Key result
A reconstituted depolarization-induced Ca2+ release platform in HEK293 cells successfully reproduced skeletal muscle excitation-contraction coupling, enabling the functional validation of disease-causing Cav1.1 mutations and drug screening.
Why the study?
Functional validation of novel mutations in depolarization-induced Ca2+ release machinery is time-consuming because it must be performed in a skeletal muscle environment.
A novel reconstituted DICR platform in nonmuscle cells enables efficient functional validation of skeletal muscle disease mutations and drug screening.
Enables efficient preclinical mutation validation and drug screening; leaves open human translation and clinical utility.
In skeletal muscle excitation-contraction (E-C) coupling, depolarization of the plasma membrane triggers Ca 2+ release from the sarcoplasmic reticulum (SR), referred to as depolarization-induced Ca 2+ 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. Exome sequencing has accelerated the discovery of many novel mutations in genes encoding DICR machinery in various skeletal muscle diseases. However, functional validation is time-consuming because it must be performed in a skeletal muscle environment. In this study, we established a platform of the reconstituted DICR in HEK293 cells. The essential components were effectively transduced into HEK293 cells expressing RyR1 using baculovirus vectors, and Ca 2+ release was quantitatively measured with R-CEPIA1er, a fluorescent ER Ca 2+ indicator, without contaminant of extracellular Ca 2+ influx. In these cells, [K + ]-dependent Ca 2+ release was triggered by chemical depolarization with the aid of inward rectifying potassium channel, indicating a successful reconstitution of DICR. Using the platform, we evaluated several Cav1.1 mutations that are implicated in malignant hyperthermia and myopathy. We also tested several RyR1 inhibitors; whereas dantrolene and Cpd1 inhibited DICR, procaine had no effect. Furthermore, twitch potentiators such as perchlorate and thiocyanate shifted the voltage dependence of DICR to more negative potentials without affecting Ca 2+ -induced Ca 2+ release. These results well reproduced the findings with the muscle fibers and the cultured myotubes. Since the procedure is simple and reproducible, the reconstituted DICR platform will be highly useful for validation of mutations and drug discovery for skeletal muscle diseases. Summary Mutations in essential components for depolarization-induced Ca 2+ release (DICR) are implicated into various skeletal muscle diseases. Murayama et al. establish a reconstituted DICR platform in nonmuscle cells for evaluation of disease-causing mutations and drug discovery.
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Murayama et al. (2022) studied Skeletal muscle diseases (Malignant hyperthermia, myopathy). Reconstituted DICR platform (Cav1.1, β1a, Stac3, JP2, Kir2.1 in HEK293 cells) vs. Cells without essential components or wild-type vs mutant was evaluated on Depolarization-induced Ca2+ release (DICR) measured by R-CEPIA1er fluorescence. A reconstituted depolarization-induced Ca2+ release platform in HEK293 cells successfully reproduced skeletal muscle excitation-contraction coupling, enabling the functional validation of disease-causing Cav1.1 mutations and drug screening.
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