| Background:The current report from World Health Organization has indicated that myocardial infarction after ischemia/reperfusion is one of the most important cause of death in the world. Persistent myocardial ischemia in myocardial infarction without reperfusion inevitably results in myocardial cell death from endocardium to epicardium, thus, myocardial reperfusion is necessary to salvage tissue from ultimate death. However, paradoxically reperfusion after even brief periods of ischemia also triggers new pathophysiological changes that induce myocardial cell death[1,2]. With loss of cardiomyocytes, increasing replacement and reactive fibrosis resulting in scar formation lead to opposing effect on cardiac function after MI/R. Recently, growing evidence shows that blocking the signal transduction leading to cell death significantly reduces myocardial infarct size and ameliorates cardiac function。Ischemia/reperfusion (I/R) injury induces necrotic and apoptotic cell death in the myocardium. The contribution of necrosis to infarct size is large, but apoptosis as a programmed cell death is generally considered to be more controllable than necrosis. Recently, numerous studies have substantiated that apoptosis is not exclusive programmed cell death. For example, autophagic cell death as a necrosis-like cell death has been confirmed that is a novel programmed cell death (termed programmed cell death II).Programmed cell death II is characterized with cytosolic massive autophagosome,which can be suppressed by downregulating the formation of autophagosome. Autophagy was considered as cell self-salvaged mechanism in response to evil condition, which has a protective role within a certain threshold; whereas, exceeding the threshold will result in cell death. However, recent some studies found that autophagic cell death induced MI/R could be not inhibited by blocking autophagy pathway. Therefore, we considered that upstream mechanism of autophagy might be a critical event mediating cell death during MI/R, but further investigation was limited by lack of suitable tool.In 2005, Degterev et al. discovered a small tryptophan-based molecule called necrostatin-1 (Nec-1), can specifically inhibit a nonapoptotic programmed cell death characterized with autophagy, and protect the cerebral cortex against ischemia-reperfusion (I/R) injury. Although the cell death morphologically resembles autophagic cell death, inhibition of autophagy had no effect on final demise of cell. Therefore, the author considered that Nec-1 inhibited a necroptotic signaling step upstream of autophagy but did not inhibit autophagy; researching mechanism of Nec-1 could reveal signaling pathway of the cell death. However, we firstly need determine effect of Nec-1 in MI/R.Objective:1. To determine the effects of Nec-1 at rats subjected to myocardial ischemia and reperfusion.2. Through analyzing the correlation between myocardial damage and autophagy to search the time window about Necroptosis.Methods:Male Sprague-Dawley rats (280±10g) underwent a protocol of 30min LAD ischemia followed by 12h, 24h, 48h and 72h of reperfusion, respectively. The rats were randomized to receive vehicle (0.05% DMSO) or necrostatin-1 (0.6mg/kg, Sigma-Aldrich, Inc. USA) via caudal vein at the onset of reperfusion. Cardiac function were measured at different time points after MI/R. The myocardial infarct size and ventricular remodeling were determined by Masson trichromatic stains at 72h after reperfusion. Then, serum creatine kinase (CK) and myocardial MAP-LC3Ⅱwere measured at different time points after MI/R, and observed myocardial ultrastructure under transmission electron microscope (TEM) at 48h after reperfusion. Finally, investigate correlation between the expression of MAP-LC3Ⅱand serum CK levels.Results:1. Effect of Nec-1 on serum CK level at different time points after MI/RThere was no significant difference among all rats of CK level before ischemia (232±34 U/L); after MI/R, serum CK levels were significantly elevated at different time points, which arrived to a peak at 24h after reperfusion and then gradually descend. Nec-1(0.6mg/kg) administered at the onset of reperfusion could significantly reduce serum CK levels at 12h and 24h after reperfusion, compared with relative vehicle group (722±111 and 929±271 U/L versus 1433±124 and 1438±174 