Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by a loss of motor neurons and, as a result, skeletal muscle atrophy (van Es et al., 2017). Muscle atrophy is the decrease in muscle size/mass that results from loss of contractile and structural proteins and muscle cells (Brooks and Myburgh, 2014; Fanzani et al., 2012). The progressive motor neuron loss, denervation-induced and primary muscle dysfunction and wasting in ALS culminates in paralysis and death (van Es et al., 2017; Loeffler et al., 2016). Unfortunately, there are currently no effective curative treatments or therapeutic strategies for ALS (van Es et al., 2017).
Muscle atrophy is prevalent across many chronic diseases (Powers et al., 2016a), in individuals subjected to prolonged bedrest and immobility (Brooks and Myburgh, 2014), and in astronauts in spaceflight (Fitts et al., 2010). In fact, the neuromuscular system is seen as one of the most heavily impacted physiological systems in microgravity (Fitts et al., 2001). Muscle atrophy in microgravity has been previously linked to a lack of exercise opportunities (Fitts et al., 2010). However, the use of resistive exercise countermeasures programs by astronauts was only able to partially mitigate muscle atrophy in microgravity (Fitts et al., 2010; Tanaka et al., 2017). Given the engineering challenge to generate artificial gravity in spaceflight, and the inability of resistance exercise to prevent atrophy in microgravity, it is essential to continue to delineate the molecular mechanisms underpinning muscle loss in space to enable the development of effective interventions to preserve and/or restore muscle mass. We were provided an opportunity by the Student Spaceflight Experiments Program (SSEP), a space education program aimed at students from elementary school to college level administered through the National Center for Earth and Space Science Education (NCESSE) and the Arthur C. Clarke Institute for Space Education, to conduct an experiment on the International Space Station (ISS). We decided to investigate the underlying causes of muscle atrophy in microgravity. This research may bring us one step closer to developing an effective treatment for muscle wasting and weakness in both astronauts during spaceflight and in individuals with chronic diseases such as ALS.
Oxidative stress contributes to the development of skeletal muscle atrophy in both chronic disease states and inactivity via a myriad of cellular signaling networks (Cutler et al., 2002; Powers et al., 2016b), Microgravity has been shown to induce oxidative stress (Takahashi et al., 2017). Sphingomyelin and ceramide accumulation have been linked to both ALS pathogenesis and to oxidative stress (Cutler et al., 2002). Furthermore, in the SODG86R ALS mouse model, there is evidence of increased gene expression of some sphingomyelinasesenzymes that catalyzes the hydrolysis of sphingomyelin to phosphorylcholine and ceramide (Henriques et al., 2018). This could contribute further to ceramide accumulation. Acid sphingomyelinase (ASM) levels are elevated and activity is increased in the presence of oxidative stress, infection, and inflammation (Kornhuber et al., 2015). However, ASM also releases ceramide, which in itself induces oxidative stress (Jana et al., 2009). These data together suggest that ASM dysregulation may be involved in the pathology of ALS and muscle wasting of spaceflight
We therefore undertook a study to determine ASM levels in
In this study,
This experiment was conducted within a Type 3 Fluids Mixing Enclosure (FME) system (NanoRacks, Houston, TX, USA), designed for space research. FMEs have been used for all previous SSEP experiments (Warren et al., 2013). As depicted in Figure 1, the FME is a flexible silicone tube, divided into three 2.8 mL compartments by plastic clamps. Sterilization of the FME minilab was achieved by autoclaving the tube at 121°C for 20 minutes, spraying the caps and clamps with 70% ethanol followed by exposure to ultraviolet (UV) light for 1 hour.
Type 3 Fluids Mixing Enclosure (FME) Mini-Lab. The FME is 17 cm in length with a total volume of 8.4 mL divided into three 2.8 mL compartments. Volume 1 and 3 contained 2.4 mL of CeMM and Volume 2 contained 2.4 mL of CeMM housing 5,000
Timeline of the experiment. The letter A stands for arrival (at the ISS) and the letter U stands for undock from the ISS.
