Inhibition of mechanosensitive cation channels inhibits myogenic differentiation by suppressing the expression of myogenic regulatory factors and caspase‐3 activity

Abstract

Mechanosensitive cation channels (MSC) are ubiquitous in eukaryotic cell types. However, the physiological functions of MSC in several tissues remain in question. In this study we have investigated the role of MSC in skeletal myogenesis. Treatment of C2C12 myoblasts with gadolinium ions (MSC blocker) inhibited myotube formation and the myogenic index in differentiation medium (DM). The enzymatic activity of creatine kinase (CK) and the expression of myosin heavy chain-fast twitch (MyHCf) in C2C12 cultures were also blocked in response to gadolinium. Treatment of C2C12 myoblasts with gadolinium ions did not affect the expression of either cyclin A or cyclin D1 in DM. Other inhibitors of MSC such as streptomycin and GsTMx-4 also suppressed the expression of CK and MyHCf in C2C12 cultures. The inhibitory effect of gadolinium ions on myogenic differentiation was reversible and independent of myogenic cell type. Real-time-polymerase chain reaction analysis revealed that inhibition of MSC decreases the expression of myogenic transcription factors MyoD, myogenin, and Myf-5. Furthermore, the activity of skeletal alpha-actin promoter was suppressed on MSC blockade. Treatment of C2C12 myoblasts with gadolinium ions prevented differentiation-associated cell death and inhibited the cleavage of poly (ADP-ribose) polymerase and activation of caspase-3. On the other hand, delivery of active caspase-3 protein to C2C12 myoblasts reversed the inhibitory effect of gadolinium ions on myogenesis. Our data suggest that inhibition of MSC suppresses myogenic differentiation by inhibiting the caspase-3 activity and the expression of myogenic regulatory factors.

Regulation of transient receptor potential canonical channel 1 (TRPC1) by sphingosine 1-phosphate in C2C12 myoblasts and its relevance for a role of mechanotransduction in skeletal muscle differentiation

“…On this basis, and in consideration of our previous findings that Ca 2+ influx is a prerequisite for S1P-induced myoblast differentiation (Squecco et al, 2006), and the evidence reported here that TRPC1 localized especially after S1P stimulation into lipid microdomains, where key Ca 2+ signalling proteins are concentrated (Murata et al, 2007), we have next explored the potential involvement of TRPC1 in skeletal myogenesis. Of interest, the silencing of TRPC1 expression dramatically hampered the ability of S1P to promote Journal of Cell Science 122 (9) the expression of muscle differentiation markers and myoblastmyotube transition, in agreement with the results of our group and others showing that the pharmacological inhibition of SACs prevents skeletal myogenesis (Formigli et al, 2007b;Wedhas et al, 2005). Interestingly, we have also shown that TRPC1 expression was tightly regulated during myogenesis and modulated by known factors involved in skeletal myogenesis: the promyogenic agent, S1P , and a potent inhibitor of skeletal muscle specific gene expression, TGFβ (Olson et al, 1986).…”

“…Since previous reports of our group and others (Formigli et al, 2007b;Wedhas et al, 2005) have demonstrated that the pharmacological inhibition of SACs blocks skeletal myogenesis and that S1P exerts its pro-myogenic action through SAC activation, we finally examined the relevance of TRPC1 in skeletal muscle differentiation and the ability of the bioactive lipid to modulate the channel expression. To this purpose, native, SCR-and TRPC1-siRNA-treated C2C12 cells were cultured in differentiation medium (DM) and assayed for the accomplishment of skeletal muscle differentiation in the presence or absence of S1P.…”

“…It has been recently reported that activation of stretch-activated channels (SACs) may also play a role in muscle biology and myogenesis (Formigli et al, 2007b;Wedhas et al, 2005). SACs are non-selective ion channels that are present in a huge variety of cells and organisms, where they participate in mechanotransduction, converting membrane stretch into electrical Ca 2+ , K + and Na + current across the plasma membrane, and biochemical signals (Ambudkar, 2006;Sachs and Morris, 1998).…”

“…In particular, SAC expression and activity are prevalent in neonatal skeletal muscle tissue and undifferentiated myoblasts, and decline as myoblasts mature and fuse into myotubes (Formigli et al, 2007b;Haws and Lansman, 1991). Moreover, the inhibition of these channels with several pharmacological compounds, including gadolinium chloride (GdCl 3 ), streptomycin and the spider venom toxin (GsMTx4), has been shown to suppress the expression of the typical myogenic markers and the myoblast-myotube transition, suggesting that SAC activation may lead to increased Ca 2+ entries necessary for skeletal muscle differentiation (Formigli et al, 2007b;Wedhas et al, 2005). Notably, there is also evidence supporting a role for SACs in the Ca 2+ -handling abnormalities observed in muscle diseases such as mdx mouse and human Duchenne dystrophies, stretch-induced muscle damage and cardiac hypertrophy (Allen et al, 2005;Lammerding et al, 2004).…”

Role for stress fiber contraction in surface tension development and stretch-activated channel regulation in C2C12 myoblasts

“…Taking into consideration the emerging evidence of the roles played by SACs (9,43) and cytoskeletal remodeling in skeletal muscle differentiation (9,29), this mechanism of SAC activation may have profound implications in muscle development, regeneration, and diseases, underlying the need to understand the functions of the subplasmalemmal acto-myosin network and its ability to transmit tensional forces to the cell membrane.…”