Inhibition of protein kinase C{221} as a novel strategy to combat diabetic cardiomyopathy and myocardial ischemic insult

Abstract

Hyperglycemia-induced oxidative stress plays a critical role in the development of diabetic cardiovascular complications including diabetic cardiomyopathy and ischemia heart disease. The β isoform of protein kinase C (PKC β), a family of serine/threonine kinases is preferentially over-expressed and activated in the myocardium of diabetic subjects, accompanied with increased activation of the pro-oxidant enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which may further exacerbate oxidative stress. However, the role and mechanism of PKC β in the development and progressing of diabetic cardiomyopathy and the subsequently decreased myocardial tolerance to ischemic insult has not been studied. We hypothesized that PKC β is a key upstream mediator of oxidative stress and that PKC β inhibition can attenuate myocardial cardiomyopathy and enhance myocardial tolerance to ischemia in diabetes. Diabetes was developed by injection of streptozotocin(STZ) in Sprague Dawley rats and was pre-treated with the selective PKC β inhibitor ruboxistaurin(RBX) or the antioxidant N-acetylcysteine(NAC) for 4 weeks. Cardiomyocyte cross-sectional area was significantly increased in diabetic rats that was associated with concomitantly increased myocardial PKC β protein expression and increased production of superoxide anion(O2-) and 15-F2t-isoprostane, an index of oxidative stress. Echocardiography revealed that the left ventricular mass/body weight ratio was significantly increased in diabetic rats in parallel with an impaired left ventricular relaxation. RBX normalized these changes with concomitant inhibition of PKCβ2 activation and prevention of NADPH oxidase activation. The effects of RBX were comparable with that of NAC, except that NAC was inferior to RBX in attenuating diastolic dysfunction. These results indicate that RBX processes the antioxidant properties and is superior to NAC for the treatment of cardiomyopathy at early stage of diabetes.

Diabetes is prone to develop ischemia and reperfusion (I/R) injury. I/R can enhance oxidative stress that further exacerbates myocardial I/R injury. We further tested the hypothesis that PKC β inhibition can reduce oxidative stress during myocardial I/R and subsequently ameliorate post-ischemic myocardial injury in diabetes. STZ-induced diabetic rats were pre-treated with RBX for 4 weeks. Myocardial production of O2- was significantly increased accompanied with increased Cu/Zn and Mn SOD activity resulting in increased levels of 15-F2t-isoprostane in plasma and myocardium of diabetic rats. Consequently, the bioavailability of myocardial nitric oxide (NO), a key cardioprotective molecule, was remarkably decreased. These changes were reversed by RBX. Myocardial I/R injury were induced in vivo by coronary artery occlusion and reperfusion, and ex vivo in isolated Langendorff-perfused rat hearts. RBX significantly reduced post-ischemic myocardial infarct size in diabetic rats in vivo and ex vivo by 40% and 37% respectively. The load-independent end-systolic pressure-volume relation(ESPVR) and load-dependent max dP/dt as assessed with pressure-volume(PV) loop, were lower in diabetic rats after ischemia and were restored by RBX. In Langendorff-perfused heart, RBX also significantly improved recovery of contractile function in diabetes rats. RBX mediated cardioprotection was associated with enhancement of post-ischemic protein levels of the oxygen sensitive hypoxic inducible factor-1(HIF1)α and pro-survival p-Akt in diabetic rats.

In summary, inhibition of PKC β may represent a novel therapy to combat diabetic cardiomyopathy and myocardial ischemic insult.