Study of calcium sparks in skeletal and smooth muscle cells in normal and pathological conditions

mTOR signaling influence a wide range of cellular process including protein synthesis (Iadevaia et al., 2012; Ma and Blenis, 2009; Thoreen et al., 2012), lipids synthesis (Lamming and Sabatini, 2013), transcription (Dibble and Manning, 2013; Vazquez-Martin et al., 2011), nucleotides biosynthesis (Ben-Sahra et al., 2013; Robitaille et al., 2013) and cellular energetics (Albert and Hall, 2015; Duvel et al., 2010; Inoki et al., 2012). In muscle, suppression of mTORC1 signaling results in several phenotypic changes including decreased life expectancy, increased glycogen deposits and alterations of the twitch kinetics of slow fibres (Bentzinger et al., 2008), however it is unclear what is its specific role in the excitation contraction (EC) coupling. Likewise, the ryanodine receptor (RyR), the calcium release channel of the sarcoplasmic reticulum, plays a fundamental role in calcium homeostasis in a variety of cells and particularly in muscle (Lanner et al., 2010). Mutations in the gene encoding this channel have been associated with a number of debilitating or life-threatening neuromuscular pathologies including malignant hyperthermia (Kolb et al., 1982; Rosenberg et al., 2015; Treves et al., 2005), but little or no knowledge is known about their pathophysiological influence in mild bleeding disorders. In this thesis we investigated in greater detail 1) the effect of the mTORC1 signalling pathway on the integrity of the protein participants in skeletal muscle EC coupling and calcium homeostasis by using a muscle specific Raptor KO mouse model. 2) The calcium homeostasis of vascular smooth muscle cells of an MH mouse model and its association to mild bleeding disorders as also observed in MH patients.
As far as the mTOR is concerned, we found that in raptor knockout (RamKO) mice, the bulk of glycogen phosphorylase (GP) is mainly associated in its cAMP-non-stimulated form with sarcoplasmic reticulum (SR) membranes. In addition, radio ligand binding assay showed a ryanodine to dihydropyridine receptors (DHPRs) ratio of 0.79 and 1.35 for wild-type (WT) and raptor KO skeletal muscle membranes respectively, which was confirmed by Western Blot analysis. Peak amplitude and time to peak of the global calcium transients evoked by supramaximal field stimulation were not different between WT and raptor KO. However, the increase in the voltage sensor-uncoupled RyRs leads to an increase of both frequency and mass of elementary calcium release events (ECRE) induced by hyper-osmotic shock in flexor digitorum brevis (FDB) fibres from raptor KO. These findings together with previous reports should be taken into consideration in the clinical practice when rapamycin or its analogs (rapalogs) is administrated to patients.
As far as RYR1–mutations in human patients and its relationship to bleeding abnormlities is concerned, 8/20 mutation carriers revealed abnormal bleeding scores compared with their healthy relatives (0/11). Similarly, MHS RYR1Y522S knock in mice exhibited 3 times longer bleeding times compared to their wild type littermates. The bleeding defect of MHS mice could be reversed by pre-treatment with the ryanodine receptor 1 antagonist dantrolene. Primary vascular SMCs from RYR1Y522S knock-in mice exhibited a higher frequency of subplasmalemmal Ca2+ sparks leading to a more negative resting membrane potential. Furthermore, Ca2+ sparks were blocked by pre-treatment with ryanodine or dantrolene. These results stimulated us to generate a model that could explain how impaired calcium homeostasis addressed by RyR1 mutation could affect bleeding without influencing platelet or coagulation factor function. Our results on impaired calcium homeostasis caused by RyR1 mutations could extend to other tissues that functionally express this channel.
In conclusion, the present study shows that the protein composition and function of the molecular machinery involved in skeletal muscle excitation–contraction (EC) coupling is affected by mTORC1 signaling and that RYR1 mutations cause prolonged bleeding by altering vascular SMC function and emphasize the potential therapeutic value of dantrolene in the treatment of such bleeding abnormalities.