Introduction

Skeletal muscle must sense and adapt to mechanical loads to maintain its size and strength, a process known as mechanotransduction. The dystrophin glycoprotein complex (DGC), a group of proteins at the muscle cell membrane, is a key player in this process, providing both structural support and a hub for cellular signaling. When components of the DGC, such as γ-sarcoglycan, are absent, it leads to severe muscular dystrophies. This study investigates the specific role of γ-sarcoglycan in translating mechanical force into biochemical signals, focusing on how its absence disrupts critical pathways that regulate muscle mass.

Research Objective

The primary goal of this research was to define the role of γ-sarcoglycan in mechanosensitive signaling within skeletal muscle. The specific objectives were to:

Investigate how the absence of γ-sarcoglycan alters signaling pathways in response to passive mechanical stretch.
Determine the activation patterns of key signaling proteins, particularly p70S6 kinase (p70S6K), in both normal and γ-sarcoglycan-deficient muscles.
Assess the influence of calcium and the mTOR signaling pathway on the observed differences in mechanotransduction.

Key Findings

Basal activation of the growth-promoting protein p70S6K was significantly elevated in resting muscles from mice lacking γ-sarcoglycan (γ-SG-/-) compared to normal mice.
This heightened p70S6K signaling was found to be independent of both extracellular and intracellular calcium, distinguishing it from other aberrant signaling pathways (like ERK1/2) that are calcium-dependent in dystrophic muscle.
In normal muscle, passive stretch induced a transient increase in p70S6K activation, which returned to near-baseline levels after 90 minutes of stimulation.
In contrast, γ-SG-/- muscle exhibited a prolonged and sustained p70S6K activation in response to stretch, suggesting γ-sarcoglycan is essential for the inactivation or “turn-off” signal for this pathway.
When the upstream activator mTOR was blocked with rapamycin, stretch-induced phosphorylation of the downstream target S6RP was eliminated in normal muscle but persisted in γ-SG-/- muscle, indicating the activation of an alternate, compensatory signaling pathway.

Methodology

Organisms: The study utilized isolated extensor digitorum longus (EDL) muscles from wild-type C57BL/6 mice and γ-sarcoglycan-null (γ-SG-/-) mice, which serve as a model for limb-girdle muscular dystrophy.
Experimental Conditions: Isolated muscles were subjected to ex vivo cyclic passive stretch (15% strain for up to 90 minutes) in an organ bath. This approach simulated mechanical loading without the confounding factors of active muscle contraction.
Key Techniques: Immunoblotting was used to quantify the phosphorylation levels of key signaling proteins (p70S6K, S6RP, ERK1/2, Akt), which indicates their level of activation. Pharmacological inhibitors like rapamycin (mTOR inhibitor) were used to dissect the signaling cascade.

Importance for Space Missions

Muscle atrophy due to mechanical unloading is a significant health risk for astronauts on long-duration missions. This research provides fundamental insights into mechanotransduction, the core process by which muscle cells sense and respond to physical forces. The p70S6K pathway is a central regulator of muscle protein synthesis and growth. This study’s finding that a structural protein, γ-sarcoglycan, is critical for properly terminating this growth signal after stimulation is a major step forward. An inability to deactivate this pathway could lead to dysfunctional signaling and contribute to poor adaptation to changes in load, as experienced in microgravity. Understanding these molecular “off-switches” is vital for developing targeted countermeasures to preserve muscle mass and function during spaceflight.

Knowledge Gaps & Future Research

The precise molecular mechanism by which γ-sarcoglycan’s presence leads to the deactivation of p70S6K is not yet understood. Identifying the specific phosphatases or inhibitory proteins involved is a critical next step.
The identity of the alternative, mTOR-independent signaling pathway that phosphorylates S6RP in γ-SG-/- muscle needs to be determined.
Further research is required to translate these findings from a muscular dystrophy model to healthy muscle experiencing the mechanical unloading of microgravity.
The potential interplay and crosstalk between sarcoglycan-mediated signaling and other load-sensing systems in the muscle cell, such as integrins, remains an open area of investigation.

Results

This study identifies a novel and critical function for the γ-sarcoglycan protein as a negative regulator of the p70S6K mechanosignaling pathway. By demonstrating that its absence leads to uncontrolled, prolonged signaling in response to mechanical stress, the research uncovers a key “braking” mechanism essential for normal muscle homeostasis. These findings not only deepen our understanding of the pathology of muscular dystrophies but also illuminate the intricate signaling networks that govern muscle adaptation to load, providing valuable insights for addressing muscle atrophy in space.

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