Calcium is a universal second messenger regulating different biological functions from muscle contraction and neuronal excitability, to gene transcription and cell death. Physiologically, Ca²⁺ signals result both from the release of Ca²⁺ from intracellular stores (endo/sarcoplasmic reticulum) as well as influx from the extracellular environment, via the opening of channels on the plasma membrane. In skeletal muscle, Ca²⁺ regulates contraction and relaxation and alterations in its intracellular concentration can lead to several neuromuscular disorders among which Central Core disease, Malignant Hyperthermia, Multimini core disease, King-Denborough Syndrome, Brody’s disease and some forms of Centronuclear myopathy.

Type 1 ryanodine receptor (RyR1) is a key protein involved in the regulation of the intracellular Ca²⁺ concentration in skeletal muscle cells, playing a crucial role in muscle contraction by releasing Ca²⁺ from the sarcoplasmic reticulum after plasma membrane depolarization. RyR1 is preferentially expressed in skeletal muscles but recent data has shown that it is also expressed in some areas of the central nervous system, in some cells of the immune system (B-lymphocytes and dendritic cells) and in smooth muscle cells. These results imply that mutations in RYR1 (the gene encoding RyR1) may lead to alterations of Ca²⁺ homeostasis not only in skeletal muscle, but also in other tissues expressing this intracellular calcium release channel. Indeed ryanodinopathies have recently been implicated in other clinical conditions such as sepsis and intensive care polyneuropathiy, broadening the clinical spectrum of disorders linked to altered RyR1functions.

The aims of this project are: (i) Determine the mechanism by which recessive mutations in RYR1 linked to some forms of Multiminicore Disease, Centronuclear myopathy and Congenital Fiber Type disproportion lead to a drastic decrease in RyR1 content in muscle biopsies. (ii) Determine the expression profile of major proteins involved in calcium homeostasis, the protein content of the sarcoplasmic reticulum and the characteristics of calcium homeostasis of human eye muscles This is important in order to understand why some patients affected by neuromuscular disorders linked to recessive RYR1 mutations exhibit eye muscle involvement (ptosis, ophthalmoplegia) but other patients, particularly those with dominant RYR1 mutations which alter channel function, do not. (iii) What are the causes of the prolonged bleeding times seen in some patients with dominant RYR1 mutations linked to Malignant Hyperthermia. While examining immune functions of the mouse animal model carrying the RYR1_Y522S mutation linked to Malignant Hyperthermia, we observed prolonged bleeding times without alterations in platelet number or function. Prolonged bleeding times have also been reported in humans carrying some dominant RYR1 mutations. Since smooth muscle cells are thought to express RyR1, but not much is known about its specific role in these cells, we will investigate if freshly isolated smooth muscle cells from the RYR1_Y522S knock in mouse differ in calcium spark frequency compared to their wild type counterpart. (iv) Extend the studies on the functional properties of muscle cells from patients with neuromuscular disorders (including Central Core disease, Multi-mini core disease, Malignant Hyperthermia) harboring endogenous mutations in genes encoding for proteins involved in Ca²⁺ homeostasis by monitoring both the “global” increase of the [Ca²⁺]_i and Ca²⁺ influx by conventional and TIRF (total internal reflection fluorescence) microscopy, monitor if there are alterations in the production of reactive nitrogen species, in the subcellular distribution of NFAT and in the circulating levels of  the pro-inflammatory cytokines IL-1ß, IL-6, TNFa. We think that the results of this study will further our understanding the impact of RYR1 mutations in patients with neuromuscular disorders and will help devise therapeutic strategies for patients with ryanodinopathies.