Archive | 2021
Ryanodine receptor calcium regulation in healthy and compromised skeletal muscle
Abstract
The pathogenesis of several skeletal muscle myopathies has been associated with dysregulation of the ryanodine receptor (RyR) Ca2+ channel, and more specifically the leak of Ca2+ through the RyR. However, increased RyR activity and Ca2+ leak is not always considered pathological in mammalian skeletal muscle. It has been proposed that the generation of heat in resting mammalian skeletal muscle is largely attributable to RyR Ca2+ leak and the subsequent activity of the sarcoplasmic reticulum (SR) Ca2+ pump. Accordingly, this thesis examines the fundamental properties of RyR Ca2+ handling in healthy and compromised skeletal muscle.The major difference between mammals and lower vertebrates in their thermoregulatory capacity may lay within the RyR isoforms present on the SR. Changes in the leak of Ca2+ through the SR ryanodine RyR may create an increased cytoplasmic Ca2+ concentration ([Ca2+]cyto) in the resting muscle to activate the SR Ca2+ pump for heat generation. In amphibian skeletal muscle, a local increase in [Ca2+]cyto triggers Ca2+ release via Ca2+-induced Ca2+ release (CICR) due to activation of the Ca2+-sensitive RyR isoform. The presence of CICR in mammalian muscle is controversial and would de-stabilize a heat regulatory system based on increases in RyR Ca2+ leak and [Ca2+]cyto. In this study, the rapid opening of RyRs in amphibian skeletal muscle by abruptly raising local [Ca2+]cyto in the presence of a RyR agonist was due to CICR, whereas human muscle with a gain-of-function malignant hyperthermia RyR mutation displayed resistance to opening under identical ionic conditions. Instead, delayed Ca2+ release in human muscle was a function of SR [Ca2+] and agonist concentration, differentiating mammalian RyRs from amphibian by its sensitivity to cytoplasmic agonists. Consistent with sympathetic activation under low ambient temperatures, the downstream β-adrenergic activator cAMP increased RyR Ca2+ leak in healthy mammalian muscle. Additionally, the application of dantrolene (a RyR inhibitor) resulted in a reduction in RyR Ca2+ leak, which is consistent with previously reported findings whereby dantrolene reduced the maintenance of core body temperature during cold exposure. These results demonstrate that the isolation of a single, Ca2+-resistant RyR isoform (RyR1) in mammalian muscle allow increases in [Ca2+]cyto via RyR-dependent leak following cytoplasmic agonist activation, without risk of Ca2+ release that would be detrimental to endothermy.Elevated thermogenic Ca2+ cycling in obese individuals is thought to act as an adaptive mechanism in response to high energy stores, but the presence of RyR Ca2+ leak, if any, has not been established. In an 8 week diet induced obesity model, no alterations in t-system Ca2+ handling or Ca2+ regulatory proteins were observed when compared to non-obese controls, however, an increase in RyR Ca2+ leak was shown. This is consistent with the proposal of enhanced Ca2+ cycling during thermogenesis as an adaptation to combat obesity by upregulating muscle-based energy expenditure. Though, in a severe obesity model (36 week high fat diet) alterations in t-system Ca2+ handling, an incredibly leaky RyR and disrupted SR buffering capacity was observed. Despite the potential for positive Ca2+ cycling, these massive alterations in skeletal muscle Ca2+ handling would most likely result in impaired EC coupling. On this basis, the identification and use of novel RyR modulators represents a valuable tool in the study of such pathologies. In this thesis, two compounds (chloroxine and myricetin) were identified using a FRET-based high-throughput screening assay. Both compounds decreased RyR1 Ca2+ leak in human skeletal muscle fibres but only slightly affected Ca2+ release in EC coupling. Finally, the examination of RyR functional properties throughout this thesis provided a foundation that allowed the development of a novel malignant hyperthermia diagnostic assay. The preliminary assay was able to determine agonist sensitivity thresholds in single skeletal muscle fibres and accurately characterise malignant hyperthermia susceptible and negative populations.