioning) activate molecular self-protection mechanisms in the target organ, the heart. Remote conditioning, that is ischaemic remote preconditioning (RIC) through short repeated cycles of ischaemia/reperfusion at a limb, as well as non-invasive electrical stimulation of the ear (ATS) through periodic very short electrical stimulation impulses of the heart, have been established in experimental studies and successfully transferred to patients. Remote conditioning is a systemic reaction. There are three levels of remote conditioning and the resulting cardioprotection: (I) the stimulus level; (II) the transmission level, neuronal and humoural mediators transmit the protective signal from the periphery to the heart and (III) the target level, receptor activation and intracellular signal transduction ultimately influence myocardial protection, as observed in various animal models and in humans/patients. Novel findings on signal transduction at different levels are highlighted: for example, the spleen as a relay organ for cardioprotective signal transduction and the non-neuronal cholinergic cardiac system at the target level.

cardioprotection – infarct size – myocardial ischaemia – remote conditioning – signal transduction

Endothelin-1 (ET-1) plays a critical role in pulmonary arterial hypertension (PAH) and is currently targeted by ET-1 receptor antagonists. However, strategies aimed at reducing cellular ET-1 expression have been less thoroughly explored. In preliminary experiments, we targeted multiple signalling pathways to identify those regulating endothelin-1 gene (Edn1) expression in A7r5 vascular smooth muscle cells. Based on these findings, we selected the guanylate cyclase stimulator riociguat (RIO) and the β2-adrenergic receptor agonist terbutaline (TER) for experimental treatment in a rat monocrotaline (MCT) model of PAH. Rats were sacrificed either after 4 weeks (MCT4w) or prematurely when reaching humane endpoints due to acute health deterioration (ptMCT). Drug treatment commenced 2 weeks after MCT injection. In the PAH model, we observed increased right ventricle and lung weights, along with elevated heart and respiratory rates, accompanied by increased Edn1 expression in the pulmonary artery, right ventricle and left ventricle. Plasma ET-1 protein levels were elevated in both monocrotaline groups. Although the treatments did not significantly improve biometrical indicators of organ damage, TER reduced heart rate and RIO decreased respiratory rate. At the molecular level, both treatments favourably influenced the expression of the physiological myosin isoform Myh6 and atrial natriuretic peptide in the right ventricle. RIO did not affect elevated Edn1 expression in the ventricles and only partially reduced Edn1 overexpression in the pulmonary artery. Interestingly, TER-treated MCT4w rats did not show Edn1 overexpression in the ventricles, and Edn1 overexpression was attenuated in terminally ill TER + ptMCT rats. With the exception of TER +ptMCT, neither RIO- nor TER-treated rats showed increased plasma levels of ET-1 protein. In conclusion, riociguat and terbutaline reduced Edn1 gene expression in vascular smooth muscle cells and in the monocrotaline model of PAH. Therapies aimed at reducing ET-1 levels may offer therapeutic benefits in PAH.

endothelin-1 – monocrotaline – pulmonary arterial hypertension – riociguat – terbutaline

Supported by APVV-19-0458 and APVV-23-0502.

Radiation-induced heart disease (RIHD) is a known harmful side effect of mediastinal radiotherapy applied in oncological diseases. RIHD involves structural and functional abnormalities of the pericardium, coronary vessels, myocardium, valves and conduction system. The underlying pathological mechanisms are complex and mainly related to endothelial cell damage, oxidative stress and inflammation. These can cause cardiomyocyte disorders, tissue fibrosis and ultimately heart failure. To overcome these complications, it is necessary to look for specific therapeutic interventions, which are still lacking. Studies have shown that molecular hydrogen (H₂) possesses radioprotective effects in different experimental models, including RIHD. H₂ could be effective in alleviating RIHD through various mechanisms, for example selective neutralization of hydroxyl radicals, protection against inflammatory and apoptotic damage, antifibrotic and antihypertrophic effects, etc. Available information shows that microRNAs (miRNAs) might be involved in the radioprotective effect of H₂. In this work, our research team investigated the effect of 6-weeks of H₂ administration to 1-year-old Wistar rats irradiated in the mediastinal area with a single dose of 10 Gy (4–5 Gy/min). The results show that the inhalation of H₂ effectively reduced heart damage (lactate dehydrogenase), improved oxidative stress (malondialdehyde, superoxide dismutase activity) and inflammatory (pro-inflammatory cytokines) parameters, as well as had a positive impact on heart function (infarct size, left ventricular end-diastolic pressure) parameters measured 6 weeks after irradiation. This study also reveals that H₂ administration on the RIHD rat model also normalizes dysregulated expression of selected miRNAs (miRNA-21, -30e, -208 and miRNA-142). Based on these results, we conclude that H₂ administration represents a promising treatment strategy for patients undergoing mediastinal irradiation with radiation-induced cardiac damage. However, to clarify other mechanisms of H₂ action and to verify the effectiveness of H₂ therapy in clinical practice, further studies are needed, especially focusing on the still unclear mechanism of its biological effect.

antioxidant – heart disease – irradiation – molecular hydrogen – oxidative stress

This work was funded by grants VEGA 2/0092/22 and APVV-19-0317.

Alterations in cardiac energy metabolism are an important contributor to heart failure severity. Despite this, there is considerable confusion as to what changes in cardiac energy metabolism occur in heart failure with preserved ejection fraction (HFpEF). To assess this, we measured cardiac fatty acid oxidation and glucose oxidation in a mouse model of HFpEF [which involved administering 13-month-old C57BL/6N female mice a 60% high-fat diet and L-NAME (0.5 g/L/day) for 8 weeks to induce HFpEF]. We observed an increase in fatty acid oxidation in isolated working hearts from HFpEF mice, which was accompanied by a marked decrease in cardiac glucose oxidation. An increased reliance of the heart on fatty acid oxidation as a source of ATP production results in a decrease in cardiac efficiency (cardiac work/O₂ consumed). Our goal was therefore to inhibit fatty acid oxidation in HFpEF mice as an approach to normalize cardiac energy metabolism. These studies were performed in a mouse model of diabetic cardiomyopathy (DbCM), which displays HFpEF symptoms and has high cardiac fatty acid oxidation rates and decreased glucose oxidation rates. A significant contributor to the increased fatty acid oxidation observed in DbCM hearts is the weakening of the regulatory control of mitochondrial fatty acid oxidation by malonyl-CoA. We therefore treated DbCM mice with an inhibitor of malonyl-CoA decarboxylase (MCD), which increases malonyl-CoA in the heart. A 4-week treatment with the MCD inhibitor, CBM-30011 (10 mg/kg/day), resulted in a significant decrease in cardiac fatty acid oxidation and a parallel increase in glucose oxidation in DbCM mice. An improvement in diastolic function was also observed in the DbCM mice (as assessed by echocardiographic E/e⊠). We conclude that in HFpEF mice the heart switches from glucose oxidation to fatty acid oxidation as the main source of ATP production. However, inhibiting fatty acid oxidation can increase the contribution of glucose oxidation to ATP production and lessen the severity of HFpEF.

fatty acid oxidation – glucose oxidation – HFpEF – diabetic cardiomyopathy

Supported by CIHR Foundation Grant.

Heart failure is an important cause of mortality and morbidity globally and carries a significant economic burden. The advances in pharmacotherapy have led to improved outcomes for patients with heart failure. However, there remain patients who do not respond to the treatment; their condition deteriorates and ultimately results in untimely death or heart transplantation. Heart failure resulting from varied aetiologies is characterized by heterogeneous patterns of cardiac tissue remodelling that are not adequately reflected in the current therapies. Addressing this heterogeneity will be key for improving outcomes. Single-cell RNA sequencing allows addressing this unmet clinical need by studying transcriptomic phenotypes of individual cells contained in heart tissue in health and in disease at a large scale. Spatial transcriptomic technologies allow the localization of cells in niches, which enables inferences about cell-cell interactions. In patients with advanced heart failure, we have been able to interrogate the cell type abundance shifts and perturbations to cellular gene expression programmes that associate with heart failure in general but also with individual aetiologies and genotypes. With refined understanding of gene activation heterogeneity in heart failure, we will be able to validate putative disease mechanisms that may be amenable to therapeutic targeting.

heart failure – RNA – transcriptomics

This work was supported by The British Society for Heart Failure Research Fellowship, the British Heart Foundation Clinical Research Training Fellowship, the Rosetrees Trust and Alexander Jansons Myocarditis UK.

The endoplasmic reticulum (ER) plays a central role in cellular stress responses via mobilization of ER stress coping responses, such as the unfolded protein response (UPR). Activation of UPR is associated with multiple cardiac conditions and has been shown to be either protective or detrimental to the heart. The serine/threonine protein kinase/endoribonuclease (IRE1α) is the most evolutionarily conserved ER stress sensor and a key UPR stress signal transducer. Impaired calcium homeostasis activates ER stress responses, resulting in severe cardiac fibrosis that is prevented by the inhibition of IRE1α. There are two pools of IRE1α in cardiac muscle cells: one is localized to perinuclear ER responding to cellular stresses and the other is at the junctional sarcoplasmic reticulum (SR), a subspecialized membrane system involved in the regulation of excitation-contraction (E-C) coupling. The junctional SR containing ryanodine receptor/calcium channel forms membrane contacts with the T-tubules containing the L-type calcium channel. Deletion of the exons 20 and 21 of the IRE1a gene (encoding the RNase domain of IRE1α) in adult cardiac myocytes caused severe heart dilation and mild fibrosis. We discovered that cardiomyocytes with deleted exons 20 and 21 of the IRE1a gene have prolonged calcium transients that can be corrected with L-type calcium channel inhibitors. Our results support an alternative function of IRE1α associated with L-type calcium channel function. These findings provide new and unexpected insights into the link between E-C coupling and stress coping responses in muscle cells.

ER stress – E-C coupling – calcium channel – calcium signalling – dyads – sarcoplasmic reticulum

This work was funded by the Canadian Institutes for Health Research.

The nuclear genome is exposed to numerous internal and external stressors, which affect the structure and function of DNA. It is estimated that each mammalian cell develops 104–105 lesions in the nuclear genome every day. The stressors cause DNA oxidation, alkylation and cross-linking, among others, which disturb the delicate homeostatic balance that maintains genomic stability. The DNA lesions also affect the replicative function of the DNA and RNA transcription. A consequence of the replicative and transcriptional stress is the generation and accumulation of double-stranded breaks (DSBs) in the DNA, which are the most dangerous form of DNA lesions. The genomic stressors are increased in pathological conditions such as heart failure. Likewise, the DNA repair mechanisms are impaired. Collectively, these changes lead to the accumulation of DNA lesions, including DSBs. The increased DSBs in the nucleus activate the nuclear component of the DDR, including the tumour protein 53 (TP53) and its downstream target cyclin-dependent kinase inhibitor 1A (CDKN1A), leading to cell cycle arrest and cell death. Likewise, a subset of the DSBs are released into the cytosol, where they are sensed by the DNA-sensing proteins, leading to activation of the nuclear factor-kappa B1 (NFkB1) and interferon regulatory factor 3 (IRF3) and consequent expression of the senescence-associated secretory phenotype (SASP). Expression of SASP contributes to the pathogenesis of heart failure by inducing senescence, inflammation, cell cycle arrest and cell death. Genetic blockade of the cytosolic or nuclear DNA imparts salubrious effects in the experimental models of heart failure. The stage is set to test potential pharmacological inhibitions of these pathways, initially in large animals and subsequently in humans with heart failure.

DNA damage – double-stranded breaks – heart failure – inflammation

This work was supported by the National Institutes of Health; National Heart, Lung and Blood Institute (Awards R01 HL151737 and R01 HL132401) and National Institute of Aging (R01AG082751).

Atrial fibrillation (AF) is the most common sustained arrhythmia in the clinical setting, which is associated with significant morbidity and mortality. AF is also associated with severe cellular calcium (Ca²⁺)-handling abnormalities in atrial myocytes that may contribute to the initiation and maintenance of the arrhythmia. This so-called remodelling is supposed to involve abnormal phosphorylation of key Ca²⁺-handling proteins. In addition, previous studies suggest that impaired phosphodiesterase (PDE)-dependent regulation of spatiotemporal distribution of cAMP may contribute to Ca²⁺-handling abnormalities associated with AF. We investigate how PDE8 controls the spatiotemporal response of intracellular cAMP in key relevant Ca²⁺-handling nanodomains by measuring real-time cAMP, ICa, L, spontaneous Ca²⁺ releases from the sarcoplasmic reticulum, protein expression and localization using highly sensitive and specifically targeted cAMP biosensors, patch-clamp, confocal Ca²⁺ imaging, contractility measurements and biochemical experiments.

atrial fibrillation – phosphodiesterases – cAMP – calcium handling

Supported by DFG, Heisenberg programme, Gertraud und Heinz Rose Stiftung.

While calcium signalling of SR by Ca2 transporters of the surface membrane (ICa and INCX) has been well studied, possible regulation of mitochondrial and nuclear Ca2 signalling by these proteins remains not fully explored. Here we investigate whether there are privileged interactions between Ca2 transporters of surface membrane and mitochondria, SR or nuclear envelope (NE), by comparing their Ca2 activity with cytosolic Ca2+, using genetically engineered fluorescent probes GCamP3/GCamP6 targeted to mitochondria, nuclear Lamin B, and SR lumen. In whole-cell clamped and TIRF- or confocal-imaged cardiomyocytes, caffeine-triggered SR Ca2 release caused rapid rise in cytosolic calcium (△F/F ~0.5: fura-2), followed by delayed and slower mitochondrial Ca2 uptake (△F/F =~1–2: mito-GCamp6), and much larger NE Ca2 uptake (△F/F ~10: LaminB-GCaMP6). Calcium-Induced Calcium Release (CICR)-triggered SR Ca2 release induces Ca2 uptake by NE that follows and outlasts cytosolic Ca2 signal significantly. Long-duration application of 5 mM caffeine to empty SR of its calcium content first transiently increases mitochondrial calcium, then triggers mitochondrial pacing despite the absence of SR calcium release. Lowering Na concentration in Ca2+-depleted SR increased the baseline calcium concentrations of SR, mitochondria and NE, enhancing the mitochondrial pacing rate. Spontaneous pacing was rapidly suppressed by blocking Ica but not by suppressing SR calcium uptake with CPA. We suggest that while activation of CICR is critical in triggering SR Ca2 release that replenishes NE Ca2 and initiates spontaneous pacing, calcium influx on NCX regulates mitochondrial pacing that sustains autorhythmicity in the absence of SR calcium release.

mitochondria – hiPSCs – Nuclear envelope – Ca2+ signalling – sarcoplasmic reticulum

Overweight and obesity represent serious health concerns whose prevalence and severity have been constantly rising worldwide. The global threat of the obesity pandemic is related to the high risk of cardiometabolic comorbidities/complications. Increased body fat leads to vascular and adipose tissue dysfunction mediated via increased oxidative stress and chronic systemic inflammation. Overwhelming experimental and clinical studies have reported the association of vitamin D deficiency with metabolic and cardiovascular pathologies, including chronic venous disease. Monoamine oxidases (MAOs) with two isoforms, A and B, are mitochondrial enzymes that catalyse the deamination of biogenic amines and neurotransmitters with the constant generation of hydrogen peroxide in both vessels and adipose tissue. We here present evidence that (i) suboptimal vitamin D status is associated with increased reactive oxygen species in visceral adipose tissue and vascular samples (mesenteric artery branches and varicose vein explants), (ii) serum levels of vitamin D are inversely correlated with the magnitude of local oxidative stress, (iii) in vitro administration of the active vitamin D (1,25(OH)2-D3) alleviated both oxidative stress and vascular dysfunction and (iv) the protective effects of vitamin D are related, at least partly, to a decrease in MAO expression. In both types of vascular samples, acute incubation with 1,25(OH)2-D3 was responsible for the upregulation of eNOS and downregulation of iNOS in both obese and non-obese patients. Further research is required to elucidate the signal transduction pathways responsible for the antioxidant effects of vitamin D in human samples.

vitamin D – antioxidant – monoamine oxidase – vascular and adipose tissues

This research supported by the university internal funds awarded to the Centre for Translational Research and Systems Medicine.

Regular physical exercise has well-known beneficial effects on health; however, increasing evidence indicates that extreme sports activities – although very rare – can induce various cardiac arrhythmias, such as atrial fibrillation or even sudden cardiac death. A significant number of sudden cardiac deaths among athletes have been attributed to ischaemic origins or other deviations revealed by autopsy. However, the underlying cause of the remaining cases remains unclear. Cardiac alternans indicate a periodic, regular oscillation of the action potential (short-long pattern) and parallel intracellular Ca2 release (large-small pattern). Alternans have been clearly associated with the development of ventricular fibrillation and sudden cardiac death. However, the potential role of cardiac alternans in the ‘athlete heart’ is unknown. In this study, the potential role of alternans was assessed in arrhythmia development in trained dogs. Beagle dogs underwent a vigorous 16-week long training program and were compared to a sedentary group. Cardiac action potentials were measured by the conventional microelectrode technique from right ventricular tissue samples. Ionic currents were recorded by the whole-cell configuration of the patch-clamp technique. Ca2 transients were monitored by employing fluo-4AM fluorescent dye. All experiments were performed at 37°C. Trained dogs exerted increased susceptibility to ventricular fibrillation as a response to burst pacing. At the cellular level, the Ito was downregulated in trained animals. Action potential alternans in endocardial and midmyocardial tissue layers were increased in trained animals. Purkinje-ventricle dispersion during alternans was found to be larger in trained dogs. Ca2 transient alternans were enhanced in trained animals. Ca2+ content of the sarcoplasmic reticulum was decreased in exercised animals, while cell shortening was identical between groups. Alternans may contribute to the development of ventricular fibrillation in intensive training-associated arrhythmias. The underlying mechanism of alternans could be SERCA downregulation in trained animals.

exercise – arrhythmia – remodelling – alternans – fibrillation

Supported by NKFIH-OTKA-142949.

Molecular hydrogen (H₂) has emerged as a promising therapeutic and preventive medical gas. In 2007, we overturned the conventional wisdom that H₂ has no effect on mammalian cells, leading to birth to the field of ‘hydrogen medical biology’. H₂ has many advantages, including high efficacy, lack of toxic effects, and diverse functions such as antioxidant, anti-inflammatory, anti-cell death, and energy metabolism stimulation. In addition, H₂ is beneficial not only for severe chronic and acute diseases but also for healthy people. The target molecule of H₂ was identified as the oxidized form of porphyrin, which acts as a catalyst to stimulate the selective reaction of H₂ with hydroxyl radicals. Various types of porphyrins are widely and abundantly distributed throughout the body. Therefore, the repair of oxidized porphyrins by H₂ may bring multiple benefits to various cells. H₂ suppresses free radical chain reactions and modifies signalling mediators involved in lipid peroxidation. We present the findings from animal experiments to clinical trials. The best example of H₂'s effectiveness is recovery after cardiac arrest. Inhalation of H₂ gas has reduced the mortality rate by more than half and more than doubled the number of patients discharged without sequelae. The molecular mechanics will be discussed. The future mission of H₂ medicine is to apply H₂ for treatment, prevention, and quality of life across a wide range of fields. Furthermore, the use of H₂ in agriculture has the potential to produce safe, productive and high-quality foods. H₂ medicine could be practiced with simple and low-cost treatments without adverse effects and contribute to many unsolved serious problems facing today's society. H₂ has the potential to address a wide range of issues, including cardiac arrest, cerebral infarction, metabolic syndrome, advanced cancer, inflammatory cytokine storm, Alzheimer's dementia, healthcare, beauty and agriculture.

cardiac arrest – ischaemia-reperfusion – molecular hydrogen – multiple functions – no adverse effects

Cardiogenic shock is a critical circulatory condition in which a severely failing cardiac pump is unable to generate sufficient cardiac output, leading to tissue hypoperfusion in vital organs. Despite advances in intensive cardiology care, mortality from cardiogenic shock remains high. During the past years, mechanical circulatory support, including extracorporeal membrane oxygenation (ECMO), has become a standard therapy for cardiogenic shock with the aim of bridging the most critical conditions. Current evidence supporting the use of ECMO (and other mechanical circulatory support systems) in cardiogenic shock remains insufficient. The initiation of ECMO leads to rapid restoration of total circulatory output, blood pressure and tissue perfusion. On the other hand, it may be associated with a possible negative effect on the already severely damaged left ventricle as well as several specific complications. Furthermore, the haemodynamic effect of ECMO also depends on the cause of cardiogenic shock and modulating factors, such as the presence of valvular heart disease (e.g. aortic stenosis or mitral regurgitation) or mechanical complications (e.g. ventricular septal defect). The negative effect of ECMO could be at least partially prevented by synchronized pulsatile extracorporeal flow. Randomized clinical trials have reported a neutral effect of the use of ECMO on clinical outcomes in cardiogenic shock. However, these trials had significant limitations, particularly in the inclusion criteria. Several recently published analyses indicate that detailed haemodynamic examination allows for the identification of subjects who may benefit from mechanical support. Currently, numerous clinical questions remain unanswered, and further research in this field is fundamentally needed.

cardiogenic shock – mechanical circulatory support – extracorporeal membrane oxygenation

This work was supported by the Charles University Research Program ‘Cooperation Cardiovascular Sciences’ and Ministry of Health, Czech Republic – Conceptual Development of Research Organization – MH CZ DRO, Motol University Hospital, Prague, Czech Republic, 00064203.

Experimental and clinical studies have clearly demonstrated significant sex differences in myocardial structure and function, both under physiological and pathological conditions. The best example is significant sex differences in cardiac tolerance to ischaemia/reperfusion injury: pre-menopausal adult female hearts are more resistant than male myocardium. The importance of these findings is supported by the fact that the number of studies dealing with this issue has increased significantly in recent years. Detailed molecular and cellular mechanisms responsible for sex differences are yet to be elucidated; however, it has been stressed that the differences cannot be explained only by the effect of oestrogens. In recent years, a promising new hypothesis has been developed, suggesting that mitochondria may play a significant role in the sex differences in cardiac tolerance to oxygen deprivation. However, one thing is clear already today: sex differences are so important that they should be taken into consideration in clinical practice for the selection of the optimal diagnostic and therapeutic strategy in the treatment of ischaemic heart disease. The present review attempts to summarize the progress in cardiovascular research on sex-related differences in cardiac tolerance to oxygen deprivation during the last 40 years, that is from the first experimental observation. Particular attention was paid to the sex-related differences of the normal heart, sex-dependent tolerance to ischaemia-reperfusion injury, the role of hormones and, finally, to the possible role of cardiac mitochondria in the mechanism of sex-dependent differences in cardiac tolerance to ischaemia/reperfusion injury.

female heart – cardiac hypoxic tolerance – ischaemia-reperfusion injury – sex difference

This study was supported by the Charles University Research Program ‘Cooperation Cardiovascular Sciences’; Ministry of Health, Czech Republic–Conceptual Development of Research Organization, Motol University Hospital, Prague, Czech Republic (No. 00064203); Czech Science Foundation (project No. 24-10497S); project National Institute for Research of Metabolic and Cardiovascular Diseases (Programme EXCELES, ID project No. LX22NPO5104) – Funded by the European Union – Next Generation EU.

Mutations in genes encoding certain myocardial proteins result in hypercontractility of the left ventricle (LV) and are thought to drive pro-hypertrophic signalling in hypertrophic obstructive cardiomyopathy (HOCM). Accordingly, targeting the myosin heavy chain-β by small molecules appears to be an attractive approach to prevent the development of HOCM. Indeed, clinical evidence now supports that modulation of the myosin motor can be an effective treatment option for well-defined HOCM patient groups. Moreover, the potential benefit of myosin inhibitors has also been implicated for patients with heart failure with preserved ejection fraction (HFpEF) in the absence of known genetic alterations. However, it is unclear if cardiomyocytes on different genetic backgrounds respond identically to myosin inhibitors or not. Here, we tested the contractile effects of aficamten, a second-generation myosin inhibitor agent, in cardiac preparations of healthy rodents, dogs and humans. Moreover, these effects were also contrasted to those obtained in permeabilized cardiomyocytes isolated from HOCM patients following myectomy. Aficamten reduced maximal Ca²⁺- activated tensions (Fmax) and evoked robust rightward shifts in the Ca²⁺ sensitivities of isometric force production of permeabilized cardiomyocytes of all sources. Nevertheless, aficamten-induced Ca²⁺ desensitizations were higher in HOCM cardiomyocytes than those in cardiomyocytes of healthy animals or humans. Our results illustrate largely comparable contractile effects upon myosin inhibitor applications, irrespective of the myosin isoform composition or the presence or absence of mutated myocardial proteins in cardiomyocytes of different species. These data also implicate that pro-hypertrophic signalling can also be limited to similar degrees by aficamten in cardiomyocytes on different genetic backgrounds.

myosin inhibitors – aficamten – cardiomyocyte – hypertrophic obstructive cardiomyopathy

This work was supported by a grant from the National Research, Development and Innovation Office (K147173 to ZP). Project no. K147173 has been implemented with the support provided by the Ministry of Culture and Innovation of Hungary from the National Research, Development and Innovation Fund, financed under the K_23 ‘OTKA’ funding scheme.

