The Eeffect Of Aging On RNS In Myocardial Ischemia Reperfusion Injury Of Rat

Introduction:The progressive myocardial cell loss during aging is a critical cause of aggravated cardiac function and increased susceptibility to ischemia/reperfusion(I/R)injury in aged heart.However, the mechanisms involved in which remain largely unknown.Recent studies have illustrated that Reactive Nitrogen Species(RNS)and reactive derivatives of Nitric Oxide(NO)are important mediators in cell injury and have both pro- and anti- apoptotic properties.It is generally considered/ accepted that low concentrative /active RNS(NO catalyzed by eNOS)are cell protective,high concentrative/ active RNS(NO catalyzed by iNOS and ONOO~-,the production of NO with free radicals)are cell toxic and abnormal cells are more susceptible to the toxicity of ONOO- compared to normal cells.Growing evidence from both basic research and clinical observations indicated that aged heart was more susceptible to ischemic and other heart diseases. However,the mechanisms responsible for the enhanced susceptibility are still being investigated. The content of NO was reported to be enhanced in aged heart compared to young one. Nevertheless,the pathophysiological significance of the increased NO and whether it contributes to the increase of RNS and enhanced susceptibility to ischemia/reperfusion injury in aged heart have not been reported.Objective:1.To determine the changes in the contents of NO and its active derivatives(RNS)in the myocytes of aged heart.2.To investigate the mechanism of the change in the RNS content in aged cardiomyocytes and its relation with enhanced susceptibility to ischemia/reperfusion injury in aged heart.Methods:Male Sprague-Dawley adult(4-6 months,n=72)and old(22-24months,n=90)rats were randomly divided into 9 groups:(1)In vitro young adult group(n=6):in vitro perfusion without intervention; (2)In vitro old adult group(n=6):in vitro perfusion without intervention;(3)In vitro young adult + Adnosine group(n=6):the administration of 5μmol/L adnosine solution in vitro perfusion;(4)In vitro old adult + Adnosine group(n=6):the administration of 5μmol/L adnosine solution in vitro perfusion;(5)Young Normal Group(n=36):Non-treatment or measuring the cardiac function by inserting into left ventricular cavity through the right carotid artery and the suture was not tied;(6)Old Normal Group(n=36):Non-treatment or measuring the cardiac function by inserting into left ventricular cavity through the right carotid artery and the suture was not tied;(7)Young Ischemia/ Reperfusion Group(Young I/R Group,n=24):Placing a 6-0 silk suture slipknot around the left anterior descending coronary artery.After 30 min of ischemia,the slipknot was released and the myocardium was reperfused for 3 h;(8)Old Ischemia/ Reperfusion Group(Old I/R Group,n=24):Placing a 6-0 silk stature slipknot around the left anterior descending coronary artery.After 30 minutes of ischemia,the slipknot was released and the myocardium was reperfused for 3 h;(9)Old Ischemia/ Reperfusion Group+1400W(Old I/R + 1400 W Group,n=18): Ischemia 30 min,reperfusion 3h.Administrate 1400W(2mg/kg)via intraperitoneal injection 24 h before ischemia and 25 minutes before reperfusion.Cardiac function was continuously measured in vitro and at the end of the experiment the myocardial infarct sized was stained with Even's blue and TTC and then the area at risk were determined.Myocardial cell apoptosis was measured by greatly sensitive TUNEL staining and relatively specific caspase-3 activity assay.Myocardial NOx was detected by Nitrate/Nitrite Colorimetric Assay Kit(Lactate Dehydrogenase Method).Nitrotyrosine content,a footprint of in vivo ONOO- formation,was determined using an ELISA method.The Myocardial content of iNOS was measured by Westem-blot.Results:1.The changes of RNS in aged hearts1.1 Increased basal,but diminished agonist-stimulated NO production in aged hearts.Our results demonstrated that young adult hearts had a low basal NOx,and infusion of adenosine markedly increased NO_x production(Figure 1,P＜0.01).In contrast,aged hearts had a markedly increased basal NOx compared to young adult hearts(Figure 1,P＜0.01). However,infusion of adenosine only slightly increased NOx production. 