Characterisation of calcium kinetics in muscle cells of mouse models of malignant hyperthermia and central core disease

Abstract

Malignant hyperthermia (MH) is a potentially lethal pharmacogenetic disorder. Numerous mutations of the ryanodine receptor 1 (RyR1) found in skeletal muscles have been identified to be causative for the susceptibility to the hypermetabolic status called MH crisis. This life-threatening state can be triggered by volatile anaesthetics, depolarising muscle relaxants and excessive body exercise in individuals carrying these mutations. Susceptibility to MH shows a certain overlap with the congenital myopathy central core disease (CCD), which is also mainly based on RyR1 mutations. An increased resting calcium concentration and a reduced sarcoplasmic reticulum (SR) calcium concentration were found in muscle cells expressing RyR1 and carrying so-called leaky channel mutations, leading to an inordinate calcium outflow from the SR and to store depletion. A reduced SR calcium concentration was found in some representatives of the so-called excitation contraction (EC)-uncoupling mutations, possibly caused by reduced induction of the sarcoplasmic/endoplasmic reticulum calcium-ATPase (SERCA) gene. Furthermore, the phenomenon of store operated calcium entry (SOCE) was discovered. It describes the activation of calcium channels in the plasma membrane after store depletion in the SR. This work investigated the influences of the lack of extracellular calcium on RyR1 mutants, preventing possible effects of SOCE. The Y524S (YS) mouse model served as a representative model for leaky channel mutations, the I4895T (IT) mouse model for EC-uncoupling mutations. I compared muscle cells of wild type and heterozygous mice in calcium free and calcium containing solutions, respectively. This study confirmed findings of a reduced maximal calcium concentration and a slowed calcium release in mice heterozygous for IT and gave evidence for a similar behaviour in heterozygous YS mice. Moreover, resting calcium concentration was decreased in calcium free solution compared to calcium containing solution in the YS mouse model. Looking at calcium removal, it appeared enhanced in IT mutants in calcium free solution. On the contrary, YS mutants showed a significantly slower calcium removal than their wild type littermates. Regarding the evaluation of single-pulse induced calcium transients, the stretched exponential function was introduced as an alternative method to describe and to compare calcium removal by simple exponential models.