Complex Regulation of PKCβ2 and PDK-1/AKT by ROCK2 in Diabetic Heart

Objectives: The RhoA/ROCK pathway contributes to diabetic cardiomyopathy in part by promoting the sustained activation of PKC beta 2 but the details of their interaction are unclear. The purpose of this study was to investigate if over-activation of ROCK in the diabetic heart leads to direct phosphorylation and activation of PKC beta 2, and to determine if their interaction affects PDK-1/Akt signaling. Methods: Regulation by ROCK of PKC beta 2 and related kinases was investigated by Western blotting and co-immunoprecipitation in whole hearts and isolated cardiomyocytes from 12 to 14-week diabetic rats. Direct ROCK2 phosphorylation of PKC beta 2 was examined in vitro. siRNA silencing was used to confirm role of ROCK2 in PKC beta 2 phosphorylation in vascular smooth muscle cells cultured in high glucose. Furthermore, the effect of ROCK inhibition on GLUT4 translocation was determined in isolated cardiomyocytes by confocal microscopy. Results: Expression of ROCK2 and expression and phosphorylation of PKC beta 2 were increased in diabetic hearts. A physical interaction between the two kinases was demonstrated by reciprocal i mmunoprecipitation, while ROCK2 directly phosphorylated PKC beta 2 at T641 in vitro. ROCK2 siRNA in vascular smooth muscle cells or inhibition of ROCK in diabetic hearts reduced PKC beta 2 T641 phosphorylation, and this was associated with attenuation of PKC beta 2 activity. PKC beta 2 also formed a complex with PDK-1 and its target AKT, and ROCK inhibition resulted in upregulation of the phosphorylation of PDK-1 and AKT, and increased translocation of glucose transporter 4 (GLUT4) to the plasma membrane in diabetic hearts. Conclusion: This study demonstrates that over-activation of ROCK2 contributes to diabetic cardiomyopathy by multiple mechanisms, including direct phosphorylation and activation of PKC beta 2 and interference with the PDK-1-mediated phosphorylation and activation of AKT and translocation of GLUT4. This suggests that ROCK2 is a critical node in the development of diabetic cardiomyopathy and may be an effective target to improve cardiac function in diabetes.