Pregnancy is a dynamic process associated with significant physiological changes in the cardiovascular system. During pregnancy, there is an increased metabolic demand on the mother and foetus to ensure adequate uteroplacental circulation for foetal growth and development. Insufficient haemodynamic changes can result in maternal and foetal changes to the cardiovascular system. Shift work during pregnancy results in significant changes to the circadian rhythm of both mother and foetus. Furthermore, shift work is linked to increased risk for developing cardiac disease. Therefore, engaging in shift work during pregnancy may adversely affect the cardiovascular health of the mother and have negative implications for the offspring, including increased risk for cardiovascular disease, obesity, and diabetes. While the link between shift work and negative pregnancy outcomes, such as miscarriage, preterm birth and reduced foetal growth, has been previously established, the outcomes of pregnancy on cardiovascular health in shift working mothers are not well understood. Herein, we utilize a night shift work model in mice and provide new evidence that shift work during pregnancy results in changes in genes controlling endothelial function and vascular regulation, coincident with increased cardiac haemodynamic demand. Furthermore, in silico analysis revealed that genes responsible for hemodynamic and endothelial functions are regulated by the circadian genes Clock and BMAL1. The downregulation of these genes following shift work leads to failed vascular response and insufficient metabolic supply to the maternal-foetal system, resulting in placental insufficiency and restricted foetal growth. Our findings provide the first evidence for a direct link between haemodynamic function and circadian regulation during pregnancy. Understanding the key genes and networks that are associated with circadian rhythms and cardiac haemodynamics during pregnancy may contribute to developing new therapeutic strategies to mitigate the adverse effects of shift work during pregnancy.

women's heart health – pregnancy – circadian rhythm – endothelial function

Cardiac adipose tissue plays a crucial role in maintaining healthy cardiac function. It communicates with the myocardium through paracrine signalling and provides free fatty acids for beta-oxidation. However, an increase in cardiac adipose tissue has been associated with a higher risk of arrhythmias. This risk is linked to the release of adipokines, which signal fibroblasts to increase fibrosis and alter ion channel profiles in cardiomyocytes. These changes can lead to prolonged action potentials and re-entry circuits. Additionally, intramyocardial adipocytes can act as conduction blocks, slowing down conduction velocity and further increasing the risk of arrhythmias. In this study, we examine the adipose tissue of human atrial appendages in terms of fibrosis, adipose tissue volume and adipocyte size. The left and right atrial appendages were collected from 40 patients undergoing cardiac surgery at Rigshospitalet, Copenhagen (Denmark). Formalin-fixed and paraffin-embedded tissue was sectioned at 4 μm and stained with Picro Sirius Red. The sections were imaged using the AxioScanZ1 and subjected to automated intelligent image segmentation using the QuPath software in order to quantify fibrosis, adipose tissue and adipocyte size. We found that the content of fibrosis was significantly higher in the right atrial appendage (54.3%) compared to the left (35.9%) (P<0.0001). In opposition, we found that the left appendage had a significantly higher amount of epicardial fat (35.0%) compared to the right (11.5%) (P<0.0001). Infiltration of intramyocardial fat was also found to be higher in the left appendage (0.68%) compared to the right (0.21%) (P=0.02). Furthermore, we also observed that the intramyocardial adipocytes are smaller compared to epicardial adipocytes in the left atrial appendage (990.6 vs 1791.9 μm², respectively, P<0.0001) with a trend in the right appendage (901.5 vs 1214.9 μm², P =0.09). Surprisingly, adipocytes in the left epicardial adipose tissue are larger compared to those in the right epicardial adipose tissue (1791.9 vs 1214.9 μm², P<0.0001). A reason for larger adipocytes observed in the left could be that we see a strong correlation between body mass index and adipocyte size in the left (P=0.0007) that is not found in the right appendage (P=0.65). We show structural differences between the left and right human atrial appendages in terms of fibrosis, epicardial adipose tissue and infiltrating adipocytes. Furthermore, adipocyte size also varies drastically between the atrial appendages, which could be partly explained by the increase in left atrial adipose cell size with body mass index, whereas body mass index has no effect on the right appendage. Future work will leverage the unique opportunity of having both left and right atrial appendages for comparative analysis in our patient cohort. We will first investigate if the structural characteristics have a predictive value for post-operative atrial fibrillations.

atrial fibrillation – cardiac adipose tissue

Patients with occlusive coronary artery disease can be treated by coronary artery bypass surgery (CABG) using cardiopulmonary bypass (CPB) and cardioplegic arrest. However, cardioplegic arrest renders the heart ischaemic and susceptible to reperfusion injury. This injury is augmented by the effects of CPB, which can be largely avoided by performing surgery on a beating heart. However, despite extensive clinical research, adopting beating heart surgery remains controversial. Therefore, an approach to reduce ischaemia reperfusion (I/R) injury associated with cardioplegic arrest remains a major target. A significant number of additives to cardioplegia have thus far failed to translate to clinical practice. Several early studies reported cardioprotection by the general anaesthetic propofol against I/R. Our research aimed to investigate whether propofol cardioplegia is cardioprotective when used during CPB. Work in our unit started in 1996 and showed that the addition of propofol to cardioplegia is protective during cold or warm arrest and in hearts from different species and with different pathologies. Furthermore, CPB surgery on a pig model showed cardioprotection with propofol. Inhibition of the mitochondrial permeability transition pore seems to be the mechanism. Preparations for clinical research started in 2007/8. This involved approval by several regulatory and health agencies/committees. A detailed description of the trial has been published (1). Patient recruitment started in 2010 and finished in 2012. The primary analysis showed that cardiac injury using propofol (6 μg/mL) cardioplegia was not statistically different from that of the intralipid vehicle (2). This inconclusive outcome (P <0.051) promoted an ongoing second trial (3) that involves comparing control cardioplegia (not intralipid) to propofol cardioplegia. Interestingly, the secondary analysis of data provided extensive and seminal information relating to metabolic stress, cardiac injury, pathology and associated molecular changes (e.g. 4).

propofol – cardioplegia – cardiac surgery – ischaemia

Supported by NIHR, BHF, NHRF, BRC and BRU.

Autonomic imbalance is one of the key components in the pathophysiology of heart failure (HF), with an increase in the sympathetic tone and a decrease in parasympathetic activity. Acetylcholinesterase (AChE) inhibitors constitute a large and chemically diverse group of compounds that inhibit AChE. Some of them are used to treat myasthenia gravis, such as pyridostigmine (PYR) or Alzheimer's disease (such as rivastigmine and donepezil), and they have been shown to exhibit some favourable cardiovascular effects. By using the isoprenaline (ISO) model of chronic HF (two doses of 85 mg/kg sc, 24 h apart), we aimed to investigate the potential protective effects of PYR, a peripheral AChE inhibitor. It was administered to Wistar rats in drinking water for 2 weeks at a daily dose of 20 mg/kg, while the control rats drank plain tap water instead. It was shown that PYR caused the attenuation of maladaptive cardiac remodelling, which was noted in histopathological preparations of the myocardium. PYR treatment also led to the preservation of ejection fraction (EF). In the untreated group, EF decreased significantly (from 68.71% to 53.20%), while in the PYR-treated group, EF remained stable (68.76% vs72.10% after treatment). Slowing down of progressive left ventricle dilatation was also noted, with a significant difference in the left ventricular internal diameter end-systole (LVIDs) being increased in the untreated group (from 4.43 mm to 6.8 mm). In contrast, in the PYR-treated group, there was no significant difference between the starting and end values of LVIDs. Similar changes were found in end-systolic volume (ESV), which showed a 259.37% increase in the untreated group.

Thus, by attenuating the decline in systolic function of the left ventricle and slowing the maladaptive cardiac tissue remodelling, PYR represents a promising agent in the treatment of HF.

heart failure – isoprenaline – pyridostigmine – rats – cardiac remodelling

Supported from intramural sources of the Faculty of Medicine, University of Banja Luka.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by increased fat accumulation in liver tissue and is recognized as a hepatic manifestation of metabolic syndrome. Mitochondrial dysfunction and oxidative stress play significant roles in the progression of MASLD. Molecular hydrogen (H2) has been recognized as a medical gas with antioxidant, anti-inflammatory and antiapoptotic effects, and it can also stimulate energy metabolism. This study aimed to evaluate the effect of adjuvant therapy with hydrogen-rich water (HRW) on platelet mitochondrial bioenergetics and endogenous coenzyme Q10 (CoQ10) levels in patients with MASLD. The study included 30 patients with MASLD and 15 healthy volunteers. Over 8 weeks, 17 patients (H2 group) drank water (3 330 mL/day) enriched with HRW tablets (>4 mg/L H2), while 13 patients (P group) drank water with placebo tablets producing CO2. Mitochondrial bioenergetics in platelets isolated from whole blood was assessed using high-resolution respirometry. The concentration of CoQ10-TOTAL in platelets and plasma was determined by HPLC, and the oxidative stress parameter TBARS in plasma was measured spectrophotometrically. Patients with MASLD exhibited relatively higher CI-linked LEAK respiration, lower CI-linked OXPHOS, and reduced CII-linked ET capacity compared to the control group, along with lower CoQ10 concentrations. Plasma TBARS concentration was higher in the H2 group. After 8 weeks of HRW adjuvant therapy, CoQ10 concentration in platelets increased, plasma TBARS decreased, and the impaired parameters of platelet mitochondrial respiration improved. Our results support the evidence that mitochondria are the primary target of H2 therapy. The application of H2 appears to be a promising new strategy for the targeted treatment of mitochondrial disorders. Additional and longer-term studies are needed.

MASLD – hydrogen-rich water – platelets – mitochondrial bioenergetics – CoQ10

This work was supported by grants APVV-19-0317, VEGA 2/0063/18, 2/0092/22, 2/0148/22 and HRW Natural Products Inc.

It is now well established that obesity is a global epidemic and a major health hazard. Obesity is known to contribute to an increase in the risk of cardiovascular disease. Unhealthy dietary practices affect human health, and excessive fructose intake has been linked to metabolic syndrome and the occurrence of obesity and concomitant cardiac dysfunction. This study was undertaken to test the hypothesis that long-term consumption of a high fructose (HF) diet induces cardiac abnormalities during obesity in metabolic syndrome. Accordingly, our investigations, conducted in male SD rats fed an HF (60% w/w) diet plus a moderate level of saturated fat (10% w/w lard) for up to 20 weeks, revealed that systolic blood pressure and serum levels of insulin, triglycerides and total cholesterol were significantly increased in the HF fed group. Furthermore, an increase in visceral fat mass and reduced levels of the anti-inflammatory and anti-atherosclerotic cytokine, IL-10, were observed. Others have demonstrated an increase in cardiomyocytes size, left ventricular wall thickness and reduced ejection fraction and fractional shortening subsequent to high-fructose feeding of mice for 12 weeks. These data suggest that long-term consumption of HF can lead to cardiovascular dysfunction. Thus, an effective intervention to reduce HF intake may attenuate the risk for obesity-related morbidities.

high fructose diet – metabolic syndrome – obesity – cardiovascular abnormalities

The infrastructure support for this study was provided by the St. Boniface Hospital Albrechtsen Research Centre.

Novel cardioprotective antidiabetic medications include treating patients with dual or multiple receptor agonists to have better cardiovascular benefits with consistently reduced major adverse cardiac events, improvement in heart failure, and diminished cardiovascular and all-cause mortality. Especially in recent years in this field, intensive studies have suggested that active incretin hormone glucagon-like peptide-1 amide (GLP-1) and its analogs have cardioprotective effects by modulating cardiomyocyte metabolism. In addition, a glucose-dependent insulinotropic polypeptide (GIP) is a hormone produced in the upper gut and supports the physiological and pharmacological relevance of GIP in metabolic disorders. Tirzepatide (TZPD), as a new generation dual agonist of GIP and GLP-1 receptors, has been shown to have metabolic effects in type 2 diabetic (T2DM) patients, as well as acting as an unbalanced and disproportionate agonist of GIP and GLP-1 receptors, and thereby showing a biased effect on GLP-1 receptors both in experimental animal studies and in cultured cells. In addition to the fact that TZPD administration affects the expression levels of these 2 receptors. It has been suggested that TZPD has a “biased pharmacologic” effect on GLP-1 receptors, and thus TZPD administration with a biased pharmacologic effect has the potential to affect other targets while activating some specific target proteins. Moreover, it has been proposed that GLP-1R activation in pancreatic beta-cells stimulates insulin secretion and activates adenylate cyclase (AC)-mediated cAMP production, cellular Ca2+-regulation, and modulates Gas protein activation. Although there are no clear studies to demonstrate a direct target effect of TZPD administration on heart function, the current data on pancreatic beta-cells strongly suggest a similar pathway is also possible in cardiomyocytes associated with its action. However, it is well accepted that off-target activity is the biological activity of a drug that is different from and not at that of its intended receptor. In some cases, off-target activity can be taken advantage of for therapeutic purposes. Therefore, we aimed to investigate both on-target effects and possible target effects of this drug in cardiomyocytes by considering its action in cAMP signaling. We first demonstrated its two receptors (GIP and GLP-1 receptors) localizations in cardiomyoblast (H9c2 cell line) and responses to TZPD application (40 nM). There was a significantly high response to the TZDP application in GIP-R under hyperglycemia and hyperinsulinemia mimicking cells compared to the GLP-1R. Furthermore, this application also induced a significant increase in the depressed cAMP level parallel to the increases in the expression levels of both depressed β1- and β2-adrenergic receptors. Moreover, the TZDP application significantly decreased the overexpressed β3-adrenergic receptor level in hyperglycemia and hyperinsulinemia mimicking cells parallel to a decrease in increased cGMP level. Our study overall demonstrated that a dual-action receptor agonist antidiabetic drug TZDP can provide both on-target receptor action and an off-target action via the cAMP signaling pathway in cardiomyoblasts under pathological stimuli such as in hyperglycemia and hyperinsulinemia, while both types of actions can include important therapeutic approaches fort he heart dysfunction under pathological conditions.

metabolic heart – antidiabetic drugs – beta-adrenergic receptors – glucose metabolism – mitochondria

This study was supported by The Scientific and Technological Research Council of Turkiye (No. 124Z217).

Although the double and triple hits (i.e., hypertension, diabetes/diet/obesity, ageing) contribute to HFpEF, the contribution of a single-hit high-fat dysbiotic diet (HFD) to HFpEF is unclear. To test whether the HFD causes HFpEF, we administered HFD to normal wild-type (WT, C57) mice and measured HFpEF. Previous studies from our laboratory have observed gut dysbiosis instigating disruption in epigenetic memory (i.e. rhythmic methylation/de-methylation cycle on H3K4, K9, H4K20) via folate 1-carbon metabolism (FOCM) pathways by gene writer (DNMT), eraser (TET/FTO) and homocysteine (Hcy). We observed a link between mitochondrial TCA cycle and gene writer, eraser, and Hcy. A disruption in recycling Hcy back to methionine and impaired clearance by transsulfuration—specifically, mitochondrial sulfur metabolic pathways involving 3MST, CBS, and CSE—leads to a reduction in mitochondrial bioenergetics, ATP, OCR, H₂S, PGC1α, and TFAM, along with an increase in the fission/fusion ratio, ultimately instigating cardiac dysfunction. The hypothesis is that the HFD causes epigenetic dysregulation and contributes to cardiac dysbiosis by HHcy and a decrease in H2S promoting HFpEF. To determine whether the lactobacillus lowers homocysteine and increases H2S, bidirectionally producing folate and lactone/ketone, we created a chronic dysbiotic environment by utilizing WT and DNMT1KO mice, treated w/o HFD and w/o lactobacillus. The in vivo data with lactobacillus show that lactobacillus (a folate and lactone/ketone body producer) mitigated HFD-induced HFpEF and cardiac remodelling. The results indicate that HFD-led HHcy negatively impacts cardiac function. The alteration in 1-carbon metabolism leads to cardiac pathological etiologies. The probiotic treatment, a folic acid and Hcy lowering agent producing probiotic mitigates epigenetic defects and HFpEF. The treatment with Lactobacillus rhamnosus significantly reduced the levels of cardiac MMP-2 and MMP-9, fibrosis and hypertrophy in the hearts of HFD mice, thus mitigating the HFpEF.

HFpEF – microbiota – probiotics – homocystene – epigenetics – 1-carbon metabolism

Immune checkpoint molecules are physiological regulators of the adaptive immune response. Immune checkpoint inhibitors (ICIs), such as anti-PD-1 or anti-CTLA-4 monoclonal antibodies, have revolutionized the treatment of cancer, and their clinical application is rapidly increasing, as reflected by thousands of ongoing clinical trials. However, ICIs cause various immune-related side effects, including acute and chronic cardiovascular toxicities. Among these, ICI-induced acute fulminant myocarditis is the most studied direct cardiotoxicity, although emerging clinical and preclinical data suggest that other ICI-related chronic cardiac toxicities are important, such as accelerated atherosclerosis, heart failure, arrhythmias and additional forms of cardiac dysfunction. These chronic cardiovascular toxicities are often hidden, as they may only occur in the presence of preexisting injuries (ischaemic necrosis, hypertrophy) and comorbidities (metabolic disease). It is therefore of high clinical importance to identify the risk factors and comorbidities that may precipitate ICI-induced cardiotoxicity. The mechanisms of ICI-induced myocarditis and cardiac dysfunction appear to be distinct: myocarditis develops as a specific autoimmune response against cardiac myocytes involving expansion of autoreactive CD8 T cells to cardiac self-antigens (e.g. against alpha myosin heavy chain), whereas cardiac dysfunction/heart failure following ICI therapy is associated with disturbances in cardiac transcriptomic and metabolic effects, likely due to increased expression of pro-inflammatory cytokines. The occurrence of these profound effects on the heart suggests a possible role for immune checkpoint molecules in the maintenance of cardiovascular homeostasis, and therefore, the disruption of physiological immune checkpoint signalling may lead to cardiac diseases, including heart failure.

heart failure – inflammation – immune checkpoint – T cell – cardiotoxicity

Ino-lusitropic therapy would be highly beneficial in heart failure (HF), even at its early stages, but it is underexploited because of the undesirable effects (mainly proarrhythmia) of currently available agents. In the early 2000, we came across a Na⁺/K⁺ pump blocker, named PST2744, in which we detected an ancillary SERCA2a stimulatory effect. After a thorough bench characterization, this compound, now known as istaroxime, has been tested in large animal models and patients with advanced heart failure, showing ino-lusitropic effects and an excellent safety profile (no proarrhythmia). Since then, we have synthetized and validated a series of derivatives that overcome istaroxime limitations: 1) lack of selectivity for SERCA and 2) pharmacokinetics suitable for acute use only. The outcome of this endeavour is a toolbox of ‘pure’ SERCA2a stimulators suitable for chronic HF treatment. My talk will go through the development process, what we have learned from it and where it is going.

SERCA2a stimulators – heart failure – lusitropy – inotropy – drug development

This work was supported by the Windtree Therapeutics, INC. (https://windtreetx.com/) 2021, PI Antonio Zaza - PRIN 2022-NAZ-0595, PI Francesco Lodola.

Periods of biological rhythms in the cardiovascular system (CVS) range from milliseconds to days. Disruption of rhythmicity can have fatal consequences, and understanding physiological and molecular mechanisms is necessary to prevent and treat chronodisruption. Circadian rhythms (about 24 h) are important in CVS functioning. The disrupted daily blood pressure (BP) profile (non-dipping) can increase cardiovascular risk, but underlying mechanisms are not understood. We showed that hypertensive patients with the non-dipping BP profile had lower nighttime melatonin levels than dippers. However, the melatonin profile was not suppressed in transgenic rats with the upregulated renin-angiotensin-aldosterone system [TGR(mRen-2)27], which is accepted as the animal model of non-dipping. Aldosterone concentrations were upregulated, the rhythmicity of sympathetic activity was phase-shifted and expression of clock genes in the area postrema, a sensory transducer between blood and brain, was in antiphase in TGR compared to controls. Artificial light at night (ALAN) has increased dramatically, and epidemiological studies suggest its adverse effects on health, which can be mediated by disruption of circadian rhythms. We explored the consequences of ALAN on cardiovascular parameters in normotensive and spontaneously hypertensive rats. Control rats were kept in a light:dark cycle of 12:12, and ALAN groups were exposed to dim light (~2 lux) during the night. Heart rate, BP and locomotor activity were measured by telemetry, and gene expression in the heart was quantified by qPCR. The power of circadian oscillations in BP and heart rate decreased, and the rhythmic pattern of clock and clock-controlled genes and selected metabolic genes was disturbed by light at night. ALAN eliminated rhythmic expression of Nos3, Pkc1ɛ and glucocorticoid receptors and suppressed amplitude in Pdk4 and Pparγ daily rhythms. Results suggest that ALAN attenuates circadian rhythms in CVS, disrupts the expression of genes controlling heart metabolism and interferes with the ability of the heart to anticipate and compensate for altered workload.

circadian – blood pressure – ALAN – heart – metabolism

This work was supported by APVV-21-0223 and VEGA 1/0309/23.

Poor adherence to lifestyle changes and antihypertensive drugs increases the risk of malignant arrhythmias and heart failure in chronic hypertension. Structural remodelling and altered Cx43 in the myocardium promote instability and dysfunction, but it is unclear if these changes are limited to the left ventricle (LV) or differ by sex. The aim of this study was to investigate key factors involved in the development of malignant arrhythmias and progression to heart failure in both the left and right ventricles of wild-type and hairless spontaneously hypertensive rats (SHR) males and females acclimated to ambient temperature. Notably, 6-month-old male and female wild SHR, hairless SHR, and normotensive Wistar strains were housed at 22°C. Biometric, electrocardiographic, and echocardiographic parameters were recorded. LV and right ventricle (RV) tissues were used for Cx43 immunolabeling, protein level analysis of Cx43, protein kinase C-ɛ (PKC-ɛ), protein kinase C-δ (PKC-δ), and extracellular markers (transforming growth factor-β (TGF-β), SMAD2/3, and matrix metalloproteinase-2 (MMP-2). Susceptibility to electrically inducible ventricular fibrillation (VF) was tested ex vivo in perfused hearts. Corrected QT interval was prolonged in male SHR strains compared to Wistar rats, with a lesser increase in hairless SHR. Ejection fraction was reduced in wild SHR males but not in hairless SHR compared to normotensive Wistar rats. Cx43 downregulation and abnormal topology were seen in the LV of wild SHR males and to a lesser extent in hairless SHR but not in females. Cx43 levels and topology in the RV were consistent across strains and sexes. Phosphorylated Cx43 at ser368 was reduced in the LV but not in the RV of SHR males. MMP2, PKC-ɛ, and PKC-δ levels were higher in the RV compared to the LV in both SHR strains. Hairless SHR males and females had reduced susceptibility to inducible VF compared to wild SHR males. Findings indicate that hypertension-induced pro-arrhythmic changes in Cx43 are primarily associated with the left, but not the right, ventricle in both hypertensive strains. Additionally, acclimation of hairless SHR appears to offer some protection against malignant cardiac arrhythmias and mechanical dysfunction.

hairless SHR – heart – Cx43 – arrhythmias

This study was supported by grants VEGA 2/0133/24, 2/0006/23, 2/0002/20, and APVV 21-0410.

