ABSTRACT
Endothelial cell (EC) dysfunction is implicated in cardiovascular diseases, including diabetes. The decrease in nitric oxide (NO) bioavailability is the hallmark of endothelial dysfunction, and it leads to attenuated vascular relaxation and atherosclerosis followed by a decrease in blood flow. In the heart, decreased coronary blood flow is responsible for insufficient oxygen supply to cardiomyocytes and, subsequently, increases the incidence of cardiac ischemia. In this study we investigate whether and how reactive oxygen species (ROS) in mitochondria contribute to coronary endothelial dysfunction in type 2 diabetic (T2D) mice. T2D was induced in mice by a high-fat diet combined with a single injection of low-dose streptozotocin. ACh-induced vascular relaxation was significantly attenuated in coronary arteries (CAs) from T2D mice compared with controls. The pharmacological approach reveals that NO-dependent, but not hyperpolarization- or prostacyclin-dependent, relaxation was decreased in CAs from T2D mice. Attenuated ACh-induced relaxation in CAs from T2D mice was restored toward control level by treatment with mitoTempol (a mitochondria-specific O2(-) scavenger). Coronary ECs isolated from T2D mice exhibited a significant increase in mitochondrial ROS concentration and decrease in SOD2 protein expression compared with coronary ECs isolated from control mice. Furthermore, protein ubiquitination of SOD2 was significantly increased in coronary ECs isolated from T2D mice. These results suggest that augmented SOD2 ubiquitination leads to the increase in mitochondrial ROS concentration in coronary ECs from T2D mice and attenuates coronary vascular relaxation in T2D mice.
Subject(s)
Coronary Vessels/physiopathology , Diabetes Mellitus, Experimental/metabolism , Endothelium, Vascular/physiopathology , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Acetylcholine , Animals , Culture Media , Endothelial Cells/drug effects , Endothelial Cells/physiology , Glucose/pharmacology , Humans , Immunoprecipitation , Male , Mice , Mice, Inbred C57BL , Mitochondria/genetics , Nitric Oxide , Palmitic Acid/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Superoxide DismutaseABSTRACT
A decrease in capillary density due to an increase in endothelial cell apoptosis in the heart is implicated in cardiac ischemia in diabetes. The voltage-dependent anion channel (VDAC) plays a crucial role in the regulation of mitochondrial metabolic function and mitochondria-mediated apoptosis. This study is designed to examine the role of VDAC in coronary endothelial dysfunction in diabetes. Endothelial cells (ECs) were more apoptotic in diabetic left ventricle of diabetic mice and mouse coronary ECs (MCECs) isolated from diabetic mice exhibited significantly higher mitochondrial Ca(2+) concentration and VDAC protein levels than control MCECs. The expression of VDAC-short hairpin RNA (shRNA) not only decreased the resting mitochondrial Ca(2+) concentration but also attenuated mitochondrial Ca(2+) uptake in diabetic MCECs. Furthermore, the downregulation of VDAC in diabetic MCECs significantly decreased mitochondrial superoxide anion (O(2)(-)) production and the activity of the mitochondrial permeability transition pore (mPTP) opening (an indirect indicator of cell apoptosis) toward control levels. These data suggest that the increased VDAC level in diabetic MCECs is responsible for increased mitochondrial Ca(2+) concentration, mitochondrial O(2)(-) production, and mPTP opening activity. Normalizing VDAC protein level may help to decrease endothelial cell apoptosis, increase capillary density in the heart, and subsequently decrease the incidence of cardiac ischemia in diabetes.
