Calpain 3 deficiency affects SERCA expression and function in the skeletal muscle.

Ivan Toral-Ojeda,Adolfo López de Munain,Ainara Vallejo-Illarramendi,Garazi Aldanondo,Roberto Fernández-Torrón,Haizpea Lasa-Fernández,Jaione Lasa-Elgarresta

doi:10.1017/erm.2016.9

Abstract

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a form of muscular dystrophy caused by mutations in calpain 3 (CAPN3). Several studies have implicated Ca2+ dysregulation as an underlying event in several muscular dystrophies, including LGMD2A. In this study we used mouse and human myotube cultures, and muscle biopsies in order to determine whether dysfunction of sarco/endoplasmatic Ca2+-ATPase (SERCA) is involved in the pathology of this disease. In CAPN3-deficient myotubes, we found decreased levels of SERCA 1 and 2 proteins, while mRNA levels remained comparable with control myotubes. Also, we found a significant reduction in SERCA function that resulted in impairment of Ca2+ homeostasis, and elevated basal intracellular [Ca2+] in human myotubes. Furthermore, small Ankyrin 1 (sAnk1), a SERCA1-binding protein that is involved in sarcoplasmic reticulum integrity, was also diminished in CAPN3-deficient fibres. Interestingly, SERCA2 protein was patently reduced in muscles from LGMD2A patients, while it was normally expressed in other forms of muscular dystrophy. Thus, analysis of SERCA2 expression may prove useful for diagnostic purposes as a potential indicator of CAPN3 deficiency in muscle biopsies. Altogether, our results indicate that CAPN3 deficiency leads to degradation of SERCA proteins and Ca2+ dysregulation in the skeletal muscle. While further studies are needed in order to elucidate the specific contribution of SERCA towards muscle degeneration in LGMD2A, this study constitutes a reasonable foundation for the development of therapeutic approaches targeting SERCA1, SERCA2 or sAnk1.

Highlights

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a neuromuscular disease caused by mutations in the gene encoding calpain 3 (CAPN3), a nonlysosomal cysteine protease necessary for normal muscle function and regeneration (Refs 1, 2)
Our results indicate that CAPN3 knockdown myotubes display reduced SERCA1/2 protein levels and Ca2+ clearance capacity compared to controls, which is likely related with a higher basal intracellular Ca2+ concentration observed in CAPN3-deficient human myotubes
We have demonstrated that silencing CAPN3 expression in mouse and human myotubes leads to a decrease in SERCA activity because of reduced SERCA1 and SERCA2 protein levels, the main forms differentially expressed in fast and slow muscle fibres, respectively (Refs 9, 10)

Summary

Introduction

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a neuromuscular disease caused by mutations in the gene encoding calpain 3 (CAPN3), a nonlysosomal cysteine protease necessary for normal muscle function and regeneration (Refs 1, 2). Previous studies performed on Capn[3] knockout mice describe a reduced expression of the ryanodine receptor type 1 (RyR1) and reduced Ca2+ release from the sarcoplasmic reticulum (SR) to cytoplasm (Refs 3, 4), suggesting that dysregulation of Ca2+ homeostasis plays a role in the pathogenic mechanisms involved in this form of muscular dystrophy (Ref. 5). Reinforcing this line of evidence, we have recently contributed to a study demonstrating a reduction of RyR1 expression and βCamKII signalling in LGMD2A muscles (Ref. 6). We sought to characterise more in detail the pathway leading to abnormal Ca2+

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Results

Conclusion