Helichrysum italicum (immortelle) is a Mediterranean plant from the Asteraceae family known for its anti-inflammatory, antioxidant, and antibacterial properties. Traditionally, immortelle essential oil has been used for treating wounds and burns, but scientific validation for these claims is lacking. This study aimed to formulate a hydrogel containing immortelle essential oil and assess its effectiveness in promoting wound healing in excision wounds on streptozotocin-induced diabetic rats. Thirty-two Wistar albino rats with induced diabetes mellitus type 1 were used to evaluate the in vivo wound healing effects of the immortelle hydrogel. Wounds were created 1 week after diabetes confirmation. The rats were anesthetized, and their backs were shaved before creating a 2 cm × 2 cm excision wound using a scalpel and scissors. The rats were randomly divided into the following four groups: Group I (untreated), Group II (hydrogel base), Group III (0.5% immortelle hydrogel), and Group IV (1% silver sulfadiazine, positive control). The effectiveness of the treatments was evaluated through macroscopic and biochemical analyses. The immortelle hydrogel demonstrated the highest wound contraction and the highest hydroxyproline content compared to all examined groups. The immortelle hydrogel achieved significant wound contraction from day 7 to day 21. On day 21, the immortelle group exhibited an average wound contraction of 95.24%; whereas, the untreated and vehicle control groups showed mean wound contractions of 71.36% and 75.14%, respectively. Our findings revealed that the immortelle hydrogel approach might serve as a promising and innovative tool for wound healing.

Helichrysum italicum – essential oil – wound healing – hydrogel

Oxylipins are bioactive lipid molecules that are involved in multiple signalling pathways regulating inflammation, blood flow, or cardioprotection, and form an efficient signalling system across various cells and tissues. However, the understanding of oxylipins and their pathological site-of-action/molecular crosstalk is very limited. Given the activation of inflammatory pathways during ischaemia -reperfusion (I/R) injury, we aimed to investigate the oxylipidome across the systemic circulation to determine the impact of I/R injury on oxylipins in various blood pools. Arterial (Art), venous (Ven), and coronary sinus (CS) blood samples were obtained at baseline, 60 min of ischaemia and 120 min of reperfusion. Oxylipins were measured by LC-MS/MS. We detected 45 oxylipins in plasma. There were neither significant differences in total nor individual oxylipins among Art, Ven, and CS. At 60 min of ischaemia, we detected a significant decrease in total oxylipin levels in Ven and CS blood, as well as altered levels of individual oxylipins (9-HETE, 12-HETE, 12,13-diHOME, and TXB2). Following the reperfusion, TXB2 was significantly decreased. There were no differences between Art and CS blood. In conclusion, there are no significant differences in pig plasma oxylipidome between three blood groups during stable non-pathological conditions. Oxylipin levels were altered during cardiac I/R in Ven and CS blood. Further depiction of more comprehensive oxylipin mechanisms in I/R injury is required.

oxylipins – lipidomics – ischaemia-reperfusion

This study was supported by the Heart and Stroke Foundation of Canada.

Inhibition of a transient receptor potential vanilloid 2 channels (TRPV2) has been described as a promising tool to affect the course and outcome of several diseases, from biomarker role in various types of cancer to modulation of immunological, neurological, and cardiovascular conditions such as heart failure and adverse remodelling. However, the lack of selective inhibitors has been a significant limitation. Therefore, we aimed to characterise the pharmacokinetics of drug probe SET2, the first selective TRPV2 inhibitor. Healthy Wistar rats underwent left carotid artery cannulation for arterial blood access. SET2 pharmacokinetics were assessed following a single intraperitoneal dose (25 mg/kg), with urine excretion monitored for 48 h in metabolic cages. Plasma and urine concentrations were measured using an UHPLC-MS method. Pharmacokinetic parameters such as maximum plasma concentration (Cmax), time to reach maximum plasma concentration (Tmax), biological half-life (T1/2), mean residence time (MRT), and clearance (CL) were analysed by non-compartmental analysis. Acute cardiovascular effects, including electrocardiogram (ECG) and blood pressure (BP), along with plasma markers of cardiac and renal damage, were also evaluated. SET2 reached Tmax at 2 min with a Cmax of 1,428 ng/mL. The estimated T1/2 was 65.1 min, and MRT was 70.5 min. The clearance rate was 0.448 mL/min/kg. Importantly, the compound maintained its inhibitory concentration for nearly 3 h without causing any acute changes in ECG or BP. This study is the first to characterise the pharmacokinetics and acute cardiovascular effects of SET2, demonstrating its safety and favourable pharmacokinetic profile following intraperitoneal administration.

SET2 – TRPV2 inhibitor – pharmacokinetics

This study was supported by grant VEGA 1/0775/21.

Heart failure is a progressive, multifactorial clinical syndrome, leading to development of several life threating arrhythmias such as ventricular fibrillation. A possible mechanism contributed to arrhythmia development in heart failure is the so-called alternans, indicating periodic short-long oscillation of the action potential that leads to repolarisation inhomogeneity. Alternans are considered suitable predictors of sudden cardiac death; however, there is no specific pharmacological intervention to avoid or reduce alternans. In this study, we tested a novel, selective Na⁺/Ca²⁺ exchanger inhibitor ORM-10962 on alternans development in human heart failure and in undiseased human hearts. Left and right ventricular papillary muscles and trabeculae were obtained from explanted hearts of heart failure patients undergoing cardiac transplantation. Undiseased human hearts were obtained from an organ donor after removal of pulmonary and aortic valves for transplant surgery. Action potentials were measured by conventional microelectrode technique. Alternans were evoked by rapid pacing from 500 ms to 190 ms. All experiments were made at 37°C. Results show that ventricular action potential duration (APD) alternans in heart failure were larger than alternans recorded in undiseased ventricle. In failed hearts, left ventricle exerted larger alternans compared to right ventricle. Importantly, failing hearts – especially the left ventricle – often exerted considerable action potential amplitude alternans, typically in higher pacing rates that amplify APD alternans and ultimately lead to 2:1 conduction block. Data indicate that 1 μmol/L ORM-10962 shortens APD in the failed hearts although does not influence APD alternans magnitude, however, delays the development of action potential amplitude alternans and conduction block. These data support animal models claiming increased APD alternans in heart failure. Additionally, human failing hearts often exert large action potential amplitude alternans leading to conduction block that could be an important link between sudden cardiac death and alternans. Selective NCX inhibition may shift the development of amplitude alternans presumably due to APD shortening; however, other Ca²⁺-dependent mechanism could also contribute but these require further experiments.

heart failure – arrhythmia – alternans – Na+/Ca2+ exchanger – ORM-10962

This study was supported by grants TKP2021-EGA-23, RRF-2.3.1-21-2022-00003, K134939, and Nemzeti Tudós Akadémia.

Diazepam, primarily known for its central anxiolytic and muscle relaxant effects, also exhibits vasodilating and hypotensive properties. However, its potential effect on vascular contractility, particularly in the human umbilical artery model, remains relatively unexplored. The aim of this study was to examine the effect of diazepam on human umbilical artery contractility and to identify the mechanisms behind these effects, such as the role of potassium channels, calcium channels and endothelial function. To examine the mechanisms of diazepam-induced relaxation (10⁻⁸–10⁻⁴ M), a series of functional tests were applied to the organ bath system. Human umbilical artery rings were incubated with different selective blockers: 4-Aminopyridine (10⁻⁵ M), TEA (10⁻⁵ M), and glibenclamide (10⁻⁵ M) to examine the role of potassium channels, amlodipine (10⁻⁵ M) to examine the role of voltage-gated calcium channels, and L-NAME (10⁻⁴ M) and indomethacin (10⁻⁴ M) to examine the role of endothelium. Diazepam caused a significant percentage of relaxation of human umbilical arteries (about 90%, expressed in relation to serotonin-induced precontraction). No significance was found in the relaxation responses of diazepam in the presence of potassium channel blockers, as well as endothelium-related relaxation mediators. However, the percentage of contraction of rings treated with BaCl₂ was significantly lower in the presence of diazepam (Emax 9.95%) compared to control rings (Emax 87.358%). The relaxation effects of diazepam on human umbilical arteries indicate its vasodilation potential, which can be significant in conditions of pronounced use of diazepam during pregnancy. The presented functional tests suggest that the relaxation effects of diazepam are primarily mediated through calcium channels, but additional research is needed to explain the exact mechanisms.

diazepam – mechanism – contractility – umbilical arteries

It is well known that cholinergic potentiation by acetylcholinesterase inhibitors (AChEi) has reduced proinflammatory cytokine production and slowed the progression of heart failure. In this study, chronic cholinergic potentiation with the peripherally acting AChEi pyridostigmine (PYR) was studied in male: (a) Normotensive Wistar-Kyoto (WKY) rat, (b) spontaneously hypertensive rat (SHR), a well-studied model of hypertension and cardiac hypertrophy with autonomic nervous system (ANS) imbalance, and (c) SHR-CRP rats, a transgenic SHR strain with systemic inflammation due to expression of human C-reactive protein (CRP). Mean arterial pressure (MAP), cardiac ischaemic tolerance, ANS balance, and splenic cytokine gene expressions were determined in rats chronically treated with PYR at 25 mg/kg/day for 8 weeks in drinking water and untreated controls. In both hypertensive strains, ANS balance was reduced, and PYR treatment markedly improved it by augmenting parasympathetic tone. SHR-CRP rats had significantly higher MAP than SHR (by 15 ± 1 mmHg) regardless of daytime. PYR significantly reduced MAP in SHR and SHR-CRP strains, and this effect was greater in SHR-CRP. Infarct sizes were similar among all groups. PYR treatment slightly (but not significantly) reduced the infarct size normalised to the area at risk in SHR-CRP rats (by 9%). Gene expression of tumour necrosis factor-1α (TNF-1α) was significantly higher in the spleens of control SHR-CRP rats compared to WKY and SHR groups, which were similar levels. Interleukine-10 (Il-10) gene expression was comparable between SHR and SHR-CRP groups, and WKY rats showed higher expression regardless of treatment. Therefore, chronic PYR treatment reduced the TNF-α/Il-10 gene expression ratio in SHR-CRP rat spleens by reducing TNF-α gene expression. In conclusion, our data suggest that chronic peripheral cholinergic potentiation has the potential to significantly slow the progression of cardiovascular diseases associated with systemic inflammation state through immunomodulation.

hypertension – cytokines – ischaemic tolerance

This study was supported by the National Institute for Research of Metabolic and Cardiovascular Diseases (Programme EXCELES, ID Project No. LX22NPO5104) – Funded by the European Union – Next Generation EU.

Despite its extensive traditional use in treating various disorders, the potential of Helichrysum arenarium in preventing cardiovascular diseases remains underexplored. This study aimed to evaluate the effects of H. arenarium (HE) extract on redox status and cardiac function in isolated rat hearts subjected to ischaemia-reperfusion (I-R) injury. Sixteen male Wistar albino rats were divided into the following two groups: Control group and HE group, which received H. arenarium extract orally for 14 days. After this period, hearts from both groups were isolated and subjected to ex vivo I-R injury protocol. Parameters of cardiac function were measured, including the maximum and minimum rates of pressure development, systolic and diastolic left ventricular pressures, and heart rate. Coronary flow was assessed flowmetrically. In plasma samples, levels of superoxide anion radicals, hydrogen peroxide, nitrites, and lipid peroxidation were quantified spectrophotometrically. Additionally, in erythrocyte lysates, the antioxidative defence system was evaluated by measuring reduced glutathione levels and the activities of catalase and superoxide dismutase. Our results indicated that a 14-day intake of H. arenarium extract preserved cardiac contractility and relaxation and prevented the decline of systolic function induced by ischaemia. Furthermore, the extract consumption was associated with an enhanced antioxidative defence activity. These findings suggest that H. arenarium extract possesses cardioprotective properties, although further research is needed to fully understand its therapeutic potential.

H. arenarium – heart – ischaemia-reperfusion

3′,5′-cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger, which acts in nanodomains to regulate cardiac excitation-contraction coupling by activating phosphorylation of Ca²⁺-handling proteins. The sarcoplasmic reticulum (SR) Ca²⁺ATPase2a (SERCA2a) is a key regulator of Ca²⁺ reuptake into the SR during diastole, and thus, alterations of protein phosphorylation in this specific nanodomain could trigger atrial fibrillation (AF)-associated mechanisms of the Ca²⁺-handling leading to arrhythmias. Our aim was to investigate the cAMP-dependent regulation of SERCA2a in the human atrium. A total of 119 atrial myocytes were isolated from tissues of 80 patients in sinus rhythm. Föster-resonance energy transfer (FRET) was used to measure cAMP levels. Cells were transfected with an adenovirus (E1-PLN) to express the phospholamban FRET-based cAMP sensor and cultured during 48 h. Inhibition of phosphodiesterases (PDEs) 1 to 4 promoted a huge increase in cAMP levels in SERCA2a nanodomain. Surprisingly, this increase was reduced to half when a PDE8 inhibitor (PF-04957325, 30 nmol/L) was applied on top. Same tendency was observed when 1 mmol/L Ca²⁺or Bay-K 8644 (an L-type Ca²⁺ channel agonist, 1 mmol/L) were applied on top of 3-isobutyl-1-methylxanthine, while ryanodine receptor inhibition after PF-04957325 increased cAMP levels even further. Notably, 10 μmol/L forskolin was used at the end of the protocol as positive control. Stimulation with the beta-adrenergic agonist isoprenaline (ISO, 100 nmol/L) was also performed in a number of cells before applying the aforementioned PDE inhibitors. This regulation of cAMP levels in SERCA2a nanodomain indicates a possible Ca²⁺-dependent phosphorylation of SERCA2a modulated by L-type Ca²⁺ channel activity.

atrial fibrillation, SERCA2a, cAMP

Myocardial infarction (MI) is the most serious manifestation of coronary artery disease defined as the death of cardiomyocytes due to prolonged ischaemia. Cardiovascular disease is often associated with obesity, which increases inflammation and oxidative stress, negatively affecting the bioavailability of nitric oxide (NO), a key vasodilator synthesized by nitric oxide synthase (NOS). The aim of this study was to examine the effect of a high-fat diet on NO bioavailability in the context of oxidative stress and inflammation after MI. Nine weeks old Wistar Kyoto rats were divided into two groups, one fed high-fat diet while the other continued with standard lab chow for 4 weeks. In week 13, the induction of MI was performed by the reversible ligation of the left descending coronary artery. The occlusion lasted 20 min. Seven days after surgery, animals were sacrificed. Plasma levels of proinflammatory cytokines and superoxide dismutase (SOD) activity were measured using a Bioplex kit and a colorimetric assay using a commercially available kit, respectively. The total NOS activity was assessed by converting [3H]-L-arginine to [3H]-L-citrulline. Protein expression levels of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and nuclear factor kappa B (NFκB) were analysed by Western blot. Concentration of conjugated dienes in the heart was measured spectrophotometrically. The results showed that a high-fat diet significantly decreased total NOS activity and eNOS expression, while conversely increasing the expression of iNOS and inflammatory markers such as tumor necrosis factor-α, interleukin-6, and NFκB. Likewise, the increased concentration of conjugated dienes in the heart and the trend of decreased activity of the antioxidant system SOD demonstrates oxidative stress. This suggests that a high-fat diet negatively affects physiological NO bioavailability after MI and contributes to further myocardial damage via inflammation and oxidative stress.

myocardial infarction – nitric oxide – high-fat diet – oxidative stress – inflammation

This study was supported by grants VEGA 2/0131/24, APVV-22-0271, and VV Bujnová.

Ventricular stiffness is a crucial diagnostic and prognostic indicator of diastolic function, influenced by both physiological and pathological factors. However, assessing ventricular stiffness and diastolic function in a clinical setting remains challenging. Cardiac Multifrequency Magnetic Resonance Elastography (MMRE) has emerged as a promising tool for evaluating stiffness in both left ventricle (LV) and right ventricle (RV). This study aimed to explore the capability of cardiac MMRE to non-invasively quantify the diastolic ventricular stiffness in adult healthy controls (HCs) and its physiological variation with age and sex. We prospectively included 40 HCs who underwent cardiac MMRE. Shear wave speed (SWS) maps were reconstructed for the LV and RV as surrogate for tissue stiffness. No significant correlations were found between age and SWS in either the LV (R² = 0.076, P = 0.090) or RV (R² = 0.086, P = 0.070) across the entire HC cohort. However, an interaction effect between age and sex was identified. Specifically, female exhibited a significant increase in ventricular stiffness with advancing age in both the LV (R² = 0.2573, P = 0.0377) and RV (R² = 0.3550, P = 0.0116); whereas, male did not demonstrate a similar age-related stiffness progression (R² = 0.0104, P = 0.6510 for LV and R² = 0.0109, P = 0.6433 for RV). Ventricular stiffening with natural ageing affects female but not male. Cardiac MMRE shows promise as a clinical tool for the non-invasive quantification of diastolic ventricular stiffness.

magnetic resonance elastography – ventricular stiffness – MMRE – ageing – sex

Magnolol (MAGNO) is a hydroxylated biphenyl compound, isolated from the bark of Magnolia officinalis. This product, as a traditional Chinese medicine, demonstrated potential resources for natural antioxidants to treat a variety of disorders via scavenging of free radicals, protecting DNA, providing growth performance, and disease resistance at the cellular and system levels in mammalians. Since previous studies have shown that magnolia bark extract administration provided a significant antioxidant effect in isolated rats, protected the myocardium against ischaemic injury, and suppressed ventricular arrhythmia, in this study, we aimed to evaluate the cardioprotective effects of administration of MAGNO (100 mg/kg; a cumulative dose of 15 mg/kg in 2-week) in Doxorubicin (DOX)-treated rat hearts. One group of adult male Wistar rats was injected with DOX (DOX-group) or saline (CON-group), while other group of DOX-treated rats was administered with MAGNO before DOX (Pre-MAGNO group) or after DOX (Post-MAGNO group). The MAGNO administration, before or after DOXO, provided full survival of animals during 12–14 weeks and significant recoveries in the systemic parameters of animals such as high systolic and diastolic blood pressures. This treatment also significantly improved heart function including recoveries in end-diastolic volume, left ventricular end-systolic volume, heart rate, and cardiac output. Furthermore, the MAGNO administrations improved the structure of left ventricles such as recoveries in loss of myofibrils, degenerative nuclear changes, fragmentation of cardiomyocytes, and interstitial oedema, while all of these benefits were observed in the pre-MAGNO treatment group, more prominently. Overall, one can point out the beneficial antioxidant effects of MAGNO in chronic heart diseases as a supporting and complementing agent to conventional therapies.

magnolol (MAGNO) – doxorubicin (DOX)

This study was supported by the Lokman Hekim University Scientific Research Projects Coordination Unit. Project No. 202AP404, 2022.

Flecainide and other class Ic antiarrhythmic drugs (AADs) are widely used in Andersen-Tawil syndrome type 1 (ATS1) patients with life-threatening arrhythmias but structurally normal hearts. However, class Ic AADs may fail and exhibit proarrhythmic effect in some ATS1 patients through mechanisms that remain poorly understood. We aimed to provide novel insight into the molecular mechanisms of AAD-induced arrhythmias and whether they might increase the risk of life-threatening ventricular arrhythmias and sudden cardiac death (SCD). A review of data from 48 ATS1 patients reported in the literature showed that 50% of patients responded partially to flecainide, with ventricular arrhythmias (VA) reduction in only 18.2% and of those patients, VA persisted in 20%–50%. Flecainide was ineffective in 29.5% of patients, and of those, 13.5% suffered a non-fatal cardiac arrest. In AAV-mediated cardiac-specific mouse models expressing five different mutations (Kir2.1Δ314-315, Kir2.1C122Y, Kir2.1G215D, Kir2.1R67W, and Kir2.1S136F), treatment with flecainide or propafenone (40 mg/kg) differentially altered the cardiac conduction velocity by prolonging the duration of the P wave, PR, QRS, and QTc intervals compared to Kir2.1WT, with Kir2.1S136F showing milder effects. Flecainide also increased VA inducibility in all mutant mice except Kir2.1S136F, which exhibited a significant reduction in VA. Voltage-clamp studies revealed a mutation-dependent effect on inward rectifier K⁺ (IK1) and Na⁺(INa) current densities. Kir2.1G215D cardiomyocytes showed a higher impact on IK1, followed by Kir2.1C122Y, Kir2.1R67W, and Kir2.1S136F. Additionally, Kir2.1C122Y cardiomyocytes had a significant decrease in INa. Superfusion of flecainide (10 μmol/L) slightly increased IK1 density in Kir2.1WT and Kir2.1S136F, while Kir2.1C122Y and Kir2.1R67W showed a decrease in both IK1 and INa densities, despite normal trafficking of mutant channels. Optical mapping experiments in ATS1 patient-specific iPSC-CM monolayers expressing Kir2.1C122Y, Kir2.1G215D, and Kir2.1R67W mutations showed an increase in rotor incidence at baseline and under flecainide treatment, confirming the drug's proarrhythmic effect. Lastly, in silico molecular docking studies predict that the Kir2.1-Cys311 pharmacophore binding site is altered in Kir2.1C122Y heterotetramers, reducing flecainide accessibility and leading to channel closure and arrhythmias. Class Ic AADs are only partially effective and might be proarrhythmic in some ATS1 patients. Our findings indicate that the efficacy of AADs may vary depending on the specific ATS1 mutation. We submit that mutations impacting both the resting membrane potential and cellular excitability potentially create a substrate for life-threatening arrhythmias. These data raise concern about the widespread use of class Ic AADs in ATS1 patients, urgently calling for further mechanistic studies to advance personalised therapy.

arrhythmias – sudden cardiac death – ion channel diseases – antiarrhythmic drugs – flecainide

This study was supported by the National heart, Lung and Blood Institute, NIH grant number R01HL163943; La Caixa Banking Foundation project code HR18-00304 (LCF/PR/HR19/52160013); grants PI-FIS-2020 # PI20/01220 and PI-FIS-2023 # PI23/01039 from Instituto de Salud Carlos III (ISCIII) and co-funded by Fondo Europeo de Desarrollo Regional (FEDER); and by the European Union, respectively; grant PID2020-116935RB-I00 and BFU2016-75144-R funded by MCIN/AEI/10.13039/501100011033; Fundación La Marató de TV3 (736/C/2020) ‘amb el suport de la Fundació La Marató de TV3’; CIBERCV (CB16/11/00458; CB/11/00222 to CV); European Union's Horizon 2020 grant agreement GA-965286; and Program S2022/BMD7229 -CM ARCADIA-CM funded by Comunidad de Madrid to JJ.

Growing evidence suggests that rigorous exercise regimens in elite athletes increase the risk of arrhythmias, including ventricular fibrillation, which can lead to sudden cardiac death. Our results have demonstrated that 4 months of sustained, high-intensity endurance training in dogs prolonged the corrected QT interval and lengthened APD90, accompanied by a decrease in transient outward potassium current [I(to)] density. This training also increased repolarisation variability and decreased the resting heart rate. Left ventricular hypertrophy developed in trained dogs. Compared to the sedentary dogs, trained dogs exhibited significantly higher level of fibrosis in the left ventricle, which may contribute to an increased ectopic activity and heightened arrhythmia susceptibility. In this study, we have examined the expression of ion channel subunits potentially involved in cardiac arrhythmic remodelling induced by sustained, high-intensity endurance training. Western blot and immunocytochemistry were used to analyse the expression and density of I(to) subunit Kv4.3 and KChiP2 in left ventricular tissue and isolated cardiomyocytes from both sedentary and trained dogs. Hyperpolarization-activated cyclic nucleotide-gated channels (HCN)-1, HCN2, and HCN4 were immunolabeled in left ventricular cardiomyocytes, and the ultrarapid delayed-rectifying potassium current [I(Kur)] subunit Kv1.5 expression was measured in atrial cells. High-intensity training did not alter Kv4.3 or KChiP2 expression. Interestingly, HCN4 density significantly increased in trained dogs (10391 ± 616 a.u.; n = 30 cells/6 dogs) compared to sedentary dogs (6453 ± 1149 a.u.; n = 30 cells/6 dogs). The expression of HCN1 and HCN2 remained unchanged. Kv1.5 fluorescence was significantly reduced in the trained group (2654 ± 559 a.u.; n = 12 cells/4 dogs) compared to the sedentary group (5190 ± 442 a.u.; n = 12 cells/4 dogs). Unchanged Kv4.3 and KChiP2 expression suggests that other auxiliary subunits may influence I(to) function. Sustained, high-intensity endurance training increased HCN4 channel expression in the ventricular myocardium, which, along with increased fibrosis, may contribute to the heightened arrhythmia sensitivity. Reduced atrial Kv1.5 expression, by altering APD, might contribute to the electrical remodelling.

arrhythmia – ion channels – endurance training

This work was supported by the National Research Development and Innovation Office (NKFIH K 135464, K 142738, K-147212, GINOP-2.3.2.-15-2016-0006, GINOP-2.3.2.-15-2016-00047 GINOP-2.3.2.-15-2016-00040, and TKP2021-EGA-32), the Ministry of Human Capacities Hungary (EFOP-3.6.2-16-2017-00006), the Albert Szent-Györgyi Medical School institutional grant (SZTE AOK-KKA 2021 and SZTE AOK-KKA 2022), and by Hungarian Research Network (HUN-REN TKI project). This study was also supported by Project RRF-2.3.1-21-2022-00003 ‘National Heart Laboratory, Hungary’ implemented with the support provided by the European Union and by the Pharmaceutical and Medical Device Developments Competence Centre of the Life Sciences Cluster of the Centre of Excellence for Interdisciplinary Research, Development and Innovation of the University of Szeged, Hungary.

