Functional effects of mutations in the skeletal muscle ryanodine receptor type 1 (RYR1) linked to malignant hyperthermia and central core disease

Malignant hyperthermia (MH) is a pharmacogenetic disorder with autosomal dominant inheritance. In susceptible individuals, a MH crisis may be triggered by commonly used halogenated anaesthetics (halothane, isoflurane) or muscle relaxants (succhinylcholine). The main symptoms are hypermetabolism and muscle rigidity. Without treatment, death will occur in more than 80% of cases. Although a genetic-chip based diagnostic approach is under development, the invasive in vitro contracture test (IVCT) remains the “gold standard” to diagnose the disorder. Central core disease (CCD) is a slowly progressive myopathy characterised by muscle weakness and hypotonia; affected individuals show delayed motor development and remain physically compromised. Multi-minicore (MmD) disease is a more severe, rare, autosomal recessive myopathy characterised histologically by the presence of multi-minicores in only a small number of sarcomeres. So far, no effective therapy has been developed to treat muscle weakness in CCD and MmD patients and their diagnosis is difficulton the basis of clinical findings alone and a histological examinationof muscle tissue is essential. MmD, CCD and MH are thought to result from a defect in the components involved in excitation-contraction mechanisms and all three diseases are linked to point mutations in the gene encoding the sarcoplasmic reticulum ryanodine receptor calcium release channel (RYR1). The aim of the thesis is to increase our knowledge of the underlying mechanisms which lead to the three different pathologies from mutations in the same gene, namely the ryanodine receptor type 1. Cultured skeletal muscle cells as well as immortalized B-lymphocyte cell lines were used to assay the underlying functional effects of RYR1 mutations, both cell types having the advantage of naturally expressing the ryanodine receptor type 1. The first element of my thesis reports our investigations of the functional characteristics of the ryanodine receptor in cells carrying the following RYR1 mutations: (i) V2168M mutation linked with Malignant hyperthermia; (ii) 2 substitutions, I4898T and R4893W and 1 deletion R4214-F4216 associated with central core disease and (iii) 3 substitutions P3527S, V4849I and R999H associated with CCD/MmD mixed phenotypes. The second aim of my thesis deals with the downstream effects of Ca2+ dysregulation, in particular, the possible role of the ryanodine receptor in the immune system. For this purpose, we have established whether RYR1 mutations influence the release of two cytokines:
interleukin-1β and interleukin-6 and if so whether the latter effect may influence the clinical symptoms of MH, CCD or MmD. In the long run, this work may help to develop a non-invasive approach for the diagnosis of MH susceptibility as well as new concepts for the treatment of these muscular pathologies.