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 (EC 50) for the test compounds in each myotube and calculated cutoff 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 EC 50 values for caffeine, halothane and 4-chloro-m-cresol of the accelerated group were significant lower than those of the nonaccelerated group (P <0.001, P <0.001 and P <0.001, respectively). The calculated cutoff points of EC 50 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 Ca 2+ homeostasis may identify the predisposition to malignant hyperthermia.

The effects of dantrolene in the presence or absence of ryanodine receptor type 1 variants in individuals predisposed to malignant hyperthermia

“…However, the CICR rate test can reveal abnormalities associated with Ca 2+ release from the SR, a process mainly controlled by RYR1 in skeletal muscle. 9 The EC 50 values of 4CmC and caffeine in participants with accelerated CICR rates in this study were lower than the previously reported cut-off values to diagnose MH, 9 which confirmed that the participants in this study had MH predisposition. The RYR1 variants identified in this study are shown in Table 1.…”

“…As described previously, 9 myoblasts were obtained from the surplus muscle pieces after CICR examination and cultured in Dulbecco’s modified Eagle’s medium and F12 medium (DMEM/F12; Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma–Aldrich, St Louis, MO, USA). The myoblasts were then frozen and stored at –70°C.…”

Effects of Remimazolam on Intracellular Calcium Dynamics in Myotubes Derived from Patients with Malignant Hyperthermia and Functional Analysis of Type 1 Ryanodine Receptor Gene Variants

“…The isometric tension was measured using a force transducer to determine the CICR rate. MH predisposition was achieved if CICR was increased and defined as a CICR rate > two standard deviations (SD) above the mean of the control (IVCT and CHCT negative) [16] CICR values [17,18].…”