Dysfunctional Ca 2+ dynamics in primary skeletal myocytes from R2509C-RYR1 mice.

The skeletal muscle contracts when depolarization is transmitted to the dihydropyridine receptor in the T-tubule, and Ca2+ is released through type 1 ryanodine receptor (RYR1) from the sarcoplasmic reticulum (SR). Mutations in RYR1 cause severe muscle diseases, such as malignant hyperthermia (MH), a disorder of Ca2+-induced Ca2+ release (CICR) through RYR1 from the SR. We recently reported that volatile anesthetics (e.g., isoflurane) induce MH-like episodes via enhanced CICR in heterozygous R2509C-RYR1 mice. However, the characterization of Ca2+ dynamics has yet to be investigated in skeletal muscle cells from homozygous mice because these animals die in utero. In the present study, we generated primary cultured skeletal myocytes from R2509C-RYR1 mice. We analyzed morphological and functional changes in myocytes from R2509C-RYR1 mice. In homozygous R2509C-RYR1 myocytes, Ca2+ release by depolarization or caffeine was decreased and resting intracellular Ca2+ concentrations ([Ca2+]cyt) was increased, indicating a Ca2+ leak and a reduced SR Ca2+ content. Also, in homozygous R2509C-RYR1 myocytes, sarcomeres were remarkably shortened to the A-band length, even in the resting condition, which was associated with abnormal Ca2+ homeostasis, particularly an increase in the resting [Ca2+]cyt. In heterozygous R2509C-RYR1 muscle cells, heat sensitivity for intracellular Ca2+ release via the heat-induced Ca2+ release mechanism was increased, and the cellular temperature was elevated in the presence of isoflurane. Our findings suggest that the R2509C mutation in RYR1 causes dysfunctional Ca2+ dynamics in a mutant-gene dose-dependent manner in the skeletal muscles, in turn provoking MH-like episodes and embryonic lethality in heterozygous and homozygous mice, respectively.