| BackgroundDiabetes mellitus is an independent risk factor for both mortality and morbidity of ischemic heart disease caused by coronary atherosclerosis. Importently, hyperglycemia, by itself, is associated with increased risk of heat failure and death after myocardial infarction in patients with and without established diabetes, even if myocardium was timely reperfused through the restoration of blood flow by thrombolysis or percutaneous coronary intervention. This phenomenon suggests that hyperglycemia may directly sensitize myocardium to ischemia/reperfusion (I/R) injury independent of coronary atherosclerosis, which is still controversal in animal studies, and thus the issue remains to be investigated. Strong evidence exists that oxidative stress is the common denominator in the development and progression of both I/R injury and diabetes complications. Increased oxidative stress and modification of cellular targets by free redicals produced may alter the response of diabetic myocardium to I/R injury. Posttranslational modification of proteins by free radicals has been demonstrated to be affecting key proteins at nearly all molecular levels, related to gene expression, signal transduction, and antioxidant defense. Of prominent note is the process of protein tyrosine residue nitration, a well-documented pathologic modification, which alters protein structure and function, and is the potential etiology of many cardiovascular diseases. The hyperglycemic condition may exhibit pronounced myocardial injury as a result of protein nitration, but target responsible protein and underlying mechanisms remain to be identified. Thioredoxin (Trx) is an important cellular survival and antioxidant protein. Recent evidence shows that Trx functions as a key regulator of cardiovascular homeostasis directly via antioxidant effects and indirectly by affecting molecules in signaling pathway. We have previously demonstrated its susceptibility to nitrative modification, which results in the irreversible inhibition of its activity. The present study investigated the role of Trx nitration in diabetic myocardial ischemia/reperfusion (MI/R) injury both in vivo and in vitro.Aims1. To examine if hyperglycemia directly enhances myocardium vulnerability to I/R injury;2. To establish a causative link between protein nitration and increased sensitivity of myocardium to diabetic I/R injury;3. To determine the role of Trx nitration in MI/R injury under hyperglycemic condition; and to determine the signaling mechanism by which Trx nitration exacerbates diabetic MI/R injury. Methods1. Diabetes was induced by 5 days of daily intraperitoneal injection of low-dose streptozotocin (STZ) in mice. Mice were subjected to following experiments after successful establishment of diabetes (5 days after the final STZ injection), which was confirmed by markedly elevated whole-blood glucose levels;2. MI/R was produced by placing a silk suture slipknot at the left anterior descending coronary artery. After 30 min of ischemia, the slipknot was released, and the myocardium was reperfused. At 10 min before reperfusion, mice were randomized to receive either vehicle (0.9% NaCl), EUK134 (ONOO- scavenger), exogenous Trx by intraperitoneal injection;3. After 3 h reperfusion, myocardial apoptosis was determined by TUNEL staining and caspase-3 activity assay; p38 MAPK activity assay was performed by an assay kit per manufacturer's instructions; nitrotyrosine content, indicating protein nitration, in cardiac tissue was measured by immunochemistry staining and an ELISA assay kit; Trx activity assay was done by insulin disulfide reduction; Trx nitration and Trx-ASK1 interaction detection were performed by immunoprecipitation and immunoblotting;4. At the end of the 24 h reperfusion period, left ventricular ejection fraction (LVEF) was determined by echocardiography; Left ventricular end-diastolic pressure (LVEDP) and first derivative of the left ventricular pressure (±dP/dtmax) were obtained by invasive hemodynamic evaluation methods; After completion of functional determination, the ligature around the coronary artery was retied, and myocardial infarct size was determined by the Evans blue/TTC double staining method; 5. Cardiomyocytes were isolated by collagenase perfusion using Langendorff machine, and then cultured with normal glycaemic (control) or high glucose (HG) medium for 12 h; Expression of inducible nitric oxide synthase (iNOS) and gp91phox (a major component of NADPH oxidase) in cardiomyocytes were determined by Western Blot; Then cardiomyocytes were subjected to glucose-free medium in hypoxia incubator for 3 h, followed by immediate treatment with vehicle (PBS), 1400W (an iNOS inhibitor) or Apocynin (a NADPH oxidase inhibitor);6. After 3 h reoxygenation, termed as simulated ischemia/reperfusion (SI/R), cardiomyocyte apoptosis was determined by Caspase-3 activity assay; Trx activity assay was done by insulin disulfide reduction; Trx nitration were measured by immune precipitation and immunoblotting.Results1. Blood glucose was measured 5 days after the final injection, and diabetic condition was confirmed by markedly elevated whole-blood glucose level above normal (10mmol/L). The mean glucose value in STZ-treated mice was significant higher than that in control mice (20.8±1.5 mmol/L vs. 7.5±1.3 mmol/L);2. Compared with that in control mice, after 3 h reperfusion, the percentage of TUNEL-positive nuclei or caspase-3 activity was significantly higher in diabetic mice; MI/R-induced nitrotyrosine production, Trx nitrative inactivation, disassociation of Trx with ASK1, and consequent p38 MAPK activation were enhanced in the diabetic mice; after 24 h reperfusion, infarct size was enlarged and LVEDP was further increased in diabetic mice; MI/R-induced decrease in LVEF value and±dP/dtmax were exacerbated in diabetic mice; 3. Treatment with EUK134 significantly reduced nitrotyrosine content and Trx nitration, preserved Trx activity, restored Trx-ASK1 interaction, and inhibited p38 MAPK activity in cardiac tissues from diabetic mice subjected to MI/R; Treatment with EUK134 or exogenous Trx significantly attenuated myocardial apoptosis, evidenced by both decreased caspase-3 activity and percentage of TUNEL-positive nuclei, reduced infarct size, improved LVEF value and lower LVEDP in diabetic mice subjected to MI/R;4. Freshly isolated adult mouse cardiomyocytes were rod shape with clearly defined sarcomeric striations, and had no frequent spontaneous contractile waves. The viability was over 80%, evidenced by Trypan blue staining;5. Culturing cardiomyocytes with HG for 12 h significantly increased iNOS and gp91phox expression. Compared with cells in control medium, cardiomyocytes pre-cultured in HG had significantly higher apoptosis when subjected to SI/R, as evidenced by increased caspase-3 activity to a greater extent; SI/R-induced nitrotyrosine production and Trx nitrative inactivation were amplified in HG group;6. In HG pre-cultured cardiomyocytes subjected to SI/R, administration with 1400W or Apocynin significantly reduced nitrotyrosine content and Trx nitrative inactivation. Treatment with 1400W, Apocynin or exogenous Trx significantly inhibited apoptosis, demonstrated by decreased Caspase-3 activity.Conclusion1. Hyperglycemia directly sensitized cardiomyocytes to I/R injury; Apoptosis plays a critical role in cardiomyocyte loss and the subsequent exacerbated myocardial injury in diabetic mice after MI/R, showed by enlarged infarct size and magnified cardiac dysfunction; 2. MI/R-induced nitrotyrosine production was further elevated in diabetic mice, accounting for myocardial apoptosis and the development of cardiac injury. HG-induced iNOS and gp91phox expression is causatively related to intensified protein nitration and cardiomyocyte apoptosis after SI/R;3. Diabetes potentiated MI/R-induced Trx nitrative inactivation and disassociation of ASK1 from Trx, thereby faciliating myocardium to I/R injury by exaggerated downstream p38 MAPK signaling pathway- mediated cardiomyocyte apoptosis.
|
PDF Full Text Request