Defective SERCA1protein causing bovine Pseudomyotonia and human Brody disease: from pathogenic mechanism to a novel therapeutic approach

Cattle congenital pseudomyotonia (PMT) is an inherited recessive autosomal muscular disorder due to missense mutations in ATP2A1 gene, encoding SERCA1 protein (Sarco-endo-plasmic reticulum Ca2+-ATPase, isoform1) [1]. SERCA1 is responsible for transporting Ca2+ from cytosol back into the lumen of sarcoplasmic reticulum (SR) playing a crucial role in muscle relaxation. Bovine PMT has been described in the Chianina and Romagnola italian breeds. Even though clinical symptoms were homogeneous, PMT has been found genetically heterogeneous. All PMT Chianina animals were homozygous for a single point mutation leading to an Arg164His (R164H) substitution, while most of PMT Romagnola cases were compound heterozygous, carrying a mutation identical to that of Chianina breed, in addition to two point mutations leading to Gly211Val and Gly286Val (G211V/G286V) substitutions [2]. In spite of this heterogeneity of SERCA1 gene mutations, a striking selective reduction of SERCA1 protein has been described in SR membranes isolated from bovine muscles of different PMT-affected cattle breeds. Recently we have clarified the pathogenic mechanism underlying Chianina PMT: the R164H SERCA1 mutation generates a protein functionally active but corrupted in proper folding that was ubiquitinated and prematurely degraded by the ubiquitin-proteasome system [3]. The relevance of cattle PMT is based on phenotypic and genotypic similarities with human Brody myopathy, a “rare” genetic muscular disorder. Clinical symptoms genetic and biochemical findings, clearly demonstrated that cattle PMT is the true animal model of Brody disease. This is not surprising since, in the last years, the counterparts of human pathologies have been found in many domestic mammalian species. Using the heterologous cellular model HEK293 overexpressing SERCA1 mutants, we have deeply investigated SERCA1 mutations found in Romagnola breed. G211V and G286V mutations were introduced separately or together into bovine SERCA1 cDNA. Using both immunofluorescence and western blot analyses we have found that only the G211V mutation is responsible for the PTM phenotype of Romagnola and the treatment with proteasome inhibitor rescues the expression level of G211V mutated SERCA1 at ER membranes in HEK293 cell model. At present, neither specific therapy nor mouse model for Brody myopathy exists. Our findings opened new perspectives for a therapy of this rare disease. To this aim, in the same cell model we have tested small molecules known as “CFTR correctors” specifically developed for rescuing type II Cystic Fibrosis Transmembrane Regulator (CFTR) mutants, causing Cystic Fibrosis. We have observed, by western blot analysis, that treatments with these compounds induce an increase of SERCA1 mutant content. So, a possible pharmacological therapy could be hypothesized for the specific population of Brody patients in which, as in bovine PMT, ATP2A1 mutations impair SERCA1 protein folding causing its rapid degradation. [1] Sacchetto et al. A defective SERCA1 protein is responsible for congenital pseudomyotonia in Chianina cattle, Am J Pathol ,174:565-573, 2009. [2] Murgiano, Sacchetto et al. Pseudomyotonia in Romagnola cattle caused by novel ATP2A1 mutations, BMC Vet. Res, 8:186. 2012. [3] Bianchini et al. Inhibition of ubiquitin proteasome system rescues the defective sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) protein causing Chianina cattle pseudomyotonia, J Biol Chem. 289:33073-33082, 2014.