Abstract
The molecular mechanisms involved in myogenic differentiation are relatively well-known. Myogenic differentiation is regulated by the sequential activation of the basic helix-loop-helix myogenic regulatory transcription factors (MRFs), and biomechanical signals play an important role in the regulation of myogenesis. In this study, we sought to determine whether simulated microgravity culture using Gravite® may affect myoblast differentiation and expression of MRF genes. Although rat myoblasts, L6 cells were differentiated to myotubes in an incubation period-dependent manner, myogenesis of L6 cells was significantly attenuated under simulated microgravity (10-3G) conditions. Real-time Reverse transcription polymerase chain reaction (RT-PCR) showed that expressions of Myog, Myf6, Mef2c, Des, and Ckm under 1 G conditions increase in an incubation period-dependent manner, and that Myod1 expression was specifically observed to increase transiently in the early phase. However, expressions of Myod1 and Myog were significantly inhibited under simulated microgravity conditions. To clarify the molecular mechanisms, L6 cells were treated with 5-AzaC, and further incubated with differentiation medium under 1 G or 10−3 G conditions. The results showed differences in expression levels of Myod1, Myog, and, as well as those of myotube thickness between 1 G and 10−3 G conditions, completely disappeared in this experimental condition. Modified HpaII tiny fragment enrichment by ligation-mediated PCR (HELP)-assay showed that kinetic changes of DNA methylation status were attenuated in simulated microgravity conditions. These results indicate that microgravity regulates myogenesis and Myod1 expression by controlling DNA methylation.
Highlights
Sarcopenia is defined as an age-related loss of skeletal muscle mass and strength
Gene expression analyses of myogenesis-relating genes under microgravity conditions In order to clarify the molecular mechanisms of attenuation of myogenesis under microgravity conditions, expression levels of myogenesis-related genes were evaluated using real-time Reverse transcription polymerase chain reaction (RT-PCR) (Fig. 2): Expression of Myog, Myf[6], Mef2c, Des, and Ckm under 1 G conditions increased in an incubation period-dependent manner
Previous reports have suggested that biomechanical signals play an important role in the regulation of myogenesis
Summary
Sarcopenia is defined as an age-related loss of skeletal muscle mass and strength. Beginning with the 4th decade of life, and symptoms progress with age.[1] If muscle mass accounts for up to. 60% of body weight, pathological changes in skeletal muscle can cause serious effects on older adults. The therapeutic outcome of age-related skeletal muscle atrophy and weakness remains unknown.[2,3,4] The understanding of molecular mechanisms of myogenic differentiation process will result in better treatment outcomes, since impaired regulation of myogenic differentiation is closely associated with age-related skeletal muscle dysfunction.[5,6,7,8] It is well known that myogenic differentiation is regulated by the sequential activation of the basic helixloop-helix myogenic regulatory transcription factors (MRFs): MyoD, Myf[5], myogenin, and MRF4 (Myf6).[9,10,11] MyoD in particular, is involved in the commitment of cells to the myogenic lineage.[12,13,14]
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