Efficient pharmacological modulation of autophagy in isolated muscle fibers: impact on excitation-contraction coupling

Macroautophagy/Autophagy is a key regulator of muscle mass and of muscle adaptation to stress and defective Autophagy is a feature of many muscle disorders. Still, how changes in autophagic flux influence the integrity and function of differentiated muscle fibers remains under-documented. Specifically, links between Autophagy and mechanisms involved in CA²⁺ homeostasis and excitation-contraction coupling are largely unexplored. We developed an assay to monitor Autophagy modulation in mouse muscle fibers maintained in culture. Exposure to 3-methyladenine, an inhibitor of Autophagy initiation, reduced the density of autophagic vesicles. Conversely, hydroxychloroquine, a blocker of autolysosome formation, as well as two mTOR inhibitors that activate Autophagy, rapamycin and torin-1, enhanced the vesicle density. The density of lysosomal vesicles was increased by mTOR inhibitors and by hydroxychloroquine, but insensitive to 3-methyladenine. Measurements of CA²⁺ signals associated with contractile activation revealed that voltage-activated sarcoplasmic reticulum CA²⁺ release was unaffected by torin-1 but was depressed in 3-methyladenine- and in hydroxychloroquine-exposed fibers, suggesting that restraining autophagic flux is detrimental to excitation contraction coupling. The density of the inner plasma membrane network that carries the electrical excitation was depressed by 3-methyladenine and hydroxychloroquine, likely contributing to the function defect. Results establish that autophagic flux is preserved and can be manipulated in cultured muscle fibers, and revealed the power of the approach to tackle the cellular and subcellular consequences of Autophagy modulation. They also uncover the possibility that Autophagy is a determinant of maintenance and/or function of excitation-contraction coupling, with a potential role in several muscle disease situations.Abbreviations: 3-MA: 3-methyladenine; EC: excitation-contraction; HCQ: hydroxychloroquine; MTM1: myotubularin 1; RYR1: ryanodine receptor 1.