Myostatin Inhibits Myoblast Differentiation by Down-regulating MyoD Expression

Abstract

Myostatin, a negative regulator of myogenesis, is shown to function by controlling the proliferation of myoblasts. In this study we show that myostatin is an inhibitor of myoblast differentiation and that this inhibition is mediated through Smad 3. In vitro, increasing concentrations of recombinant mature myostatin reversibly blocked the myogenic differentiation of myoblasts, cultured in low serum media. Western and Northern blot analysis indicated that addition of myostatin to the low serum culture media repressed the levels of MyoD, Myf5, myogenin, and p21 leading to the inhibition of myogenic differentiation. The transient transfection of C 2 C 12 myoblasts with MyoD expressing constructs did not rescue myostatin-inhibited myogenic differentiation. Myostatin signaling specifically induced Smad 3 phosphorylation and increased Smad 3⅐MyoD association, suggesting that Smad 3 may mediate the myostatin signal by interfering with MyoD activity and expression. Consistent with this, the expression of dominant-negative Smad3 rescued the activity of a MyoD promoterreporter in C 2 C 12 myoblasts treated with myostatin. Taken together, these results suggest that myostatin inhibits MyoD activity and expression via Smad 3 resulting in the failure of the myoblasts to differentiate into myotubes. Thus we propose that myostatin plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in animals that lack functional myostatin is because of deregulated proliferation and differentiation of myoblasts.Expression of a differentiated skeletal muscle phenotype involves the initial determination of mesoderm cells toward the myogenic lineage followed by their proliferation and irreversible withdrawal from the cell cycle. Concomitant with this is the expression of muscle-specific genes, culminating in the formation of differentiated multinucleated myotubes. Specification of the myogenic lineage and differentiation of skeletal muscle cells are critically dependent on the basic helix-loop-helix transcription factors MyoD, Myf5, myogenin, and MRF4, collectively known as the MyoD family muscle regulatory factors (MRFs)

Association of the K153R polymorphism in the myostatin gene and extreme longevity

“…For instance, under starvation conditions, TORC1 is responsible for up-regulating autophagy, a process that generates energy by degrading portions of the cytoplasm (Neufeld 2010;Ravikumar et al 2010). A number of studies have demonstrated that myostatin acts as a negative regulator of mTOR-directed signalling, which is consistent with its inhibitory effect on protein synthesis (Amirouche et al 2009;Langley et al 2002;Lipina et al 2010;McFarlane et al 2006;Rios et al 2004;Sartori et al 2009;Trendelenburg et al 2009) and more specifically on protein translation in skeletal muscle (Trendelenburg et al 2009). On the other hand, myostatin deletion is beneficial for bone density, insulin sensitivity and heart function in senescent mice (Morissette et al 2009), and inhibition of ActRIIB, the cell membrane receptor for which myostatin has the highest affinity (Sakuma and Yamaguchi 2012), increases survival (by 17 %) in myotubularin-deficient mice, due to a delay in the point at which animals experience weight loss or complete hind limb paralysis (Lawlor et al 2011) (X-linked myotubular myopathy is a severe form of congenital myopathy which most often manifests with severe perinatal weakness and respiratory failure).…”

“…The mTOR pathway is an evolutionarily conserved nutrient-sensing pathway that adjusts metabolism and growth to amino acid availability, growth factors, energy status and stress (Fenton and Gout 2011;Sengupta et al 2010). A number of studies have demonstrated that myostatin acts as a negative regulator of mTOR-directed signalling (Amirouche et al 2009;Langley et al 2002;Lipina et al 2010;McFarlane et al 2006;Rios et al 2004;Sartori et al 2009;Trendelenburg et al 2009), which provides support for a potential role of myostatin inhibition in longevity. Myostatin deletion is in fact beneficial for bone density, insulin sensitivity and heart function in senescent mice (Morissette et al 2009).…”

Modulation of reactive oxygen species in skeletal muscle by myostatin is mediated through NF‐κB

“…Myostatin is an inhibitor of myogenesis regulating both the proliferation and the differentiation of myoblasts (Thomas et al. , 2000; Langley et al. , 2002) and overexpression of Mstn in vivo results in cachexia in mice (Zimmers et al.…”

Key signalling factors and pathways in the molecular determination of skeletal muscle phenotype

“…This inhibition is mediated, at least in part, through the p38 MAPK stress response pathway (Philip et al, 2005) (Figure 4). Mst also appears to directly inhibit muscle differentiation by interfering with the activity of MyoD (Langley et al, 2002). Muscle wasting conditions, such through disease or disuse atrophy, are associated with elevated Mst in affected muscles (McCroskery et al, 2003).…”