Absence of γ-sarcoglycan alters the response of p70S6 kinase to mechanical perturbation in murine skeletal muscle

Abstract

BackgroundThe dystrophin glycoprotein complex (DGC) is located at the sarcolemma of muscle fibers, providing structural integrity. Mutations in and loss of DGC proteins cause a spectrum of muscular dystrophies. When only the sarcoglycan subcomplex is absent, muscles display severe myofiber degeneration, but little susceptibility to contractile damage, suggesting that disease occurs not by structural deficits but through aberrant signaling, namely, loss of normal mechanotransduction signaling through the sarcoglycan complex. We extended our previous studies on mechanosensitive, γ-sarcoglycan-dependent ERK1/2 phosphorylation, to determine whether additional pathways are altered with the loss of γ-sarcoglycan.MethodsWe examined mechanotransduction in the presence and absence of γ-sarcoglycan, using C2C12 myotubes, and primary cultures and isolated muscles from C57Bl/6 (C57) and γ-sarcoglycan-null (γ-SG-/-) mice. All were subjected to cyclic passive stretch. Signaling protein phosphorylation was determined by immunoblotting of lysates from stretched and non-stretched samples. Calcium dependence was assessed by maintaining muscles in calcium-free or tetracaine-supplemented Ringer’s solution. Dependence on mTOR was determined by stretching isolated muscles in the presence or absence of rapamycin.ResultsC2C12 myotube stretch caused a robust increase in P-p70S6K, but decreased P-FAK and P-ERK2. Neither Akt nor ERK1 were responsive to passive stretch. Similar but non-significant trends were observed in C57 primary cultures in response to stretch, and γ-SG-/- cultures displayed no p70S6K response. In contrast, in isolated muscles, p70S6K was mechanically responsive. Basal p70S6K activation was elevated in muscles of γ-SG-/- mice, in a calcium-independent manner. p70S6K activation increased with stretch in both C57 and γ-SG-/- isolated muscles, and was sustained in γ-SG-/- muscles, unlike the transient response in C57 muscles. Rapamycin treatment blocked all of p70S6K activation in stretched C57 muscles, and reduced downstream S6RP phosphorylation. However, even though rapamycin treatment decreased p70S6K activation in stretched γ-SG-/- muscles, S6RP phosphorylation remained elevated.Conclusionsp70S6K is an important component of γ-sarcoglycan-dependent mechanotransduction in skeletal muscle. Our results suggest that loss of γ-sarcoglycan uncouples the response of p70S6K to stretch and implies that γ-sarcoglycan is important for inactivation of this pathway. Overall, we assert that altered load-sensing mechanisms exist in muscular dystrophies where the sarcoglycans are absent.

The Dystrophin Complex: Structure, Function, and Implications for Therapy

“…Expression of WT γ-sarcoglycan was able to restore the ERK-1 response while expression of γ-sarcoglycan engineered with Y6A mutation failed to do so despite its normal sarcolemma localization, supporting that tyrosine 6 phosphorylation mediates ERK-1 response (20). Recently, γ-sarcoglycan was shown to regulate another mechanical-responsive kinase, p70S6 (84, 114). Upon stretch, p70S6K was activated in both Sgcg null and WT muscles but failed to return to normal level in mutant muscle.…”

Focal adhesion kinase and its role in skeletal muscle

“…This decrease was seen at 2 h post-exercise but not 48 h post-exercise (Graham et al 2015). In C2C12 cells, 30 min of static stretch decreases phosphorylated FAK by roughly 50 % while FAK phosphorylation was not altered in isolated primary muscle fibers from control or γ-sarcoglycan knockout mice over the course of 4 h of static stretching (Moorwood et al 2014). In proliferating C2C12 cells, cyclic strain activates FAK 15 min post-strain, followed by a return to baseline at 30 min post-strain (Kumar et al 2004).…”

Optimizing Mechanical Stretching Protocols for Hypertrophic and Anti-apoptotic Responses in H9c2 Cardiomyocytes

“…Furthermore, two primary mechanosensitive intracellular pathways have been associated with the IGF-1 actions in skeletal and cardiac muscle physiology [26,34]; the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, the activation of which is involved in cellular processes such as protein synthesis, hypertrophy and protection from apoptosis, and the Ras/Raf/MEK/Erk 1/2 signaling pathway, which has been shown to increase muscle cell proliferation. The outcomes of these two pathways are based on complex interactions that require comprehensive identi cation, since in some cell types, PI3K and Erks appear to act in concert, e.g., both PI3K and Erks are possibly required for the myogenic differentiation of myoblasts [26,41].…”

“…Given the complexity of the in vivo models of cardiac adaptations [33] in response to mechanical loading, ex vivo and in vitro models of muscle mechanical loading [34] applied on myocardial cells are crucial for understanding the cellular and molecular mechanisms that mediate loading-induced adaptations.…”