U/L, P < 0.05 and P < 0.01, respectively). However, serum CK levels of Nec-1 group at 48h after reperfusion were markedly increased, compared with relative vehicle group (559±175 versus 1238±158, P < 0.05) (Fig. 2).2. Effect of Nec-1 on myocardial infarct size after MI/RMyocardial ischemia 30min/ reperfusion 72h resulted in loss of massive cardiomyocytes, myocardial reactive fibrosis from endocardium to epicardium; Nec-1(0.6mg/kg) administered at the onset of reperfusion inhibited myocardial fibrosis and reduced significantly infarct size from 15.1±1.7% (% of LV area) to 5.3±2% (% of LV area) (Fig. 3).3. Effect of Nec-1 on myocardial ultrastructure at 48h after reperfusionMyocardial ultrastructure at 48h after reperfusion was severely damaged, in which cardiomyocytes were disintegrated and formed massive vacuolus but almost no autophagosome. Inversely, treatment with Nec-1 markedly maintain myocardial ultrastructural integrity, however, there was a few autophagosome diffused distribution in some cardiomyocytes (Fig. 4).4. Effect of Nec-1 on myocardial infarct size after MIAfter 2 weeks of ischemia, myocardial reactive fibrosis in infarct zone ran through whole left ventricular wall, and myocardial infarct size were significantly greater than those in sham. However, the administration of Nec-1 (0.6mg/kg) at the onset of myocardial ischemia can obviously inhibit myocardial remodeling, and reduced myocardial infarct size in compared with control (Fig. 5).5. Effect of Nec-1 on Cardiac function at different time points after MI/RThere was no significant difference among all rats of cardiac function before ischemia; after 30min of ischemia, both left ventricular systolic and diastolic functions were markedly degraded (Fig. 6). However, Nec-1(0.6mg/kg) administered at the onset of reperfusion could markedly improve cardiac function, especially left ventricular diastolic function (LVDP and LVEDP, P<0.05) (Fig. 6A. 6B) at 12h and 48h after reperfusion, compared with vehicle. Moreover, treatment with Nec-1 could significantly elevate +dp/dtmax (P<0.05) and -dp/dtmax (P<0.01) at 12h after reperfusion, compared with vehicle (Fig. 6C. 6D).6. Effect of Nec-1 on expression of LC3Ⅱat different time points after MI/RThere was no significant difference among all rats of expression of LC3Ⅱbefore ischemia; after MI/R, expression of LC3Ⅱwere significantly elevated at different time points, which arrived to a peak at 24h after reperfusion and then gradually descend. Nec-1(0.6mg/kg) administered at the onset of reperfusion could significantly reduce content of LC3Ⅱat 12h and 24h after reperfusion, compared with relative vehicle group (Fig. 7A.7B). However, content of LC3Ⅱof Nec-1 group at 48h after reperfusion were markedly increased, compared with relative vehicle group. Then, expression of LC3Ⅱhad no significant difference between Nec-1 and vehicle group at 72h after reperfusion.7. Correlation analysis between LC3Ⅱand CKThe result of correlation analysis showed significantly positive correlation between expression of LC3Ⅱand serum CK level (r=0.89, p<0.001) (Fig. 7B). Besides, the results of correlation analysis showed significantly positive correlation about respectively vehicle (r=0.80, p<0.002) (Fig. 7C) and Nec-1 group (r=0.91, p<0.001) (Fig. 7A).Conclusion1. The administration of Nec-1(0.6mg/kg) at the onset of myocardial reperfusion can extenuate the reperfusion injury by reducing the area of myocardial infarction and the level of interstitial fibrosis. Moreover, Nec-1 can relieve the cardiac dysfunction induced by MI/R injury, especially the diastolic function of left ventricle.2. The administration of Nec-1 (0.6mg/kg) at the onset of myocardial ischemia can obviously inhibit myocardial remodeling, and reduced myocardial infarct size in chronically myocardial ischemia. It is suggested that Necroptosis takes part in the chronically myocardial ischemia injury and myocardial remodeling.3. In the process of MI/R, autophagy increased. Autophage displayed a time-dependent change. After the reperfusion, autophage gradually increased, achieved the peak value at 24 hours, and then tapered. This change of autophage displayed positive correlation with the cardiac injury induced by MI/R. It is suggested that Necroptosis takes part in the MI/R injury. |
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