Untreated fresh cardiac tissue is the optimal tissue material for investigating DNA methylation patterns of cardiac biology and diseases. However, fresh tissue is difficult to obtain. Therefore, tissue stored as frozen or formalin-fixed, paraffin-embedded (FFPE) is widely used for DNA methylation studies. It is unknown whether storage conditions alter the DNA methylation in cardiac tissue. In this study, we compared the DNA methylation patterns of fresh, frozen, and FFPE cardiac tissue to investigate if the storage method affected the DNA methylation results. We used the Infinium MethylationEPIC assay to obtain genome-wide methylation levels in fresh, frozen, and FFPE tissues from nine individuals. We found that the DNA methylation levels of 21.4% of the examined CpG sites were overestimated in the FFPE samples compared to that of fresh and frozen tissues, whereas 5.7% were underestimated. Duplicate analyses of the DNA methylation patterns showed high reproducibility (precision) for frozen and FFPE tissues. In conclusion, we found that frozen and FFPE tissues gave reproducible DNA methylation results and that frozen and fresh tissues gave similar results.

DNA methylation – cardiac tissue – epigenetics – tissue storage

This project was internally funded by the Department of Forensic Medicine.

The precise effect of acute unilateral damage of cardiac vagus nerve (CVN) on the spontaneous electrical activity of contralateral vagus nerve at the cardiac level is not well comprehended. We examined the transient neuroelectrical response to a transection of the right or left cardiac vagal nerve (VNTx) by measuring the spiking activity of each CVN. Fourteen male Gottingen minipigs were subjected to sternotomy. Multiple electrode cuffs were placed below the cut level to capture vagal electroneurographic signals while monitoring electrocardiogram and hemodynamics, both before and immediately after cardiac vagotomy (left: L-cut, n = 6; right: R-cut, n = 8). The results showed that left cardiac VNTx drastically decreased the firing rate of multi-unit electrical activity (MUA) in the vagal stump and the intact right VN at the heart level. These reductions were observed without any impact on heart rate, heart rate variability, or hemodynamics. On the contrary, right cardiac VNTx did not immediately change the MUA in either VN, but it did modestly raise (P < 0.022) the root mean square of consecutive RR interval differences (rMSSD), which is a measure of parasympathetic outflow, without impacting hemodynamics. In conclusion, our findings reveal an early left-lateralized pattern in vagal spiking activity after unilateral cardiac vagotomy. These results improve comprehension of the neuroelectrical reaction to vagal damage and offer valuable considerations for maintaining vagal outflow following unilateral cardiac vagotomy. The rapid changes in physiological electrical activity along the intact right VN at the cardiac level can also serve as early predictors of left vagal fatigue.

vagus nerve – heart function – ageing

This study was supported by H2020-FETPROACT-2018-2020 NEUHEART project #824071.

Ossabaw minipigs differ from Göttingen minipigs in their unique genetic predisposition to develop a full metabolic syndrome and their non-responsiveness to cardioprotective interventions, even before the development of their diseased phenotype. Previous DNA sequencing data revealed differences in a cluster of mitochondrial genes between the two pig strains. Obviously, alterations in their protein composition affect the function of mitochondria, i.e., Krebs cycle, oxidative phosphorylation, mitochondrial biogenesis, and mitochondrial dysfunction play a crucial role in the development of metabolic syndrome as well as in myocardial ischaemia-reperfusion injury and cardioprotection.

Aim: To compare the cardiac mitochondrial proteome of adult, lean Ossabaw minipigs with that of adult, and lean Göttingen mini-pigs.

Mitochondria were isolated from cardiac left ventricular tissue samples of both pig strains (n = 4, each) by differential centrifugation and density-gradient ultracentrifugation. The mitochondrial proteome was analysed by LC-MS/MS. Proteins were classified as of mitochondrial origin and intra-mitochondrial function according to MitoCarta3.0 and Beyond MitoCarta.

A total of 810 proteins were identified in at least three of four samples, and 654 were classified as mitochondrial proteins. Among them, 22 proteins were differentially expressed between both pig strains. Most of them were classified as related to mitochondrial metabolism (9 higher/7 lower expressed in Ossabaw minipigs), four (two higher/two lower expressed) as related to mitochondrial transcription and translation, and three as related to small molecule transport (one higher/two lower expressed), two as related to oxidative phosphorylation (one higher/one lower expressed), and one as related to dynamic and surveillance (lower expressed).

The mitochondrial proteome is largely comparable between Ossabaw and Göttingen minipigs. However, the proteins that were differentially expressed are related to mitochondrial metabolism, which play a crucial role in the development of metabolic syndrome and ischaemic heart disease. In the next step, the results of the proteomic approach must be validated via protein biochemistry.

cardioprotection – metabolic syndrome – genetic predisposition – mitochondrial proteome

This work was supported by the German Research Foundation [SFB 1116 B08 to G.H. and P.K.] and the European COST ACTION in CARDIOPROTECTION [CA16225 to G.H. and IG16225 to G.H. and P.K.]. S.I., L.K. and A.S. acknowledge the support by the‘Ministerium für Kultur und Wissenschaft des Landes Nordrhein-Westfalen’ and ‘Der Regierende Bürgermeister von Berlin, Senatskanzlei Wissenschaft und Forschung’.

In severe cases of COVID-19, autoantibodies appear in the circulation, targeting lung and heart tissues. The pathophysiological role and long-term effects of these antibodies remain unknown, and there is little information on the transient or permanent persistence or detectability of these antibodies. Lung-specific autoantibodies developed during COVID-19 may reappear or increase in titre due to tissue damage accompanying lung surgery, which may affect the wound healing and recovery from surgery. The objective of this study was to investigate the long-term behaviour of anti-cardiac and anti-lung autoantibodies in patients who had recovered from COVID-19. Autoantibodies were detected by a Western blot-based method in patients who had undergone lung surgery for various reasons with partial lung resection; all relevant clinical data were also collected. Patients who underwent lung surgery (n = 20; females n = 13 and males n = 7) were recruited to assess the presence of anti-cardiac and anti-lung autoantibodies. The average age was 59.91 ± 10.86 years. Among the 20 patients, 9 had a previous COVID-19 infection, where we expected to observe autoantibodies. The remaining 11 patients served as controls. Autoantibodies developed during a COVID-19 infection were still present, but no new autoantibody appeared during our investigation. We observed no reactivation of existing autoantibodies. The possible reason behind this may be that the lung tissue damage during the thoracic surgery was not as extensive (lactate dehydrogenase (LDH) before surgery 208.47 ± 54.88, LDH after surgery 217.61 ± 51.64 U/L, P = 0.6057), which might not have been a sufficient trigger for the immune system. Autoantibodies are less likely to reactivate after lung surgery because of the lower degree of tissue damage, but existing autoantibodies persistently remain.

autoantibodies – anti-cardiac autoantibodies – anti-lung autoantibodies – COVID-19 – SARS-CoV-2

This study was supported by the EKÖP-24-3-II-DE-302 (E.E.E.), EKÖP-24-3-II-DE-277 (A.Á.Sz.) University Research Scholarship Program (EKÖP) of the Ministry of Culture and Innovation from the source of the National Research, Development and Innovation Fund.

Ischaemic heart disease represents a major ailment leading to heart remodelling, cardiac hypertrophy, and subsequent heart failure. Cardioprotective adaptive interventions can mitigate the deleterious consequences of ischaemia/reperfusion (I/R) injury to the heart. Intermittent hypoxic preconditioning (IHPC) is a relatively well-established cardioprotective non-pharmacological intervention in various models, such as cardiac injury and I/R injury in particular. However, its mode of action is currently not completely understood. The study aimed to investigate the mechanisms and cellular cardioprotective signalling of specific IHPC protocol in rat hearts. For this purpose, we subjected the male Wistar rats of age 3 months to IHPC (10% O2, normal pressure) for 8 h once per week for four weeks. Subsequently, the rat hearts were isolated for Langendorff perfusion and subjected to 20-min stabilization, 30-min ischaemia, and 40-min of reperfusion to evaluate post-ischaemic contractile dysfunction and reperfusion arrhythmias and to harvest the tissue samples for analysis by WB and mass spectrometry. The recovery of contractile function after I/R injury was significantly increased in IHPC animals compared to non-IHPC controls. Overall activity of reperfusion injury salvage kinase (RISK) pathway components was increased in IHPC hearts, suggesting the role in cellular cardioprotective signalling mediated by intermittent IHPC. On the contrary, the activity of survivor activating factor enhancement (SEFE) pathway components were non-affected in IHPC hearts compared to controls. The cardioprotective effect of intermittent IHPC in rat hearts can be mediated by the upregulation of RISK but not SAFE pathway signalization.

heart – cardioprotection – hypoxic preconditioning – ischaemia/reperfusion injury

This study was supported by grants APVV-19-0540; APVV-22-0264; VEGA 2/104/22; and VEGA 1/0775/21.

Cardiovascular system diseases belong to the most prevalent civilization diseases. To date, several cardioprotective effects of quercetin (QCT) have been confirmed in young healthy individuals. The aim of our work was to reveal the effects of QCT on cardiovascular system in elderly animals with type 2 diabetes. In this study, QCT was orally administered for 6 weeks at a dose of 20 mg/kg/day to 6-month-old/1-year-old lean (fa/+) and obese (fa/fa) Zucker diabetic fatty (ZDF) rats. Blood pressure measurement and echocardiography were performed. Oxidative stress markers were analysed from blood plasma, and the expression of the selected proteins were monitored in myocardium. Isolated hearts of ZDF rats were subjected to I/R (30 min/120 min). The total collagen content was determined in the left ventricle. Our data showed that administration of QCT significantly reduced blood pressure in 6-month-old rats, while it had no effect in 1-year-old rats. QCT significantly increased the antioxidant activity parameter in blood plasma of obese 1-year-old rats. Administration of QCT did not have cardioprotective effects against I/R damage in any group. Complex activation of the RISK pathway was not induced by QCT. In 1-year-old obese ZDF rats, administration of QCT normalized the E/A ratio (marker of diastolic dysfunction), reduced the thickness of the left ventricular wall, and significantly decreased the collagen content. QCT has been shown to have beneficial effects on blood pressure in a model of type 2 diabetes, but increasing age and/or development of diabetes impair its antihypertensive effects. In the prevention of I/R damage, QCT was not effective in both ages. Potential explanation of failing of QCT cardioprotection in increasing age and diabetes may be the failure to activate the RISK pathway. On the contrary, QCT suppressed diabetes-induced diastolic dysfunction and reduced the collagen content in the left ventricle in elderly individuals.

quercetin – diabetes mellitus – cardioprotection – ageing

This study was supported by grants APVV-21-0194, VEGA 1/0775/21, VEGA 1/0775/21, APVV-20-0242, and VEGA 2/0148/22.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a highly arrhythmogenic syndrome triggered by stress, primarily linked to gain-of-function point mutations in the cardiac ryanodine receptor (RyR2). This Ca²⁺ release channel in the sarcoplasmic reticulum (SR) plays a fundamental role in the Ca²⁺ cycling essential for heart contraction. CPVT is effectively suppressed by flecainide. This antiarrhythmic agent is a known inhibitor of the surface-membrane Na⁺channel while also inhibiting the intracellular RyR2 channel. Initially, the flecainide-RyR2 interaction was considered therapeutically irrelevant since RyR2 activity was affected only when the channel conducted ions in the cytosol-to-lumen direction, opposite to physiological SR Ca²⁺ release. This statement has been revisited as the RyR2 channel is able to carry charge-compensating countercurrent in the cytosol-to-lumen direction, and reducing this current may in fact suppress pathologically increased SR Ca²⁺ release. Monitoring single-channel properties, we examined whether flecainide can target the RyR2-mediated countercurrent. Additionally, we assessed the impact of flecainide on the SR Cl-channel as it may also play a role in conducting a charge-compensating countercurrent. RyR2 and Cl- channels isolated from ventricles of the rat heart were incorporated into planar lipid membranes (BLMs) and examined under voltage-clamp conditions. Flecainide was added to the RyR2 cytosolic face in a dose-dependent manner, while Ca²⁺ current flowed via the channel pore from lumen to cytosol, and the countercurrent carried by Tris + cations was in the opposite direction. These conditions more closely mimic the cell situation. The activity and conductive properties of the Cl- channel were assessed with a Cl- gradient applied across the BLM. We demonstrate that flecainide significantly reduced the RyR2-mediated countercurrent without affecting channel activity. In contrast, the SR Cl- channel was completely resistant to flecainide. Based on these findings, it is reasonable to propose that the primary intracellular target of flecainide in vivo is the RyR2-mediated countercurrent.

CPVT – cardiac arrhythmia – ryanodine receptor – flecainide

This study was supported by grants VEGA 2/0018/21 and APVV-22-0061.

Thromboembolic disorders represent a significant cause of mortality leading to life threatening conditions such as myocardial infarction, pulmonary embolism, and deep vein thrombosis. Factor Xa (fXa) plays a key role in blood coagulation cascade and thrombus formation. It is a serine protease that catalyses hydrolysis of prothrombin into thrombin, which further promote conversion of fibrinogen into fibrin, a final stage of blood clotting. In recent years, fXa has been identified as a promising target for the design of efficient and safer antithrombotics, compared to conventional warfarin therapy. Today, several (fXa) inhibitors has been approved, such as rivaroxaban, apixaban, betrixaban, edoxaban, and fondaparinux, but research in this area is still in the focus of pharmaceutical industry. To identify the most important structural determinants that influence fXa inhibitory activity, 3D-quantitative structure–activity relationship (QSAR) studies were performed on dataset consisting of 54 structural analogues of betrixaban. Experimentally determined IC50 activities (expressed as pIC50) were collected from literature, and all structures were previously optimized using the semiempirical PM3 method. The 3D-QSAR model was created using partial least squares regression analysis. Internal (R² = 0.93; Q² = 0.75) and external (R²pred = 0.66) validation parameters confirmed that the model can be used for prediction of fXa inhibitory activity. The 3D-QSAR model revealed most important variables that influence activity (distances between H-bond donors, acceptors, hydrophobic, and steric regions), and this information will be further used for the design of new selective fXa inhibitors.

3D-QSAR – Thromboembolic disorders – Factor Xa inhibitors – Betrixaban analogues

The authors did not receive any financial support from any organization for the submitted work.

Cardiovascular diseases are the leading cause of morbidity and mortality worldwide, imposing significant health and economic burdens on the individual, families, and societies. Accordingly, a substantial effort is exerted for finding novel effective therapeutics for these diseases. Recently, inhibitors of the poly(ADP-ribose) polymerase (PARPi), originally developed for cancer therapy, show promise for repurposing in cardiovascular diseases. These drugs' cytoprotective, antioxidant, and anti-inflammatory properties can be pivotal in treating heart conditions characterised by tissue damage and inflammation. Studies suggest that PARPi could protect cardiac cells from ischaemia-reperfusion injury and reduce adverse remodelling post-myocardial infarction. Additionally, their anti-inflammatory properties might mitigate atherosclerosis progression. Although prolonged administration of PARPi could lead to malignant transformation due to DNA damage accumulation, short-term application of these drugs may be beneficial in post-infarct cardiac remodelling. We are presenting experimental evidence for the use of PARPi in the aforementioned conditions, and we provide mechanistic background for the protective effects of PARPi including the effects of these drugs on protein kinase B/Akt and mitogen-activated protein kinase signalling.

PARP inhibitors – Akt, MAPK – repurposing – cardiovascular diseases

Clinical studies suggest an increased incidence of atrial fibrillation (AF) in endurance athletes. However, the underlying mechanisms of AF associated with endurance training are not understood. In this study, we examined the electrophysiological role of pulmonary veins in a canine model of high-intensity endurance exercise. Beagle dogs were randomized into sedentary group (SG, n = 7) and trained group (TG, n = 7). The TG dogs ran on a treadmill for 280 min a day for 16 weeks. At termination, epicardial atrial stimulation, concomitant pulmonary vein (PV) monophasic action potential (MAP), and multielectrode-array (MEA) mapping of the left atrial (LA)-PV junction were performed on opened-chest animals. Atrial fibrillation was induced by 10 s long 800/min atrial burst stimulation in the presence and absence of 5 μg/kg acetylcholine (ACh). Ex vivo studies on LA-PV preparations paced at 2 Hz and 4 Hz in the presence or absence of 1 μmol/L isoproterenol were also carried out to examine spontaneous and pacing-induced AP generation and conduction properties of PVs and LA-PV junction in both groups. Atrial APD90 was significantly shorter in TG than in SG (115.2 ± 20.2 ms vs 159.2 ± 34.5 ms P < 0.05). Compared to SG, TG animals showed significantly higher occurrence (6 out of 7 vs 2 out of 7 animals, P < 0.05) and log duration (0.85 ± 18 vs 0.25 ± 0.17 P < 0.05) of burst stimulation-induced AF in the presence of Ach. The duration of PV-MAP showed no significant difference between groups, even in the presence of Ach. However, the direction of conducted stimuli recorded by MEA on PV-LA junction differed significantly between groups. Compared to SG, LA-PV preparations isolated from TG showed a higher occurrence of spontaneous AP-generating activity (4 out of 6 vs 0 out of 6 preparations, P < 0.05) in the presence of 1 μmol/L isoproterenol. The spontaneous AP activities in TG were also significantly longer and exhibited higher frequency than in SG. In this study, we demonstrated the increased susceptibility of athletes to AF. The increased propensity for AF may be related to increased PV firing pattern and altered conduction properties of the PV-LA junction.

atrial fibrillation – athletes heart – cardiac electrophysiology – pulmonary vein

Recent studies have highlighted the receptor-interacting protein kinase 3 (RIP3) as a critical regulator of necroptosis, oxidative stress, and inflammation in various tissues, including the heart. This study investigates the pleiotropic effects of RIP3 inhibition under ischaemic conditions, focusing on its role in myocardial protection. Utilising various models of ischaemia-reperfusion injury (IRI) ex vivo (30-min I, 10-min R) and in vivo (1-h I, 7 days-R), we administered a specific RIP3 inhibitor – GSK'872 during the whole respective R period. Under ex vivo conditions, RIP3 inhibition significantly reduced myocardial necrosis-like cell death evidenced by decreased LDH activity after IRI; however, without altering the expression of the main pro-necroptotic proteins – pRIP3 and pMLKL. Interestingly, although this intervention reversed the IRI-induced increase in the extent and rate of mitochondrial swelling, it did not affect the level of RIP3-associated mitochondrial proteins – PGAM5 and pDrp1. In an in vivo IRI model, a 7-day administration of GSK'872 prevented the likely formation of the necroptosis, but not pyroptosis-associated membrane pores, proven by decreased pRIP3 and pMLKL and unchanged NT-GSDMD expression, respectively. Furthermore, RIP3 inhibition was associated with a likely reduction of mitochondrial damage based on the PGAM5 and pDrp1 expression limitation. Interestingly, consistently with human patients with an ischaemic insult to the heart, such rats also exhibited elevated plasma RIP3 levels, while GSK'872 mitigated such an increase. However, such intervention was unable to preserve cardiac function since it did not affect any echocardiographic parameters after IRI. This research indicates RIP3 as a promising target for cardiovascular protection and recovery following ischaemic events and underscores the potential of RIP3 inhibition as a therapeutic strategy for mitigating post-ischaemic cardiac damage.

RIP3 – myocardial ischaemia/reperfusion – mitochondria – cardioprotection

This study was supported by grants APVV-20-0242; APPV-15-0607, and VEGA 1/0016/20.

Cell loss due to necrosis-like cell death is a crucial mechanism underlying the pathogenesis of heart failure (HF). However, it is unknown whether a role of various types of programmed necrosis differs depending on the HF aetiology. In this study, we aimed to examine necroptosis and pyroptosis in failing hearts due to myocardial infarction (MI) or pressure overload induced genetically or surgically. The effects of a pharmacological inhibitor of receptor-interacting protein kinase 3 (RIP3), a molecule connecting both these pro-death pathways, were also studied. As a clinical translational approach targeting necrosis-like events, a plasma screening of certain circulating molecules in the human HF subjects was performed. All three HF models showed active necroptosis as documented by the increased activation of RIP3 and mixed lineage kinase domain-like pseudokinase (MLKL). On the contrary, only hearts failing due to MI were positive for the markers of pyroptosis (NLR family pyrin domain containing 3, caspase-1, gasdermin D, and interleukin-1β). In post-MI HF, the RIP3 inhibitor was able to reduce the activation of both RIP3 and MLKL, advocating for its protective effect against necroptosis, but it had no effect on pyroptosis and did not modulate the compromised cardiac function. A two-fold increase in the serum levels of high mobility group box 1 (HMGB1) was found in HF patients regardless of the HF aetiology or severity, while they positively correlated with the serum C-reactive protein. This is the first study showing that different necrosis-like cell death pathways likely underlie the development of HF of various origins. The RIP3 inhibition mitigates the necroptotic cell loss in post-MI HF, highlighting its potential clinical perspective in HF management. The serum HMGB1 was suggested as a novel promising marker in the HF diagnosis.

necroptosis – pyroptosis – heart failure – cell death

This study was supported by grants VEGA 1/0016/20, APVV-20-0242, APPV-15-0607, NU21J-02-00039, and LX22NPO5104.

A lot of work was done dealing with insulin regulation of cardiomyocyte function. However, little is known about the remodelling of morphology and ionic homeostasis of ventricular heart cells. The non-essential amino acid taurine, which is present in seafood and produced by the heart and the brain, contributes to the excitation-contraction coupling of cardiomyocytes. However, whether taurine prevents the remodelling of heart cells is to be explored. In the first series of experiments, we studied the effect of chronic high concentrations of insulin in hypertrophy and intracellular concentrations of sodium and calcium in cultured adult rat heart cells using quantitative 3D confocal microscopy. In the second series of experiments, we verified whether taurine prevents insulin-induced remodelling of cardiomyocytes. In this study, we show that treatment with a high concentration of insulin induces an increase in the volume of ventricular cardiomyocytes that is associated with an increase of cytosolic and nuclear sodium and calcium as well as a decrease in the ratio of pCREB/tCREB. Long-term treatment with taurine prevented insulin-induced remodelling of ventricular cardiomyocytes. This effect of taurine was prevented by the taurine-sodium symporter blocker. In conclusion, our results show that insulin induces ventricular cardiomyocyte hypertrophy via downregulation of the pCREB/tCREB level and that chronic taurine treatment prevents this effect.

taurine – insulin – cardiomyocytes – hypertrophy – CREB

This work was funded by NSERC and CHIR.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder affecting women of reproductive age, often linked to a range of cardiometabolic complications, including obesity, insulin resistance, and dyslipidemia. Despite the high incidence of these metabolic disturbances, the underlying mechanisms remain poorly understood. Dietary approaches that enhance antioxidant levels by incorporating polyphenol-rich foods may offer promising therapeutic options for the prevention of chronic metabolic diseases. This study aimed to evaluate the effects of standardised Aronia extract (SEA) and metformin (MET) on metabolic regulation in a rat model of PCOS. This research involved female Wistar albino rats, aged 6 weeks, with body weights ranging from 150 g to 170 g. PCOS was induced through subcutaneous administration of dehydroepiandrosterone during 5 weeks. Following successful confirmation of PCOS, the rats were categorized into the following four treatment groups: PCOS + distilled water, PCOS + MET, PCOS + SEA, and PCOS + MET + SEA. Results demonstrated that treatment with MET and SEA, both independently and in combination, significantly decreased final body weight, ovarian weight, total cholesterol, triglycerides, low-density lipoprotein (LDL), glycaemia, and insulin levels, while increasing high-density lipoprotein (HDL) levels compared to the PCOS control group. Additionally, the PCOS group showed a prolonged diestrus phase in the estrous cycle; whereas, the other treatment groups exhibited regular estrous cycles lasting 4–5 days. Cystic follicles significantly decreased, while corpora lutea significantly increased in all treated groups vs untreated PCOS. Furthermore, both SEA and MET activated AMPK; staining intensity was weakest in the PCOS group, significantly higher in all treated groups. These findings indicate that SEA, whether used alone or alongside MET, alleviated metabolic, such as lipid metabolism and glycoregulation, as well as reproductive disturbances in the rat model of PCOS at least by AMPK activation. Future clinical studies will be supported by these findings, providing a strong foundation for exploring therapeutic interventions in PCOS.

polycystic ovary syndrome – metformin – Aronia Melanocarpa L. – lipid status – glycoregulation

Diabetic nephropathy is a complication of diabetes mellitus type 2 (DM2) that affects transmembrane enzymes such as Na,K-ATPase (NKA) and angiotensin-converting enzyme (ACE)-2. This study aimed to assess the effect of hyperglycaemia and quercetin (QCT) on these enzymes and the degree of renal tissue fibrosis. We used 6-month-old obese Zucker diabetic fatty (ZDF) rats as the model of DM2 and included lean ZDF rats as the control group. QCT (20 mg/kg/day) was orally administered to the rats for 6 weeks. The ZDF model exhibited varying glucose concentrations, resulting in the categorisation into groups with lower (9–11 mmol/L, D-↓, DQ-↓) and higher (> 11 mmol/L, D-↑, DQ-↑) hyperglycaemic levels. Glucose levels were positively correlated with both the Vmax value of NKA and hydroxyproline (a marker of fibrosis). We observed a significant upregulation in the expression levels of NKA (α1, β1) in the D-↓ group relative to the control group. However, this alteration did not significantly impact the expression of ACE2. QCT reduced the NKA activity in both control and DQ-↓ rats. The DQ-↑ group showed an increase in NKA and ACE2 expression compared to DQ-↓, while D-↑ had significantly lower NKA and ACE2 expression than D-↓. Administration of QCT reduced hydroxyproline in the DQ-group more significantly than in the DQ-↓ group. Administration of QCT to lean control ZDF rats and obese diabetic rats with a milder form of hyperglycaemia partially reduced NKA activity but mainly ACE2 and NKA expression. On the contrary, administration of QCT to obese diabetic rats with such severe hyperglycaemia significantly increased the expression of both enzymes, ACE2 and NKA. We found that QCT administration to obese ZDF rats had an anti-fibrotic effect regardless of the degree of hyperglycaemia, which we consider positive.

diabetes mellitus type 2 – Na,K-ATPase – ACE2 – renal fibrosis – quercetin

This project was supported by grants VEGA: 2/0148/22, 2/0104/20, 2/0159/24, 2/0123/24, APVV-20-0421, and APVV-21-0194.

