Characterization of the effect of Calsequestrin 1 mutations identified in tubular aggregate myopathies on calcium homeostasis in mouse FDB fibers

Calsequestrin 1 (CASQ1) is a Ca2+ binding protein of the sarcoplasmic reticulum (SR) of skeletal muscle, where it participates to regulate Ca2+ homeostasis, by buffering Ca2+, interacting with triadic proteins, like triadin and RYR1, and with STIM1, a component of the store operated calcium entry (SOCE) mechanism. Here we report the characterization of three CASQ1 missense mutations identified in patients with tubular aggregate myopathy (TAM). These mutations affect conserved amino acids in position 44 (Asp44Asn), 103 (Gly103Asp), and 385 (Ile385Thr). We previously demonstrated that, in non-muscle cells, these mutations affect the Ca2+ store content and SOCE (ref.). Here we characterize the effects of GFP-tagged CASQ1 mutants expressed in skeletal muscle fibers from Flexor Digitorum Brevis (FDB) of CASQ1-KO mice. Single fiber analysis showed a significant reduction in the SR Ca2+ content of fibers expressing mutant CASQ1 compared to fibers expressing wild type CASQ1, confirming the reduced ability of all these mutants to store Ca2+. Experiments regarding the effects of these mutations in SOCE, performed using the manganese quenching technique, indicate a reduced ability of regulating it from all the three mutations in exams, showing a constitutive calcium influx even in rest condition.These results strengthen the knowledge about the CASQ1 complex and delicate functions and indicate that these mutations affect properties critical for correct Ca2+ handling in skeletal muscle fibers.