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

G.D. Lamb,1 T.L. Dutka,1 D. Horvath,2 J.R. Bell,3 L.M.D. Delbridge3 and R.M. Murphy,1 1Department of Zoology, La Trobe University, Melbourne, VIC 3086, Australia, 2Department of Human Biosciences, La Trobe University, Melbourne, VIC 3086, Australia and 3Department of Physiology, University of Melbourne, Melbourne, VIC 3010, Australia.

Excessive increases in intracellular [Ca2+] in skeletal muscle fibres can cause prolonged muscle weakness by disrupting normal communication between the dihydropyridine receptors (DHPRs) in the transverse tubular (T-) system and the Ca2+ release channels (RyRs) in the sarcoplasmic reticulum (SR), but the exact basis of this effect is unknown. Our previous work suggested a possible role of Ca2+-dependent proteolysis in this uncoupling process (Lamb et al., 1995; Murphy et al., 2006; Verburg et al., 2009), but found no proteolysis of the DHPRs, RyRs or triadin. Junctophilin1 (JP1) (∼90kDa) stabilizes close apposition of the T-system and SR membranes in adult skeletal muscle fibres; its C-terminal end is embedded in the SR and its N-terminal associates with the T-system membrane (Takeshima et al., 2000). Junctophilin-2 (JP2) has an analogous role in cardiac cells. This study examined whether JP1 and JP2 undergo Ca2+-dependent proteolysis, and whether this occurs in conjunction with the disruption of EC-coupling in Ca2+-treated skinned fibres and in other intact muscle situations.

Extensor digitorum longus (EDL), tibialis anterior and diaphragm muscles were dissected from rats and mice killed by isoflurane overdose, and whole heart obtained from adult rats anaesthetized with sodium pentobarbitone (60mg/kg i.p.) and injected with sodium heparin (200IU) via the femoral vein. Depolarization-induced force responses were recorded in mechanically-skinned fibres of rat EDL muscle as previously described (Lamb et al., 1995). Hearts were retrogradely perfused with oxygenated bicarbonate buffer in non-recirculating Langendorff mode at a constant pressure equivalent to 73mmHg. Left ventricular pressure measurements were performed using a fluid-filled balloon connected to a pressure transducer and recorded on a data acquisition system. Hearts were subjected to 20 min global ischemia (37˚C) and 60 min reperfusion. Control hearts were perfused aerobically throughout. Western blotting of proteins was performed as in Murphy et al. (2011).

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 μ-calpain. JP1 cleavage occurs close to the C-terminal, yielding a ∼75kDa diffusible fragment and a fixed ∼15kDa fragment. Depolarization-induced force responses in rat skinned fibres were abolished following 1min exposure to 40μM Ca2+, with accompanying loss of full-length JP1. Supra-physiological stimulation of rat skeletal muscle in vitro, by repeated tetanic stimulation in 30mM caffeine, also produced marked proteolysis of JP1 (and not RyR1). In dystrophic mdx mice, there was marked proteolysis of JP1 in limb muscles at 4 and not at 10 weeks of age, and in diaphragm at >6 months of age. JP2 also underwent Ca2+-dependent proteolysis and JP2 levels were reduced following cardiac ischaemia-reperfusion. It is concluded that junctophilin proteolysis may contribute to skeletal muscle weakness and cardiac dysfunction in a range of circumstances.

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