Hyperglycemia-induced protein kinase C β2 activation induces diastolic cardiac dysfunction in diabetic rats by impairing caveolin-3 expression and Akt/eNOS signaling.
Diabetes.2013 Jul; 62(7): 2318-28
Lei S, Li H, Xu J, Liu Y, Gao X, Wang J, Ng KF, Lau WB, Ma XL, Rodrigues B, Irwin MG, Xia Z
Department of Anesthesiology, University of Hong Kong, Hong Kong, China.
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.
Diabetic cardiomyopathy is a leading cause of mortality in diabetic patients. Clinically, this complication is characterized initially as left ventricular diastolic dysfunction, which then progresses to systolic dysfunction and ultimately cardiac failure. Excessive activation of protein kinase C (PKC) β2 is implicated in the development and progression of diabetic cardiomyopathy. Caveolae are specialized plasma membrane microdomains, which modulate transduction pathways via signaling molecules docked within them. However, it is not clear whether or not PKCβ2 activation induced by hyperglycemia is modulated by cavoelae or Caveolin (Cav)-3, which is essential for the formation of cardiomyocyte caveolae.
We first investigated the relationship between PKCβ2 and Cav-3 or caveolae. We found that excessive activation of PKCβ2 was accompanied with decreased Cav-3 expression in cardiomyocytes isolated from control and streptozotocin-induced diabetic rats. Our immunoprecipitation experiments further demonstrated that diabetic condition increased the association of PKCβ2 with Cav-3, which was confirmed by confocal immunofluorescence staining. We then found that disruption of caveolae by methyl-β-cyclodextrin affected PKCβ2 activation, indicating hyperglycemia-induced PKCβ2 activation involves caveolae.
We next investigated the impact of PKCβ2 activation by hyperglycemia in the downstream signaling molecules Akt and eNOS, both of which are modulated by caveolins. Our results demonstrated that inhibition of PKCβ2 activation by the selective PKCβ2 inhibitor compound CGP53353 or gene knockdown of PKCβ2 expression via siRNA attenuated the reductions of Cav-3 expression and Akt/eNOS phosphorylation in cardiomyocytes exposed to high glucose. Similar results were also found in in vivo experiments.
Collectively, our study demonstrates that hyperglycemia induced PKCβ2 activation was associated with caveolae dysfunction and, consequently, deranged Akt/eNOS signaling. Inhibition of PKCβ2 activation attenuated cardiac diastolic dysfunction by restoring Cav-3 expression and subsequently rescuing Akt/eNOS/NO signaling.
This is the first study examining the relationship between PKCβ2 and Cav-3 in cardiomyocytes subjected to hyperglycemic conditions. The major novel finding of this study improves our understanding of the pathophysiology of diabetic cardiomyopathy and, PKCβ2 blockade may therefore serves as a novel therapeutic avenue in the treatment of diabetic cardiomyopathy.