Characterization and pharmacological modulation of calcium handling proteins in Limb-Girdle Muscle Dystrophy type R1

Páginas 114-122 confidenciales. Tesis completa174 p.-- Tesis censurada 167 p.
Limb-girdle muscle dystrophy type R1 (LGMDR1) is the most common form of limb-girdle muscle dystrophy, currently with no effective treatment. LGMDR1 is caused by mutations in the CAPN3 gene and is characterized by reduced mobility, making daily live activities challenging. CAPN3 gene encodes calpain 3 protein (CAPN3), a non-lysosomal cysteine protease necessary for proper muscle function. Although the main function of CAPN3 in skeletal muscle and the pathophysiological mechanisms underlying the disease remain to be clarified, previous studies suggest that dysregulation of calcium (Ca2+) homeostasis is involved in the pathogenic mechanisms of this muscular dystrophy. In fact, our previous data suggest that CAPN3 deficiency may trigger calcium handling proteins dysfunction such as Sarco-Endoplasmic Reticulum ATPase pumps (SERCA) due to its over-ubiquitination and its consequent exacerbated degradation by Ubiquitin-Proteasome System (UPS). In the present work, we have characterized SERCA expression in different human in vitro LGMDR1 models observing that CAPN3 dysfunction generates SERCA protein downregulation, leading to increase basal intracellular calcium levels. Moreover, CAPN3-deficient LHCN-M2 human myotubes show upregulated sarcoplasmic reticulum (SR) stress markers. We have targeted SERCA degradation inhibiting UPS by Bortezomib (BTZ) treatment, which recovers not only SERCA2 protein expression but also mutated CAPN3 expression, restoring at least its structural function, and consequently, normalizing resting cytosolic Ca2+ levels. Despite the mild phenotype observed in C3KO mouse model, SERCA protein expression is reduced particularly in the diaphragm, as in C3 null rats, which show slightly more severe phenotype. However, due to the general modest phenotype of LGMDR1 animal models, the narrow therapeutic window between effectiveness and toxicity of BTZ treatment, and the high susceptibility of the rat model to Bortezomib, SERCA protein expression is not resto