Using a novel STAC3 mouse model to bridge the gap in myopathy and malignant hyperthermia mechanisms

A number of hereditary muscle disorders cause muscle weakness coupled with susceptibility to malignant hyperthermia (MH), a potentially fatal reaction to the most commonly used general anaesthetics. These disorders which include the core myopathies, and the recently described STAC3 myopathy, affect childhood development and quality of life, and shorten life expectancy.

All involve genetic variants affecting constituents of the calcium signalling machinery required for normal function of skeletal muscle, but the mechanisms by which these variants cause progressive muscle weakness or malignant hyperthermia are poorly understood.

In the last 10-years, STAC3 protein has been found to be a key protein in skeletal muscle development, with a role in excitation-contraction coupling. Much of the progress in understanding the role of STAC3 led from the discovery that a missense variant in the STAC3 gene caused a congenital myopathy associated with MH. However, the possibility of further advances with the previously available tools is limited.

Our aims are to use a new transgenic mouse model of STAC3 myopathy to define the specific role of the STAC3 protein in muscle calcium signalling, and the mechanisms by which dysregulated calcium signalling lead to myopathy and to MH. Our hypothesis is that the clinical features of STAC3 disorder result from skeletal muscle cellular calcium dysregulation and consequent mitochondrial dysfunction, both of which represent viable targets for drug treatments for STAC3 myopathy, for which there are currently no specific treatments.

The work encompassed within this application builds on our successful bid for a Stac3 knock-in mouse under the MRC 'Genome Editing Mice for Medicine' call. Here, we propose to execute the work described in the justification of our GEMM application.

We will characterise the morphological, histological and MH susceptibility phenotypes of the Stac3 mice and detail the developmental changes that result in the post-partum phenotypes. We will perform detailed studies of the skeletal muscle molecular pathophysiology in myotubes derived from Stac3 pW280S homozygous, heterozygous and wild type mice.

The first strand of myotube studies will focus on the detailed mechanism of the primary derangement of Ca2+dysregulation. Using appropriate fluorescent Ca2+ indicators we will measure changes in cytosolic Ca2+ concentration, sarcoplasmic reticulum (SR) stores, SR leak, evoked SR release and different mechanisms of Ca2+ entry. As part of this work we will establish whether dantrolene, the clinical antidote for MH associated with the more prevalent RYR1 variants, is likely also to be of benefit in STAC3-associated MH.

The second strand of myotube studies will focus on the mitochondria, as targets for secondary effects of skeletal muscle Ca2+ dysregulation. We will use confocal microscopy with appropriate indicators to quantify mitochondrial mass and turnover, and to measure mitochondrial membrane potential. Respirometry (Seahorse FX96) will be used to measure mitochondrial oxygen consumption and evaluate respiratory chain complex function.

We will add to the evolving understanding of the intimate relationship between cytosolic Ca2+, SR function and mitochondrial bioenergetics by measuring the effects of exaggerated Ca2+ signalling in Stac3 myotubes on mitochondrial Ca2+uptake, oxygen consumption and free radical production.

Finally, we will use transcriptomic analyses of muscle from 2 stages of embryonic development to integrate coordinated changes in mRNA and protein expression with functional and histological data to identify potential novel therapeutic targets.