Lichens represent a distinctive symbiotic association between fungi (mycobiont) and algae (photobiont). While lichens have been employed in traditional medicine and specific secondary metabolites have been explored for their effects, research on their cardioprotective properties remains limited. This study investigated the potential cardioprotective effects of the acetonic extract of Xanthoparmelia stenophylla (XSA) lichen in a rat model of doxorubicin-induced cardiotoxicity. The lichen sample was collected from the Stara Planina region. The acetone extract of XSA was prepared using cold maceration. This study involved 40 male Wistar albino rats, with the extract administered orally at a dose of 125 mg/kg for 28 days. Following the treatment, doxorubicin was administered intraperitoneally at a cumulative dose of 15 mg/kg. Three days after the final dose of doxorubicin, the hearts were isolated and examined ex vivo using the Langendorff apparatus. Coronary venous effluent samples were collected to assess oxidative stress markers via spectrophotometric analysis. Additionally, myocardial tissue samples were analysed using histological staining with haematoxylin and eosin. The administration of XSA at a dose of 125 mg/kg for 28 days significantly reduced oxidative stress parameters in the coronary venous effluent in a model of doxorubicin-induced cardiotoxicity. Furthermore, the architecture of the myocardial tissue was preserved, and degenerative changes were reduced in the group treated with XSA, indicating its potential as a cardioprotective agent. Our findings demonstrate that XSA possesses significant cardioprotective and antioxidant properties, suggesting its potential utility as a cardioprotective agent. Further investigation is required to elucidate the underlying mechanisms through which XSA or some of its ingredients exert its effects'.

lichen – Xanthoparmelia stenophylla – oxidative stress – doxorubicin – cardiotoxicity

This work was supported by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Agreement No: 451-03-65/2024-03/200111 and 451-03-65/2024-03/200124)

Despite many improving in medical procedures and techniques, heart failure (HF) still remains a global health issue with high rates of mortality, morbidity, and reduced quality of life. Among the key mechanisms implicated in HF development are oxidative stress and inflammation, resulting in myocardial damage, blood congestion, and poor tissue perfusion. Molecular hydrogen (H₂) has emerged as a potential therapeutic agent mainly due to its strong antioxidant and anti-inflammatory effects. Intraperitoneal administration of isoproterenol is considered a reliable and proven model of HF induction in experimental animals. In this study, six-month-old male Wistar rats were divided into the following four groups: Control (C), isoproterenol-induced HF (ISO, 50 mg/kg for 5 consecutive days), HF with H₂ treatment (ISO + H₂, inhalation of 4% H₂-enriched air for 30 min daily for 6 weeks), and HF with vitamin C treatment (ISO + C, 75 mg/kg in drinking water ad libitum). Biochemical markers such as lipid profile, glucose, aspartate aminotransferase (AST), alanine transaminase (ALT), and uric acid were colorimetrically analysed in blood plasma, while the expression of pro-inflammatory proteins, such as interleukins (IL-6), tumour necrosis factor alpha (TNF-α), and nuclear factor kappa B (NF-κB) in the heart tissue was assessed using the Western blot method. Isoproterenol administration led to a non-significant rise in uric acid, ALT, and AST levels, alongside a significant increase in blood glucose. In contrast, mostly H₂ treatment slightly reduced all these parameters, approaching values seen in the control group. Expression of inflammatory proteins was notably elevated in the ISO group, while both treatment groups exhibited reduced protein levels, especially H₂ treatment showing a more substantial decrease. Conclusion: H₂ treatment attenuated inflammatory protein expression and improved biochemical parameters in ISO-induced HF, suggesting its therapeutic potential in cardiovascular diseases. These findings indicate that H₂ inhalation offers a promising approach for novel HF treatment strategies.

heart failure – isoprotenerol – molecular hydrogen – oxidative stress

This study was supported by grants VEGA 2/0092/22 and APVV 19-0317

Cardiovascular disease (CVD) remains a leading cause of mortality and disability worldwide. Myocardial infarction (MI), a critical manifestation of CVD, arises from an imbalance between myocardial oxygen supply and demand. Isoprenaline (ISO), a synthetic non-selective β-adrenergic receptor agonist, has been utilized in experimental models to induce myocardial infarction in rats at high doses by disrupting the balance between free radical production and antioxidant defences. Urolithins, derived from the intestinal enzymatic breakdown of ellagic acid (EA), are notable gut metabolites. Among these, urolithin A is the most extensively studied and is recognized for its significant health benefits, including anti-inflammatory, anti-cancer, and antioxidant effects. Despite these promising findings, the specific effects of urolithin A on oxidative stress and myocardial injury in the isoprenaline rat model have not been thoroughly investigated. The aim of this study was to explore the cardioprotective effects of urolithin A against isoprenaline-induced myocardial injury in rats. All the animals were divided into the following four groups: control group (Con) (n = 10) received DMSO i.p. for 7 days + saline s.c. on days 6 and 7. Urolithin A group (UrA) (n = 10) received urolithin A 5 mg/kg i.p. for 7 days + saline s.c. on days 6 and 7. The isoprenaline group (ISO) (n = 10) received DMSO i.p. for 7 days + isoprenaline 85 mg/kg s.c. on days 6 and 7. Rats in urolithin A + isoprenaline group (UrA + ISO) (n = 10) received urolithin A 5 mg/kg i.p. dissolved in DMSO for 7 days + isoprenaline 85 mg/kg s.c. on days 6 and 7. Urolithin A pretreatment reduced isoprenaline-induced oxidative stress, by lowering nitrite (NO₂^-) and hydrogen peroxide (H₂O₂) levels and increasing catalase (CAT) and superoxide dismutase (SOD) and reduced glutathione (GSH) levels. The histological evaluation of heart tissue also confirmed that urolithin A had protective effects on isoprenaline-induced myocardial injury preventing interstitial oedema, inflammation, bleeding, and cardiomyocyte fragmentation. Urolithin A pretreatment has a significant antioxidative and anti-inflammatory impact in the isoprenaline-induced myocardial injury.

cardioprotection – urolithin A – isoprenaline-induced myocardial injury – oxidative stress

Hydrogen gas has demonstrated selective antioxidant properties, specifically against hydroxyl radicals, and possesses both antioxidant and anti-inflammatory effects. This study explored the therapeutic potential of hydrogen in a monocrotaline-induced model of pulmonary hypertension, which is characterized by oxidative stress—a common feature in cardiovascular diseases. Male Wistar rats were administered a subcutaneous injection of a water-alcohol solution containing monocrotaline to induce pulmonary hypertension or a control solution. One group of monocrotaline-treated rats was exposed to 4% molecular hydrogen in atmospheric air, while other groups were ventilated with standard atmospheric air. After 21 days, hemodynamic parameters, including systemic and pulmonary pressures, were measured under urethane narcosis. The results indicated that hydrogen inhalation did not significantly alter the primary markers of pulmonary hypertension. However, there was a significant reduction in systemic blood pressure, particularly in the systolic component. Furthermore, hydrogen exposure led to a decrease in TGF-β expression and a reduction in tryptase-positive mast cells, suggesting anti-inflammatory and vascular protective effects. These findings highlight hydrogen's potential as a therapeutic agent in mitigating cardiovascular complications associated with oxidative stress and inflammation.

hydrogen gas inhalation – ROS – pulmonary hypertension – inflammation – mast cells

This study was supported by the Russian Science Foundation, grant number 22-13-00111 (accessed on 11 October 2023) (Oleg Medvedev).

Atrial fibrillation (AF) is a common arrhythmia in both horses and humans, sustained by electrical, structural, and metabolic changes that compromise treatment efficacy. This study investigates the molecular and electrophysiological remodelling associated with chronic AF and evaluates the potential of metformin, an indirect activator of AMP-activated protein kinase (AMPK), in mitigating these pathological changes. Twenty retired racehorses underwent AF induction via two weeks of right atrial tachypacing and were randomized to receive either metformin (n = 10) or placebo (n = 10). Four additional sham-operated controls were maintained in sinus rhythm. Baseline effective refractory periods (ERPs) were measured, and right atrial (RA) biopsies were collected. After four months, bi-atrial epicardial mapping was performed for AF complexity analysis, and ERPs were measured again. Terminal biopsies were collected from both atria. We employed data-independent acquisition mass spectrometry and RNA-sequencing on atrial biopsies to compare expression profiles between groups. The metformin-treated horses required significantly longer tachypacing to achieve self-sustained AF compared to controls. After four months of AF, metformin-treated horses had longer RA ERPs and AF cycle length (AFCL) but demonstrated fewer RA re-entries. Integrative transcriptomic and proteomic analyses revealed right atrial-specific alterations in ion channel gene expression, along with changes in metabolic pathways and the extracellular matrix proteome in metformin-treated horses. These molecular changes were associated with increased activation of AMPKα2. Metformin delayed early AF stabilization and reduced right atrial AF complexity after 4 months of chronic AF in a horse model. These findings highlight the potential of metformin as a cost-effective, upstream therapy for AF prevention.

The project was funded by the Danish Independent Research Foundation (grant number 1032-00053B). SLH was funded by a research grant from the Danish Cardiovascular Academy, which is funded by the Novo Nordisk Foundation (grant number NNF20SA0067242) and the Danish Heart Foundation. MJS was funded by a research grant from the Danish Cardiovascular Academy, which is funded by the Novo Nordisk Foundation (grant number PhD 2022002-HF) and the Danish Heart Foundation.

This study aimed to analyse the vascular reactivity of great saphenous vein (GSV) segments harvested using conventional, endoscopic, and No-Touch (NT) techniques in coronary artery bypass grafting (CABG). A total of 416 vein rings from 41 patients were studied, including 136 rings from the conventional technique (without perivascular fat), 104 from the NT technique (with perivascular fat), and 176 from the endoscopic technique (without perivascular fat). Vascular reactivity was evaluated based on the contractile response to phenylephrine, and nitric oxide (NO) bioavailability was assessed using the NO synthase inhibitor L-NAME. Results showed that vascular reactivity was significantly lower in the NT technique compared to the conventional (CV) and endoscopic (EVH) methods. The application of L-NAME revealed that the NT technique resulted in lower vascular contraction due to the influence of perivascular fat, which promoted vascular relaxation. NO detection was significantly higher in the NT technique, while the CV and EVH techniques showed limited NO presence and no significant change after L-NAME application. Morphological analysis confirmed the presence of perivascular fat in the NT technique and vessel stretching in the CV and EVH techniques due to hydraulic distention. In conclusion, the NT technique demonstrated reduced vasoconstrictive response and greater NO bioavailability, suggesting better vascular function and potentially lower risks of acute graft thrombosis compared to the CV and EVH techniques. The findings support the benefits of preserving perivascular fat in improving graft outcomes in CABG.

myocardial revascularization – saphenous vein – vascular endothelium – adipose tissue – blood vessels

Doxorubicin (DOX) is an anticancer drug with toxic effects on normal cells. Important role in DOX-induced cytotoxicity plays in increased production of reactive oxygen species (ROS) and oxidative stress. Increased ROS production can also induce autophagy, process playing an important role in maintaining normal cellular homeostasis through degradation of macromolecules. Autophagy can have a dual role in the cellular responses to pathological conditions. It may represent a beneficial adaptive response to stress but can also lead to maladaptive responses, which are linked to disease pathogenesis and cell-death induction. It has been found that excessive autophagy may lead to cell death. Several proteins, such as Beclin-1, p62, LC3A/B, and AuTophaGy-related (Atg), play an essential role in the regulation of autophagosome formation and the proper functioning of autophagy. To investigate the influence of short- and long-term exposure of HEK293 cells to effects of DOX on proteins involved in autophagy regulation. Human embryonic kidney cells HEK293 were used in studies. The cells were exposed to the effects of DOX for 3 h or 21 h. Protein samples obtained after cell lysis were used for the determination of levels of specific proteins using Western blot analysis. We found that DOX induced time-dependent changes in protein levels of studied autophagy markers. Prolonged exposure of HEK293 cells to 2.5 μmol/L DOX was associated with significant up-regulation of all studied autophagy markers (Atg5, Atg12, LC3A/B, and Beclin-1). Similar to the effects observed with long-term cell exposure to doxorubicin, Atg5 increases after 3-h of exposure to 2.5 μmol/L to DOX. Doxorubicin induced also an increase in LC3A/B, Atg5, and Atg3 protein levels after 3 h, with the most pronounced effects observed at a DOX concentration of 1 μmol/L. The results point to a significant role of proteins involved in autophagy processes in the mechanisms of DOX action on HEK293 cells. The effects of DOX were for some autophagy markers depended on the concentration of used DOX and the duration of cell exposure to this cytostatic agent.

autophagy – doxorubicin – oxidative stress

This study was supported by grants VEGA 2/0169/24 and APVV-18-0548.

Left ventricular (LV) dilatation following myocardial infarction (MI) is an important predictor of prognosis and indicates which patients may eventually develop heart failure (HF). The hallmark of systolic HF is a reduced LV ejection fraction (LVEF) due to impaired LV contractility, which can occur after MI as a result of cardiac remodelling and LV dilatation. Up-regulation of lncRNA Plasmacytoma Variant Translocation 1 (PVT1) has been observed in hypertrophy and myocardial fibrosis, and circulating PVT1 is a potential diagnostic biomarker for chronic HF. The aim of this preliminary study was to investigate the potential effects of the PVT1 gene variant rs4410871 on the parameters of LV structure and function, i.e., LV end-systolic volume (LVESV), LV end-diastolic volume (LVEDV), and LVEF, in patients after first acute MI. This study included 126 patients. The rs4410871 C > T variant was genotyped using Applied Biosystems TaqMan® technology. Statistical analyses were performed using Statistica 8 software. LVESV and LVEDV values > 62 mL/m² and > 156 mL/m² in men and > 47 mL/m² and > 121 mL/m² in women, respectively, were defined as dilatation. Patients with LVEF < 50% were defined as having impaired LVEF, i.e., systolic dysfunction. We found that genotypes carrying the T allele according to the dominant model (CC vs CT + TT) were significantly more frequent in patients with dilated LVESV (55.7%) than in patients without dilatation (37.5%) (adjusted OR = 2.18, 95% CI = 1.05–4.55, P = 0.04). The same genotypes were more frequent in patients with impaired LVEF (52.9%) than in patients with normal and borderline low LVEF (30.8%) (adjusted OR = 2.56, 95% CI = 1.01–6.48, P = 0.04). OR was adjusted for gender. rs4410871 was not associated with LVEDV. Our preliminary results suggest that PVT1 rs4410871 may be associated with LV dilatation and systolic dysfunction after MI. Further research is warranted.

LncRNA PVT1 – rs4410871 – myocardial infarction – LVESV – LVEF

This study was funded by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Agreement No. 451-03-66/2024-03/200017).

An arteriovenous fistula (AVF) is a surgically created connection between an artery and a vein that is used to provide vascular access for haemodialysis in patients with end-stage renal disease. Compared to a central venous catheter (CVC), AVF offers numerous benefits, including lower infection rates, reduced thrombosis, and improved patient survival. However, AVF is also associated with issues such as maturation failure, posing a significant medical challenge. AVF surgery triggers a complex interplay of cellular processes, including inflammation, proliferation, and differentiation. Muscle dissection and adjacent structural changes during AVF creation may have paracrine effects via vascular cuffing and inflammation, potentially influencing AVF maturation. Myogenic Differentiation 1 (MYOD1) is a key regulator of muscle differentiation, and its expression increases after muscle injury. This study aimed to examine the expression of MYOD1 and pro-inflammatory genes and proteins in an AVF swine model and assess the effect of a TREM-1 antagonist on their expression and AVF maturation. AVF was surgically created between the femoral artery (FA) and femoral vein (FV) using a side-to-side anastomosis in Yucatan miniswine. Swine were randomly divided into the following three groups: (I) Control with no treatment, (II) treatment with the TREM-1 antagonist, LR12 peptide, and (III) treatment with a scrambled peptide. LR12 or the scrambled peptide was administered at the AVF site. Three months after AVF creation and the application of LR12 or scrambled peptide, FA, FV, and surrounding tissues were harvested for histology, immunostaining, and qPCR to analyse the expression of various molecules, including MYOD1, MMP9, NF-κB, and TGFβ3. AVF maturation was assessed by ultrasound and angiography. Increased expression of mRNA transcripts and proteins for MYOD1, MMP9, NF-κB, and TGFβ3 was observed in the control and scrambled peptide groups. LR12 treatment significantly reduced the expression of all these molecules and was associated with less fibrosis and improved AVF patency, as revealed by ultrasound and angiography. The findings of this study suggest that targeting perivascular cuffing and muscle injury during AVF creation may offer potential therapeutic targets or biomarkers for early fibrosis in AVF maturation failure.

arteriovenous fistula – muscle injury – perivascular cuffing – impaired maturation – fibrosis

I am recipient of the Martin Morad Young Investigator Exchange Award.

Regulated forms of necrosis-like cellular demise have been shown to contribute to cardiac ischaemia/reperfusion (I/R) injury. Nonetheless, the underlying mechanisms occurring in the very first minutes of cardiac I/R remain partially elucidated. Thus, this study was conducted to provide a comprehensive protein screening focusing on pro-pyroptotic and mitochondrial damage-associated events. In Langendorff-perfused rat hearts subjected to 30 min I followed by ultrashort 10 min R. The proteomics was examined utilising liquid chromatography with mass spectrometry (LC-MS/MS) and immunoblotting techniques. Furthermore, calcium-induced mitochondrial swelling and lactate dehydrogenase (LDH) release were assessed to prove mitochondrial stress and necrosis-like phenotype, respectively. An assay determining adenosine 5′-triphosphatase (ATPase) activity was used to assess energy metabolism and overall mitochondrial function. About 160 proteins related to cell-death pathways, cellular metabolism, and post-translational modifications exhibited significant differential expression in hearts affected by such a short R. In spite of the signs of the plasma membrane structure disruption, conventional proteins involved in pyroptotic signalling, both canonical and non-canonical, were unchanged by this R interval. Importantly, this brief I/R was linked to the upregulation of p25 fragment of poly (ADP-ribose) polymerase 1 (PARP1 p25) and mature apoptosis-inducing factor (AIF) proteins, alongside nitrosative stress and mitochondrial swelling, while ATPase activity was lower. Conversely, a receptor-interacting protein kinase 3 (RIP3) inhibitor (GSK'872, 250 nmol/L) mitigated the elevation in PARP1 p25 and AIF expression, as well as prevented such a reduction of ATPase activity due to short R in previously ischaemic hearts. This study showed for the first time that during early I/R, necrosis-like injury is likely associated with RIP3-mediated mitochondrial, non-pyroptotic events, while the inhibition of RIP3 confers cardioprotective effects.

cell death – inflammation – myocardial ischaemia/reperfusion – pyroptosis – receptor-interacting protein kinase 3 inhibition

This study was supported by grants VEGA1/0016/20, APVV-20-0242, APVV-22-0264, and VEGA 2/0016/23

The isoprenaline (ISO) model of heart failure is a long-standing model. However, since its introduction in 1965, a wide range of approaches has been implemented. With the array of used doses and different application durations, various heart failure or myocardial damage models have been derived. This raises a challenge of how to choose the appropriate dose and duration, as well as the question of comparability and reproducibility of the results. This study aimed to compare time-related functional and structural changes of the ISO-induced model of chronic heart failure. Male Wistar rats, weighing 300–350 g, were used. The baseline echocardiogram was performed, and afterwards, an 85 mg/kg/day dose of ISO was applied subcutaneously for two consecutive days. Repeated echocardiograms and histopathology samples were obtained. At the 2-week time point, significant changes in left ventricular internal diameter end diastole and end systole (LVIDd and LVIDs) and end-diastolic and end-systolic volume (EDV and ESV) were noted. At the same time point, histological examination showed inflammatory infiltrate with initial fibrotic changes. However, no significant change in ejection fraction (EF) was found. After an additional week, further progression of heart failure was noted; however, there was no significance when compared to the two-week time point, and therefore, no samples for histopathology were obtained. Five weeks after the ISO application, the echocardiographic assessment showed a further increase of LVIDd, LVIDs, EDV, and ESV, as well as a significant reduction of the ejection fraction (< 55%). Furthermore, histological examination showed the progression of the fibrotic changes in the myocardium. Based on the results, a five-week period was found to be sufficient for the development of chronic heart failure using the isoprenaline model of 85 mg/kg/day subcutaneously for two consecutive days.

heart failure – isoprenaline – rat – echocardiography

Atrial fibrillation (AF) is the most common arrhythmia in humans, affecting 59 million people worldwide. It is difficult to predict AF since initial symptoms are mild and passing. Once the disease progresses, symptoms become more prominent, and treatment is initiated. These challenges highlight the importance of researching the underlying mechanisms leading to AF, to start treatment earlier and improve quality of life. Studies show that the hearts of AF patients are characterised by remodelling of the tissue, and with the use of biomarkers, this remodelling can be examined. Based on preliminary results, the biomarker periostin is suggested as a new existing marker for structural remodelling in the heart. The project aimed to determine if periostin levels in heart biopsies and blood samples from patients undergoing open heart surgery can help predict AF. Heart biopsies and blood samples from 17 patients were included and grouped according to whether the patients developed post-operative AF (POAF) or stayed in sinus rhythm (SR) (SR: n = 9, POAF: n = 8). Heart biopsies from right atrial appendages (RAA) and blood samples (serum) were included in a Western blot and ELISA analysis, respectively. A correlation calculation between biopsies from RAA and blood samples is to be performed with the hypothesis that blood analysis alone can help predict AF through more time-effective and less invasive analysis than to biopsies analysis. Heart biopsies from RAA showed significantly elevated periostin levels in the POAF group compared to SR (SR: 0.78 ± 0.37, POAF: 1.39 ± 0.61 P = 0.022). Results from the ELISA showed that serum samples from POAF and SR patients have similar periostin levels (SR: 18.43 ± 8.22 ng/mL, POAF: 16.63 ± 5.71 ng/mL, P = 0.66). Periostin levels in RAA tissue are significantly upregulated in POAF patients compared to SR. Similar periostin levels were found in the blood samples of both groups.

atrial fibrillation – atrial fibrosis – biomarkers – periostin – human

Elevated plasma levels of homocysteine (HCY) represent a risk factor for the development of several cardiovascular diseases. Better understanding of mechanisms how HCY can induce endothelial dysfunction are needed for prevention and treatment of these diseases. The aim of study was to investigate negative effects of HCY in the following two models: (1) Human endothelial cells isolated from the vein of human umbilical cord (HUVEC) and (2) hybrid immortalised endothelial cells (EA.hy926). We treated these endothelial cells with the indoleamine melatonin to attenuate HCY-induced endothelial damage. The cell viability was significantly decreased after 48 h HCY treatment in dose-dependent manner (1–6 mmol/L) in HUVEC. In contrast, only higher doses of HCY (4–6 mmol/L) decreased the viability of EA.hy926 cells. HCY significantly inhibited cell proliferation in HUVEC, and this negative effect was reversed by cotreatment with melatonin. Surprisingly, proliferation of EA.hy926 cells increased after 1–3 mmol/L HCY, and coincubation with melatonin did not influence cell proliferation. Morphology and cytoskeletal remodelling were analysed after 48 h exposure to 5 mmol/L HCY by immunocytochemistry. HUVEC and EA.hy926 responded differently to the treatment, and their morphology was dramatically changed compared to control cells. We observed an altered actin cytoskeleton and reduced cytoplasm in both types of endothelial cells. However, actin remodelling and structure change were not prevented by melatonin treatment. Based on results, we can conclude that both models of endothelial cells respond to hyperhomocysteinemia in partially different ways, and EA.hy926 cells are less sensitive. Findings highlight the importance of considering the specific characteristics of both models. The role of melatonin as a protective agent against hyperhomocysteinemia requires further investigation, especially in relation to its differential effects in various endothelial cell types.

homocysteine – endothelial cells – endothelial dysfunction – melatonin

This study was supported by grant APVV-21-0223.

