Metabolic Flexibility and Mitochondrial Bioenergetics in the Failing Heart. Therapeutic Approaches

The formation of new mitochondria (mitochondrial biogenesis) is supported by synthesis of mitochondrial proteins and replication of mitochondrial DNA, both processes under the control of the transcription factor peroxisome proliferator-activated receptor γ (PPAR γ) and its co-activator α (PGC1α). PGC1α is considered the master regulator of mitochondrial biogenesis due to the activation of nuclear respiratory factors 1 and 2, (NRF1 and 2) as well as mitochondrial transcription factor A (TFAM), all targeting genes encoding for mitochondrial proteins and mtDNA^22,23. PGC1α is downregulated in humans with HF leading to decreased mitochondrial density^24,25. PPARα is an isoform predominantly regulating FA oxidation enzymes, and is downregulated in both animal models and humans with HFrEF²⁶ but is increased in HFpEF associated with metabolic syndrome, which is associated with an increase in FA oxidation¹⁹.

There is plethora of evidence that specific activities of individual ETC complexes are decreased in HF²⁰. ETC complexes aggregate into functional supercomplexes²⁷, and this form of organization provides a more efficient electron transport and is protective against excessive mitochondrial reactive oxygen species (ROS) generation²⁷. A decrease in mitochondrial supercomplexes has been reported in HF²⁸.

Cardiolipin (CL) is an anionic phospholipid with four acyl chains that are enriched in linoleic acid ((C18:2)4-CL), which resides in the inner mitochondrial membrane. CL provides structural and functional support to ETC components²⁹, and its depletion results in reduced activities of ETC complexes. CL is also proposed to maintain the structural integrity of ETC supercomplexes, as it may act as a molecular ‘glue’ to hold the complex protein subunits together in a supramolecular organization^30,31. In humans, reduced (C18:2)4-CL, due to defective CL remodeling (Barth syndrome), causes dilated cardiomyopathy associated with destabilization of all supercomplexes containing complex IV, loss of complex I from the supermolecular assembly and decrease in the individual enzymatic activities of complexes I, III and IV³¹, indicating that CL is essential for the function of cardiac mitochondria. Myocardial ischemia³² and HF^33,34,35 are associated with mitochondrial dysfunction and CL peroxidation, loss of total CL content and decrease in (C18:2)4-CL. Approaches that target cardiolipin are likely to improve electron transport across the ETC and, by correcting mitochondrial function, might be beneficial in treating HF.

Classic examples of redox reactions are the transfer of electrons between reduced and oxidized subunits within the mitochondrial ETC according to their redox potential. Mitochondria have multiple redox couples (redox players)⁴⁴ including NAD⁺ (oxidized)/NADH (reduced), NADP⁺/NADPH, GSSG (glutathione disulfide)/GSH (glutathione) (Figure 3). Energized mitochondria have a high NADH concentration to provide electrons for oxidative phosphorylation⁴⁵. In contrast, in the extra-mitochondrial space, the NADPH/NADP⁺ ratio is maintained in a reduced state (the reduced NADPH > the oxidized NADP) via several enzymatic reactions in order to drive reductive biosynthesis and maintain antioxidant defense. The cytosolic GSH/GSSG couple is also maintained in a reduced state that is needed for ROS detoxification. These redox couples are interconnected (Figure 4). In mitochondria, the inner membrane nicotinamide nucleotide transhydrogenase (NNT) reduces NADP⁺ at the expense of NADH oxidation, utilizing the mitochondrial inner membrane protonmotive force to drive this process. NNT is a physiologically relevant source of mitochondrial NADPH⁴⁶. The NADPH/NADP⁺ couple supplies electrons to keep mitochondrial GSH pool in order to scavenge H₂O₂, a strong ROS⁴⁷. In conclusion, redox signaling regulates metabolism while metabolic state influences redox signaling. The NAD⁺/NADH redox couple is a critical node integrating metabolic and signaling events.

