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

Key points If skeletal muscle fibres are subjected to excessive activation, or stretched whilst contracting, they subsequently display long‐term reductions in their force response, apparently due in part to structural or molecular changes at the triad junction, where excitation of the surface membrane triggers Ca 2+ release from the internal Ca 2+ store. The changes appear to be due to excessive or prolonged increases in intracellular Ca 2+ levels, which activate Ca 2+ ‐dependent proteases known as calpains, but their target proteins are currently unknown. This study shows that excessive muscle stimulation, or directly raising intracellular Ca 2+ levels, causes calpain activation in tandem with proteolysis of junctophilin, a key protein thought to hold the triad junction together. Proteolysis of junctophilin is also seen in muscle of mice with muscular dystrophy and in cardiac muscle following ischaemic damage. Proteolysis of junctophilin may be a major factor causing muscle weakness and cardiac dysfunction in a range of circumstances. Abstract Excessive increases in intracellular Ca 2+ in skeletal muscle fibres cause failure of excitation–contraction coupling by disrupting communication between the dihydropyridine receptors in the transverse tubular system and the Ca 2+ release channels (RyRs) in the sarcoplasmic reticulum (SR), but the exact mechanism is unknown. Previous work suggested a possible role of Ca 2+ ‐dependent proteolysis in this uncoupling process but found no proteolysis of the dihydropyridine receptors, RyRs or triadin. Junctophilin‐1 (JP1; ∼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 Ca 2+ revealed that JP1 undergoes Ca 2+ ‐dependent proteolysis over the physiological Ca 2+ range in tandem with autolytic activation of endogenous μ‐calpain. Cleavage of JP1 occurs close to the C‐terminal, yielding a ∼75 kDa diffusible fragment and a fixed ∼15 kDa fragment. Depolarization‐induced force responses in rat skinned fibres were abolished following 1 min exposure to 40 μ m Ca 2+ , with accompanying loss of full‐length JP1. Supraphysiological stimulation of rat skeletal muscle in vitro by repeated tetanic stimulation in 30 m m 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 Ca 2+ ‐dependent proteolysis, and junctophilin‐2 levels are reduced following cardiac ischaemia–reperfusion. Junctophilin proteolysis may contribute to skeletal muscle weakness and cardiac dysfunction in a range of circumstances.