Maternal-offspring interaction, behaviour, and the quantity and quality of maternal milk during early postnatal period can significantly impact the risk of cardiovascular disease later in life. In our study, hypertensive homozygous transgenic (mRen2) 27 rats (TGR) and normotensive Hannover Sprague-Dawley (HanSD) rats were used, with litters culled to eight pups per dam and cross-fostered, resulting in the following four groups: HanSD male pups fostered with HanSD mothers (HanSD), HanSD male pups fostered with TGR mothers (HanSDx), TGR male pups fostered with TGR mothers (TGR), and TGR male pups fostered with HanSD mothers (TGRx). Post-weaning, we implanted telemetry to measure blood pressure (BP) and heart rate in 5 to 12-week-old offspring. BP and heart rate were consistently higher in TGR than in HanSD offspring at all ages, regardless of fostering. On the base of heart rate beat-to-beat variability (marker of the autonomic nervous system), we found that TGR had a higher LF/HF ratio, indicating increased sympathetic activity, while HanSD showed a developmental decrease in LF/HF ratios into adulthood. Cross-fostering partially restored the normal decreasing LF/HF ratios in TGRx. At week 12, a salt load (2%) was introduced to assess its effects on BP and renal function. TGRx exhibited exaggerated BP responses to salt load, and cross-fostering in this strain accelerated renal injury. Cross-fostering did not prevent genetically determined hypertension in TGRx, although it reduced sympathetic activity. TGRx showed increased BP responses to salt, more severe renal damage, and altered sodium handling, possibly influenced by differences in the ionic composition of maternal milk between hypertensive and normotensive mothers. These findings highlight the importance of early maternal factors in hypertension development.

hypertension – cross-fostering – telemetry – early life programming – rats

This study was supported by APVV-17-0178, APVV-21-0223, and ACCORD (the Operation Program of Integrated Infrastructure for the project, Advancing University Capacity and Competence in Research, Development and Innovation, ITMS2014 + : 313021 × 329, co-financed by the European Regional Development Fund) projects.

Influence of natural polyphenol resveratrol on cardiorenal injuries induced by malignant hypertension (MH) has not been completely elucidated. The aim of this study was to examine long-term resveratrol treatment on cardiac and kidney structure, glomerular filtration, oxidative stress, and antioxidant defence in MH rats.

This study was performed on 6-month-old female spontaneously hypertensive rats (SHRs) with induced MH. MH was induced by 4-week L-NAME (10 mg/kg/day) treatment. Resveratrol groups (R) received resveratrol (10 mg/kg/day) during the same period. Animals were randomly divided into the following four groups: SHR (n = 10); SHR + R (n = 10); MHR (n = 10): SHR rats treated with L-NAME; and MHR + R (n = 10): SHR rats treated with the combination of L-NAME and resveratrol.

Results: Resveratrol significantly reduced blood pressure as well as plasma creatinine, urine protein, and urine protein/creatinine ratio in both SHR + R and MHR + R groups. Besides, chronic resveratrol treatment significantly reduced total oxidant status and prooxidant-antioxidant balance as well as increased total antioxidant capacity in the SHR + R. A similar trend was observed in the MHR + R group. Renal histopathological examination showed decreased tubular epithelial cell swelling and degeneration in the MHR + R, followed by lower renal damage score. Inhibited inflammation in the heart tissue of both SHR + R and MHR + R as well as attenuated Bax and increased Bcl-2 expression in these groups revealed anti-inflammatory/apoptotic effects of resveratrol.

Resveratrol attenuates heart and kidney damages in MH rats at the same way but less intensive, than in SHR, mainly due to its antioxidant activity. This promotes it as therapeutic agent against cardiorenal damages induced by malignant hypertension.

experimental hypertension – resveratrol – reactive oxygen species

This study was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Grant No. 451-03-68/2022-14/200015.

Lemon balm (Melissa officinalis) is a well-regarded medicinal plant known for its therapeutic benefits, including potent antioxidant and anti-inflammatory effects that may offer cardioprotection. This study explores the cardioprotective potential of an ethanolic extract of lemon balm in mitigating doxorubicin-induced cardiotoxicity in a rat model, with a particular focus on potential sex-based differences. In this study, thirty Wistar albino rats (8–10 weeks old, 160 ± 20g), including both males and females, were divided into the following three groups: A control group of untreated, healthy rats; an experimental group receiving doxorubicin (15 mg/kg i.p.); and an experimental group treated with both doxorubicin and an ethanolic extract of lemon balm (200 mg/kg/os). The lemon balm treatment lasted for 10 days, with doxorubicin administered on the eighth day of treatment. Doxorubicin administration led to a reduction in fractional shortening (FS) and ejection fraction (EF) compared to the control group. However, the addition of lemon balm to doxorubicin treatment notably improved FS and EF values in both male and female rats. Additionally, doxorubicin increased intraventricular septum diameter in diastole (IVSDd), left ventricular internal diameter in systole (LVIDs), and left ventricular end-systolic volume (LVESV) in both sexes. The concurrent administration of lemon balm counteracted these increases, bringing the values closer to those seen in the control group. Furthermore, the combination of lemon balm and doxorubicin was associated with a significant enhancement in total antioxidant capacity, as demonstrated by elevated levels of reduced glutathione and increased superoxide dismutase activity. These results highlight the potential cardioprotective effects of lemon balm extract against doxorubicin-induced cardiotoxicity, with a particular emphasis on the somewhat more pronounced cardioprotective effects observed in female rats.

lemon balm – cardiotoxicity – doxorubicin – antioxidants capacity – sex differences

Xention/Servier pharmaceutical companies developed XEN-D0103 as a selective inhibitor of IKur/Kv1.5 current to treat atrial fibrillation. The design of XEN-D0103 was motivated by the general consensus that IKur is expressed and functional primarily in the atrial tissue, with a minimal presence in the ventricular tissue. Consequently, it was anticipated that XEN-D0103 would circumvent the proarrhythmic side effects commonly linked to the currently available therapies. However, a previous study casted doubt on whether IKur is actually absent from the ventricles and started a contentious debate that remains unresolved within the scientific community. To provide further clarity on the expression of Kv1.5 channels within cardiac ventricular muscle and to elucidate the impact of Kv1.5 channel inhibition on the dynamics of repolarisation both on cardiac ventricular muscle and Purkinje fibres. Immunocytochemistry was utilised to detect the potential expression of Kv1.5 channels in isolated canine ventricular myocytes. Action potentials were recorded using standard conventional microelectrode techniques from both canine right ventricular papillary muscle and Purkinje fibres. Patch-clamp measurements were conducted to explore the impact of XEN-D0103 on isolated canine ventricular myocytes. Immunocytochemical analysis revealed abundant expression of Kv1.5 channels in canine ventricular myocytes. Action potential recordings demonstrated a significant prolongation of APD90 in both canine ventricular (244 ± 26 ms vs 264 ± 15 ms P < 0.05) and Purkinje fibres (230 ± 9 ms vs 258 ± 9 ms P < 0.05) after treatment with XEN-D0103 at concentrations below 1 μmol/L. Patch-clamp experiments showed that XEN-D0103 does not inhibit IKr, IKs, Ito, or IK1 currents at concentrations of 1 μmol/L or less. However, at higher concentrations (3 μmol/L and 10 μmol/L), XEN-D0103 showed some inhibitory effects on Ito, IKr, IKs, and IK1 currents. Contrary to the prevailing belief, our experimental findings suggest that IKur is in fact expressed within the ventricular muscle, and its inhibition can lengthen cardiac ventricular repolarisation.

IKur inhibitor, XEN-D0103, arrhythmias

Short QT Syndrome Type 3 (SQTS3) is a rare arrhythmogenic channelopathy caused by gain-of-function mutations in KCNJ2, the gene coding the inward rectifier potassium channel Kir2.1 responsible for IK1. We aimed to enhance the understanding of SQTS3 by investigating its electrophysiologic and arrhythmogenic mechanisms. It is characterized by variable expressivity, which led me to hypothesised that the phenotype depends on the specific gain-of-function mechanisms triggered by each mutation. We used in vivo mouse models of three different pathogenic SQTS3 mutations (Kir2.1D172N, Kir2.1E299V and Kir2.1M301K). The last two were identified in patients presenting the most extremely abbreviated QT intervals described in the literature. On ECG, the QT interval was significantly shortened in all SQTS3 models. Intracardiac stimulation showed variable inducibility among SQTS3 subtypes. Kir2.1D172N animals presented the mildest phenotype, but Kir2.1E299V mice manifested atrial-specific arrhythmias, whereas Kir2.1M301K mice presented the most severe ventricular episodes. Patch-clamping demonstrated extremely abbreviated action potentials in Kir2.1E299V and Kir2.1M301K cardiomyocytes due to lack of inward-going rectification and increased IK1 at voltages positive to -80mV. The interaction of Kir2.1 with other proteins contributed to the explicit cardiac manifestations in each SQTS3 subtype. The functional interactions of Kir2.1 with the voltage-gated cardiac sodium channel NaV1.5 responsible for sodium current (Ina) stand out. First, Kir2.1E299V increased INa in cardiac conduction system cells and shifted ventricular INa activation and inactivation, increasing the excitability and protecting the ventricle against arrhythmia. Second, Kir2.1M301K reduced the availability of NaV1.5, reducing INa density and cardiac excitability. Altogether, my research served to improve the understanding of SQTS3, and to demonstrate that its cardiac manifestations are different depending on the causative mutation. Moreover, spermine demonstrated to be effective in prolonging the QT interval and reducing the arrhythmogenicity of SQTS3 models. Therefore, our results may contribute to identify novel antiarrhythmic drugs in an inheritable cardiac disease with no defined therapy.

Short QT Syndrome, Kir2.1, arrhythmia, channelopathy, spermine

Ministerio de Universidades (Spanish Government) awarded me the ‘Formación del Personal Universitario’ or FPU Fellowship [FPU20/01569] to conduct my PhD.

Multiple sclerosis (MS) is a chronic immune-mediated disease characterized by inflammation, demyelination, and axonal degeneration in the CNS, leading to motor impairment, visual disturbances, sensory problems, fatigue, and cognitive impairment. Patients with MS are at an increased risk of metabolic and cardiovascular comorbidities as well. Glucagon-like peptide-1 (GLP-1), an incretin secreted from the GIT after food intake, is not only involved in maintaining body weight, glucose, and lipid metabolism but also affects cognitive functions and acts as a neurotransmitter in the CNS. GLP-1 receptor agonists (GLP-1RA) are used in Type 2 DM treatment and have cardioprotective, neuroprotective, neurotrophic, anti-inflammatory, and antioxidant properties. The aim of our study was to assess effects of concomitant therapy with GLP-1RA on atherosclerosis risk and oxidative damage in treated people with MS. A total of 28 people (age: 39.4 ± 8.7 years, BMI: 25.0 ± 3.9 kg/m2) with relapse-remitting MS treated with natalizumab participated in this study. The subjects were randomly divided into the following two groups: – GLP1 group (n = 15) receiving dulaglutide (0.75 mg, s.c. once weekly) for one year and control group without the GLP-1RA treatment. Endothelial function, expressed as reperfusion hyperaemia index (RHI), did not change in the GLP1 group but significantly decreased in the control group (P = 0.002). Markers associated with an increased risk of atherosclerosis decreased significantly (tissue-type Plasminogen Activator (tPA): P = 0.038, Plasminogen Activator Inhibitor-1 (PAI-1): P = 0.009, and Intercellular Adhesion Molecule-1 (ICAM-1): P < 0.001) in the GLP1 group, while they remained unchanged in the control group. Markers associated with oxidative damage (lipoperoxide, TEAC) improved (P < 0.05) in the GLP-1RA treated subjects but not in controls. Markers of atherosclerosis correlated positively with markers of oxidative damage and negatively with antioxidant capacity in the GLP1 group only. Our results suggest that GLP-1RA has a positive effect on endothelial function and in preventing atherosclerotic changes, probably due to the decrease in oxidative damage in MS.

multiple sclerosis – GLP-1 analogues – atherosclerosis – antioxidant capacity

This study was supported by grant APVV-21-0261.

Heart failure (HF) is a complex syndrome often associated with oxidative stress. Empagliflozin and sacubitril/valsartan have shown individual benefits in HF management. In this study, we investigated whether the combination of empagliflozin and sacubitril/valsartan provides enhanced effects on oxidative stress in HF. Forty Wistar Albino rats were divided into control group (CTRL, n = 8) and an experimental HF group (eHF, n = 32). HF was induced with isoproterenol (5 mg/kg, subcutaneously) for 7 days and confirmed after 4 weeks by transthoracic echocardiography with a left ventricular ejection fraction below 55%. The eHF rats were then randomly assigned to one of four treatment groups for the next 4 weeks: no treatment (HF group), empagliflozin (10 mg/kg, HF-EMPA), sacubitril/valsartan (68 mg/kg, HF-S/V), or both (HF-EMPA + S/V). Oxidative stress markers in systemic and heart tissues were assessed at the end of this study. Significantly higher TBARS, O2-, and H2O2 levels were observed in the HF vs CTRL group, while HF-EMPA + S/V had the most pronounced effect on TBARS and H2O2 levels. HF-S/V and HF-EMPA + S/V had nearly comparable O2- levels to the CTRL group. The HF group significantly decreased superoxide dismutase (SOD), catalase (CAT), and (glutathione) GSH levels compared to CTRL. SOD and GSH levels significantly increased in HF-EMPA + S/V compared to the HF group. The HF group had significantly higher cardiac TBARS levels comparing to CTRL, while only HF-EMPA + S/V had significantly decreased TBARS levels compared to the HF group. CAT, SOD, and GSH cardiac levels were significantly lower in the HF group compared to CTRL. CAT and SOD levels were significantly higher in treated groups compared to the HF group, while only HF-S/V and HF-EMPA + S/V significantly increased GSH levels compared to the HF group. This combined treatment significantly improves oxidative status in HF rats, suggesting potential advantage in managing HF.

heart failure – empagliflozin – sacubitril/valsartan – oxidative stress – rats

This work was supported by the Faculty of Medical Sciences, University of Kragujevac (JP 01/24) and the Ministry of Science, Technical Development, and Innovation of the Republic of Serbia (contract No. 451-03-65/2024-03/200111).

Exercise is a widely recognised intervention for preventing and treating cardiovascular diseases. One potential mediator of these benefits could be brain-derived neurotrophic factor (BDNF), which is released during exercise and may contribute to cardioprotection. While the role of BDNF is well known in neurocognition, its involvement in cardioprotection is underexplored. This study aimed to test the hypothesis that exercise-induced BDNF signalling via the tropomyosin receptor kinase B (TrkB) receptor in the heart is one of the mechanisms underlying improved cardiac function, particularly in hypertensive conditions. This study was performed in adult male Wistar (n = 16) and spontaneously hypertensive (SHR) rats (n = 16), which were randomly assigned to the control (sedentary groups) (n = 8/strain) and voluntary wheel running (exercise) groups (n = 8/strain). After 5 weeks of exercise intervention, left ventricular myocardial tissue samples were collected to detect gene expression of BDNF, TrkB receptor, nerve growth factor inducible (VGF) (non-a cronymic), and cAMP response element-binding protein (CREB) by RT-PCR. The Kruskal–Wallis test assessed differences between groups, and the Bonferroni test was applied for post hoc comparisons. We found that exercise intervention increased heart function (cardiac output), expression of TrkB receptor, VGF, and CREB levels in normotensive Wistar rats but not in SHR rats. The expression of these factors was not significant between the control SHR vs running SHR rats and the control Wistar vs running Wistar rats. The findings suggest that SHR and Wistar rats exhibit different responses to exercise, with distinct activation of BDNF/TrkB/CREB/VGF signalling pathways potentially improving cardiac function and contributing to cardioprotection in normotensive but not hypertensive conditions. A deeper understanding of these molecular mechanisms could help us better explain benefits of exercise for the heart that might lead to development of new treatments for cardioprotection.

exercise – cardioprotection – BDNF – SHR – Wistar

This study was supported by grants Slovak Grant Agency VEGA 2/0158/22, VEGA SR 2/0104/22, APVV-19-0540, and APVV-20-0242.

Despite the time passed since the SARS-CoV-2 pandemic, its negative effects persist, with patients experiencing complications linked to the infection and other health disorders. In terms of long-term outcomes, it became crucial to define a condition encompassing all such complications, now termed post-COVID syndrome. The wide range of these complications has led to the adoption of a multidisciplinary approach that identified organ-specific effects, though the underlying mechanisms of post-COVID remain unclear. This study aimed to explore potential immunological mechanisms in cardiac tissue that may contribute to the development of post-COVID syndrome.

Materials and Methods: Sera from 148 COVID-positive patients, collected during ambulance tests, were analysed using Western blot. After SDS-PAGE electrophoresis and transfer to nitrocellulose membranes, the membranes were incubated with the patients' sera. Both human IgG and IgM (at a 1:13000 dilution) were then applied, and chemiluminescence was used to detect suspected autoantibodies. Among the 148 samples, IgG autoantibodies were found in 33 samples (22%) and IgM autoantibodies in 39 samples (26%). Many positive samples exhibited multiple autoantibodies (49 IgG and 48 IgM in total). Some molecular patterns were identified based on the presumed molecular weight of the autoantibodies.

The significant presence of autoantibodies in the samples suggests that they may play a role in the pathophysiology of post-COVID syndrome, potentially involving autoimmune responses. Additionally, the large number of detected autoantibodies points to potential issues with isotype-switching or other immune dysfunctions, raising hypotheses about immune system disruption or the alteration of proteins into autoantigens.

post-COVID syndrome – autoantibodies – SARS-CoV-2 – immune dysfunction

This study was supported by the University of Debrecen Scientific Research Bridging Fund (DETKA).

This research aimed to evaluate the potential of using a combination of neprilysin inhibitor (sacubitril) and an AT1 receptor antagonist (valsartan) to induce white adipocyte browning and to evaluate whether this combined approach is more effective than cold exposure in promoting the formation of beige adipocytes. Forty male Wistar albino rats (8 weeks old; body weight: 200 ± 20g) were included in this study and further divided according to diet regime and applied treatment into the following four equal groups: CTRL + 4°C – healthy untreated rats; CTRL + E + 4°C – healthy rats treated with S/V; MetS + 4°C – rats with metabolic syndrome (MetS); and MetS + E + 4°C – rats with MetS treated with S/V. All rats were exposed to a low temperature (+4°C) for 4 h daily over a period of 4 weeks. Experimental groups of rats were administered per os a drug combination at a dosage of 68 mg/kg daily for 4 weeks. On completion of the experimental protocol, white adipose tissue was isolated for further analyses. The results of our study singled out combined cold exposure with S/V treatment as a more effective approach in inducing multilocular morphology of white adipocytes compared to untreated rats exposed to low temperatures alone. A significantly higher gene expression of thermogenic molecules was observed in white adipocytes of rats with MetS treated with S/V compared to expression found in animals from the MetS + 4°C group. In line with these results, the percentage of UCP1 stained adipocytes in MetS + E + 4°C was significantly higher in comparison to MetS + 4°C rats. In summary, due to more noticeable promotion of browning achieved by sacubitril/valsartan treatment in cold environment compared to untreated rats, the findings of this study indicate strong potential of this fixed drug combination to induce browning, making it an agent of quite similar potency in the formation of beige adipocytes as exposure to cold temperatures, which is defined as the gold standard.

browning – cold exposure – sacubitril – valsartan – adipose tissue

This study was supported by the Faculty of Medical Science, University of Kragujevac, Grant/Award Number: 04/22; Ministry of Science, Technological Development and Innovation, Republic of Serbia, Grant/Award Number: 451-03-47/2023-01/200111.

An enhanced inflammatory response is commonly observed in patients with atrial fibrillation (AF) contributing to increased cardiac fibrosis. Recent studies have identified that the expression of S100A8 and S100A9, calcium binding proteins, is significantly enhanced in the atrial tissue of patients with AF, with a 2.8-fold increase for S100A8 and 5.3-fold increase for S100A9. These proteins act as endogenous ligands for toll-like receptor 4 (TLR4) and, on binding, may downstream activate the NLRP3 inflammasome, a known key player in the pathogenesis of AF. However, the specific mechanisms by which S100A8/A9 proteins activate the inflammasome and their involvement in AF-related structural remodelling remains unclear. The aim of this project was to develop a macrophage-fibroblast co-culture system to explore the potential role of S100A8/A9 in fibroblast activation and their downstream pro-inflammatory effects. Given that S100A8/A9 proteins are released in response to enhanced inflammatory signals, selectively inhibiting atrial inflammation through these proteins could constitute novel therapeutic strategies to prevent or slow the progression of fibrosis driven AF. This study will examine the role S100A8/A9 in NLRP3 inflammasome activation and fibroblast activation using commercial mouse macrophage and fibroblast such as cell lines, human macrophages differentiated from isolated peripheral blood mononuclear cells (PBMCs), and human atrial fibroblasts. To activate the NLRP3 inflammasome and initiate macrophage-fibroblast interactions, cells will be treated with lipopolysaccharide from Escherichia coli (LPS) and Nigericin. Inhibition of S100A8/A9 will be evaluated using the commercially available inhibitor, ABR-238901, to confirm suppression of S100A8/A9 signalling. Preliminary data from these experiments will be presented at the conference as the experiments are ongoing.

inflammation – macrophages – fibroblasts – co-culture

This study aimed to assess the impact of a 2-week administration of wild garlic (Allium ursinum) essential oil (WG) and allitridin (a biologically active compound found in wild garlic) on doxorubicin (DOX)-induced cardiotoxicity in a rat model. The experiment involved the following four groups: Healthy untreated rats (CTRL, n = 8), rats injected with a single dose of doxorubicin (15 mg/kg ip) on the 14th day (DOX, n = 8), rats treated with 100 mg/kg/day of Allium ursinum essential oil for 2 weeks orally, with 15 mg/kg doxorubicin ip on the 14th day (DOX + WG, n = 8), and rats treated with 16 mg/kg/day of allitridin for 2 weeks orally, with 15 mg/kg doxorubicin ip on the 14th day (DOX + AL, n = 8). Three days after DOX treatment, the rats were sacrificed. In vivo hemodynamic parameters were measured via echocardiography, and ex vivo cardiodynamic parameters of isolated rat hearts were assessed using the Langendorff apparatus. Histological examination of the myocardium was performed to evaluate structural changes. Our results highlighted that DOX-induced prominent depression of cardiac function whereas examined treatment markedly recovered heart contractility and relaxation force. Treatment with allitridin and wild garlic essential oil resulted in a slight increase in ejection fraction in the DOX group. Histological evaluation revealed significant structural injuries in the cardiac tissue of the DOX group, which were mitigated by both treatments. This study suggests that allitridin and wild garlic essential oil may serve as effective cardioprotective agents against doxorubicin-induced cardiotoxicity, potentially improving myocardial performance and preserving cardiac structure in rats.

diallyl trisulfide – echocardiography – Langendorff apparatus – rat model

Electrocardiography (ECG) is an invaluable tool in diagnosing cardiovascular diseases. This study introduced a novel method to reconstruct the heart's Three-Dimensional Electrical Current Vector (ECV3) by combining a subset of the standard 12-lead ECG leads. ECV3 was then used to estimate the perfusion angle and any other leads. This study aimed to investigate how ECV3 and perfusion angle could be used to assess electrophysiological changes in myocardial infarction. For this purpose, an open-source online ECG dataset from 45152 subjects was utilised. ECV3 was estimated using three independent components (x, y, and z) from the available ECG recordings. Lead I was marked as the x component, lead aVF as the z counterpart, and lead V2 as the y component. Based on the specifications of each lead measurement, trigonometric equations were applied to reconstruct ECV3 and use it to estimate the electrical activity in the other leads. The perfusion angle was measured as the mean angle between the S and T waves of the ECV3. ECV3 estimated leads yielded a significant cross-correlation for 94% of all patients in the database with a median correlation of 0.999 for limb leads and 0.829 for chest leads. The mean perfusion angle significantly decreased from an average of 140.7° for subjects with normal sinus rhythm (n = 123) to 126.7° for patients who suffered from a previous myocardial infarction (n = 123). This study provided the first proof of concept to reconstruct ECV3 and reliably estimate the other leads in real time. Patients with previous myocardial infarction have reduced perfusion angle, indicating persistent electrophysiological abnormalities. Analysis of the perfusion angle from ECV3 could offer a potential diagnostic tool to assess ischaemic myocardial diseases. More data are required to establish the relationship between myocardial infarction distribution and perfusion angle using a comprehensive dataset including records of CT angiography and blood markers.

