Establishment of reconstituted depolarization-induced Ca²⁺ release platform for drug discovery of skeletal muscle diseases

Abstract

In skeletal muscle, depolarization of the plasma membrane triggers Ca²⁺ release from the sarcoplasmic reticulum (SR),referred to as depolarization-induced Ca²⁺ release (DICR). DICR occurs via the type 1 ryanodine receptor (RyR1), which physically interacts with the dihydropyridine receptor Cav1.1 subunit in specific machinery formed with additional essential components including β1a, Stac3 adaptor protein and junctophilins. It has recently become clear that mutations in these components cause various skeletal muscle diseases. However, no specific treatment has been developed yet. In this study, we established a high-throughput platform of the reconstituted DICR in HEK293 cells. The essential components were effectively transduced using baculovirus vectors, and Ca²⁺ release was quantitatively measured with R-CEPIA1er, a fluorescent ER Ca²⁺ indicator. High [K⁺] depolarization triggered rapid Ca²⁺ release, indicating successful reconstitution of DICR. We tested several known drugs modulating DICR. Whereas RyR1 inhibitors, dantrolene and Cpd1, suppressed DICR, twitch potentiators, e.g., perchlorate, accelerated DICR. These results well reproduced the findings with the muscle fibers and the cultured myotubes. The reconstituted DICR platform will be highly useful for drug discovery for skeletal muscle diseases.