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

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 {"type":"entrez-protein","attrs":{"text":"CGP53353","term_id":"875191971","term_text":"CGP53353"}}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. {"type":"entrez-nucleotide","attrs":{"text":"LY333531","term_id":"1257370768","term_text":"LY333531"}}LY333531 treatment (for a duration of 4 weeks) prevented excessive PKCβ₂ activation and attenuated cardiac diastolic dysfunction in rats with streptozotocin-induced diabetes. {"type":"entrez-nucleotide","attrs":{"text":"LY333531","term_id":"1257370768","term_text":"LY333531"}}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.Cardiovascular disease is the leading cause of diabetes-related death (1). While most diabetic heart failure etiology concerns coronary disease associated with atherosclerosis, a diabetes-associated cardiomyopathy has been reported in humans (2) and animal models of type 1 (3) and type 2 (4) diabetes. Numerous studies by our group (5,6) and others (7,8) suggest the involvement of excess expression or activation of protein kinase C (PKC)β₂ in the development and progression of diabetic cardiomyopathy. Moreover, inhibition of PKCβ activation improves cardiac function in diabetic animals (9,10). Despite these observations, the underlying mechanism by which PKCβ₂ activation exerts deleterious effects in the diabetic myocardium remains unclear.PKCβ₁ and PKCβ₂ are two of the classical isoforms (α, β, and γ) of PKC (11). Of the two isoforms, PKCβ₂ is preferentially overexpressed in the myocardium of patients (12) or animals (10) with diabetes. PKCβ₂ activation has been implicated in diabetes-associated abnormalities via inhibition of Akt-dependent endothelial nitric oxide (NO) synthase (eNOS) activity (13), and restoration of Akt-eNOS-NO signaling has been shown to attenuate diabetic cardiomyopathy and myocardial dysfunction (14). Altered caveolae formation may potentially be the root cause of such inhibition. Caveolae, lipid rafts formed by small plasma membrane invaginations, serve as platforms modulating signal transduction pathways (e.g., PKC isoforms [15]) via molecules docked with caveolin (Cav), a major constituent protein associated with caveolae. Of the three Cav isoforms identified in mammalian caveolae, Cav-3 is mainly expressed in cardiac muscle and is essential for proper formation of cardiomyocyte caveolae (16). Interestingly, in cardiomyocytes, eNOS localizes to Cav-3 (17), permitting eNOS activation by cell surface receptors and cellular surface NO release for intercellular signaling (17). Therefore, NO is an endogenous inhibitor of hypertrophic signaling (18), and Cav-3 is important for maintaining NO function. Additionally, Cav-3 has been demonstrated to inhibit growth signaling in the hearts of nondiabetic subjects (19). Thus, any alteration in Cav-3 expression in the diabetic condition may participate in the pathogenesis of diabetic cardiomyopathy, which is supported by findings that decreased cardiac Cav-3 expression is detected in rats with chronic streptozotocin (STZ)-induced diabetes (20,21). In the current study, we hypothesize that PKCβ₂ activation induced by hyperglycemia promotes caveolae dysfunction with associated signaling abnormality. Our data suggest that excessive PKCβ₂ activation during diabetes reduces Cav-3 expression, with subsequent decreased Akt/eNOS signaling, which ultimately and negatively affect cardiac remodeling and function.