In adults, skeletal muscles make up approximately 30 to 40% of body mass. Along with adipose tissue, skeletal muscles growth and maintenance are the main sites of energy utilization. To gain a better understanding adult muscle build-up and its associated energy metabolism, we employed a mouse model with transgenic expression of myostatin propeptide and dramatic muscle growth. The results demonstrated continuous and significant muscle buildup at 12 months of age, with 76% to 153% increase in individual muscles of transgenic mice over the wild-type mice. In muscle tissue, the basal lamina is extremely thin, it is relatively difficult to determine if the nuclei are outside the basal lamina, which can be a good indicator of active satellite cells. In principle, muscle fiber nuclei or "centralized" nuclei can be a good indicator of muscle tissue in active developing stage. From the muscle fiber histology, the transgenic mice also showed the increased nuclei in the central region of muscle fiber, indicating active myofiber fusions in adult stages of the transgenic mice. Previous studies showed that a lack of myostatin increased muscle regeneration through enhanced satellite cell activation and self-renewal, leading to better muscle healing and reduced fibrosis after injury [14
]. We believe that the enhanced muscle mass of the transgenic mice is initiated by muscle-specific expression of myostatin propeptide at early stage of muscle development. It is continuously maintained until adult stage.
In relation to myogenic regulatory factors, the results from microarray and qRT-PCR analysis showed increased expression of myogenin, Cdk inhibitor p21, Fstl1 and rock1. During myogenesis, MyoD and Myf5 are redundant in myoblast specification whereas myogenin with either MyoD or Mrf4 are required for differentiation [24
]. Myogenin is associated with terminal differentiation and fusion of myogenic precursor cells to new or existing fibers. When satellite cells are activated, cell-cycle markers, MyoD and Myf5 transcripts are detectable. Subsequent satellite cell differentiation is marked by the appearance of myogenin [24
]. In this study, we did not observe significant changes of MyoD, Myf5 and Mrf4 gene expressions. Cdk inhibitor p21 is induced during early stage of skeletal muscle differentiation, and a high level of p21 expression is sustained when myotubes are re-exposed to high mitogen media, its expression is critical for myocyte viability [25
]. Cdk inhibitor p21 was significantly up-regulated in the transgenic mice. Regarding to Fstl1, it is not known about its definite biological role in muscle tissue. Although Fstl1 is classified as a protein similar to follistatin, it only share 7% sequence homology with follisatin [26
]. Follistatin binds to activin to neutralize its activity by prevent its binding to type II receptor, therefore blocking myostatin activity when transgenic expression of follistatin specifically to skeletal muscle [5
]. Follistatin also directly antagonize myostatin during myogenesis [27
]. During the embryonic stage, Fstl1 mRNA is strongly depressed by MyoD induction [28
]. In adult muscle, Fstl1 appears to behave like a myokine that acts on vascular endothelial cells as it is secreted into the media by cultured skeletal muscle cells [26
]. Fstl1 has direct action on endothelial cell signaling pathways as it was upregulated by muscle ischemia, and its over-expression enhance endothelial cell differentiation and migration. Fstl1 can stimulate revascularization in response to ischemic insult through its ability to activate Akt-eNOS signaling [29
]. The increased expression of Fstl1in the adult muscle in the transgenic mouse model may simply result from muscle hypertrophic status. It is certainly worth further investigation. In regard to Rock1, it is a down-stream effector of Rho GTPase or RhoA, playing a critical role in myoblast fusion [30
]. RhoA is progressively and specifically down-regulated for execution of tissue-specific morphogenetic events such as fusion into multinucleated syncitia, and maintenance of the terminally differentiated phenotype. Rock appears to concur in keeping myoblasts cycling and in preventing commitment to terminal differentiation [31
]. A further study of Fstl1 and Rock1 in this model is likely to yield new information regarding their importance and specific roles in muscle myogenesis in adult stages.
In adults, muscle regeneration requires coordinated actions of capillary morphogenesis, satellite cells, and interactions between inductive signals from myogenic factors and ECM [32
]. The identified ECM genes such as procollagens had been reported to be up-regulated during adult skeletal muscle regeneration [33
]. The increased expression of MMP2 is consistent with a report that demonstrated that increased MMP2 expression and activation is concomitant with regeneration of new myofibers [34
]. Biglycan is a leucine-rich proteoglycan involving matrix organization, as well as modulation of growth factors [35
]. Biglycan, along with periostin, was highly up-regulated during adult muscle regeneration, and modulated by transformation growth factor β1 [33
]. The up-regulation of ECM components further complements the enhanced expression of myogenic regulatory factors of the skeletal muscle. Taken together, these results support a distinct regulatory mechanism of myofiber formation in adult skeletal muscle, resulting from the over-expression of myostatin propeptide.
The balance of protein synthesis and degradation determines net protein deposition of skeletal muscle. In skeletal muscle tissue, four proteolytic systems are possibly involved in protein degradation, including the calpain system, the caspase system, the lysosomal system, and the proteasome. The expression levels of calpain 3, 7 and caspase 7 was down-regulated in the transgenic mice. Caspase 7 is a member of the cysteine-aspartic acid protease family, which plays a central role in the execution-phase of cell apoptosis. Caspases cleave and activate other caspases that subsequently degrade cellular targets, leading to cell death. Caspase 7 is one of the effector caspases, or downstream activator caspases. Skeletal muscles with neurogenic atrophy showed distinct up-regulation of caspase 7 and 9. Expression of caspase 7 was restricted to atrophic fibers, and up-regulated by caspase-9 in proteolytic cascade of degradation of denervated muscle fibers [36
]. These results may suggest that enhanced muscle build-up of the adult stage in the transgenic mice is also supported by decreased activities of apoptosis.
It has been well established that myostatin is implicated in the induction of muscle cachexia. Increased levels of myostatin have been implicated in AIDS patients, sarcopenia, chronic human muscle disuse atropy, glucocorticoid-induced muscle atrophy [37
] A systemic administration of myostatin also induced cachexia [43
] while functional blockage of myosatin by intraperitoneal injection of its antibody increases muscle mass and strength in dystrophic mouse model [44
]. The mechanism of myostatin action on muscle cachexia appears to activate the ubiquitin proteolytic system independently of cytokine-tumor necrosis factor-α (TNF-α) and transcription factor-κB (NF-κB) pathway [45
]. Myostatin induced muscle atrophy through up-regulation of Forkhead box O (FoxO1) transcription factor, and atrophy-related genes such as atrogin-1. FoxO1 have recently beenidentified as a key activator of the atrophy process downstreamof AKT in the IGF-1 signaling pathway [46
]. Transgenic over-expression of active FoxO1 in skeletal muscle was also shown to inhibit protein synthesis, causing severe skeletal muscle atrophy [48
]. In the transgenic mice with possibly depressed myostatin function by its propeptide, we detected a decreased level of atrogin-1 expression. No significant changes in TNF-α and NF-κB expressions were detected in the muscle tissues from the transgenic mice, which is consistent with the observations of myostatin-knockout mice [45
]. The role of Fox O1 in the current myostatin propeptide transgenic model is under investigation.