dinoprost and ruboxistaurin

Activation of PKCβ (protein kinase Cβ) plays a critical role in myocardial I/R (ischaemia/reperfusion) injury in non-diabetic rodents. In the myocardium of diabetes, PKCβ2 overexpression is associated with increased vulnerability to post-ischaemic I/R injury with concomitantly impaired cardiomyocyte Cav (caveolin)-3 and Akt signalling compared with non-diabetic rats. We hypothesized that myocardial PKCβ overexpression in diabetes exacerbates myocardial I/R injury through impairing Cav-3/Akt signalling. Streptozotocin-induced diabetic rats were treated with the selective PKCβ inhibitor ruboxistaurin (RBX, 1 mg/kg per day) for 4 weeks, starting from 1 week after diabetes induction, before inducing myocardial I/R achieved by occluding the left descending coronary artery followed by reperfusion. Cardiac function was measured using a pressure-volume conductance system. In an in vitro study, cardiac H9C2 cells were exposed to high glucose (30 mmol/l) and subjected to hypoxia followed by reoxygenation (H/R) in the presence or absence of the selective PKCβ2 inhibitor CGP53353 (1 μmol/l), siRNAs of PKCβ2 or Cav-3 or Akt. Cell apoptosis and mitochondrial membrane potential were assessed by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) and JC-1 staining respectively. RBX significantly decreased post-ischaemic myocardial infarct size (35±5% compared with 49±3% in control, P<0.05) and attenuated cardiac dysfunction, and prevented the reduction in cardiac Cav-3 and enhanced phosphorylated/activated Akt (p-Akt) in diabetic rats (P<0.05). H/R increased cardiomyocyte injury under high glucose conditions as was evident by increased TUNEL-positive and increased JC-1 monomeric cells (P<0.05 compared with control), accompanied with increased PKCβ2 phosphorylation/activation and decreased Cav-3 expression. Either CGP53353 or PKCβ2 siRNA significantly attenuated all of these changes and enhanced p-Akt. Cav-3 gene knockdown significantly reduced p-Akt and increased post-hypoxic cellular and mitochondrial injury despite a concomitant reduction in PKCβ2 phosphorylation. PKCβ2 inhibition with RBX protects diabetic hearts from myocardial I/R injury through Cav-3-dependent activation of Akt.

Topics: Animals; Apoptosis; Caveolin 3; Cell Line; Diabetes Mellitus, Experimental; Dinoprost; Enzyme Activation; Indoles; Isoprostanes; Male; Maleimides; Membrane Potential, Mitochondrial; Mitochondria, Heart; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Phosphorylation; Phthalimides; Protein Kinase C beta; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Rats, Sprague-Dawley; RNA Interference; Signal Transduction; Time Factors; Transfection; Ventricular Function, Left

Oxidative stress plays critical roles in the development of diabetic cardiovascular complications, including myocardial hypertrophy. The β isoform of PKC (protein kinase C) is preferentially overexpressed in the myocardium of diabetic subjects accompanied with increased activation of the pro-oxidant enzyme NADPH oxidase, which may exacerbate oxidative stress. We hypothesized that myocardial PKCβ is a major upstream mediator of oxidative stress in diabetes and that PKCβ inhibition can attenuate myocardial hypertrophy and dysfunction. Control or streptozotocin-induced diabetic rats were treated with the selective PKCβ inhibitor RBX (ruboxistaurin; 1 mg/kg of body weight per day) or the antioxidant NAC (N-acetylcysteine) for 4 weeks. LV (left ventricular) dimensions and functions were detected by echocardiography. 15-F2t-isoprostane (a specific index of oxidative stress) and myocardial activities of superoxide dismutase as well as protein levels of NADPH oxidase were assessed by immunoassay or Western blotting. Echocardiography revealed that the LV mass/body weight ratio was significantly increased in diabetic rats (P<0.01 compared with the control group) in parallel with the impaired LV relaxation. A significant increase in cardiomyocyte cross-sectional area was observed in diabetic rats accompanied by an increased production of O2- (superoxide anion) and 15-F2t-isoprostane (all P<0.05 compared with the control group). RBX normalized these changes with concomitant inhibition of PKCβ2 activation and prevention of NADPH oxidase subunit p67phox membrane translocation and p22phox overexpression. The effects of RBX were comparable with that of NAC, except that NAC was inferior to RBX in attenuating cardiac dysfunction. It is concluded that RBX can ameliorate myocardial hypertrophy and dysfunction in diabetes, which may represent a novel therapy in the prevention of diabetic cardiovascular complications.

Topics: Acetylcysteine; Animals; Diabetes Mellitus, Experimental; Diabetic Cardiomyopathies; Dinoprost; Drug Evaluation, Preclinical; Enzyme Activation; Free Radical Scavengers; Hypertrophy, Left Ventricular; Indoles; Isoprostanes; Male; Maleimides; Myocytes, Cardiac; NADPH Oxidases; Oxidative Stress; Protein Kinase C; Protein Kinase C beta; Rats; Rats, Sprague-Dawley; Superoxides; Ultrasonography