ECV3 – perfusion angle – cardiac axis – myocardial infraction

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been shown to improve the clinical outcome of chronic heart failure (CHF) and atrial fibrillation (AF) and reduce the risk of arrhythmias and sudden cardiac death (SCD). The current ESC guidelines recommend the use of SGLT2 inhibitors as first-line agents in the treatment of CHF. Despite the outstanding clinical efficacy of SGLT2 inhibitors, the underlying mechanisms for their antiarrhythmic effects are poorly understood. Therefore, we aimed to investigate the cardiac electrophysiological effect of the following two SGLT2 inhibitors: Empagliflozin (EMPA) and Dapagliflozin (DAPA). Action potential (AP) measurements were carried out by conventional microelectrode technique using healthy canine and failing human (explanted) cardiac preparations. The effect of EMPA and DAPA on AP parameters [AP duration at 90% of repolarisation (APD 90), maximal rate of depolarisation (V max), AP amplitude (AMP), and conduction time (CT)] and their potential frequency dependency were measured. All data shown as mean ± SEM. Compared to control, 1 μmol/L and 3 μmol/L EMPA increased the maximal rate of depolarisation (166.5 ± 14.5 V/s vs 194.5 ± 15.7 V/s and 207 ± 15.5 V/s, both p< 0.05, n = 9) and the AP amplitude (115.1 ± 3.1 mV vs 118.9 ± 3 mV and 122.1 ± 2.7 mV, both p< 0.05, n = 9) while decreased the conduction time (6.49 ± 1.35 ms vs 6.19 ± 1.41 ms and 5.94 ± 1.36 ms, both p< 0.05, n = 7) and slightly lengthened APD 90 (219.5 ± 4.9 ms vs 226.2 ± 4.1 ms and 225.6 ± 6.1 ms, both p< 0.05, n = 12) in canine preparations. Of note, the APD 90 prolonging effect of 3 μmol/L EMPA was more pronounced at cycle lengths of 300 ms and 400 ms, indicating a frequency-dependent mode of action. Tendencies for an increased V max (n = 4) and decreased CT (n = 5) were also observed in the explanted human samples, but the differences were not significant. The effect of DAPA (1 μmol/L and 10 μmol/L) on AP parameters – both in canine and human samples – showed similar tendencies as in the case of EMPA; however, the differences were not significant. In line with the results of recent works using cellular expression systems to assess the electrophysiological characteristics of SGLT2-inhibitors, the observed changes in AP parameters (increased V max and AMP, decreased CT and frequency-dependent APD 90 prolongation primarily at low cycle lengths) in our work could indicate that EMPA – and maybe other SGLT2 inhibitors as well – has the potential to activate the peak sodium current (I Na) in more complex model systems (in canine or human tissues) with better translational value as well. Theoretically, this effect on the sodium channels could increase the impaired conduction velocity in HF, which could contribute to the SGLT2 inhibitors' overall antiarrhythmic effect.

SGLT2 inhibitors – cardiac electrophysiology – arrhythmia – heart failure – sodium channel

Data on the effects of different types of training activities on the functional recovery of the heart after ischaemia/reperfusion damage are still very inconsistent. An additional problem is the fact that it is still not known which type of training and its intensity (aerobic or anaerobic) achieves the best results in myocardial preconditioning. The aim of this study was to investigate the effects of exercise preconditioning on an animal model of ischaemia/reperfusion (I/R) injury in rat myocardium, specifically to determine the impact of different types of training processes on I/R, as well as the role of oxidative stress in the observed effects. This study was conducted on 40 rats divided into one control group (sedentary rats) and four experimental groups. Rats in the experimental groups underwent a training protocol for preconditioning through running and swimming (in both aerobic and anaerobic forms). After the training protocols, the animals were sacrificed, and the isolated hearts were perfused using the Langendorff retrograde perfusion method at a constant coronary perfusion pressure. Following stabilization, the hearts were subjected to global ischaemia for 20 min and then reperfusion for 30 min. Oxidative stress parameters were assessed in samples of coronary venous effluent as well as in the blood of rats using spectrophotometric methods. Both types of training were associated with increased production of cardiac pro-oxidants, with anaerobic intensity leading to the greatest increase in their production.

aerobic and anaerobic preconditioning – swimming – running – rat – myocardial ischaemia

Nowadays, the neprilysin inhibitor sacubitril in combination with an angiotensin II receptor antagonist is successfully used in the treatment of heart failure patients, which illustrates the prominent role of neprilysin (NEP) in the pathogenesis of cardiovascular diseases. By inhibiting the NEP enzyme, sacubitril significantly reduces the number of hospitalisations and mortality from cardiovascular causes. There are significant individual differences in the efficacy of the medication and the response to therapy, and we wanted to study the mechanisms underlying this. Serum NEP concentration was determined by commercially available ELISA kits and serum NEP activity by end-point fluorescence assay. The NEP gene was analysed by Sanger sequencing and the detected genetic variations by restriction fragment length polymorphism analysis. Serum NEP concentrations in cardiovascular patients showed a significant interindividual variation (mean: 35.41 ng/L [min-max: 0.38-10765 ng/L; n = 75), which was suggested to be due to genetic polymorphisms. Sequencing of the NEP gene of the individual with the highest NEP concentration (NEP = 10765 ng/L) identified a mutation at position chr3:155180618 of exon 23, in which the presence was associated with increased NEP concentration previously. Its presence was tested in several patients, but high serum NEP concentrations were measured even in the absence of the mutation. Investigation of the relationship between NEP concentration and activity suggested the presence of an endogenous NEP inhibitor in serum. Fourfold diluted serum reduced recombinant NEP activity by more than 60%. Removal of the two most abundant serum proteins (i.e., immunoglobulin G (IgG) and albumin) from the sample did not increase the activity of serum endogenous NEP, but abolished its inhibitory effect on recombinant NEP. IgG had no effect; whereas, albumin significantly reduced recombinant NEP activity with an IC50 of 15.03 g/L. Albumin is an endogenous inhibitor of serum NEP, which, in addition to reducing NEP activity, may affect the results of ELISA-based NEP concentration measurements. This sheds new light on the findings of previously published studies, as determined by the methods we used. In conclusion, the physiological regulation of NEP and the mechanism of action of NEP inhibitors hold many surprises for the times to come.

NEP – neprilysin – CD10 – HF – biomarker

This study was supported by the EKÖP-24-3-II-DE-277 (A.Á.Sz.), EKÖP-24-3-II-DE-302 (E.E.E), and University Research Scholarship Programme (EKÖP) of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund.

Oxidative stress is known to lead to cardiovascular diseases, including myocardial infarction (MI). The major antioxidant enzyme that maintains redox homeostasis and cellular response to oxidative stress is glutathione S-transferase P (GSTP), a member of the GST enzyme superfamily. Besides, new evidence suggests that this gene's polymorphisms can enable oxidative stress-related lipid abnormalities, influence lipid levels, and contribute to cardiovascular disease risk. The two most commonly occurring single nucleotide polymorphisms (SNPs), i.e., GSTPAB rs1695 and GSTPCD rs1138272, result in a deficiency of the GSTP antioxidant capacity. Therefore, we speculated that polymorphic variants of GSTP1 are related to MI risk and lipid traits in patients with MI. The case-control study comprised 75 patients with MI not receiving antilipemic therapy and 79 age and sex-matched controls. The GSTP1 genotypes were determined by qPCR using Applied Biosystems Taqman Drug Metabolism Genotyping assays. We found that SNP rs1138272 (*CT + TT) in the GSTP1 gene, associated with the lack of GSTP activity, modifies the risk for MI. Our results showed that carriers of variant GSTP1CD*CT + TT genotype are at almost five-fold increased risk of MI (OR = 4.68, 95% CI = 1.47–14.78, P = 0.009). In the case of the second investigated polymorphism of GSTP1, rs1695, carriers with at least one variant *G allele (GSTP1AB*AG + GG genotype), exhibited an elevated risk of hypertension among patients with MI (OR = 3.69, 95% CI = 1.21–11.28, P = 0.022). The haplotype consisting of variant alleles for both polymorphisms G and T was associated with 3.4-fold increased risk (OR = 3.42; 95% CI = 1.05–11.17; P = 0.043). In the patient group, there were higher LDL-C and total cholesterol levels in carriers of the GSTP1AB*AG + GG variant genotype compared to the carriers of the GSTP1AB*AA genotype (P = 0.031 and P = 0.019, respectively). We have demonstrated that GSTP1 polymorphisms may determine individual susceptibility to the MI risk. Additionally, our results may provide an unexplored link between lipid metabolism and GST homeostasis.

myocardial infarction – oxidative stress – GSTP1 – polymorphism

Necroptosis, a regulated form of necrotic cell death, is associated with the pathomechanisms of various cardiovascular diseases. However, the relevance of the necroptotic process in cardiomegaly, especially considering potential sex differences, remains elusive. In this study, we used male and female Wistar rats in which cardiomegaly was induced by constriction of the abdominal aorta 2 days after birth. Animals were sacrificed by thiopental overdose after 90 days, and the left ventricular tissue was used for molecular analysis by SDS-PAGE/Western blotting. The left ventricular weight to the body weight ratio was significantly increased in both males and females after constriction of the abdominal aorta, while the right ventricular and lung weight to the body weight ratio was higher only in males. Echocardiographic analysis revealed decreased fractional shortening in both sexes, but ejection fraction was decreased only in males. Both sexes exhibited higher levels of the main necroptotic markers in the left ventricles – phosphorylated receptor-interacting protein kinase 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL), indicating necroptosis activation. However, regarding the molecular processes associated with necroptosis, we found increased protein expression of tumour necrosis factor (TNF), interleukin 6 (IL-6), and cyclooxygenase 2 (COX-2) only in females, indicating a pro-inflammatory environment. Contrary, males were rather positive for upregulated expression of NADPH oxidase 2 and 4 (NOX2 and NOX4) and inducible NO synthase (iNOS), indicating the presence of pro-oxidative damage. Concerning the upstream receptors, females had higher expression of TNFR1 while had increased protein expression of TLR4 and MyD88. Conclusively, we indicated for the first time that although necroptosis is potentially involved in the pathomechanisms in both sexes, males exhibited more pronounced cardiac enlargement and pro-oxidative stress, while females were positive for a pro-inflammatory environment. Such data might advocate for a need for an individual pharmacological approach between the two sexes.

cell death – necroptosis – cardiomegaly – RIP3 – MLKL

This study was supported by grants APVV-20-0242; APVV-15-0607, and VEGA 1/0016/20.

Cardiovascular diseases, especially ischaemic heart disease (IHD) as the leading cause of heart failure and mortality, will not reduce over the coming decades despite the progress in pharmacotherapy, interventional cardiology, and surgery. This represents a danger, especially in the elderly with hypertension and metabolic disorders. The infarct-sparing effect of ischaemic ‘preconditioning’ (IPC) is the most robust form of innate cardioprotection based on heart adaptation to moderate stress. However, its translation to clinical practice is limited. There are novel forms of adaptive interventions, such as ‘remote’IPC (RPC) and other non-invasive approaches, such as exercise-induced preconditioning (EPC) or adaptation to hypoxia (HPC). We explored that these non-pharmacological interventions in the adult male Wistar rats in vivo aimed to increase cardiac resistance to I/R ex vivo. RPC was induced by three cycles of 5-min inflation (200 mmHg)/5-min deflation of pressure cuff on the hind limb. For EPC, rats were placed in cages equipped with wheels for 2 weeks of free running. In a hypoxic chamber, HPC was induced by staying animals (10% O2, 4 weeks). The efficacy of these interventions was tested in the Langendorff-perfused hearts exposed to 30 min global ischaemia/2 h reperfusion, focused on the post-I/R functional recovery, arrhythmogenesis, and lethal injury (infarct size, IS). In parallel groups, heart tissue samples were processed (WB) to investigate the levels and activity of proteins involved in the ‘pro-survival’ RISK cascade. All interventions reduced contractile dysfunction, IS, and incidence and severity of reperfusion-induced arrhythmias. Cardioprotection was associated with significant up-regulation of selected pro-survival RISK proteins, such as PKB, PKCɛ, eNOS, and anti-apoptotic and anti-oxidative effects. Protective effects were maintained under conditions of hyperglycaemia and in the hearts of SHR rats. The beneficial effects of these ‘conditioning’ interventions suggest their potential that could be used in the management of ischaemic heart disease in patients with comorbidities.

ischaemic heart injury – adaptation – cardioprotection – molecular mechanisms

This study was supported by grants Slovak Grant Agency VEGA SR 2/0104/22, APVV-19-0540, and APVV-20-0242.

Atrial fibrillation (AF), the most common arrhythmia, is associated with altered Ca²⁺-handling in atrial myocytes. Up-regulation of adenosine A2A receptors in AF has been linked to increased protein kinase A (PKA)-mediated phosphorylation of RyR2, leading to spontaneous sarcoplasmic reticulum Ca²⁺ release. It is well accepted that 3′5′-cyclic adenosine monophosphate (cAMP)/PKA signalling is compartmentalised into specific nanodomains due to the action of phosphodiesterases (PDEs), the enzymes that degrade cAMP. The aim of this study was to characterise cAMP dynamics in key Ca²⁺-handling nanodomains on A2A receptor activation and to study the roles of PDE3, PDE4, and PDE8 in human atrial myocytes (HAMs) from patients in sinus rhythm (SR) and with AF. A total of 198 HAMs were isolated from atrial samples of 45 patients undergoing cardiac surgery. Cells were transfected with adenoviral vectors encoding FRET biosensors targeted to measure cAMP specifically in main Ca²⁺-handling nanodomains: The sarcolemma, cytosol, RyR2, or phospholamban (PLN)/SERCA2a. After 48 h of culture to allow for biosensor expression, FRET live imaging was used to measure cAMP levels. Our results revealed larger cytosolic cAMP increases in AF on A2A receptors activation with CGS. However, cAMP increase in the RyR2 nanodomain was lower in AF, indicating that the reported increase in PKA-mediated phosphorylation of RyR2 in AF might be surprisingly mediated by cytosolic cAMP. PDE3 and PDE4 were key regulators of cytosolic and PLN/SERCA2a compartments, though PDE4 regulation was reduced in AF. The overall response in the sarcolemma was low, and only PDE4 seemed to regulate this compartment. Finally, PDE8 emerged as a novel regulator of the A2A response in RyR2, aligning with recent electrophysiological studies in murine models. These findings highlight the complex PDE-mediated regulation of cAMP in the adenosine A2A response in HAMs, offering new insights into how cAMP signalling is altered in AF. Understanding these nanodomain-specific disruptions could provide potential therapeutic targets for restoring normal cAMP signalling in patients with AF.

atrial fibrillation – cAMP – adenosine, GPCR

The advent of human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) technology may revolutionise cardiac medicine. However, the use of hiPSC-CMs has been restricted due to their immature phenotype, more similar to foetal than adult cardiomyocytes. Multiple strategies have been proposed for improving hiPSC-CM maturation, but the most promising are those that mimic the cardiac microenvironment, provided in part by the extracellular matrix (ECM). In this study, we proposed a human cardiac decellularized ECM to enhance the structural and functional maturation of hiPSC-CMs compared to other commercial matrices. Our matrix derives from the deposition of ECM components of human cardiac fibroblasts aligned in micropattern on polydimethylsiloxane (PDMS) for 7 days, providing heart stiffness (≈ 10 kPa), ECM components, and scaffold to mature hiPSC-CMs. We also tested on our matrix that an excess of ECM deposition, becoming a more fibrotic ECM, is counterproductive to hiPSC-CM functional maturation. The human cell matrix significantly increases sarcomere length and alignment, cell circularity index, anisotropy, and the expression of structural genes such as CTnT and actinin in hiPSC-CMs. Also, the provided scaffold and stiffness of our matrix improve Conexin 43 and N-Cadherin location. Most importantly, this decellularized human cardiac matrix improves hiPSC-CM function in terms of metabolism and electrophysiology, which is a principal limitation of these cells. Patch-clamp and optical mapping experiments revealed an adult-like cardiomyocyte phenotype: maximum diastolic potential (MDP) (≈-78 mV), amplitude (≈115 mV), upstroke velocity (≈130 V/s), and AP duration at 90% repolarization (APD90) (≈300 ms). NaV1.5 expression and sodium current (INa) are similar to the adult cardiomyocyte. In addition, action potential conduction velocity (≈40 cm/s) in hiPSC-CM monolayers cultured in our matrix is close to the adult heart. In conclusion, we present a cost-effective and easy-to-make platform that mimics the ECM cardiac microenvironment. This platform represents an optimal strategy for hiPSC-CM maturation, which will help investigate molecular mechanisms of inheritable arrhythmogenic diseases.

hiPSC-CMs – ECM – cardiac diseases – structure – electrophysiology

A magnolia bark extract with the active component mainly magnolol (MAGNO) is in a group of bioactive phytochemicals. Studies mentioned that this compound has antioxidant-like actions, known as an uncommon antioxidant. The action of MAGNO includes a marked protection against increased oxidative stress in cells through various signalling pathways. In addition, it has been demonstrated that the magnolia bark extract radicals do not react with molecular oxygen and produce no superoxide radical under the typical settings of inhibited autoxidation. Therefore, in this study, we treated either insulin-resistant male elderly (24-month-old) rats/mice or doxorubicin-applied male adult rats with MAGNO (100 mg/kg) for 12–14 weeks besides their standard chow. When compared with untreated groups (elderly or adult animals), MAGNO treatment significantly provided antioxidant-like effects on biochemical parameters of heart tissues such as increases in the activity of arylesterase and total antioxidant levels as well as significant decreases in the activity of the mitochondrial enzyme succinate dehydrogenase, one of the sources of reactive oxygen species and the total oxidant level with a significant increase in the ratio of ATP to ADP. MAGNO treatment of doxorubicin-applied rats (before or after) provided significant recoveries in the systemic parameters of animals such as plasma levels of manganese and zinc, total oxidant, and antioxidant statuses. Furthermore, we determined important cardioprotective effects of this treatment such as improvements in the redox regulation of the hearts, such as marked improvements in activities of glutathione peroxidase and glutathione reductase and oxygen radical-absorbing capacities in these doxorubicin-treated animals. Overall, our data presented significant myocardial ameliorations in physiological ageing-associated biochemical alterations in the metabolic heart with MAGNO supplementation as well as significant cardioprotection against doxorubicin toxicity. Finally, one can propose the beneficial antioxidant effects of MAGNO in chronic heart diseases as a supporting and complementing agent to conventional therapies.

Magnolol (MAGNO) – oxidative stress

This study was supported by the Lokman Hekim University Scientific Research Projects Coordination Unit. Project No. 202AP404, 2022.

Activation of the autonomic nervous system (ANS) and local sympathetic hyperinnervation plays a pivotal, yet poorly understood role in the initiation and maintenance of atrial fibrillation (AF). Changes in the intricate three-dimensional (3D) network of nerves are difficult to characterise using traditional histological methods. To gain insights into the role of innervation in cardiac arrhythmia, non-destructive imaging techniques capable of visualising large tissue samples from large animal models of AF are crucial. In this study, we aimed to investigate the feasibility of 3D light sheet fluorescence microscopy (LSFM) in equine atrial tissue and provide a detailed 3D characterisation of the sympathetic nervous system in the epicardium, myocardium, and endocardium, while exploring innervation changes in a horse model of experimentally induced chronic AF. Biopsies from the left posterior atrial wall were harvested from horses after 4 months of induced AF (n = 9), from horses with naturally occurring AF (n = 3), and healthy control horses (n = 3). A neuronal marker (tyrosine hydroxylase) was stained to determine the local density of sympathetic nerves for computational image analysis. Whole-mount immunohistochemistry and clearing were optimised for equine heart samples by testing different depigmentation, permeabilization, and imaging protocols. We demonstrated a protocol for 3D imaging of nerves in large equine atrial samples. Optimised sample preparation with stepwise chemical and enzymatic extracellular matrix loosening and digestion enabled uniform sample labelling with antibodies against a neuronal marker. Customised autofluorescence bleaching, sample clearing, and imaging parameters made high resolution imaging of cardiac innervation feasible throughout the large tissue sample. Computational analysis of atrial innervation permitted quantitative analysis in the three groups to demonstrate spatial changes occurring in AF. Three-dimensional LSFM in large animal models can deepen our understanding of the autonomous nervous system's role in AF development through enabling single-cell resolution imaging in dense cardiac biopsies. We showed the applicability of the method to characterise innervation changes in an equine model of AF.

atrial fibrillation – autonomic remodelling – 3D light sheet microscopy

This work was supported by a research grant from the Danish Cardiovascular Academy and the Danish Heart Foundation (PhD 2022002-HF).

Heart transplantation, although routine, faces significant challenges due to ischaemia reperfusion injury (I/RI) after cold ischaemic storage. Reperfusion with warm, oxygenated blood generates reactive oxygen species (ROS) such as nitrosyl and hydroxyl radicals, exacerbating cell damage. In addition, extracorporeal circulation (ECC) induces systemic oxidative stress, further complicating transplant outcomes. This study investigated the therapeutic potential of hydrogen gas in reducing I/RI by targeting hydroxyl radicals, reducing lipid peroxidation, inflammation, and increasing antioxidant enzyme activity. Porcine hearts stored in cold ischaemia for 3 h were perfused with blood after 2% inhalation and blood saturation in an ECC oxygenator with 2% hydrogen gas in air. Pigs treated with hydrogen showed significantly less ventricular fibrillation and improved histopathological results compared to controls. Hydrogen administration significantly reduced systemic oxidative stress, markers of ischaemia, inflammation, and lipid peroxidation. Hydrogen gas delivered via conventional ventilators and ECC oxygenators shows promise as an adjunctive therapy in heart transplantation, improving post-transplant cardiac function and ameliorating systemic oxidative stress.

molecular hydrogen – oxidative stress extracorporeal circulation – transplantation reperfusion

This research was supported by grants from the Agency for Research and Development Support (APVV-0241-11, APVV-15-0376, APVV-19-0317), the Slovak Academy of Sciences (VEGA 2/0092/22 and 2/0063/18), the Structural Funds of the European Union (ITMS 26230120009), and the Ministry of Health of the Slovak Republic (2019/4-CEMSAV-1).

