Junctophilin-2 is a target of matrix metalloproteinase-2 in myocardial ischemia–reperfusion injury
Junctophilin-2 is a structural membrane protein that tethers T-tubules to the sarcoplasmic reticulum to allow for coordinated calcium-induced calcium release in cardiomyocytes. Defective excitation–contraction coupling in myocardial ischemia–reperfusion (IR) injury is associated with junctophilin-2 proteolysis. However, it remains unclear whether preventing junctophilin-2 proteolysis improves the recovery of cardiac contractile dysfunction in IR injury. Matrix metalloproteinase-2 (MMP-2) is a zinc and calcium-dependent protease that is activated by oxidative stress in myocardial IR injury and cleaves both intracellular and extracellular substrates. To determine whether junctophilin-2 is targeted by MMP-2, isolated rat hearts were perfused in working mode aerobically or subjected to IR injury with the selective MMP inhibitor ARP-100. IR injury impaired the recovery of cardiac contractile function which was associated with increased degradation of junctophilin-2 and damaged cardiac dyads. In IR hearts, ARP-100 improved the recovery of cardiac contractile function, attenuated junctophilin-2 proteolysis, and prevented ultrastructural damage to the dyad. MMP-2 was co-localized with junctophilin-2 in aerobic and IR hearts by immunoprecipitation and immunohistochemistry. In situ zymography showed that MMP activity was localized to the Z-disc and sarcomere in aerobic hearts and accumulated at sites where the striated JPH-2 staining was disrupted in IR hearts. In vitro proteolysis assays determined that junctophilin-2 is susceptible to proteolysis by MMP-2 and in silico analysis predicted multiple MMP-2 cleavage sites between the membrane occupation and recognition nexus repeats and within the divergent region of junctophilin-2. Degradation of junctophilin-2 by MMP-2 is an early consequence of myocardial IR injury which may initiate a cascade of sequelae leading to impaired contractile function.
KeywordsIschemia–reperfusion injury Myocardial infarction Matrix metalloproteinase Junctophilin Excitation–contraction coupling Oxidative stress
This work was supported by grants to R.S. from the Canadian Institutes of Health Research (Foundation #143299) and the Heart and Stroke Foundation of Canada. B.Y.C. was awarded a graduate studentship from the Women and Children’s Health Research Institute, through the generous support of the Stollery Children’s Hospital Foundation and the Royal Alexandra Hospital Foundation. A.R. was awarded graduate studentships from the Faculty of Medicine and Dentistry at the University of Alberta. Some experiments were performed at the University of Alberta Faculty of Medicine & Dentistry Cell Imaging Centre, which receives financial support from the Faculty of Medicine & Dentistry, the Department of Medical Microbiology & Immunology and Canada Foundation for Innovation awards to contributing investigators.
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Conflict of interest
The authors have no conflict of interest.
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