Superior calcium homeostasis of extraocular muscles

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 (Ca2+) in Duchenne’s muscular dystrophy (DMD). Enhanced Ca2+ buffering has been proposed as a contributory mechanism to explain these properties; however, the mechanisms are not well understood. We investigated mechanisms modulating Ca2+ levels in EOM and tibialis anterior (TA) limb muscles. Using Fura-2 based ratiometric Ca2+ imaging of primary myotubes we found that EOM myotubes reduced elevated Ca2+ ˜2-fold faster than TA myotubes, demonstrating more efficient Ca2+ buffering. Quantitative PCR (qPCR) and western blotting revealed higher expression of key components of the Ca2+ regulation system in EOM, such as the cardiac/slow isoforms sarcoplasmic Ca2+-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 Ca2+ regulation in EOM. Increased expression of Ca2+/SR mRNA, proteins, differential post-translational modification of Pln and superior Ca2+ buffering is consistent with the improved ability of EOM to handle elevated intracellular Ca2+ levels. These characteristics provide mechanistic insight for the potential role of superior Ca2+ buffering in the unusual physiology of EOM and their sparing in DMD.

Research highlights
► EOM have superior capacity to regulate their Ca2+ homeostasis.
► EOM express higher levels of Ca2+ handling proteins, e.g. Serca2 and Casq2 and Pln.
► Pln in EOM is preferentially phosphorylated at Thr17.
► EOM calcium homeostatic buffering mechanisms are consistent with fast and constantly active muscles that are resistant to damage in Duchenne’s Muscular Dystrophy.