Nav1.5 channels, encoded by SCNA5, are essential for the genesis of the cardiac action potential (AP) and contribute to its duration. Gain-of-function mutations in SCNA5 are associated with long QT syndrome (LQTS); whereas, loss-of-function mutations are linked with Brugada syndrome. MOG1 was described as a partner of the Nav1.5 channelosome, increasing Nav1.5 current amplitude and membrane expression. Two loss-of-function variants in the MOG1 gene (E61X and E83D) have been found in patients with Brugada Syndrome, but no relationship with LQTS has been reported. We have identified the first gain-of-function variant in MOG1 (L18F) in a patient with LQTS. The patient presented complete atrial-ventricular (AV) block, prolonged QT intervals and ventricular tachycardia. We generated AAV9-mediated cardiac-specific mouse models expressing MOG1WT and MOG1L18F. MOG1L18F mice recapitulated the patient's phenotype, with prolonged QT interval corrected for heart rate on surface ECG, and an increased incidence of arrhythmia on programmed electrical stimulation. In current-clamp experiments, the incidence of delayed afterdepolarizations (DADs) and spontaneous APs was higher in MOG1L18F compared to MOG1WT cardiomyocytes (CMs). AP duration at 90% of repolarisation (APD90) was dramatically increased at low stimulation frequencies (0.1–2 Hz) in MOG1L18F but very similar to MOG1WT CMs at higher frequencies (from 4 Hz to 10 Hz), resulting in a very steep APD90 restitution curve in MOG1L18F, which was proarrhythmic. On voltage clamping, late sodium current (INaL) was extremely enhanced in MOG1L18F CMs compared to MOG1WT, especially at depolarised membrane potentials. This effect was mediated by Nav1.8, at least in part. The increase in INaL opposed repolarising currents, lengthening the AP and the QT, ultimately leading to ventricular tachyarrhythmias. In conclusion, we demonstrated for the first time that cardiac-specific expression of the L18F gain-of-function variant of MOG1 in live mice impairs AV conduction and increases INaL, which prolongs the APD and the QT interval and provides a new genetic basis for LQTS.

long QT syndrome – MOG1 – Nav1.5 channels – arrythmia

Heart failure (HF) continues to be a major global health challenge, characterised by impaired cardiac function and remodelling. While both empagliflozin and sacubitril/valsartan have demonstrated individual efficacy in HF, their combined effects have not been thoroughly investigated. Understanding the potential synergistic benefits of these therapies could lead to more effective management of HF. To evaluate the combined effects of empagliflozin and sacubitril/valsartan on cardiac function and myocardial remodelling in a HF rat model. Forty male Wistar Albino rats were initially allocated into the following two groups: A healthy control group (n = 8) and an experimental HF (eHF) group (n = 32). HF was induced in the eHF group by administering isoproterenol (5 mg/kg daily for 7 days). After 4 weeks, rats from the eHF group were randomly assigned to one of the four treatment subgroups for an additional four weeks: No treatment (HF group), empagliflozin (10 mg/kg, HF-EMPA), sacubitril/valsartan (68 mg/kg, HF-S/V), or both at same doses (HF-EMPA + S/V). At the end of this study, hemodynamic measurements, transthoracic echocardiography, ex vivo assessment of cardiac function, and histological examination were performed. Both HF-EMPA and HF-S/V groups had improved myocardial function and structure compared to untreated rats. The combined treatment exerted the most pronounced effects across majority of assessed parameters. The HF-EMPA + S/V group had the greatest improvement in hemodynamics and superior outcomes in both in vivo and ex vivo evaluations of cardiac function. Histological analyses revealed enhanced preservation of myocardial architecture with reduced collagen content and fibrosis. The combination of empagliflozin and sacubitril/valsartan offers a synergistic advantage in managing HF, presenting a promising approach for more effective treatment of the condition.

heart failure – empagliflozin – sacubitril/valsartan – cardiac function – myocardial remodelling

This study was supported by the Faculty of Medical Sciences, University of Kragujevac (JP 01/24) and the Ministry of Science, Technical Development, and Innovation of the Republic of Serbia (contract No. 451-03-65/2024-03/200111).

Type 1 diabetes mellitus (T1DM) is regarded as autoimmune disorder, primarily manifested during early childhood and adolescence rather than adulthood. Individuals with T1DM have greater cardiovascular and total morbidity and mortality than general population. The return of consciousness and alertness after anaesthesia may be accompanied by heart failure, high blood pressure, increased abdominal and intracranial pressure, and myocardial ischaemia. This study aimed to examine the effect of thiopental (TIO) monotherapy as well as its combination with different agents used in anaesthesia induction, on cardiac function and redox state of rats with T1DM. A total of 40 Wistar albino male rats were used in this study and randomly divided into the following five groups: TIO, fentanyl + thiopental (FEN + TIO), remifentanil + hiopental (REM + TIO), midazolam + thiopental (MID + TIO), and dexmedetomidine + thiopental (DEX + TIO). After T1DM induction was confirmed, all animals were subjected to a short narcosis of tested anaesthetic or combination of anaesthetics by intraperitoneal injection: TIO 85 mg/kg, FEN 0.005 mg/kg, REM 0.04 mg/kg, MID 2.5 mg/kg, and DEX 0.05 mg/kg of body weight. The hearts were retrogradely perfused according to Langendorff technique. The parameters of the redox status were evaluated from coronary venous effluent and blood samples. Our results demonstrated that REM in combination with TIO had the most prominent positive effect on cardiac function due to significantly improved parameters of cardiac contractility and enhanced antioxidative capacity. Moreover, combination of TIO with other drugs, especially with DEX and MID, had depressive effects on cardiac contractility, HR, and parameters of oxidative stress as well as histological changes. Given that diabetes is an increasingly common entity in the population, this research supplemented knowledge about the effects of anaesthetic agents (belonging to different pharmacological groups, with different pharmacokinetic properties) on myocardial function, oxidative stress, and histological changes in conditions of diabetes.

anaesthetics – diabetic rats – oxidative stress – cardioprotection

Hyperhomocysteinemia, characterised by elevated levels of homocysteine, is a known risk factor for vascular dysfunction, potentially leading to structural changes in blood vessels and can serve as a predictor for pre-eclampsia. While magnesium sulphate is the primary treatment for the prevention and management of seizures in severe pre-eclampsia and eclampsia, diazepam may also be used in certain situations. The aim of this study was to investigate the effects of diazepam on oxidative status and histological alterations of human umbilical arteries in a hyperhomocysteinemia model. Umbilical arteries, isolated from the umbilical cord of healthy women, were cleaned and cut into strips 2–3 mm long. These arterial rings were incubated in Krebs-bicarbonate solution for 3 h at 37°C, aerated with a mixture of 95% oxygen and 5% carbon dioxide. This study included the following four treatment groups: Control (only buffer solution), diazepam (100 μmol/L), homocysteine (1 mmol/L), and combination diazepam (100 μmol/L) + homocysteine (1 mmol/L). The impact of diazepam was assessed by measuring oxidative stress markers, including thiobarbituric acid reactive substances (TBARS), NO₂, superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT). Additionally, histological examination was performed to evaluate structural changes in the arterial wall, using classic haematoxylin-eosin, as well as pentachrome, Movat staining. Findings indicate that diazepam significantly (P < 0.05) reduced oxidative stress by lowering TBARS and NO₂-levels and enhancing antioxidant defences (CAT, SOD, and GSH) in the umbilical arteries. Histologically, diazepam treatment preserved endothelial integrity and prevented smooth muscle cell damage associated with hyperhomocysteinemia. These findings suggest that diazepam may offer protective impact against oxidative damage and vascular structural alterations induced by hyperhomocysteinemia, highlighting its potential therapeutic role in managing vascular complications associated with this condition.

hyperhomocysteinemia – diazepam – umbilical arteries – pre-eclampsia – oxidative stress

Quercetin (QCT), a plant-derived flavonoid, provides cardiovascular benefits. Previous studies showed its protective effect on ischaemia-reperfusion (I/R) injury in young, healthy animals. This study investigated its cardioprotective potential in ageing rats. We administered QCT (20 mg/kg/day orally) to 2-year-old aged rats for 6 weeks. Afterwards, the animals were anesthetised, and their hearts were excised, placed on a Langendorff apparatus and subjected to 30 min of ischaemia followed by 120 min of reperfusion. The infarct size was assessed via TTC staining, and cardiac function recovery was monitored during the first 40 min of reperfusion. Western blotting was used to evaluate the expression of reperfusion injury salvage kinase (RISK) pathway proteins (Akt, PKC-ɛ, eNOS, GSK-3β), antioxidant enzymes (SOD1, SOD2), and apoptosis markers (Bax/Bcl-2). The oxidative status was assessed using markers, thiobarbituric acid reactive substances (TBARS), advanced oxidation protein products (AOPP), ferric reducing ability of plasma (FRAP), fructosamine, and advanced glycation end-products (AGEs), in blood plasma. According to our research, there was no cardioprotective advantage from extended QCT treatment in the heart's recovery after ischaemia. In fact, the impact of QCT was associated with a tendency towards decreasing cardiac functional recovery. In line with physiological data, QCT was unable to activate the RISK signalling pathway in the aged hearts. On the contrary, QCT decreased the cardiac Bax/Bcl-2 ratio suggesting its anti-apoptotic effect in aged rat hearts. Finally, elevated FRAP and AOPP levels suggest that QCT may have a pro-oxidant impact on ageing hearts. According to our results, QCT had less cardioprotective effectiveness in older individuals, most likely as a result of insufficient RISK pathway activation and decreased antioxidant efficiency. Although QCT exerts anti-apoptotic effect, this does not confer protection against I/R injury, making its use for cardioprotection in ageing populations unpromising.

heart – quercetin – cardioprotection – ageing

This study was supported by grants APVV-21-0194, VEGA 2/0104/20, and VEGA 2/0159/24.

Black chokeberry (Aronia melanocarpa) is a rich source of dietary phytochemicals, mainly the bioactive polyphenols. This study performed in mice aortic rings was aimed to complement previous research on the vascular protective effects of phytochemicals by assessing the in vitro effects of black chokeberry extracts (from either dry or frozen berries) on vascular function and oxidative stress elicited via incubation angiotensin II (AII), lipopolysaccharide (LPS), or high glucose (GLUC). Mice aortas were acutely treated with AII (100 nmol/L), LPS (1 μg/mL), or GLUC (400 mg/dL) in the absence or the presence of increasing concentrations of two characterised black chokeberry extracts (12 h incubation: 1, 10, 50, 75, 100, and 500 μg/mL). The vascular rings were then used for reactive oxygen species (ROS) measurements (spectrophotometry – ferrous oxidation xylenol orange assay and immunofluorescence – dihydroethidium stain) and organ chamber studies (vascular reactivity studies using the myograph). Both types of extracts decreased ROS levels in a dose-dependent manner after exposure to AII, LPS, and GLUC for the concentrations between 50 μg/mL and 100 μg/mL and partially restored vascular relaxation with an effect varying from 30% to 80%. The antioxidant effect was related to their ROS scavenging properties; using catalase as positive control, we showed that concentrations of □ 100 μg/mL induced an approximately 50% ROS-direct scavenger effect. As regarding their composition, the dried extract had high levels of rutin, caffeic acid, and chlorogenic acid, while the frozen extract had high levels of cyanidin-glucoside, rutin, and caffeic acid. In conclusion, the black chokeberry extracts demonstrated vascular protective benefits in mice aorta in the ex vivo conditions that mimicked the renin-angiotensin activation, acute inflammation, and hyperglycaemia, by mitigating oxidative stress and improving vascular function.

black chokeberry (Aronia) extracts – oxidative stress – endothelial dysfunction – acute inflammation – hyperglycaemia

This study was supported by the University internal grant 5DOC/1425/03.02.2020.

MicroRNA (miRNA)-based RNA therapeutics are advancing, offering novel approaches to treat various diseases by restoring miRNA expression patterns. Although there is a growing body of literature investigating the function and biology of miRNAs, data on miRNA pharmacokinetics are severely limited, leading to unanticipated pitfalls. We have previously identified several microRNAs as ProtectomiRs against myocardial ischaemia/reperfusion injury, such as miR-125b* and miR-450a. To investigate their potential use as RNA therapeutics, we performed pharmacokinetic studies in mice. A single injection of 10 μg miR-125b* mimic or its scramble miRNA control or vehicle alone (neutral lipid emulsion, NLE) was administered to C57BL/6 mice intravenously. MiR-125b* expression was measured from plasma and left ventricle (LV) at 1, 2, 4, 8, and 24-h post-injection. MiR-125b* level in blood plasma and in LV showed a marked increase at 1-h post-injection, which was decreased at later time points. Time-matched vehicle and scramble miRNA did not alter miR-125b* expression at any time point in plasma or in LV. For the next step of investigation, the vehicle compound NLE was not available on the market anymore; therefore, we have chosen a widely used cation-based new vehicle, jetPEI®. The pharmacokinetic studies of miR-450a with jetPEI and saline have been performed. As a pilot experiment, separate groups of C57BL/6 mice were administered 0.25 mg/kg or 2.5 mg/kg miR-450a intravenously dissolved either in jetPEI or saline. MiR-450a expression was measured from blood plasma and LV at 1- and 60-min post-injection and was increased at both time points only when dissolved in jetPEI. We repeated miR-450a pharmacokinetics with jetPEI using several sampling points (at 1 min and 1, 2, 4, 8, and 24-h post-injection) only at 0.25 mg/kg dose and found that miR-450a expression was significantly increased in LV only at 1 min. Pharmacokinetic properties of miRNAs are not well-understood yet; therefore, such pioneering experiments are of utmost need.

microRNA development – miR-125b* – miR-450a – pharmacokinetic challenges – microRNA solvent

This study was supported by the National Heart Program (NVKP 16-1-2016-0017), Cooperative Doctoral Program (KDP-2020), János Bolyai Research Scholarships of the Hungarian Academy of Sciences (bo_481_21), EKÖP-24-4, and EKÖP-398.

Usnic acid (UA), a natural dibenzofuran secondary metabolite present in various lichens, has garnered extensive research interest due to its diverse biological activities. Its potential therapeutic applications are significant, spanning antimicrobial, antitumour, and antioxidant effects. Despite the historical use of lichen-derived UA in traditional medicine and the established benefits of UA, there is a notable gap in research concerning its cardioprotective properties. This study aimed to investigate the impact of UA on doxorubicin-induced cardiotoxicity in a rat model. UA was extracted from the acetonic extract of the lichen Xanthoparmelia stenophylla and identified through comparison with a standard reference. This study involved 40 male Wistar albino rats. UA was administered orally at a dose of 25 mg/kg daily for 28 days. Following this treatment period, doxorubicin was administered intraperitoneally at a cumulative dose of 15 mg/kg. Three days after the doxorubicin administration, the rats' hearts were excised and examined ex vivo using a Langendorff apparatus. Coronary venous effluent samples were collected to assess oxidative stress markers via spectrophotometry. Additionally, myocardial tissue samples were analysed using histological staining with haematoxylin and eosin. Administration of UA at a dose of 25 mg/kg for 28 days leads to the alleviation of oxidative stress parameters in coronary venous effluent in a model of doxorubicin-induced cardiotoxicity. Also, in the group treated with UA, the architecture of the myocardial tissue was preserved, and degenerative changes are reduced. Our findings demonstrate that UA possesses significant cardioprotective and antioxidant properties, suggesting its potential utility as a cardioprotective agent. Further investigation is required to elucidate the underlying mechanisms through which UA exerts its effects.

lichen – usnic acid – oxidative stress – doxorubicin – cardiotoxicity

This study was supported by the Ministry of Education, Science, and Technological Development of the Republic of Serbia (Agreement Nos. 451-03-9/2021-14/200111 and 451-03-68/2022-14/200378).

Paradoxically, endurance athletes are prone to several cardiac arrhythmias, including atrial fibrillation (AF). Current guidelines suggest ‘an approximately five-fold increased lifetime risk of atrial fibrillation compared to sedentary individuals, despite a lower prevalence of traditional atrial fibrillation risk factors’. Since no comprehensive study has been conducted in a large animal model relevant to humans; therefore, the purpose of our study was to determine the underlying cardiac changes caused by the long-term intense endurance exercise that led to increased susceptibility to arrhythmias. Male beagle dogs were randomised into a ‘Sedentary’ (SED) and an ‘Exercised’ (EXE) group (n = 14–14). The latter group was trained with an intensive treadmill-running protocol for 4 months. To establish the development of the athlete's heart, echocardiography and ECG measurements were performed. In open-chest anaesthetised dogs arrhythmia susceptibility was tested with high-frequency burst stimulation. Following heart removal, myocardial fibrotic changes were quantified, and transmembrane ionic currents and action potential duration were measured. The EXE animals that developed resting bradycardia, sinoatrial node remodelling, and altered parasympathetic and sympathetic balance were also noted. The echocardiography studies showed that EXE animals had greater atrial parameters, including increased left atrial volume (10.4 ± 0.9 vs 11.4 ± 1.4, SED vs EXE, P < 0.05) and left atrial volume index (18.7 ± 1.3 vs 22.4 ± 2.3; SED vs EXE, P < 0.05). The arrhythmia provocation study found that the incidence was higher, and the mean duration of AF was significantly longer in EXE group. Similarly, rapid stimulation in the presence of carbachol (2 ug/kg and 5 ug/kg, respectively) elicited a longer mean duration of AF in EXE animals. ECG recordings showed prolonged P-wave and PQ intervals in EXE animals. Several atrial transmembrane ionic currents' amplitudes were measured by whole-cell configuration of the patch-clamp technique. The cellular electrophysiological studies showed that ICa,L decreased significantly, while Ito increased significantly in EXE animals. Although the length of the action potential did not differ significantly between the groups, their shape differs substantially, carrying the differences due to the variation in the amplitude of the above-mentioned ionic currents. Furthermore, increased levels of fibrosis were found in both atria of EXE dogs. Our study systematically demonstrated several altered structural and electrophysiological features as potential arrhythmia substrates due to intense endurance training and underpinned endurance training-induced atrial fibrillation. However, further studies are necessary to clarify this question in more detail.

athlete's heart – atrial fibrillation – arrhythmia substrate – transmembrane ionic current

This study was supported by EKÖP-24-4 - Tudománnyal fel! Felsőoktatási Doktorvárományosi és Posztdoktori Kutatói Ösztöndíj; EKÖP-403.

The β2 adrenergic receptors (ADRB2) are expressed on the various types of cells, including cardiomyocytes and white adipocytes, playing an important role in cardiovascular system control, glucose, and insulin maintenance as well as energy expenditure and lipolysis. Among the ADRB2 gene, Arg16Gly and Gln27Glu polymorphisms have been linked to the risk of myocardial infarction (MI) and altered lipid metabolism; therefore, the aim of this study was to assess the risk of the selected polymorphisms with MI and with mean values of lipid parameters in patients from the Republic of Srpska. The case-control study comprised 75 patients with MI who were not taking lowering lipid drugs and 79 age and sex-matched healthy controls. The ADRB2 genotypes were determined by qPCR. The results have shown that in the group of patients, carriers of GA or AA genotypes among Arg16Gly polymorphism have statistically lower mean values of high-density lipoprotein (HDL) cholesterol in comparison to the carriers of GG genotype (P = 0.008), as well as lower frequency of diabetes mellitus (P = 0.022). Also, carriers of GA or AA of the same polymorphism have higher values of blood pressure in comparison to the wild-type carriers; however, after adjustment for sex, BMI and diabetes values were on the border of statistical significance (P = 0.072). Assessment of risk for MI has shown that carriers of the C allele among Gln27Glu polymorphism have a reduced risk for MI compared to the carriers of GG genotype (OR1 = 0.46, 95% CI = 0.22–0.96, P = 0.040), which were confirmed in the adjusted model (OR2 = 0.41, 95% CI = 0.19–0.90, P = 0.027). Besides an association of the Gln27Glu ADRB2 polymorphism with reduced susceptibility to MI risk, further analysis with a larger sample size should be taken into consideration for a more precise conclusion.

ADRB2 – lipid metabolism – myocardial infarction

Excessive fructose consumption is associated with metabolic changes and serious health problems. Regular drinking of fructose-sweetened beverages results in obesity, inflammation, and cardiometabolic disorders. In addition, high fructose intake increases oxidative stress. Therefore, administration of antioxidant quercetin, which naturally occurs in fruits and vegetables, could protect against fructose-induced renal impairment. This study aimed to investigate the renal consequences of a 15-week intake of 10% fructose, focusing on the properties of the enzyme Na,K-ATPase and the effect of supplementation with the antioxidant quercetin. We confirmed experimentally that when drinking a 10% fructose solution for 9 weeks, young Wistar rats developed several signs of insulin resistance, as indicated by glucose, insulin, and homeostasis model assessment of insulin resistance (HOMA-IR) values. At the same time, we also focused on monitoring the effect of quercetin administered by gavage for 6 weeks (20 mg/kg/day in 1% methylcellulose solution). Rats from control groups received methylcellulose vehicles. Our study demonstrated that elevated fructose was associated with increased body weight and plasma glucose levels in the rats. Oral administration of quercetin normalised the intake of the fructose solution to levels comparable to water intake. Furthermore, quercetin administration significantly reduced glycaemia. Through the action of polyphenols, we expected protection from renal damage during the development of metabolic disease and preservation of tubular Na,K-ATPase functionality, but this was not confirmed. Also, the increase in plasma creatinine and the decrease in the glutathione GSH/GSSG ratio in the renal tissue homogenate suggest that quercetin administration may not be beneficial in a state of pre-existing renal pathology.

quercetin – fructose – insulin resistance – kidney – Na,K-ATPase

This study was supported by grants APVV-20-0421, VEGA 2/0123/24, and VEGA 2/0148/22.

Hypertension in children is an emerging clinical issue increasingly recognised for its potential to cause early vascular damage and increase cardiovascular risk in adulthood. The pathogenesis of primary hypertension (PHT) in the paediatric population has been linked to oxidative stress, with paraoxonase 1 (PON1) playing a crucial role. PON1, primarily associated with high-density lipoproteins (HDL), hydrolyses lipid peroxides, mitigating oxidative stress and protecting against low-density lipoprotein (LDL) oxidation, which is a significant factor in endothelial dysfunction and atherosclerosis. One of the natural PON1 substrates, homocysteine thiolactone, is involved in the detrimental vascular effect of homocysteine by spontaneously reacting with proteins (protein N-homocysteinylation). Several clinical studies demonstrated the relationship among PON1 deficiency, protein N-homocysteinylation, and cardiovascular disease in adults; however, this relationship in children has not been studied so far. The aim of this study was to analyse PON1 status and N-homocysteinylation of plasma proteins in children with primary hypertension. We analysed 97 children/adolescents aged 11–17 years: 46 with primary hypertension and 51 without cardiovascular abnormalities. Surprisingly, PON1 activity and concentration were significantly higher in the hypertensive group compared to controls. HDL-cholesterol as well as apolipoproteins A-I and A-II did not differ between groups. Despite higher PON1 activity against homocysteine thiolactone in the hypertensive group, the degree of N-homocysteinylation of proteins did not differ significantly from the control group. Additionally, lipolactonase activity of PON1 (activity towards thiobutylbutyrolactone) was lower in hypertensive children with obesity than in those with normal body weight. Hypertension had no effect on PON3 concentration and activity. In conclusion, PON1 concentration and activity are higher in children and adolescents with arterial hypertension, suggesting detrimental role of the enzyme in its pathogenesis. Obesity impairs lipolactonase activity of PON1, which is crucial for its antioxidant activity.

hypertension – children – paraoxonase – N-homocysteinylation

Cardiogenic shock (CS) is a life-threatening state characterised by acutely failing heart function, and despite significant advances in therapy options, CS continues to have a high in-hospital mortality rate of nearly 50%. Lack of standardised defining features of CS leads to delay in treatment, resulting in significant morbidity and mortality. Given the rapid metabolic shifts that occur during CS, we aimed to define the plasma lipidomic shifts during acute myocardial infarction (AMI)-related CS in hemodynamically well-defined patient population. Plasma samples and detailed hemodynamic assessment by non-invasive cardiac system (NICaS) were obtained for 259 patients presenting with ST-elevation myocardial infarction (STEMI) prior to their intervention. Plasma lipidomics was performed using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The lipidomic shifts seen in patients presenting with did not correlate with systolic blood pressure or cardiac index. High lactate levels were negatively correlated with several lysophosphatidylcholine (LPC) species and positively correlated with acylcarnitines. There was a significant reduction in 18 lipid species in patients with high lactate levels, including several LPCs. Hemodynamic assessment of these patients showed that heart rate, stroke index, and Granov Goor Index (GGI, a proxy for left ventricular ejection fraction) had significant correlations with LPC levels, while cardiac output and cardiac index had few or no correlation to lipid profile, respectively. Furthermore, low LPC and high carnitine levels had a significant correlation with in-hospital mortality in patients with cardiogenic shock. In our study, lack of LPC production in setting of acute MI is a predicator of poor cardiovascular hemodynamics and, as a result, in-hospital mortality. This indicates that low plasma phospholipase activity could be used as a marker of poor clinical outcomes and can be a novel therapeutic avenue in cardiogenic shock patients.

cardiogenic shock – lipidomics

This study was supported by the Heart and Stroke Foundation.