Mitochondrial bioenergetics and intracellular calcium concentration in primary myotubes from mouse models of malignant hyperthermia

TL;DR

This study investigates how mitochondrial function and calcium levels are linked in mouse models of malignant hyperthermia, using a high-throughput assay to compare different genotypes.

Contribution

The study validates a cell-based assay for mitochondrial function in malignant hyperthermia models and reveals genotype-specific bioenergetic differences.

Findings

Ryr1 p.G2435R homozygous myotubes showed the highest basal oxygen consumption rate.

These myotubes required more respiration to produce ATP and had higher proton leak and non-mitochondrial OCR.

The assay was more sensitive to genotypic effects than traditional methods and showed no direct link between calcium and mitochondrial function in young mice.

Abstract

Malignant hyperthermia (MH) is a potentially fatal hypermetabolic reaction to general anaesthesia arising from skeletal muscle calcium dysregulation. Previous studies of resting cells support an association between MH susceptibility, mitochondrial dysfunction, and defects in fatty acid metabolism, which are understood to be downstream consequences of calcium dysregulation. We hypothesised that in mouse models of MH susceptibility, genotypes associated with higher cytoplasmic calcium concentrations would have a proportionally higher mitochondrial oxygen consumption rate (OCR). We aimed to test this and validate a cell-based assay system.

A high-throughput mitochondrial assay was used to compare OCR between myotubes derived from control and three different genotypes of mice containing ryanodine receptor 1 variants (p.G2435R heterozygous and homozygous, p.T4826I heterozygous) that confer susceptibility to MH.

Baseline comparisons showed effects of genotype on OCR (P<0.0001), with Ryr1 p.G2435R homozygous myotubes having the highest basal normalised OCR (P<0.01). Ryr1 p.G2435R homozygous required a greater proportion of basal respiration to produce adenosine triphosphate (ATP), and had a higher proton leak and greater non-mitochondrial OCR (P<0.01). All genotypes except Ryr1 p.G2435R homozygous were primarily dependent on the glucose/pyruvate pathway for achieving their maximal OCR upon uncoupling.

The high-throughput method used produced data consistent with findings in skeletal muscle fibres, but with a greater sensitivity to genotypic effects. This validates the use of cultured myotubes in lieu of muscle fibres in studying mitochondrial bioenergetics in models of MH, and indicates that mitochondrial bioenergetics are not directly affected by myoplasmic calcium concentrations in young MH mice.

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