Repeated non-invasive limb ischemic preconditioning confers cardioprotection in diabetes by reducing necroptosis through STAT3 activation that is PKCɛ/Caveolin-3 dependent

Abstract

Non-invasive limb ischemic preconditioning (NLIP) confers cardioprotection by activating myocardial STAT3. Hearts from diabetes are susceptible to myocardial ischemia injury (IRI). NLIP fails to confer cardioprotection in diabetes whose cardiac STAT3 activation (pSTAT3) is reduced that is concomitant with enhanced myocardial necroptosis (a form of programmed necrosis that plays important roles in cell death during myocardial IRI). Repeated NLIP (rNLIP) for consecutively three days before inducing ischemia increases cardiac pSTAT3(FASEB, 2014, 28: LB559) and confers cardioprotection in diabetes. However, how rNLIP activates STAT3 in diabetes and whether or not modulation of cardiac necroptosis in diabetes is a mechanism of rNLIP cardioprotection is unknown. Protein kinase C epsilon (PKCɛ), by binding to caveolin-3 (Cav3, a protein of the lipid rafts/caveolae), serves as a mediator of preconditioning cardioprotection. Moderate activation of PKCɛ up-regulates STAT3. However, in diabetes, pSTAT3 is reduced with concomitant PKCɛ over-activation and its disassociation with Cav3. Thus, we postulated that rNLIP, by enhancing PKCɛ and Cav3 interaction, activates STAT3 which subsequently reduced necroptosis and attenuates myocardial IRI in diabetes. Control (C) and eight weeks streptozotocin-induced diabetic (D) rats were subjected to sham operation or myocardial ischemia-reperfusion (30 min of coronary artery occlusion and 2 hours of reperfusion) with or without rNLIP (3 cycles of 5 min occlusion and 5 min reperfusion in a unilateral hindlimb once per day for three days). In D rats, PKCɛ was over-activated accompanied with decreased Cav3 and colocalizaiton of PKCɛ with Cav3. At the end of reperfusion, D rats exhibited more severe myocardial injury (increased post-ischemic infarction and increased troponin-1 release, P<0.05 vs. C) that was associated with reduced pSTAT3 and enhanced myocardial necroptosis [increased RIP1(necroptosis initiator) and RIP3 (necroptosis executor) proteins expression]. rNLIP attenuated post-ischemic cardiac injury, elevated Cav3, and increased colocalization of PKCɛ with Cav3 that were associated with increased pSTAT3 as well as decreased necroptosis in both C and D rats. rNLIP increased PKCɛ in C rats but reduced PKCɛ in D rats (P<0.05 vs. IR). PKCɛ inhibition (by ɛV1/2) abolished above mentioned rNLIP cardioprotective effects in both C and D rats (P<0.05 vs. IR). In cultured cardiac H9C2 cells, simulated rNLIP (hypoxic preconditioning (HPo) 24 hours before inducing hypoxia/reoxygenation (HR), attenuated cell injury, concomitant with increased pSTAT3, Cav3, and PKCɛ-Cav3 colocalization (P<0.05, vs. HR) in cells exposed to normal glucose (5 mM) or high glucose (25 mM). The effects of HPo were abolished by PKCɛ or STAT3 gene knockdown. It is concluded that rNLIP confers cardioprotection in diabetes by suppressing necroptosis via PKCɛ/Cav3 dependent STAT3 activation, which needs moderate- but not excessive PKCɛ activation.