The redox signaling network linked to the NAD⁺/NADH couple depends on the total mitochondrial NAD pools. NAD is a substrate for enzymes including the SIRT family, which continuously converts NAD⁺ to nicotinamide. As NAD is degraded, cardiomyocytes must maintain a constant pool by de novo synthesis or recycle nicotinamide to replenish NAD. In cardiomyocytes, mitochondrial NAD pool is relatively high matching its critical role in mitochondrial Krebs cycle and ETC⁴⁸. Pathological cardiac hypertrophy, the prerequisite of HF, is associated with a decrease in the cardiomyocyte NAD pool⁴⁹. Similar observation was reported in diabetic cardiomyopathy, a model of HFpEF⁵⁰.

The oxidized form, NAD⁺, is an electron acceptor in the redox reactions. Therefore, NAD⁺ and NADH interconvert but are not irreversibly consumed. NAD⁺ participates in all major energetic pathways including glycolysis, Krebs cycle, FA oxidation, ketone body metabolism, and ETC (Figure 2). NAD⁺ is a potent activator of the Krebs cycle enzymes whereas NADH is a Krebs cycle allosteric inhibitor, and increases in ETC defects^45,51,52. For example, Complex I⁴⁵ and IV⁵³ defects lead to increased mitochondrial NADH content. The deficiency of frataxin, a mitochondrial protein integral to the assembly and function of iron-sulfur proteins in ETC complexes I, II and III and aconitase (Krebs cycle), is associated with an 85-fold decrease in cardiomyocyte NAD⁺/NADH ratio and pathologic cardiac hypertrophy⁵². Approaches to correct mitochondrial ETC defects increased NAD⁺ content⁵¹. In conclusion, the disruption of the electron flow to oxygen by ETC defects increases NADH causing a highly-reduced redox environment within mitochondria. The cardiac amount of the oxidized form, NAD⁺, is reported reduced in HFrEF⁵⁴, and either unchanged⁵⁵ or altered⁵⁶ in HFpEF.

While NADH/NAD⁺ redox ratio determines the production of mitochondrial ROS, the NADPH/NADP ratio is key to antioxidant defense. They are linked by the NNT enzyme that transfers electrons from NADH to NADP⁺ (Figure 3).

Sirtuins (SIRTs) remove an acetyl group from lysine residues in an NAD⁺-dependent manner by cleaving NAD⁺ to nicotinamide⁵⁷, and are reported to prolong lifespan in mammals⁵⁸. There are seven mammalian SIRTs that differ in their cellular localization. Although all SIRTs are NAD⁺-dependent, the extramitochondrial SIRT 1 and mitochondrial SIRT3 are well-known players in the heart. SIRT 1 protects against pathologic cardiac hypertrophy, and SIRT 1 knockout mice exhibit developmental cardiac defects⁵⁹. Sustained SIRT 1 overexpression causes cardiomyopathy whereas moderate SIRT 1 expression ameliorates age-induced cardiac hypertrophy and dysfunction⁶⁰, suggesting its effect is dose-dependent. SIRT 1 also protects mitochondrial function by activating PGC-1a⁶¹ to increase mitochondrial FA oxidation⁶². Overall, NAD⁺, via SIRT 1, regulates pathological hypertrophy and mitochondrial metabolism.

SIRT3 is the major mitochondrial NAD⁺-dependent deacetylase⁶³. SIRT3 knockout causes cardiac hypertrophy and failure under stress⁶⁴ while overexpression protects against pathological hypertrophy via activating antioxidant mechanisms⁶⁵.

SIRT 1 and SIRT3 regulate bioenergetic metabolism during energetic crises. SIRT3-mediated deacetylation activates enzymes involved in glycolysis^66,67, FA oxidation^68,69,70, Krebs cycle cycle⁷¹, and the ETC⁷². By upregulating metabolic machinery during states of decreased fuel availability, SIRT3 appears to be a critical metabolic regulator of coupling substrate oxidation with the formation of reducing equivalents to ATP production thus maximizing efficiency.

There is a large variability regarding the reported mitochondrial ETC defects in HF. The causal relationship between these defects and the decrease in ATP has not been defined. For example, a severe murine complex I defect did not cause energy deficit⁴⁵ suggesting that in the murine heart ATP production is not directly related to complex 1 activity. In contrast, most studies report bioenergetic impairment in human subjects diagnosed with HF, which manifest as decrease in cardiac ATP, phosphocreatine (PCr)^73,74, or, most common, a decline in the pCr/ATP ratio^73,75,76.