Travel from Toronto to Houston (FedEx) | A-25 days | 2 days | 2–4 | None |
NanoRacks (Houston) | A-23 days | 12 days | 2–4 | None |
Travel from Ferry to Launch | A-11 days | 11 days | 2–4 | None |
Rocket travel to ISS | A-6 hours | 6 hours | 2–4 | None |
ISS | A | 6 weeks | 21–24 | A+0 days: Unclamp Clamp A, shake gently for 3 seconds U-14 days: Unclamp Clamp B, shake gently for 3 seconds |
Travel from Landing to Houston | U | 4 days | 0–10 | None |
Travel from Houston to Toronto (FedEx) | U+4 days | 24 hours | 2–4 | None |
Prior to the launch, we determined the optimal ratio of CeMM to air in each volume of the FME for maximum survival of
Following return from space, the number of
To create the
Worm lysate samples (30 μg) were separated by 10% SDS-PAGE, transferred to nitrocellulose, Ponceau-S stained, and immunoblotted with rabbit anti-acid sphingomyelinase antibody (sc-11352 Santa Cruz, CA, USA; 1:500 dilution). Prepared samples were analyzed with IRDye 800 CW fluorescent secondary antibody (Mandel Scientific, Guelph ON, Canada; 1:10,000 dilution). Quantification of the Li-Cortm fluorescent signal was performed with the LiCor Odyssey FC (Mandel Scientific). Western blotting for GAPDH (ab A245 Abcam, USA; 1:10,000 dilution detected with Mandel Scientific IRDye 680 RD, 1:10,000 dilution) was performed as a loading control. Samples were run in duplicate.
Comparisons between the space worms and control worms were conducted using the Student's t-test. Statistical significance was determined if p<0.05.
The space worms were received at the lab 70 days after initial loading into the FME. There were 72,050 live space worms, compared to 95,200 live ground worms. As illustrated in Figure 2, the space worms were significantly larger in length and in cross-sectional area than the ground worms. The mean length of the space worms was 0.42±0.13 mm, while the ground worms exhibited a mean length of 0.29±0.076 mm. The mean cross-sectional area of the space worms was 0.0065±0.0031 mm2, while the mean cross-sectional area of the ground worms was only 0.0042±0.0021 mm2. The histograms in Figure 3 demonstrate a broader range of distribution of length and cross sectional area for the space worms compared to the control ground worms.
Length and cross-sectional area (CSA) of ground and space worms. Representative images of (A) ground worms, and (B) space worms are shown. Live
Histograms of length and cross-sectional area (CSA) of space and control
As illustrated in Figure 4, the space worms exhibited reduced ASM-1 and ASM-2 expression, compared to the ground worms.
Ground and space worm lysates immunoblotted for ASM-1 and ASM-2. The space worms exhibited decreased expression of both ASM-1 and ASM-2, compared to the ground worms (left panel ASM Western blot, right panel graph of quantified fluorescent signal). GAPDH immunostaining demonstrates equivalent protein loading. Data are mean ± SD. ***p<0.05, **p<0.1.
The space worm lysate had a higher protein concentration, compared to the ground worm lysate, as determined by a BCA assay (Thermo Scientific). The space worm lysate had a protein concentration of 6.667 μg/mL, whereas the ground worm lysate had a protein concentration of 4.286 μg/mL.
This is, to our knowledge, the first study completed to assess ASM protein expression in
We found that
Interestingly, Kim and Sun (2012) report that inactivation of the ASM homologs leads to slower reproduction and increased longevity of
Of interest, we were able to demonstrate that a population of
There were a number of limitations to our study, as a result of regulations set by NanoRacks, NASA, and SpaceX. Only one tube could be sent to space, thereby permitting no experimental replicates, which limited the power of our experiment. Although we could not send multiple FME tubes to the ISS, we maximized the number of
There were also several limitations of the experimental design. We did not test for the third ASM isoform, ASM-3, which may also affect the lifespan of the nematodes and their oxidative stress levels. We did not test for ASM-3 because a commercially produced antibody was not available. The anti-ASM antibody used was generated against the human ASM, and while it recognized proteins at the correct molecular weight of ASM-1 and ASM-2, it also recognized several other bands of unknown significance. These bands may have been due to non-specific reactivity or reactivity with degradation products. It is unclear why these bands were only seen in the ground control worms, given that GAPDH levels indicated equal loading of ground and space worm lysates. Furthermore, we did not evaluate oxidative stress, ASM-1 or 2 activity, or evaluate behavioral changes in the worms, which may be influenced by oxidative stress and could have served as a surrogate marker (Possik and Pause, 2015). Although this data may have enriched our knowledge, we were mainly focused on the impacts of microgravity on ASM expression. Lastly,
In summary, as high school students working within the SSEP program, we have importantly demonstrated that