RUOLO DELLE PROTEINE MINORITARIE DEL RETICOLO SARCOPLASMATICO NELL’ACCOPPIAMENTO ECCITAZIONE-CONTRAZIONE DEL MUSCOLO SCHELETRICO

Muscle strength is restored in JP45 and CASQ1 double knock-out mice. Skeletal muscle constitutes approximately 40% of human body mass, and alterations in muscle mass and in calcium release process are implicated in decrement of strength with age, in neuromuscular diseases, myopaties as well as obesity and diabetes. The action potential leads to muscle fiber contraction and generation of mechanical force in a process called Excitation-Contraction Coupling (ECC), that takes place at the triad, a structure made up of two membrane compartments: transverse tubules, invaginations of sarcolemma and the terminal cisternae of the Sarcoplasmic Reticulum (SR). The main components of ECC machinery are the 1,4 dihydropyridine receptor (DHPR) Cav 1.1 subunit on T tubules, Ryanodine receptor (RyR1) on the SR and Calsequestrin-1, which serve as channel and voltage sensor, Ca2+ release channel and main Ca storage protein of SR respectively. ECC is activated by a bidirectional signalling: depolarization of sarcolemma induces conformational changes of the DHPR that triggers the opening of RyR1 to release calcium ions via an orthograde signalling. Cav1.1 activity is enhanced by a retrograde stimulatory signal delivered by RyR1. The myoplasmic calcium activates the contractile proteins and is subsequently pumped back into the SR by a Ca2+ ATPase (SERCA) pump leading to muscle relaxation. Yet to be identified minor protein components and other proteins aside the DHPR, RyR1 and calsequestrin are essential for the structure and regulation of the machinery involved in ECC. JP45 is a membrane protein interacting with Cav1.1 and the sarcoplasmic reticulum calcium storage protein calsequestrin (CASQ1). We hypothesized that JP45 and CASQ1 form a signalling pathway that modulates Cav1.1 channel activity. We tested this in flexor digitorum brevis (FDB) muscle fibres from JP45 and CASQ1 double knock-out mice (DKO). Our results show that calcium transient evoked by tetanic stimulation in DKO fibres, result from massive calcium influx due to enhanced Cav1.1 channel activity. This enhanced activity restores muscle strength both in vitro and in vivo. Downregulation of EC uncoupling in aged JP45 KO mice. The decline in muscular strength with age, termed sarcopenia, is caused largely by a loss of total muscle mass - but also a disproportionate loss of strength. The loss of muscle strength in old age is characterized in part by a deficit in Ca2+ release caused by activation of DHPR, a phenomenon known as excitation-contraction uncoupling (ECU). On the basis of our previous data showing that ablation of JP45 results in a significant loss of muscle strength in 3 months old mice, we hypothesized that ablation of JP45 expression will result in more marked muscle weakness in JP45KO than WT mice with aging. To our surprise however, we found that JP45KO exhibit sustained in vivo and in vitro skeletal muscle force with no further decline in ECC with aging. Calorie restriction induced by the absence of expression of JP45 in specific areas of the brain (nucleus of the solitary tract, area postrema and nucleus arcuate) associated with central regulation of food intake lead to a significant decrease of body weight in JP45KO compared with WT. The latter event inhibited downregulatation of the expression of DHPR which in turn prevented worsening of the EC uncoupling in aged JP45KO mice. Peroxisome proliferator-activated receptor-γ coactivator-1α (PCG-1α) affects calcium signals in skeletal muscle. Peroxisome proliferator-activated receptor-γ coactivator-1α (PCG-1α) is implicated in muscle plasticity promoting fibre switching towards oxidative activity. Since fibre type switch involves the activation of calcium dependent transcriptional factors, we analysed the effect of PGC-1α on calcium handling in muscles overexpressing the coactivator. We demonstrate that PGC-1α causes a reduction of maximal muscle force in vivo and ex vivo by diminishing the expression of calcium release molecules and altering calcium release and uptake process. Furthermore, we demonstrate that PGC-1α increases resistance to fatigue and drives fibre type switching partly through remodelling of calcium transients. Expression of retinaldehyde in skeletal muscle. We identified the primary structure and role of SRP35, a novel minor protein component of the sarcoplasmic reticulum. We show that SRP35 is a transmembrane component of the SR, is located near the SERCA Ca2+ATPase and is a short-chain dehydrogenase/reductase belonging to the DHRS7C subfamily. We demonstrated that retinol is the substrate of SRP35, since its transient overexpression leads to an increased production of all-trans-retinaldehyde. We show that transfection of C2C12 myotubes with the fusion protein encoding SRP35-EYFP, or adding retinoic acid to C2C12 cell culture, results in a decrease of calcium released by RyR1 and a significant reduction of RyR1 protein expression. The definition of the role of minor components which make up the ECC molecular machinery is important not only to understand how mutations in genes involved in calcium homeostasis cause myopathies, but also to define new therapeutic targets for innovative strategies aimed to treat neuromuscular disorders linked to defect of EC coupling.