Analysis of Human Cultured Myotubes Responses Mediated by Ryanodine Receptor 1

Abstract

Malignant hyperthermia is a life-threatening condition caused by autosomal dominant mutations in the ryanodine receptor type 1 gene. Identifying patients predisposed to malignant hyperthermia is done through the Ca-induced Ca release test in Japan. We examined the intracellular calcium concentration in human cultured muscle cells and compared the sensitivity of myotubes to ryanodine receptor type 1 activators based on the Ca-induced Ca release rate. We assessed the utility of this method as an identifying test for predisposition to malignant hyperthermia. Muscle specimens were obtained from 34 individuals undergoing the Ca-induced Ca release test. We cultured myotubes from residual material and monitored changes in intracellular calcium concentration after exposure to the ryanodine receptor type 1 activators caffeine, halothane and 4-chloro-m-cresol by measuring fura-2 fluorescence. We determined the half maximal effective concentrations (EC50) for the test compounds in each myotube and calculated cut-off points using receiver operating characteristic curves. Seventeen patients each were classified into the accelerated and non-accelerated groups based on their Ca-induced Ca release rate. The EC50 values for caffeine, halothane and 4-chloro-m-cresol of the accelerated group were significant lower than those of the non-accelerated group (P <0.001, P <0.001 and P <0.001, respectively). The calculated cut-off points of EC50 values for caffeine, halothane and 4-CmC were 3.62 mM, 2.28 mM and 197 μM, respectively. An increased sensitivity to ryanodine receptor type 1 activators was seen in myotubes in the accelerated group. This functional test on human cultured myotubes indicates that the alteration of their intracellular Ca2+ homeostasis may identify the predisposition to malignant hyperthermia.