Superior calcium homeostasis of extraocular muscles

Abstract

Extraocular muscles (EOMs) are a unique group of skeletal muscles with unusual physiological properties such as being able to undergo rapid twitch contractions over extended periods and escape damage in the presence of excess intracellular calcium (Ca 2+) in Duchenne’s muscular dystrophy (DMD). Enhanced Ca 2+ buffering has been proposed as a contributory mechanism to explain these properties; however, the mechanisms are not well understood. We investigated mechanisms modulating Ca 2+ levels in EOM and tibialis anterior (TA) limb muscles. Using Fura-2 based ratiometric Ca 2+ imaging of primary myotubes we found that EOM myotubes reduced elevated Ca 2+ ˜2-fold faster than TA myotubes, demonstrating more efficient Ca 2+ buffering. Quantitative PCR (qPCR) and western blotting revealed higher expression of key components of the Ca 2+ regulation system in EOM, such as the cardiac/slow isoforms sarcoplasmic Ca 2+-ATPase 2 (Serca2) and calsequestrin 2 (Casq2). Interestingly EOM expressed monomeric rather than multimeric forms of phospholamban (Pln), which was phosphorylated at threonine 17 (Thr17) but not at the serine 16 (Ser16) residue. EOM Pln remained monomeric and unphosphorylated at Ser16 despite protein kinase A (PKA) treatment, suggesting differential signalling and modulation cascades involving Pln-mediated Ca 2+ regulation in EOM. Increased expression of Ca 2+/SR mRNA, proteins, differential post-translational modification of Pln and superior Ca 2+ buffering is consistent with the improved ability of EOM to handle elevated intracellular Ca 2+ levels. These characteristics provide mechanistic insight for the potential role of superior Ca 2+ buffering in the unusual physiology of EOM and their sparing in DMD.