1.2 Nitric oxide bioavailability as measured by NO/cGMP signaling integrity markedly reduced in aged hearts.VASP is the downstream target of both cAMP- and cGMP-dependent protein kinases, and reduced VASP phosphorylation(pVASP)is a sensitive monitor of defective nitric oxide/cGMP signaling and endothelial dysfunction.As illustrated in figure 2,our data demonstrated that although total VASP levels are comparable in both groups,pVASP levels are markedly reduced in heart tissues isolated from aged rats.1.3 Increased nitrotyrosine is observed in aged myocardial tissueDetection of tissue nitrotyrosine content has been extensively used by many investigators as a marker for ONOO- production as illustrated in figure 3,no nitrotyrosine was detected in myocardial tissue from a young adult heart,but clear staining was detected in the heart from an aged rat.2.Ageing increased the susceptibility of myocardium to ischemia-reperfusion injury.2.1 The changes of apoptotic level and cardiac function indexes in both young and old normal groups2.1.1 Detection of cell apoptosisTUNEL detection:The apoptotic index in old I/R group(19.0±2.1%)was significantly higher than that in young I/R group(14.6±1.7%)(P＜0.01)(Table 1,Figure 7 and8).Caspase3 activity assay:Caspase3 activity in old I/R group(436±35μmol pNA/mg) was also significantly decreased compared with young I/R group(340±32μmol pNA/mg) (P＜0.01)(Table 1,Figure 9).2.1.2 Measurement of Cardiac functionCompared to young normal group the cardiac function indicesr in old normal group changed as follows:LVDP increased(P＜0.01)(Figure 10),LVEDP enhanced(P＜0.01) (Figure 11),CI decreased(P＜0.01)(Figure 12),dp/dtmax declined significantly(P＜0.01) (Figure 13).2.2 The changes in myocardial apoptosis,infarct size and cardiac function indexes in young and old I/R groups2.2.1 Detection of cell apoptosisTUNEL detection:Apoptotic index was obviously increased to(19.0±2.1%)in old I/R group as compared with young I/R group(14.6±1.7%)(P＜0.05)(Table 1,Figure 7 and 8).Because of the characteristics of high specificity and low specificity of TUNEL staining the myocardial apoptotic death was also measured by caspse-3 activity assay.Caspase3 activity assay:Caspase3 activity of old I/R group(436±35μmol pNA/mg) was greatly higher than that of the young I/R(340±32μmol pNA/mg)(P＜0.05)(Table 1, Figure 9).2.2.2 Measurement of myocardial infarct sizeThe ratio of area at risk to left ventricular area(AAR/LV):No significant differences were observed among these groups(Table 2,Figure 14 and 15a),which indicates that the ischemic area induced by coronary ligation is roughly identical among each experimental group so that these groups are comparable.The myocardial infarct size of old group was significantly enhanced as compared with young I/R group(P＜0.05)(Table 2,Figure 14 and 15b).2.2.3 Measurement of Cardiac functionCompared to the young I/R group the LVDP in old I/R group increased obviously (P＜0.05)(Figure 16),LVEDP increased(P＜0.01)(Figure 17),-dp/dtmax decreased at 3h of reperfusion(Figure 18),CI decreased(P＜0.05)(Figure 19)and dp/dtmax declined at 3h of reperfusion(P＜0.01)(Figure 20).3.The iNOS expression in old group increased and the inhibition of iNOS decreased the susceptibility to ischemia/ reperfusion injury of aged rats3.1.Enhancement of nitrotyrosine in aged cardiomyocytesCompared to young normal group the myocardial nitrotyrosine content in old normal group.increased significantly(P＜0.05);Compared to young I/R group the myocardial nitrotyrosine content in old I/R group increased significantly(P＜0.05)(Figure 21).3.2.The changes of iNOS in all groupsCompared to young I/R group iNOS increased significantly(P＜0.05);iNOS in old normal group enhanced compare to young normal group(P＜0.05)(Figure 22).3.3 The changes of myocardial nitrotyrosine content,apoptosis and infarct size in old MI/R+1400W3.3.1 Quantitation of myocardial nitrotyrosineAfter administration of 1400W the content of nitrotyrosine in old I/R group decreased significantly(P＜0.05)(Table 3,Figure 21).3.3.2 Determination of cell apoptosis Both the percent of TUNEL-positive cells and caspse-3 activity in old MI/R+1400W group decreased compared to old I/R group(P＜0.05)(Table 1,Figure 7-9).3.3.3 Determination of myocardial infarct sizeThe ratio of area at risk to left ventricular area(AAR/LV):No significant differences were observed among these groups(Table 2,Figure 14 and 15a),which indicates that the ischemic area induced by coronary ligation is roughly identical among each experimental group and that these groups are comparable.The myocardial infarct size in old MI/R+1400W group decreased significantly compared to the old I/R group(P＜0.05)(Table 2,Figure 14 and 15b).Conclusion:(1).Aging increased the susceptibility of myocardium to ischemia/reperfusion injury.(2).Aging increased the myocardial contents of NO and ONOO~-,especially the toxic ONOO~-.(3).Aging enhanced iNOS and led to the increased contents of NO and ONOO~- in aged heart which possibly contributed to the greater susceptibility of aged cardiomyocytes to ischemia/reperfusion injury.