Ca2+-dependent proteolysis of junctophilin-1 and junctophilin-2 in skeletal and cardiac muscle

Excessive increases in intracellular [Ca2+] in skeletal muscle fibres cause failure of excitationcontraction coupling by disrupting communication between the dihydropyridine receptors in the transverse tubular system and the Ca2+ release channels (RyRs) in the sarcoplasmic reticulum (SR), but the exact mechanism is unknown. Previous work suggested a possible role of Ca2+-dependent proteolysis in this uncoupling process but found no proteolysis of the dihydropyridine receptors, RyRs or triadin. Junctophilin-1 (JP1; approximate to 90 kDa) stabilizes close apposition of the transverse tubular system and SR membranes in adult skeletal muscle; its C-terminal end is embedded in the SR and its N-terminal associates with the transverse tubular system membrane. Exposure of skeletal muscle homogenates to precisely set [Ca2+] revealed that JP1 undergoes Ca2+-dependent proteolysis over the physiological [Ca2+] range in tandem with autolytic activation of endogenous -calpain. Cleavage of JP1 occurs close to the C-terminal, yielding a approximate to 75 kDa diffusible fragment and a fixed approximate to 15 kDa fragment. Depolarization-induced force responses in rat skinned fibres were abolished following 1 min exposure to 40 m Ca2+, with accompanying loss of full-length JP1. Supraphysiological stimulation of rat skeletal muscle in vitro by repeated tetanic stimulation in 30 mm caffeine also produced marked proteolysis of JP1 (and not RyR1). In dystrophic mdx mice, JP1 proteolysis is seen in limb muscles at 4 and not at 10 weeks of age. Junctophilin-2 in cardiac and skeletal muscle also undergoes Ca2+-dependent proteolysis, and junctophilin-2 levels are reduced following cardiac ischaemiareperfusion. Junctophilin proteolysis may contribute to skeletal muscle weakness and cardiac dysfunction in a range of circumstances.