Quantitative reduction of RyR1 protein caused by a single-allele frameshift mutation in RYR1 ex36 impairs the strength of adult skeletal muscle fibres

Abstract

Here we validated a mouse model knocked-in for a frameshift mutation in RYR1 exon 36 (p.Gln1970fsX16) that is isogenic to that identified in one parent of a severely affected patient with recessively inherited Multiminicore disease. This individual carrying the RYR1 frameshifting mutation complained of mild muscle weakness and fatigability. Analysis of the RyR1 protein content in a muscle biopsy from this individual showed a content of only 20% of that present in a control individual. The biochemical and physiological characteristics of skeletal muscles from RyR1Q1970fsx16 heterozygous mice recapitulates that of the heterozygous parent.RyR1 protein content in the muscles of mutant mice reached 38% and 58% of that present in total muscle homogenates of fast and slow muscles from WT littermates.The decrease of RyR1 protein content in total homogenates is not accompanied by a decrease of Cav1.1 content, whereby the Cav1.1/RyR1 stoichiometry ratio in skeletal muscles from RyR1Q1970fsx16 heterozygous mice is lower compared to that from WT mice. High-resolution electron microscopy revealed a 40% reduction in the number of calcium release units and a reduction of the double strip arrays of RyR1 in the junctional sarcoplasmic reticulum, as indicated by a 2.5 fold increase of the number of diads. Compared to WT, muscle strength and depolarization-induced calcium transients in RyR1Q1970fsx16 heterozygous mice muscles were decrease by 20% and 15%, respectively. Altogether our results demonstrate that an average 50% decrease of the RyR content does not correlate with similar decay of muscle functional properties.

Quantitative RyR1 reduction and loss of calcium sensitivity of RyR1Q1970fsX16+A4329D cause cores and loss of muscle strength

“…Although we do not have an exact explanation for the discrepancy between the real and expected RyR1 protein level in RyR1Q1970fsX16+A4329D compound heterozygous mice, we believed that the RyR1 p.A4329D missense mutation in combination with the frameshift mutation, are involved in the down-regulation of RYR1 gene expression by several mechanisms including the induced expression of class II HDACs. This idea is consistent with data we obtained from analyzing RyR1 expression and content in RyR1Q1970fsX16 heterozygous mice (24). In the latter mouse model, the hemizygous expression of the WT RYR1 allele is also associated with a decrease of the RyR1 protein content, nevertheless this reduction is less pronounced compared to that observed in DKI mice and did not affect the expression level of HDACs.…”

“…4) a result which likely reflects differences in fiber type composition between EDL and soleus muscles. The latter muscles are composed of: 60% slow type I fibers and by approximately 40 The decreased muscle strength observed in RyR1Q1970fsX16+A4329D mice is a feature also shared by the heterozygous RyR1Q1970fsX16 mice (24). The latter mouse model, as well as the human patient isogenic for these mutations, also shows a decrease of the RyR1 protein content in total muscle homogenates (16), and this observation may further explain the poor muscle performance of this mouse model.…”

“…Q1970fsX16 mutation of the compound heterozygous mice in not expressed (24). The consequence of such an event is (i) the monoallelic expression of the RyR1 p.A4329D missense mutation, and (ii) its quantitative reduced expression.…”

In vivo RyR1 reduction in muscle triggers a core-like myopathy

“…To explore the various pathophysiology mechanisms associated with RYR1 mutations, different mouse models have been created. They mostly reproduce amino acid substitutions in the RyR1 protein associated with mutations identified in patients in dominant (Y522S, I4898T) [9,10] or recessive pathologies (T4706M + del; Q1970fsX16 + A4329D) [11][12][13]. These models have been useful to better understand some aspects of RyR1 physiology and to confirm the pathogenicity of patients' mutations, but reveal mechanisms that are specific to individual or to a combination of RyR1 mutations.…”

Expanding the clinical-pathological and genetic spectrum of RYR1-related congenital myopathies with cores and minicores: an Italian population study

“…Recently, it has been shown in a knocked-in mouse for a RYR1 single allele frame-shift mutation, that the level of RYR1 transcript and RyR1 protein are decreased without affecting the other SR proteins. The mouse also showed a mild reduction of muscle performance and decreased muscle strength, suggesting that hypomorphic RYR1 mutation should result in a mild clinical phenotype because of a functional reserve of RyR1 protein [ 107 ]. Indeed, all six patients followed the postulated rule of a mild reduction in muscle strength.…”