Hyperglycemia‐induced Protein Kinase Cβ2 Activation Causes Diastolic Cardiac Dysfunction by Disrupting Brg1‐mediated Suppression of RIP3 in Diabetic Rats

Protein kinase C β (PKCβ) 2 is preferably over‐activated in diabetic myocardium, which contributes to the development of diabetic cardiomyopathy (DCM), a pathology that is associated with increase of receptor interacting protein (RIP) 3‐mediated programmed necrosis (necroptosis) and decrease of brahma‐related gene 1 (Brg1), a key subunit of ATP‐dependent chromatin‐remodeling enzyme. Induction of PKCβ suppresses Brg1 (Nat Commun. 2015;6:7441), while deletion of Brg1 is associated with increase of RIP3. Yet, how PKCβ2 induces RIP3‐mediated necroptosis in DCM and whether Brg1 is involved in this process are unclear. We hypothesized that PKCβ2, by reducing Brg1, increases RIP3‐mediated necroptosis, leading to DCM and that inhibition of PKCβ2 activation may, by restoring Brg1, reduce RIP3 and attenuate DCM. Control (C) or streptozotocin‐induced diabetic rats (D) were untreated (C, D) or treated with LY (LY333531, a selective PKCβ inhibitor, 1 mg/kg/day) for four weeks starting from one week after diabetes induction. Left ventricular (LV) function was recorded by echocardiography. Brg1 and RIP3 binding was assessed by chromatin immunoprecipitation (ChIP). Five weeks after diabetes induction, D rats developed cardiac hypertrophy (larger cardiomyocyte cross‐sectional area and higher heart weight to body weight ratio) and LV diastolic dysfunction (decreased E/A ratio and increased LV isovolumic relaxation time), along with elevated cardiac necroptosis (enhanced protein expression of RIP1 and RIP3) and oxidative stress (enhanced cardiac 15‐F2t‐isoprostane) (All P <0.05 vs. C), which were concomitant with increased cardiac PKCβ2 activation and decreased Brg1 protein expression. All these changes were prevented by LY (All P <0.05 vs. D). In cultured cardiac H9C2 cells, high glucose (HG, 25mM for 48 hours) exposure increased cell size, elevated oxidative stress and necroptosis that was accompanied by upregulated PKCβ2 activation and reduced Brg1 protein expression (All P <0.05 vs. normal glucose). PKCβ2 inhibition by gene knockdown abolished all abovementioned alterations, while Brg1 gene knockdown or nitric oxide synthesis (NOS) inhibition reduced the protective effects of PKCβ2 inhibition. Brg1 gene knockdown also cancelled PKCβ2 inhibition‐induced endothelial NOS and nitric oxide production, while NOS inhibition cancelled PKCβ2 inhibition‐induced reduction of RIP3 without affecting Brg1. In addition, bioinformatics analysis revealed that Brg1 may bind to the enhancer‐like area of RIP3 (occupying this area reduces RIP3 expression), which was confirmed by ChIP study. Further, in HG‐incubated H9C2 cells, RIP3 protein expression was significantly increased, which was further enhanced by Brg1 gene knockdown. It is concluded that hyperglycemia‐induced PKCβ2 activation, by decreasing Brg1, reduces Brg1‐mediated reduction of oxidative stress and disrupts the binding of Brg1 and RIP3, which jointly enhance RIP3, resulting in the induction of necroptosis and the development of diabetic cardiomyopathy. Restoration of Brg1 represents a major mechanism whereby inhibition of PKCβ2 attenuates diabetic cardiomyopathy. Support or Funding Information This study was supported, in part, by grants from the National Natural Science Foundation of China (81601722 to Haobo Li; 81670770 to Zhengyuan Xia), and by General Research Fund Grant (17123915; 17158616 to Zhengyuan Xia) from the Research Grants Council of Hong Kong.