Gain-of-Function STIM1 L96V Mutation Causes Myogenesis Alteration in Muscle Cells From a Patient Affected by Tubular Aggregate Myopathy.

Elena Conte,Marina Mora,Antonella Liantonio,Paola Imbrici,Lorenzo Maggi,Giulia Maria Camerino,Sara Gibertini,Annamaria De Luca,Alessandra Pannunzio,Mauro Coluccia,Ornella Cappellari

doi:10.3389/fcell.2021.635063

Abstract

Tubular Aggregate Myopathy (TAM) is a hereditary ultra-rare muscle disorder characterized by muscle weakness and cramps or myasthenic features. Biopsies from TAM patients show the presence of tubular aggregates originated from sarcoplasmic reticulum due to altered Ca2+ homeostasis. TAM is caused by gain-of-function mutations in STIM1 or ORAI1, proteins responsible for Store-Operated-Calcium-Entry (SOCE), a pivotal mechanism in Ca2+ signaling. So far there is no cure for TAM and the mechanisms through which STIM1 or ORAI1 gene mutation lead to muscle dysfunction remain to be clarified. It has been established that post-natal myogenesis critically relies on Ca2+ influx through SOCE. To explore how Ca2+ homeostasis dysregulation associated with TAM impacts on muscle differentiation cascade, we here performed a functional characterization of myoblasts and myotubes deriving from patients carrying STIM1 L96V mutation by using fura-2 cytofluorimetry, high content imaging and real-time PCR. We demonstrated a higher resting Ca2+ concentration and an increased SOCE in STIM1 mutant compared with control, together with a compensatory down-regulation of genes involved in Ca2+ handling (RyR1, Atp2a1, Trpc1). Differentiating STIM1 L96V myoblasts persisted in a mononuclear state and the fewer multinucleated myotubes had distinct morphology and geometry of mitochondrial network compared to controls, indicating a defect in the late differentiation phase. The alteration in myogenic pathway was confirmed by gene expression analysis regarding early (Myf5, Mef2D) and late (DMD, Tnnt3) differentiation markers together with mitochondrial markers (IDH3A, OGDH). We provided evidences of mechanisms responsible for a defective myogenesis associated to TAM mutant and validated a reliable cellular model usefull for TAM preclinical studies.

Highlights

In its primary form, Tubular Aggregate Myopathy (TAM) is a clinically heterogeneous and very rare skeletal muscle disorder, in most cases inherited in an autosomal dominant pattern (Böhm and Laporte, 2018; Michelucci et al, 2018; Morin et al, 2020)
C2C12 myoblasts transfected with STIM1 L96V-YFP displayed statistically significant STIM1 clustering regardless of sarcoplasmic reticulum (SR) Ca2+ depletion (Böhm et al, 2014)
Ca2+ homeostasis alterations associated with STIM L96V mutation have been investigated on skeletal muscle cells deriving from TAM patient’s biopsy

Summary

Introduction

In its primary form, Tubular Aggregate Myopathy (TAM) is a clinically heterogeneous and very rare skeletal muscle disorder, in most cases inherited in an autosomal dominant pattern (Böhm and Laporte, 2018; Michelucci et al, 2018; Morin et al, 2020). Myalgia and cramps have been described (Böhm et al, 2014; Hedberg et al, 2014; Walter et al, 2015) and in some cases the full picture of the multisystemic Stormorken syndrome develops (Morin et al, 2020). A consistent histopathological feature of TAM patients is represented by the presence of tubular aggregates (TAs) in skeletal muscle fibers. TAs represent a non-specific morphological alteration being present in several neuromuscular disorders associated to SR stress (Michelucci et al, 2018)

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Conclusion