Bioenergetic defects in muscle fibers of RYR1 mutant knock-in mice associated with malignant hyperthermia

Abstract

Mutations in the skeletal muscle ryanodine receptor gene (RYR1) can cause susceptibility to malignant hyperthermia (MH), a potentially lethal genetic condition triggered by volatile anesthetics. MH is associated with hypermetabolism which has directed research interest into oxidative phosphorylation (OXPHOS) and muscle bioenergetics. The most common cause of MH in the United Kingdom is the c.7300G>A RYR1 variant, which is present in ~16% of MH families. Our study focuses on the MH susceptible G2435R-RYR1 knock-in mouse model, which is the murine equivalent of the human c.7300G>A genotype. Using a combination of transcriptomics, protein expression and functional analysis, we investigated adult muscle fiber bioenergetics in this mouse model. RNA sequencing data showed reduced expression of genes associated with mitochondria and fatty acid oxidation in RYR1 mutants when compared to wild-type (WT) controls. Mitochondrial function was assessed by measuring oxygen consumption rates in permeabilized muscle fibers. Comparisons between WT and homozygous G2435R-RYR1 mitochondria showed a significant increase in complex I-facilitated OXPHOS in mutant muscle. Furthermore, we observed a gene-dose specific increase in reactive oxygen species production in G2435R-RYR1 muscle fibers. Collectively these findings provide evidence of metabolic defects in G2435R-RYR1 knock-in mouse muscle under basal conditions. Differences in metabolic profile could be the result of differential gene expression in metabolic pathways, in conjunction with mitochondrial damage accumulated from chronic exposure to increased oxidative stress.

Neuromuscular symptoms in patients with RYR1-related malignant hyperthermia and rhabdomyolysis

“…Laboratory studies on the effects of aging and altered calcium metabolism on late-onset muscle weakness in mice have shown that the higher resting cytosolic calcium concentrations, as observed in skeletal muscle cells of MH susceptible individuals, 41 , 42 may cause myofibrillar disarray, sarcolemmal rupture, structural alterations such as cores and, mitochondrial damage. 43 , 44 Interestingly, the RYR1 knock-in mouse model used in one of these studies (p.Gly2435Arg) corresponds with the human RYR1 c.7300G > A, p.Gly2434Arg variant, 44 which was also found in one of the patients with late-onset proximal muscle weakness identified in our study. Furthermore, structural alterations in skeletal muscle cells of MH susceptible mice appear to increase in severity with age.…”

An Association between OXPHOS-Related Gene Expression and Malignant Hyperthermia Susceptibility in Human Skeletal Muscle Biopsies

“…It has been hypothesized that the mitochondrial dysfunction observed in MHS muscle may be a consequence of the elevated intracellular [Ca 2+ ] and increased oxidative stress associated with RYR1 variants. Increased oxidative stress and the accumulation of reactive oxygen species (ROS) have been consistently reported in the muscle of various MH mouse models [ 10 , 13 , 14 , 27 ]. The interplay between ROS and Ca 2+ has an important role in regulating cellular metabolism and its dysregulation has detrimental effects on mitochondria [ 28 ].…”

“…The biological consequences of specific RYR1 variants have been investigated using different knock-in mouse models [ 6 , 7 , 8 , 9 ]. Mitochondrial dysfunction has been a consistent feature in MH-associated RYR1-knock in mouse models and has been speculated to be a potential influencer of the MH muscle phenotype [ 7 , 10 , 11 , 12 , 13 , 14 ]. Although limited, this parallels data from studies of human MHS muscle that have shown evidence of mitochondrial structure abnormality using electron microscopy [ 15 ], and functional deficits detected through high-resolution respirometry [ 16 ].…”

“…We have previously published transcriptomics data obtained from the MH-associated G2435R-RYR1 mouse model, presenting evidence of downregulated gene expression in fatty acid oxidation pathways in the homozygous genotype [ 14 ]. Furthermore, our recent publication on exertional heat illness (a closely related condition), included whole blood transcriptomics from MHS patients and showed downregulated gene expression in oxidative phosphorylation (OXPHOS) pathways [ 17 ].…”

Mutations in proteins involved in E-C coupling and SOCE and congenital myopathies

“…This is, initially, translated into activation of a preserving mechanism that leads to increased ATP production to support the greater demand of ATP exerted by SERCA pumps in their continuous activity to counteract Ca 2+ leak ( Lamboley et al, 2021 ). Nevertheless, in the long term, mitochondria from both Ryr1 G2434R/WT and Ryr1 T4826I/WT mice showed structural and functional alterations, mainly in fatty acid metabolisms, that resulted in lipotoxicity and an increase in oxidative stress, impacting muscle bioenergetics and muscle performance ( Yuen et al, 2012 ; Chang et al, 2020 ). Similarly, muscles from Ryr1 R163C/WT mice show an increase in excitation contraction calcium entry (ECCE) linked to delayed inactivation of L-type currents through DHPR ( Cherednichenko et al, 2008 ; Bannister et al, 2010 ; Estève et al, 2010 ); both Ryr1 R163C/WT and Ryr1 Y522S/WT also display an increase in SOCE that may contribute to Ca 2+ overload, hypercontractures, heat generation, and rhabdomyolysis ( Eltit et al, 2013 ; Yarotskyy et al, 2013 ).…”