Hyperglycemia Acutely Increases Cytosolic Reactive Oxygen Species via <i>O</i>-linked GlcNAcylation and CaMKII Activation in Mouse Ventricular Myocytes.

Abstract

Diabetes mellitus is a complex, multisystem disease, affecting large populations worldwide. Chronic CaMKII (Ca²⁺/calmodulin-dependent kinase II) activation may occur in diabetes mellitus and be arrhythmogenic. Diabetic hyperglycemia was shown to activate CaMKII by (1) <i>O</i>-linked attachment of N-acetylglucosamine (<i>O</i>-GlcNAc) at S280 leading to arrhythmia and (2) a reactive oxygen species (ROS)-mediated oxidation of CaMKII that can increase postinfarction mortality. To test whether high extracellular glucose (Hi-Glu) promotes ventricular myocyte ROS generation and the role played by CaMKII. We tested how extracellular Hi-Glu influences ROS production in adult ventricular myocytes, using DCF (2',7'-dichlorodihydrofluorescein diacetate) and genetically targeted Grx-roGFP2 redox sensors. Hi-Glu (30 mmol/L) significantly increased the rate of ROS generation-an effect prevented in myocytes pretreated with CaMKII inhibitor KN-93 or from either global or cardiac-specific CaMKIIδ KO (knockout) mice. CaMKII KO or inhibition also prevented Hi-Glu-induced sarcoplasmic reticulum Ca²⁺ release events (Ca²⁺ sparks). Thus, CaMKII activation is required for Hi-Glu-induced ROS generation and sarcoplasmic reticulum Ca²⁺ leak in cardiomyocytes. To test the involvement of <i>O</i>-GlcNAc-CaMKII pathway, we inhibited GlcNAcylation removal by Thiamet G (ThmG), which mimicked the Hi-Glu-induced ROS production. Conversely, inhibition of GlcNAcylation (OSMI-1 [(αR)-α-[[(1,2-dihydro-2-oxo-6-quinolinyl)sulfonyl]amino]-N-(2-furanylmethyl)-2-methoxy-N-(2-thienylmethyl)-benzeneacetamide]) prevented ROS induction in response to either Hi-Glu or ThmG. Moreover, in a CRSPR-based knock-in mouse in which the functional GlcNAcylation site on CaMKIIδ was ablated (S280A), neither Hi-Glu nor ThmG induced myocyte ROS generation. So CaMKIIδ-S280 is required for the Hi-Glu-induced (and GlcNAc dependent) ROS production. To identify the ROS source(s), we used different inhibitors of NOX (NADPH oxidase) 2 (Gp91ds-tat peptide), NOX4 (GKT137831), mitochondrial ROS (MitoTempo), and NOS (NO synthase) pathway inhibitors (L-NAME, L-NIO, and L-NPA). Only NOX2 inhibition or KO prevented Hi-Glu/ThmG-induced ROS generation. Diabetic hyperglycemia induces acute cardiac myocyte ROS production by NOX2 that requires <i>O</i>-GlcNAcylation of CaMKIIδ at S280. This novel ROS induction may exacerbate pathological consequences of diabetic hyperglycemia.