α-Syntrophin Modulates Myogenin Expression in Differentiating Myoblasts

Abstract

Backgroundα-Syntrophin is a scaffolding protein linking signaling proteins to the sarcolemmal dystrophin complex in mature muscle. However, α-syntrophin is also expressed in differentiating myoblasts during the early stages of muscle differentiation. In this study, we examined the relationship between the expression of α-syntrophin and myogenin, a key muscle regulatory factor.Methods and FindingsThe absence of α-syntrophin leads to reduced and delayed myogenin expression. This conclusion is based on experiments using muscle cells isolated from α-syntrophin null mice, muscle regeneration studies in α-syntrophin null mice, experiments in Sol8 cells (a cell line that expresses only low levels of α-syntrophin) and siRNA studies in differentiating C2 cells. In primary cultured myocytes isolated from α-syntrophin null mice, the level of myogenin was less than 50% that from wild type myocytes (p<0.005) 40 h after differentiation induction. In regenerating muscle, the expression of myogenin in the α-syntrophin null muscle was reduced to approximately 25% that of wild type muscle (p<0.005). Conversely, myogenin expression is enhanced in primary cultures of myoblasts isolated from a transgenic mouse over-expressing α-syntrophin and in Sol8 cells transfected with a vector to over-express α-syntrophin. Moreover, we find that myogenin mRNA is reduced in the absence of α-syntrophin and increased by α-syntrophin over-expression. Immunofluorescence microscopy shows that α-syntrophin is localized to the nuclei of differentiating myoblasts. Finally, immunoprecipitation experiments demonstrate that α-syntrophin associates with Mixed-Lineage Leukemia 5, a regulator of myogenin expression.ConclusionsWe conclude that α-syntrophin plays an important role in regulating myogenesis by modulating myogenin expression.

Nuclear Recruitment of Neuronal Nitric-oxide Synthase by α-Syntrophin Is Crucial for the Induction of Mitochondrial Biogenesis

“…However, ␣-Syntrophin and Dystrophin have been also observed in the inner nuclear membrane of cultured myocytes, and its level increases during myogenesis in nuclei (57,58). Interestingly, nNOS␣ has been revealed in the nuclear membrane of HeLa cells in association with Dystrophin and ␣-Syntrophin (46).…”

α-Syntrophin is required for the hepatocyte growth factor-induced migration of cultured myoblasts

“…Primary culture of the skeletal muscle was performed as previously described [20]. In brief, skeletal muscle cells were isolated from the forelimbs and hindlimbs of neonatal mice (0–1 day old) from C57BL6/J wild-type (C57), α-syntrophin-knockout (αKO) [22], α-syntrophin transgenic (FLA) [23], and α/β2-syntrophin-double knockout mice (AB) [24].…”

“…Transfection of α-syntrophin-specific siRNA was performed as described previously [20]. In brief, C2 myoblasts were grown in 24-well plates at a density of 5.0 x 10 4 cells/ml for 24 h. Cells were transfected with control siRNA or α-syntrophin-specific siRNA by incubation with lipofectamine 2000 for 6 h. The cells were allowed to recover from the transfection by transferring into fresh 10% FBS-containing medium and incubated for 18 h. Subsequently, cells were treated with HGF (50 ng/ml) and incubated for 1 h.…”

“…Little is known about the function of syntrophin in the early stages of muscle development. Recently, we have shown that α-syntrophin is involved in the expression of myogenin, a key myogenic regulatory factor, by interaction with MLL5 in the early stages of myogenic differentiation [20]. In this study, we have focused on the intracellular localization of syntrophin, particularly in the migrating muscle cells.…”

Myocilin Interacts with Syntrophins and Is Member of Dystrophin-associated Protein Complex

“…Five different syntrophins have been reported in mammals, and they were named ␣1-, ␤1-, ␤2-, ␥1-, and ␥2-syntrophin (28). ␣1-Syn was shown to be the predominant syntrophin type in skeletal and cardiac muscles (29,30), and its level dramatically increases in differentiating C2C12 myotubes (31). To confirm that myocilin may interact with syntrophins in mammalian cells in physiological conditions, we performed co-immunoprecipitation experiments using C2C12 myotubes and skeletal muscles.…”