Metabolic inflexibility with an excessive increase in FA oxidation seems detrimental in HFpEF⁷⁷. In this regard, the β-adrenergic receptor antagonist, carvedilol, used in HF to reduce cardiac workload and decrease oxygen consumption, also inhibited mitochondrial FA uptake, increased glucose oxidation and limited the infarct size after ischemia⁷⁸ indicating that balancing the metabolic health is beneficial for the heart.

Malonyl-CoA inhibits carnitine palmitoyltransferase (CPT)¹, the rate-limiting enzyme in mitochondrial FA uptake (Figure 2), and its amount is dependent on the balance between the synthesis via acetyl-CoA carboxylase and degradation via malonyl-CoA decarboxylase. Inhibiting malonyl-CoA decarboxylase in animal models improved ischemic-induced cardiac dysfunction, reduced cardiac FA oxidation, and increased the glycolytic flux⁷⁹. Studies of malonyl-CoA decarboxylase inhibitors are yet to be performed in human patients with HF.

Due to the observed and possibly incomplete metabolic switch towards increased glucose use in both animal models and humans with HF, it is proposed that stimulating glucose oxidation may be an attractive therapeutic strategy to compensate for the energetically ‘starved’ failing heart⁸⁰. Ketone body metabolism is altered in HF. There is an increased ketone utilization in the severely failing heart in humans^81,82. Further research is needed to understand the role of ketone oxidation in the failing heart, and to determine whether targeting ketone metabolism is an efficient approach to improve energetics in HF.

Although the increased oxidative stress is an accepted pathogenic mechanism in HF, clinical trials yielded negative results to support the long term role of ROS scavengers to alleviate HF^83,84. The lack of long-term benefits may be related to the inability to reach effective therapeutic doses to stoichiometrically scavenge the ROS due to poor absorption, decreased cellular uptake or lack of strategy to match the ROS generation which is a continuous process. Mitochondrial targeted antioxidants have been tested on experimental models of cardiac disease and HF. For example, XJB-5-131, a mitochondria-targeted ROS scavenger is reported to decrease ROS generation and maintain mitochondrial and cardiac functions in rats subjected to ischemia-reperfusion injury⁸⁵. Similar effects were obtained with another mitochondrial antioxidant, MitoTEMPO⁸⁶. The compound EUK-8, a mimetic of two major mitochondrial antioxidant enzymes (superoxide dismutase and catalase, Figure 4), suppressed the progression of cardiac dysfunction and diminished ROS production and oxidative damage in dilated cardiomyopathy in mice⁸⁷. These compounds are yet to be tested in human subjects with HF.

As cardiac⁵⁴ and circulating⁸⁸ NAD pool are reported decreased in HF, NAD-boosting strategies are expected to be beneficial to the cardiac metabolic health. For example, the food supplementation with nicotinamide riboside, the most energy efficient NAD precursor was found beneficial in a murine model of dilated cardiomyopathy and transverse aorta constriction by stabilizing myocardial NAD⁺ levels in the failing heart⁸⁹. The oral supplementation with nicotinamide riboside in patients with advanced HF decreased systemic inflammation by normalizing mitochondrial function in peripheral blood mononuclear cells⁹⁰. Elevating the NAD level suppressed mitochondrial protein hyperacetylation and cardiac hypertrophy, and improved cardiac function in responses to stresses⁹¹.

NAD⁺-dependent SIRTs have been investigated as therapeutic targets in HFpEF induced by diabetes. For example, resveratrol, a polyphenol and well-known SIRT 1 activator, alleviated diabetic cardiomyopathy via activating SIRT 1, 2, 3 and 5^92,93, improved glucose metabolism in human subjects^94,95 and decreased oxidative stress in cultured cardiomyocytes⁹⁶. In a rodent model of genetic obesity, resveratrol decreased cardiac fibrosis and improved FA metabolism⁹⁷.

