Malignant hyperthermia, environmental heat stress, and intracellular calcium dysregulation in a mouse model expressing the p.G2435R variant of RYR1

Abstract

Background: The human p.G2434R variant of the RYR1 gene is most frequently associated with malignant hyperthermia (MH) in the UK. We report the phenotype of a knock-in mouse that expresses the RYR1 variant p.G2435R, which is isogenetic with the human variant.

Methods: We observed the general phenotype; determined the sensitivity of myotubes to caffeine-, KCl, and halothane-induced Ca²⁺ release; determined the in vivo response to halothane or increased ambient temperature; and determined the in vivo myoplasmic intracellular Ca²⁺ concentration in skeletal muscle before and during exposure to volatile anaesthetics.

Results: RYR1 pG2435R/MH normal (MHS-Heterozygous[Het]) or RYR1 pG2435R/pG2435R (MHS-Homozygous[Hom]) mice were fully viable under typical rearing conditions, although some male MHS-Hom mice died spontaneously. The normalised half-maximal effective concentration (95% confidence interval) for intracellular Ca²⁺ release in myotubes in response to KCl [MH normal, MHN, 21.4 (19.8-23.1) mM; MHS-Het 16.2 (15.2-17.2) mM; MHS-Hom 11.2 (10.2-12.2) mM] and caffeine (MHN, 5.7 (5-6.3) mM; MHS-Het 4.5 (3.9-5.0) mM; MHS-Hom 1.77 (1.5-2.1) mM] exhibited a gene dose-dependent decrease, and there was a gene dose-dependent increase in halothane sensitivity. Intact animals show a gene dose-dependent susceptibility to MH with volatile anaesthetics or to heat stroke. RYR1 p.G2435R mice had elevated skeletal muscle intracellular resting [Ca²⁺]_i, (values are expressed as mean (SD)) (MHN 123 (3) nM; MHS-Het 156 (16) nM; MHS-Hom 265 (32) nM; P<0.001) and [Na⁺]_i (MHN 8 (0.1) mM; MHS-Het 10 (1) mM; MHS-Hom 14 (0.7) mM; P<0.001) that was further increased by exposure to volatile anaesthetics.

Conclusions: RYR1 pG2435R mice demonstrated gene dose-dependent in vitro and in vivo responses to pharmacological and environmental stressors that parallel those seen in patients with the human RYR1 variant p.G2434R.

Keywords: gene knock-in techniques; malignant hyperthermia; mouse; ryanodine receptor 1.