Physical activity is crucial for health¹. For athletes, physical activity is associated with a notable adaptation of skeletal muscle cells^1-4. Elite athletes routinely incorporate ergogenic aids into their training to enhance their sports performance. Improving athletic performance is the focus of active ongoing research^5,6. Studies of the basic cellular mechanisms behind the diverse reactions in the human body that are associated with the simultaneous use of ergogenic aids and physical activity have included animal models. These in vivo studies have faced various challenges.

A recent study hypothesized that a form of artificial electrical stimulation termed electrical pulse stimulation (EPS) can lead to the same reactions normally produced in vivoin skeletal muscle tissues in vitro⁷. To explore this hypothesis, the authors constructed a model of physiological electrical stimulus by determining EPS parameters including voltage, stimulus interval, and frequency. The results demonstrated that the system was appropriate to study intracellular metabolism upon acute muscle contraction⁷.

Two metabolic characteristics of skeletal muscles in response to physical activity are the increases in the number of mitochondria^8-10 and oxidative capacity^4,10,11. These characteristics are reported to occur in skeletal muscle cells stimulated by EPS. Among the ergogenic aids, the essential amino acid L-arginine enhances athletic performance by increasing the synthesis of creatine and nitric oxide (NO), which rapidly promotemaximum muscular strength^13-15. NO is synthesized under the control of NO synthase (NOS)¹⁶. There are three unique isomers of NOS expressed by three different genes. Neuronal NOS (nNOS, NOS1) is found throughout the body. Inducible NOS (iNOS, NOS2) is produced by macrophages and regulated by cytokines. Endothelial NOS (eNOS, NOS3) is found in vascular endothelial cells and participates in vascular smooth muscle relaxation^17-19. The expression of nNOS in skeletal muscles has been reported to increase during contusion²⁰, muscle activation²¹, and aging²², while it decreases during denervation²³. The expression of eNOS in skeletal muscles, in particular, has been reported to increase upon long-term exercise^21,24. The expression of iNOS increases in individuals with diseases, such as chronic heart failure^25,26 and autoimmune myopathy²⁷, and in vitrowhen cultured skeletal muscle cells are exposed to bacterial endotoxin or inflammatory cytokines^28,29. However, iNOS precursor is absent or present in trace amounts in healthy skeletal muscles of rats or mice^30-32. The NO produced by these NOS isoforms has been associated with the generation of muscular strength³³, blood flow control³⁴, myoblast differentiation³⁵, respiration³⁶, and glucose homeostasis³⁷ in skeletal muscles. Thus, as elucidated by numerous prior studies, EPS plays the same role as the nerve impulse that enhances muscle adaptation, and NO acts as the regulator in the increase of muscular strength in skeletal muscles.

This knowledge suggests that simultaneous exercise and intake of NO supplement are likely to synergistically influence athletic performance in terms of muscle adaptation and muscular strength. The present study aimed to provide the basic biochemical data of this scenario.

The loss of myofibril proteins in skeletal muscles caused by a disease or physical inactivity seriously diminishes muscular strength and function. In contrast, resistance training prevents the loss of myofibrils. Resistance training reportedly causes the differentiation and enlargement of muscles by stimulating their signaling³⁸. This is because the electrical stimulation of somatic neurons leads to a notable adaptation of skeletal muscle cells^1-4. Thus, the present study examined the effects of EPS and NO on muscle contraction, and whether the simultaneous treatment with EPS and NO enhanced muscle contraction in C2C12 mouse skeletal muscle cells.

When C2C12 cells were differentiated and treated with EPS for 4 days, the cellular length was shorter in the +E/-S group than in the CON group and in the +E/+S group than in the -E/+S group. The shortest cellular length was observed on day 4. The results indicate that EPS produced muscle contraction, which is consistent with a previous study that reported the shortened length of skeletal muscle cells as a consequence of EPS³⁹. Presently, when the cells were treated with EPS, a higher level of NO secretion was observed in the +E/-S group than in the CON group, and in the +E/+S group than in the -E/+S group, suggesting that the electrical stimulation enhanced NO production. This agrees with the reports describing the increased nNOS expression in skeletal muscles upon muscular activity²¹ and increased NOS expression upon long-term exercise^21,24. These present and prior results imply that the muscle contraction caused by EPS can increase NOS expression and the subsequent secretion of NO. The assessment of protein expression using western blotting revealed that the levels of AMPK, JNK, Akt, nNOS, eNOS, GLUT4, and PGC1α proteins were higher in the +E/-S group than in the CON group, while the expression of iNOS or ACC was not detected. This suggests that the muscle contraction caused by EPS increased NO secretion, which increases the activity of the GLUT4 sugar transporter and the consequent transport of glucose to skeletal muscles^40-43. Simultaneously glycolysis is suppressed^44,45 and the metabolic pathways in the muscle including mitochondrial respiration are regulated^46,47, which facilitates the β-oxidation of fatty acids in the mitochondria and suppresses fatty acid synthesis.

