Hyperglycemia-Induced Protein Kinase C β2 Activation Induces Diastolic Cardiac Dysfunction in Diabetic Rats by Impairing Caveolin-3 Expression and Akt/eNOS Signaling

Abstract

Protein kinase C (PKC)β2 is preferably overexpressed in the diabetic myocardium, which induces cardiomyocyte hypertrophy and contributes to diabetic cardiomyopathy, but the underlying mechanisms are incompletely understood. Caveolae are critical in signal transduction of PKC isoforms in cardiomyocytes. Caveolin (Cav)-3, the cardiomyocyte-specific caveolar structural protein isoform, is decreased in the diabetic heart. The current study determined whether PKCβ2 activation affects caveolae and Cav-3 expression. Immunoprecipitation and immunofluorescence analysis revealed that high glucose (HG) increased the association and colocalization of PKCβ2 and Cav-3 in isolated cardiomyocytes. Disruption of caveolae by methyl-β-cyclodextrin or Cav-3 small interfering (si)RNA transfection prevented HG-induced PKCβ2 phosphorylation. Inhibition of PKCβ2 activation by compound CGP53353 or knockdown of PKCβ2 expression via siRNA attenuated the reductions of Cav-3 expression and Akt/endothelial nitric oxide synthase (eNOS) phosphorylation in cardiomyocytes exposed to HG. LY333531 treatment (for a duration of 4 weeks) prevented excessive PKCβ2 activation and attenuated cardiac diastolic dysfunction in rats with streptozotocin-induced diabetes. LY333531 suppressed the decreased expression of myocardial NO, Cav-3, phosphorylated (p)-Akt, and p-eNOS and also mitigated the augmentation of O2−, nitrotyrosine, Cav-1, and iNOS expression. In conclusion, hyperglycemia-induced PKCβ2 activation requires caveolae and is associated with reduced Cav-3 expression in the diabetic heart. Prevention of excessive PKCβ2 activation attenuated cardiac diastolic dysfunction by restoring Cav-3 expression and subsequently rescuing Akt/eNOS/NO signaling.

Hyperglycemia Abrogates Ischemic Postconditioning Cardioprotection by Impairing AdipoR1/Caveolin-3/STAT3 Signaling in Diabetic Rats

“…In addition, cardioprotective effects of APN were diminished in high-fat diet-induced diabetes (10,11), in which Cav3 is downregulated (12). This, together with our recent finding that Cav3 is disrupted (13) and STAT3 inactivated (9), which deranged eNOS signaling in diabetic myocardium with concomitantly reduced APN (9), indicates that APN signaling (e.g., AdipoR1/Cav3) is impaired in diabetes. Although AdipoR1 (14,15) and STAT3 (16) have both been shown to be important procell survival factors in multiple cells, their potential interplay in affecting postischemic cell survival in general and specifically in affecting the myocardial responsiveness to IPo cardioprotection in diabetes is unknown.…”

“…At the end of treatments, cells were fixed for immunofluorescence staining, as described below, or collected and snap frozen in liquid N 2 for future analysis. Lactate dehydrogenase (LDH) release in culture medium was detected via a commercial LDH kit (Roche, Mannheim, Germany) (13). After the experiments were completed, cultured cardiomyocytes were homogenized in lysis buffer.…”

“…Caveolae were isolated by discontinuous sucrose gradient centrifugation, as previously described (13). Each heart sample gradient was separated into 12 fractions.…”

“…Equal protein amounts from isolated cardiomyocytes, H9C2 cells, rat heart, and isolated mitochondria or caveolae fractions were resolved by 7.5-12.5% SDS-PAGE and transferred to polyvinylidene fluoride membrane for immunoblot analysis, as previously described (13).…”

Transactivation of the epidermal growth factor receptor in responses to myocardial stress and cardioprotection

“…Further work is warranted in unravelling the roles of caveolae and caveolin-3 in control of cardiomyocyte EGFR signalling. Importantly, significant reductions in myocardial caveolin-3 expression/localisation with ageing (Kawabe et al, 2001;Ratajczak et al, 2003;Peart et al, 2007), infarction (Ratajczak et al, 2003), and disease (Piech et al, 2003;Penumathsa et al, 2008;Crossman et al, 2011;Sharma et al, 2011;Lei et al, 2013) have the potential to dysregulate myocardial EGFR signalling.…”