Enhancement of Sarcolemmal Calcium Influx in a Novel Mouse Model of Malignant Hyperthermia

Abstract

Malignant hyperthermia (MH) is a potential fatal skeletal muscle disorder that occurs in genetically susceptible individuals on exposure to certain anaesthetic agents. The type 1 ryanodine receptor (RYR1) missense variant c.7300G>A (p.G2434R) is the most common human MH variant. It is classed as a weak variant and is commonly associated with genotype-phenotype discordance. The isogenetic p.G2435R variant in a novel knock-in mouse model has been shown to have features similar to the human MH response. We examined how the p.G2435R variant affects resting sarcolemma Ca2+ entry (RCaE) using the manganese-quench of Fura2 in wildtype (WT), heterozygous (HET) and homozygous (HOM) RYR1G2435R myotubes. We found a gene dose-dependent increase in RCaE with the RYR1G2435R variant. The non-specific cation channel blocker gadolinium (Gd3+) produced a profound block on sarcolemmal RCaE in all three genotypes while blockade using BTP2, which inhibits both TRPC and Stim1/Orai1 entry pathways, had less of an effect. Administration of the TRPC3/6 specific blocker SAR7334 had a significant effect in only the HOM and HET myotubes. The gene dose-dependent increase in RCaE with the RYR1G2435R variant mirrors the gene dose-dependent increase in resting myoplasmic [Ca2+] we have previously observed with this variant. The mechanisms behind this increased RCaE were explored and appear to be the result of multiple factors, all of which are sensitive to Gd3+.