When C2C12 cells were differentiated and treated with various concentrations of the NO donor SNAP for 4 days, the cellular length was shorter in the -E/+S group than in the CON group, and progressively shortened as the concentration of SNAP increased. The shortest cellular length was observed on day 4. These observations support the view that muscle contraction occurs due to NO. This is also supported by the description of the increased muscle contraction capacity when cells were provided with an external source of NO, which increased the movement of calcium ions in the sarcoplasmic reticulum⁴⁷. In addition, studies have reported that NO production that occurs following the activation of NOS by insulin and muscle contraction regulates the influx of glucose into skeletal muscle and promotes improved athletic performance^21,48,49. This implies that the NO provided by the SNAP treatment may have acted analogously as the NO produced by NOS that is activated by insulin or muscle contraction. When the cells were treated with SNAP, a higher level of NO secretion was observed in the -E/+S group than in the CON group, and progressively increased as the SNAP concentration increased, suggesting that SNAP led to enhanced NO secretion. SNAP is anNO donor. Hence, the increased concentration of SNAP may have yielded levels of NO. However, comparison of the levels of NO secretion in the -E/+S and +E/+S samples treated with identical SNAP concentration revealed a significantly higher level of NO in the +E/+S group than in the - E/+S group, especially in the +E/S2.0 group, indicating that the measured level of NO was not a result of the concentration of SNAP. Western blotting showed that the levels of AMPK, JNK, Akt, nNOS, GLUT4, PGC1α proteins were higher in the -E/+S group than in the CON group. This supports the suggestion that NO can cause muscle contraction, which leads to an increased production of nNOS expression increased, thereby increasing NO secretion. The resulting increased glucose influx into the muscle cells would facilitate the β-oxidation of fatty acids in the mitochondria.

Presently, when differentiated C2C12 cells were simultaneously treated with EPS and various concentrations of SNAP for 4 days, a markedly shorter cellular length was observed in the +E/+S group than in the CON group, and the length progressively decreased as the SNAP concentration increased. On day 4, the cellular length of the +E/S2.0 group was 84.2±13.2㎛, which was the shortest among all experimental groups. When the cells were simultaneously treated with EPS and SNAP, a higher level of NO secretion was observed in the +E/+S group than in the CON group as the SNAP treatment concentration increased, with the highest level of NO observed in the +E/S2.0 group. The results concerning cellular length and NO secretion supported the view that the simultaneous treatment of EPS and NO enhanced muscle contraction. Western blotting showed that the levels of AMPK, JNK, Akt, eNOS, GLUT4, and PGC1α proteins were higher in the +E/+S group than in the CON group, with a predominantly higher level observed in the +E/S2.0 group. These observations suggest that the simultaneous treatment with EPS and NO enhanced muscle contraction, increased the eNOS expression so that NO secretion increased, and increased the influx of glucose into the muscle cells to facilitate the β-oxidation of the fatty acids in the mitochondria. However, the expression of nNOS was markedly lower in the +E/S2.0 group, which is so far supported by a single report concerning the reduction of nNOS expression during denervation23. It is conceivable that EPS and the enhanced production of NO may have created an environment similar to that of denervation in C2C12 cells.

The present results demonstrate the synergistic action of EPS and NO in skeletal muscle cells. The data should prove valuable as basic information to investigate the biological phenomena occurring in individual organs. It is essential that future in vivo studies clinically verify whether simultaneous exercise and intake of NO precursor or NO supplement enhances athletic performance and whether the simultaneous treatment has a positive influence on the enhancement of athletic performance.

This study suggests the possibility that simultaneous exercise and intake of NO precursor or NO supplement helps enhance athletic performance. Systematic studies are needed to reveal the detailed mechanisms of the signaling pathway and interaction between physical activity and NO intake. The ultimate outcome could be a treatment regimen that benefits the performance of elite athletes as well as the general public.