RATIONALE 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) O-linked attachment of N-acetylglucosamine (O-GlcNAc) at S280 leading to arrhythmia and (2) a reactive oxygen species (ROS)-mediated oxidation of CaMKII that can increase postinfarction mortality. OBJECTIVE To test whether high extracellular glucose (Hi-Glu) promotes ventricular myocyte ROS generation and the role played by CaMKII. METHODS AND RESULTS 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 O-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. CONCLUSIONS Diabetic hyperglycemia induces acute cardiac myocyte ROS production by NOX2 that requires O-GlcNAcylation of CaMKIIδ at S280. This novel ROS induction may exacerbate pathological consequences of diabetic hyperglycemia.

中文翻译：

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高血糖症通过O-关联的GlcNAcylation和CaMKII激活在小鼠心室肌细胞中急性增加了细胞内的活性氧。

理由糖尿病是一种复杂的多系统疾病，正在影响全世界的大批人群。慢性CaMKII（Ca2 + /钙调蛋白依赖性激酶II）激活可能发生在糖尿病中，并致心律失常。糖尿病高血糖症显示出通过以下方式激活CaMKII：（1）S-280上的N-乙酰氨基葡萄糖（O-GlcNAc）的O型连接导致心律不齐；（2）活性氧（ROS）介导的CaMKII氧化可增加梗死后死亡率。目的检测高细胞外葡萄糖（Hi-Glu）是否促进心室肌细胞ROS的产生以及CaMKII的作用。方法和结果我们使用DCF（2'，7'-dichlorodihydrofluorescein diacetate）和遗传靶向的Grx-roGFP2氧化还原传感器，测试了细胞外Hi-Glu如何影响成人心室肌细胞中ROS的产生。Hi-Glu（30 mmol / L）显着提高了ROS产生的速率-在用CaMKII抑制剂KN-93预处理的心肌细胞中或从整体或心脏特异性CaMKIIδKO（敲除）小鼠中阻止了ROS的产生。CaMKII KO或抑制作用还阻止了Hi-Glu诱导的肌浆网C​​a2 +释放事件（Ca2 +火花）。因此，CaMKII激活是心肌细胞中Hi-Glu诱导的ROS生成和肌浆网Ca2 +泄漏所必需的。为了测试O-GlcNAc-CaMKII途径的参与，我们抑制了由Thiamet G（ThmG）去除GlcNAcylation的过程，该过程模仿了Hi-Glu诱导的ROS产生。相反，抑制GlcNAcylation（OSMI-1 [（αR）-α-[[（（1,2-二氢-2-氧代-6-喹啉基）磺酰基]氨基] -N-（2-呋喃基甲基）-2-甲氧基- N-（2-噻吩甲基）-苯乙酰胺]）阻止了对Hi-Glu或ThmG的ROS诱导。此外，在基于CRSPR的敲入小鼠中，其中消融了CaMKIIδ上的功能性GlcNAcylation位点（S280A），Hi-Glu和ThmG均未诱导肌细胞ROS生成。因此，CaMKIIδ-S280是Hi-Glu诱导的（和依赖GlcNAc的）ROS生产所必需的。为确定ROS来源，我们使用了不同的NOX（NADPH氧化酶）2（Gp91ds-tat肽），NOX4（GKT137831），线粒体ROS（MitoTempo）和NOS（NO合酶）途径抑制剂（L-NAME） ，L-NIO和L-NPA）。只有NOX2抑制或KO阻止了Hi-Glu / ThmG诱导的ROS生成。结论糖尿病高血糖诱导NOX2引起急性心肌细胞ROS的产生，这需要在S280时对CaMKIIδ进行O-GlcNAcy化。这种新颖的ROS诱导可能加剧糖尿病高血糖症的病理后果。