Department of Pharmacology, School of Pharmacy, Key Laboratory of Inflammation and Molecular Drug Target of Jiangsu Province, Nantong University, Nantong 226001, China.
Department of Pharmacology, School of Pharmacy, Key Laboratory of Inflammation and Molecular Drug Target of Jiangsu Province, Nantong University, Nantong 226001, China; School of Medicine, Nantong University, Nantong 226001, China.
Biochem Pharmacol. 2019 May;163:194-205. doi: 10.1016/j.bcp.2019.02.022. Epub 2019 Feb 16.
Ca/calmodulin-dependent protein kinase II (CaMKII) plays an important role in the cardiovascular system. However, the potential protective role of inhibitor 1 of protein phosphatase 1 (I1PP1), which is able to regulate CaMKII, in high glucose-induced cardiomyocytes injury remains unknown. In the present study, cardiomyocytes were transfected with I1PP1 adenovirus to inhibit protein phosphatase 1 (PP1) expression. After the cardiomyocytes were subjected to high glucose stimulation for 48 h, quantitative real-time PCR was used to detect CaMKIIδ alternative splicing. Lactate dehydrogenase (LDH) release and adenosine triphosphate (ATP) level were measured to assess cell damage and energy metabolism respectively. CaMKII activity was represented as phospholamban (PLB) phosphorylation, CaMKII phosphorylation (p-CaMKII) and oxidation (ox-CaMKII). Dihydroethidium (DHE), MitoSOX and JC-1 staining were used to assess oxidative stress and mitochondrial membrane potential. Necroptosis was evaluated by receptor interacting protein kinase 3 (RIPK3) expression, TUNEL and cleaved-caspase 3 levels. RIPK3, mixed lineage kinase domain like protein (MLKL) and dynamin-related protein 1 (DRP1) expressions were also detected. We found that high glucose disordered CaMKIIδ alternative splicing. I1PP1 over-expression suppressed PLB phosphorylation, ox-CaMKII, DRP1, RIPK3 and cleaved-caspase 3 proteins expression, decreased LDH release, attenuated necroptosis, increased ATP level, inhibited oxidative stress, and elevated mitochondrial membrane potential in high glucose-stimulated cardiomyocytes. However, there was no effect on phosphorylation of MLKL (p-MLKL), p-CaMKII, and receptor interacting protein kinase 1 (RIPK1) expression. Altogether, I1PP1 over-expression alleviated CaMKIIδ alternative splicing disorder, suppressed CaMKII oxidation, reduced reactive oxygen species (ROS) accumulation and inhibited necroptosis to attenuate high glucose-induced cardiomyocytes injury.
钙/钙调蛋白依赖性蛋白激酶 II（CaMKII）在心血管系统中发挥着重要作用。然而，能够调节 CaMKII 的蛋白磷酸酶 1 抑制剂 1（I1PP1）在高糖诱导的心肌细胞损伤中的潜在保护作用尚不清楚。在本研究中，通过转染 I1PP1 腺病毒抑制蛋白磷酸酶 1（PP1）的表达，使心肌细胞受到高糖刺激 48 小时。用实时定量 PCR 检测 CaMKIIδ 选择性剪接。通过测定乳酸脱氢酶（LDH）释放和三磷酸腺苷（ATP）水平，分别评估细胞损伤和能量代谢。用磷酸化肌浆球蛋白轻链（PLB）、CaMKII 磷酸化（p-CaMKII）和氧化（ox-CaMKII）来表示 CaMKII 活性。用二氢乙啶（DHE）、MitoSOX 和 JC-1 染色评估氧化应激和线粒体膜电位。通过受体相互作用蛋白激酶 3（RIPK3）表达、TUNEL 和 cleaved-caspase 3 水平评估坏死性凋亡。还检测了 RIPK3、混合谱系激酶结构域样蛋白（MLKL）和动力相关蛋白 1（DRP1）的表达。我们发现高糖打乱了 CaMKIIδ 选择性剪接。I1PP1 过表达抑制了 PLB 磷酸化、ox-CaMKII、DRP1、RIPK3 和 cleaved-caspase 3 蛋白表达，降低了 LDH 释放，减轻了坏死性凋亡，增加了 ATP 水平，抑制了氧化应激，提高了高糖刺激的心肌细胞中线粒体膜电位。然而，对 MLKL 的磷酸化（p-MLKL）、p-CaMKII 和受体相互作用蛋白激酶 1（RIPK1）的表达没有影响。总之，I1PP1 过表达缓解了 CaMKIIδ 选择性剪接紊乱，抑制了 CaMKII 氧化，减少了活性氧（ROS）的积累，并抑制了坏死性凋亡，从而减轻了高糖诱导的心肌细胞损伤。
