In this study, we utilized an in vitro SKM cell culture model to investigate the influence of exercise-related signaling on the expression of 24 genes related to myogenesis, mitochondrial biogenesis, thyroid hormone metabolism, and cellular homeostasis. Our study is novel in that multiple SKM gene expression pathways were concomitantly analyzed and interpreted together, not only for terminal stage myotubes, but also during mid-myogenesis.
Skeletal muscle cells naturally cycle through periods of both protein synthesis and protein breakdown, all of which occurs during the various stages of myogenesis throughout life. Imbalances to these processes result in increased proteolysis, dysregulated mitigation of ROS, mitochondrial dysfunction, and eventual loss of SKM mass and function [6, 7]. Exercise has many beneficial effects on muscle health and is often prescribed as a treatment for preventing or attenuating sarcopenia and pathological atrophy. However, the cross-pathway mapping of exercise-stimulated gene expression has not been fully elucidated, especially in human tissue.
Formoterol is a long-acting β2-adrenergic receptor agonist that has been effectively used as an exercise mimetic with in vivo and in vitro animal models, stimulating many pathways within SKM tissue [21, 22, 31]. Specifically, in rodent models, formoterol increases PGC-1α, mitochondrial respiration, morphology, and biogenesis [21-23]. In rhesus macaques, formoterol treatment increases PGC-1α expression, mitochondrial DNA quantity, and improves mitochondrial respiration of unhealthy myoblasts . Stimulation of B2AR signaling has been implicated as a potential therapeutic target for skeletal muscle wasting disorders, due to this pathway’s influence on regulating SKM protein synthesis and degradation [32-33]. While the use of pharmaceutical therapy utilizing B2AR agonists is currently limited for people experiencing muscular atrophy or disease, it is clear that the B2AR pathway plays a significant role in the regulation of SKM growth , especially in the context of exercise signaling. Additionally, the B2AR pathway is an upstream signaling cascade that is activated by both endurance and resistance exercise and stimulated by epinephrine and norepinephrine. This makes the pathway ideal for in vitro exercise stimulation, rather than stimulating individual downstream molecules with mimetics such as AICAR, which only affects APMK .
The mechanisms of myogenic regulation have been well characterized [9, 10]. However, this regulation has not been well studied in human myotubes throughout the various stages of myogenesis, especially in the context of exercise stimulation. In this study, our exercise mimetic model induced a myogenic program that appears to prolong myoblast proliferation and delay maturation of myotubes, as evidenced by the changes in MYF5, MYOD, and MYOG for both D4 FORM and D6 FORM. The influence of formoterol on the MRFs in this study is unique, as increases in MYOD and MYOG gene expression following exercise have been previously reported . Based on these results, the effects of exercise signaling on myogenesis may be influenced by the timing of the stimulus in relation to what stage the myotubes are on within the myogenic program (i.e. mid- or late-stage myogenesis). Thus, exercise stimuli could interfere with the regeneration or repair of SKM tissue by delaying or prolonging myogenesis. This information may be meaningful in the context of SKM growth and regeneration, as it indicates the timing of additional exercise stimuli should be strategically considered due to its ability to influence the timeline of myotube formation and maturity. To investigate muscle growth during myogenesis, we analyzed the gene expression for two important factors that regulate protein synthesis in response to exercise signaling, including PGC-1α4 [an isoform of PGC-1α, which is a promoter of resistance exercise-induced hypertrophy  and the protein kinase mTOR. We found that formoterol treatment may allow an increase in protein synthesis, despite delayed signaling for myotube formation. These results could indicate greater cellular energetic demands directed towards myotube formation and differentiation in the CON groups, whereas formoterol may have triggered processes more geared towards protein synthesis and myoblast proliferation.
Autophagy is a normal cellular process that removes damaged organelles while recycling functional cellular components that fuse with proximal healthy cells and is an important process during cell differentiation. The regulation of autophagy in healthy cells aims to optimize tissue function as a whole and prevent the accumulation of dysfunctional cells and cellular components, performing as a quality control mechanism. ATG5, a regulator of autophagy, is typically upregulated throughout the myogenic process and is responsible for myotube fusion . Alternatively, exercise upregulates ATG5 in vivo , implicating autophagy to be a primary contributing mechanism of myogenesis and tissue repair. Regulation of the autophagy system declines linearly with age , but is augmented by chronic exercise training, highlighting the importance of regular exercise in regulating cellular processes throughout the lifespan . Interestingly, we found no changes in ATG5 between conditions in this study. However, we found differential responses for ROS mitigation. Skeletal muscle is a major site of ROS generation as a result of energy production and muscle contraction. The accumulation of ROS is detrimental to organismal and tissue health and function with profound tissue dysregulation and inflammation evident as a result . Efficient mechanisms are in place to minimize the accumulation of ROS and prevent damage to DNA, as found in aging muscle . We found reduced GSS expression for all groups compared to D4 CON, and elevated SOD2, a mitochondrial related antioxidant enzyme, for both D6 groups.
The peripheral effects of thyroid hormone signaling within SKM are substantial and play crucial roles in metabolism, function, and growth . Analyzing the expression of THRα and the deiodinases DIO2 and DIO3 is of particular interest in helping to characterize SKM homeostasis due to their regulatory roles in mitochondrial biogenesis and myogenesis . Our results indicate robust activity of TH metabolism in response to exercise mimetic stimulation within SKM. In particular, the increases in DIO2 found in the FORM groups, which indicate increased activation of T4 into T3 within SKM, lead to stimulation of nuclear targets specific to metabolism and myogenesis . To further investigate TH metabolism in connection to mitochondrial homeostasis, we analyzed estrogen-related receptor-α (ERRα), a nuclear receptor that works in tangent with TH receptors, and is co-activated by PGC-1α to regulate mitochondrial processes such as biogenesis, mitophagy, fission, and oxidative phosphorylation. Interestingly, ERRα was elevated at D6 CON but not D6 FORM, despite significant increases in PGC-1α for both groups. We can therefore hypothesize that mitochondrial biogenesis still occurred in the absence of ERRα stimulation, as increases for both PGC-1α and TFAM in the D6 FORM group, were also observed. One explanation for elevated ERRα in the D6 CON group may be related to the increased NRF2 and SOD2 expression found for this condition. This likely indicates an increased need for oxidative phosphorylation and mitigation of ROS, which may have resulted from less mitochondrial biogenesis than the FORM group.