Background/Aims: β-Dystroglycan (β-DG) is a transmembrane glycoprotein that links the intracellular cytoskeleton to the extracellular matrix and is crucial for the molecular pathway of lateral force transmission in muscle. We aimed to investigate the effect of decreasing sarcolemmal cholesterol on the distribution of β-DG, its interaction with dystrophin and the impact on the contraction efficiency of muscle. Methods: Isolated rat extensor digitorum longus muscles were incubated with methyl β-cyclodextrin (MβCD) to deplete cholesterol and with MβCD-cholesterol to restore cholesterol. Electric stimulation protocols were used to determine muscle force and fatigue. Detergent-resistant membranes (lipid rafts) were separated from isolated skeletal muscle sarcolemma. The distribution and interactions of β-DG, caveolin-3 and dystrophin were determined by an immunoreactivity analysis. Results: Cholesterol depletion in muscle results in a weakened force of contraction, which recovers after cholesterol restoration. The rate of fatigue is unaffected, but fatigue recovery is dependent upon cholesterol restoration. MβCD modifies the structures of lipid rafts obtained from MβCD-treated muscles by, displacing the membrane proteins β-DG and caveolin-3 f from the lipid raft, thus reducing the interaction of β-DG with dystrophin. Conclusion: Cholesterol depletion weakens the muscle contractile force by disturbing the sarcolemmal distribution of β-dystroglycan and its interaction with dystrophin, two key proteins in the alignment of lateral force transmission pathway.
Sarcoglycans (SGs) and sarcospan (SSPN) are transmembrane proteins of the dystrophin-glycoprotein complex. Mutations in the genes encoding SGs cause many inherited forms of muscular dystrophy. In this study, using purified membranes of wild-type (WT) and δ-SG knockout (KO) mice, we found the specific localization of the SG-SSPN isoforms in transverse tubules (TT) and sarcoplasmic reticulum (SR) membranes. Immunoblotting revealed that the absence of δ-SG isoforms in TT and SR results in a secondary deficiency of γ-SG and µSPN. Our results showed augmented ATP hydrolytic activity, ATP-dependent calcium uptake and passive calcium efflux, probably through SERCA1 in KO compared to WT mice. Furthermore, we found a conformational change in SERCA1 isolated from KO muscle as demonstrated by calorimetric analysis. Following these alterations with mechanical properties, we found an increase in force in KO muscle with the same rate of fatigue but with a decreased fatigue recovery compared to WT. Together our observations suggest, for the first time, that the δ-SG isoforms may stabilize the expression of γ-SG and µSPN in the TT and SR membranes and that this possible complex may play a role in the maintenance of a stable level of resting cytosolic calcium concentration in skeletal muscle.
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