In the mitochondrial ETC, electrons are passed from donors to acceptors according to their redox potential. As ETC defects delay or reverse the electron transport, providing alternative paths for electron transport, which bypass the ETC defect, rescued mitochondrial function in ETC defects. For example, methylene blue (MB), an FDA approved pharmacological drug used to treat various ailments in human subjects^{98,99,100,101,102,103,104,105,106,107,108,109} and rodents^{110,111,112,113,114}, may provide such an electron route. MB has a low redox potential that allows the compound to receive electrons from complex I^114,115,116 and become reduced (MBH2) while being able to be re-oxidized by cytochrome c back to MB^114,117. Therefore, MB is protective because it helps the electrons to bypass the complex I and III defects and still maintain oxidative phosphorylation (Figure 5). We recently reported that MB protected retinal photoreceptors in a murine model of mitochondrial complex I defect¹¹⁸, and improved cardiac function by shifting electron away from NADH in diabetic cardiomyopathy, a model of HFpEF¹¹⁹.

Coenzyme Q (CoQ) pool is composed by two redox coenzymes, the reduced uniquinol and the oxidized ubiquinone. CoQ is endogenously synthesized, converted to ubiquinol by two-electron reduction from energetic substrates fed into complexes I and II (i.e., pyrucate, acetylCoA), which is then oxidized back to ubiquinone by donating electrons to complex III (Figure 2). Incomplete, one-electron reduction of CoQ produces semiquinone, which is a highly reactive radical. An increase in the reduced CoQ pool (ubiquinol) causes a reverse electrons flow back to complex I resulting in ROS generation¹²⁰. Circulating CoQ is decreased in patients with HF¹²¹, which correlated with poor clinical outcome and increased mortality¹²². Q-SYMBIO clinical trial¹²³ revealed a reduction in mortality after 2 years of treatment with CoQ. Recently, CoQ analogues with more efficient penetrability into the mitochondria have been developed. The delivery to mitochondria was improved by novel quinone conjugates that are tethered to lipophilic, cationic triphenyl-phosphonium moieties, such as MitoQ and SkQ¹²⁴, which have proved efficient in experimental models of HF¹²⁵. The administration of idebenone, a short-chain synthetic CoQ analogue¹²⁶, has had promising benefits in small clinical trial of genetic mitochondrial defects¹²⁷ and HF in experimental models¹²⁸ and human subjects¹²⁹.

Cardiolipin is decreased of oxidized in HF. Maintaining the amount and integrity of cardiolipin may be an efficient therapeutic approach to improve mitochondrial bioenergetics in HF. The cell-permeable tetrapeptide MTP-131 (Bendavia or Elamipretide) localizes to the mitochondrial inner membrane¹³⁰, protects against cardiolipin oxidation and improves mitochondrial function^131,132. MTP-131 reduced pathologic hypertrophy and cardiac remodeling, and improved mitochondrial and cardiac function in murine^133,135,135, porcine¹³⁶ and canine¹³⁷ models of HF. In human subjects with HFrEF, elamipretide is safe and well tolerated, and improved left ventricular function¹³⁸.

Altered Ca²⁺ homeostasis leading to impaired excitation–contraction coupling occurs in many types of HF¹³⁹. Mitochondria are critical in regulating cardiomyocyte calcium dynamics because all membrane-bound Ca²⁺ pumps are ATP-dependent. In the myocardium, Ca²⁺ necessary for the cross-bridge actin-myosin cycles derives mostly from the extracellular space via the voltage-dependent Ca²⁺ channels with a lower contribution from the sarcoplasmic reticulum (SR) via the SR Ca²⁺ release channels, also called ryanodine receptors. Diastolic relaxation depends upon the Ca²⁺ sequestration within the SR via the SR Ca²⁺ ATPase (SERCA2a) and expulsion out of cardiomyocyte versus a Ca²⁺ pump and Na-Ca exchanger.

HFpEF is defined by impaired diastolic relaxation. Increasing the duration of the diastole by decreasing heart rate led to modest benefits in patients with HFpEF¹⁴⁰ potentially by providing more time for Ca²⁺ to move to the SR via SERCA2a. However, SERCA2a is reported downregulated in HF¹⁴¹, and SERCA2a overexpression improved cardiac function in experimental models of HF^142,143. Gene therapy through infusion of adeno-associated virus 1/SERCA2a in patients with HFrEF did not improved the clinical course¹⁴⁴ suggesting that it is the SERCA2a ATP-dependent activity rather than amount that is impaired in human HF.