LATE BREAKING NEWS E-POSTER PRESENTATION: LBP 9 Constitutive Stim1 activation impairs myogenesis in Tubular Aggregate Myopathy: defective late differentiation of patient-derived myoblasts and potential druggable targets

Abstract

Tubular aggregate myopathy (TAM) is a dysfunctional inherited muscle disorder characterized by the presence of tubular aggregates (TAs) originating from sarcoplasmic reticulum. It is caused by gain-of-function mutations in STIM1 or ORAI1, proteins responsible for Store-Operated-Calcium-Entry (SOCE). The insight into molecular mechanisms is pivotal to understand clinical phenotypes and identify therapeutic targets. Considering the role of calcium homeostasis in postnatal myogenesis we investigated for the first time if Ca2+ alteration via SOCE in TAM impacts muscle myogenesis and differentiation efficacy. We characterized myoblasts and myotubes derived from a TAM patient's biopsy carrying Leu96Val STIM1 mutant. By cytofluorimetry we confirmed a significantly higher resting Ca2+ concentration and an increased SOCE activity vs controls. By automated fluorescence microscopy, we found that differentiating Leu96Val STIM1 myoblasts persisted in a mononuclear state and resulted in a reduced myotubes multinucleation with distinct morphology and different geometry of mitochondrial network compared to controls, suggesting a defect in fusion process. RealTime-PCR analysis showed a compensatory down-regulation of genes involved in Ca2+handling (RyR1,SERCA1/Atp2a1,Trpc1) in STIM1 mutants vs controls. Moreover, early differentiation markers (Myf5,Mef2D) were increased in mutated myoblasts, while a reduction in late differentiation markers (Dystrophin/DMD, Troponin/Tnnt3) were found in mutated myoblasts and myotubes indicating an altered myogenesis in the late differentiation phase associated to TAM. Finally, the mitochondrial genes Isocitrate-Dehydrogenases (IDH3A) and 2-Oxoglutarate-Dehydrogenase (OGDH) were reduced in Leu96Val myoblasts, with defect of OGDH persisting in mutated myotubes. We provided novel evidences of a defective myogenesis in TAM; SOCE or mitochondrial biogenesis can be druggable targets to be exploited.