Dysferlin-deficient immortalized human myoblasts and myotubes as a useful tool to study dysferlinopathy.

Susanne Philippi,Andreas Marg,Simone Spuler,Anne Bigot,Ute Zacharias,Vincent Mouly

doi:10.1371/currents.rrn1298

Abstract

Dysferlin gene mutations causing LGMD2B are associated with defects in muscle membrane repair. Four stable cell lines have been established from primary human dysferlin-deficient myoblasts harbouring different mutations in the dysferlin gene. We have compared immortalized human myoblasts and myotubes carrying disease-causing mutations in dysferlin to their wild-type counterparts. Fusion of myoblasts into myotubes and expression of muscle-specific differentiation markers were investigated with special emphasis on dysferlin protein expression, subcellular localization and function in membrane repair. We found that the immortalized myoblasts and myotubes were virtually indistinguishable from their parental cell line for all of the criteria we investigated. They therefore will provide a very useful tool to further investigate dysferlin function and pathophysiology as well as to test therapeutic strategies at the cellular level.

Highlights

Muscular dystrophies comprise clinically and genetically heterogeneous disorders characterized by progressive weakness and wasting of the skeletal muscle accompanied by an increase in muscle connective tissue [1] Dysferlin gene mutations cause limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy, allelic autosomal recessive diseases characterized by limb girdle or distal weakness of early adult onset [2] [3]
After their immortalization these cell lines show a prolonged proliferation and differentiation capacity compared to primary human myoblasts in vitro and they can be transplanted into regenerating muscle in vivo [13]
The material is very precious and available in very small quantities because of the limited size of biopsy procedures that can be performed and the reduced proliferative potential of human myoblasts which will be further reduced in muscular dystrophies due to the cycles of degeneration and regeneration

Summary

Introduction

Muscular dystrophies comprise clinically and genetically heterogeneous disorders characterized by progressive weakness and wasting of the skeletal muscle accompanied by an increase in muscle connective tissue [1] Dysferlin gene mutations cause limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy, allelic autosomal recessive diseases characterized by limb girdle or distal weakness of early adult onset [2] [3]. Primary human myoblasts in culture show a limited proliferative potential and undergo changes that are linked to replicative senescence [12] To circumvent these limitations immortalized human myoblast lines were generated by retroviral transduction of primary human myoblasts harbouring different disease-causing mutations with telomerase (hTERT) and cyclin-dependent kinase 4 (CDK-4). The expression of hTERT overcomes the progressive erosion of telomeres occuring due to cell division and the overexpression of CDK-4 blocks the induction of the p16mediated cellular stress-pathway [13] After their immortalization these cell lines show a prolonged proliferation and differentiation capacity compared to primary human myoblasts in vitro and they can be transplanted into regenerating muscle in vivo [13]

Methods

Results

Conclusion