Changes Of RNS In The Myocardium Of Aged Rats And Its Potential Significance

Background and Objective:Aging is the sum of all the changes that take place in the body with the passage of time ultimately leading to functional impairment and death.Aging might also be defined as an increased vulnerability to internal and extemal stress-a gradual inability to respond to physiological challenges.The cardiac function is also a victim of the aging process.However, the mechanism responsible for this age-related pathologic change remains largely elusive.A recent study from Michael Pollack showed that there was a significant loss of cardiomyocytes during aging,both in animals and human beings.Because of the limited regeneration of cardiomyocytes the abnormal amount of cell loss might lead to cardiac dysfunction in old hearts. However,this hypothesis needs further confirmation.The two most common forms of cell death are necrosis and apoptosis.Kajstura proved that apoptosis is more common than necrosis in old rats.We and others have shown that the blockage of apoptosis can improve heart functionality in aged rats.Nitric Oxide(NO)and its reactive derivatives,referred to as Reactive Nitrogen Species(RNS),have long been known to mediate cell death.They can either stimulate or inhibit apoptosis.Data from in Vitro experiments suggested that high concentrations of ONOO might induce cell apoptosis.However,whether ONOO has any affect on apoptosis or not in aged hearts remains to be determined.It has recently been recognized that the anti-apoptotic effects of NO are in large part mediated by a new posttranslational modification called protein S-nitrosylation.Accumulating studies demonstrate that protein S-nitrosylation refers to the binding of an NO group(NO⁺)to a thiol group of protein cysteine resulting in the formation of S-nitrothiols(SNOs).Jorg Hoffmann exhibited that aged cultured endothelial cells possessed enhanced apoptosis and decreased overall S-nitrosylati0n content.The aim of the present investigation is designed to test the hypothesis that increased ONOO^- and reduced protein S-nitrosyaltion ability contribute to the enhanCed cell anoptosis in aged hearts. Methods:(1)Male Sprague-Dawley adult rats,4-6months(the young group)and aged rats,22-24months (the old group)were anesthetized with 10%chloral hydrate at 0.3ml/kg.A polyethylene catheter connected with a pressure transducer was inserted into the left ventricular cavity through the right carotid artery,and heart rate(HR),left ventricular pressure(LVP),dP/dt_max, and cardiac index(CI)and other indices were measured.(2)Myocardial apoptosis was analyzed by TUNEL(Terminal deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling)staining(n = 3 hearts per group)and caspase-3 activity assay(n = 6 hearts per group).(3)The distribution and content of S-nitrosylated proteins and eNOS were detected by immunohistochemistry(IHC)(4)The level of S-nitrosylated proteins was also measured by using Nitro-Glo kit(PerkinElmer).(5)The contents of eNOS and iNOS were determined by western-blot(6)NT marks the production of ONOO^-.The content of nitrotyrosine(NT)in the myocardium was detected by sandwich Enzyme-linked Immunosorbent Assay(ELISA)(n = 6 hearts per group).Results:1.The cardiac function is aggravated in the aged rat1.1 Cardiacsystolic indexCI(Cardiac Index)is one of the indices which reflect the systolic cardiac function.The CI in the old group(39.23±1.62 s^-1)decreased notably as compared with that in the young group (CI:46.82±3.176 s^-1)(P＜0.05);1.2 Cardiac diastolic indicesThe diastolic cardiac function indices in the old group(LVDP:0.16±0.17kPa,LVEDP: 1.25±0.35kPa)were increased significantly as comparing with those in the young group(LVDP: -1.16±0.35kPa,LVEDP:-0.70±0.43 kPa)(P＜0.01).Data from the cardiac function indicated that both systolic and diastolic cardiac functions were aggravated in aged hearts.2.The relative cardiac mass decreased in the old ratThe body and heart weights of the young group and the old group were weighed and the ratios of heart weight to body weight(heart/body weight ratio)were calculated.Compared with young group,the heart weight was markedly increased in aged rat(1.42±0.04g vs.0.61±0.04g, P＜0.01).However,the heart/body ratio in old group(0.26%±0.01%)decreased significantly as comparing with that in the young group(0.31%±0.01%,P＜0.01).3.Increased cell apoptosis in myocardium of old rats3.1 TUNEL stainingMyocardial apoptosis of the young and old groups was determined by highly sensitive TUNEL staining.Myocardial apoptosis was significantly higher in aged heart than that in young one as evidenced by increased percent of TUNEL-positive myocytes,the apoptotic index (2.86%±0.43%vs.0.86%±0.29%,P＜0.05).3.2 Caspase-3 activity assayCaspase-3 is the pivotal protein in the final pathway of cell apoptosis.Myocardial cell apoptosis of young and old groups was quantitatively analyzed by caspase-3 activity assay.The apoptotic levels of both groups were expressed in a caspase-3 activity ratio.Compared with younggroup,caspase-3 activity ratio was enhanced significantly in old group(2.21±0.327 vs. 1.00±0.028,P＜0.01).3.3 Correlative analysis between myocardial apoptosis and cardiac function in aged rats3.3.1 Negative correlation between caspase-3 activity and CIThe myocardial caspase-3 activity was negatively correlated with CI in the rats(r= -0.772,P＜0.01),indicating that compared to the young heart the aggravated systolic function may relate to the enhanced apoptosis in old heart.3.3.2 Positive correlation between caspase-3 activity and LVDPThe myocardial caspase-3 activity was positively correlated with Left Ventricular Diastolic Pressure(LVDP)in the rats(r=0.718,P＜0.01),that is the LVDP was increased in accordance with the enhanced caspase-3 activity.It is suggested that the decreased diastolic function may related to the enhanced apoptosis in aged heart compared to the young heart.4.The deregulation of pro- and anti-apoptotic effects of RNS in the myocardium of aged rats4.1 Increased content of NT in aged myocardiumNT is the marker of the production of ONOO^-.The content of NT was determined by sandwich ELISA.The result shows that myocardial NT content in the old group(1.545±0.194) increased obviously as compared with that in the young group(0.858±0.143).(P＜0.05)4.2 Negative correlation between the content of myocardial NT and caspase-3 activityThere was a positive correlation between myocardial NT content and caspase-3 activity (r=0.770,P＜0.01),indicating that the increased apoptotic level might relate to the enhanced content of NT in old heart.4.3 Decreased S-nitrosylated proteins in the myocardium of aged ratsThe distribution and content of S-nitrosylated proteins were determined by immunohistochemistry(IHC)and NitroGlo assay kit(Perkinelmer).The content of S-nitrosylated proteins in the old group(36472±1239)was diminished as compared with that in the young group(41208±492).(P＜0.01)4.4 Negative correlation between myocardial S-nitrosylation proteins and caspase-3 activityThe expression of S-nitrosylated proteins was negatively correlated with caspase-3 activity in the.myocardium of rats(r=-0.767,P＜0.01),that is,the caspase-3 activity was increased along with the decrease in the content of S-nitrosylated proteins,indicating that the increased apoptosis might relate to the declined S-nitrosylated protein in aged heart compared to the young one.5.Enhanced iNOS and reduced eNOS in the myocardium of aged rats5.1 The expression iNOS was enhanced in the myocardium of the aged ratsThe content of iNOS was measured by western-blot.The content of iNOS in the old group(235485±46070)was enhanced significantly as compared with that in the young group (125610±11765)(P＜0.01). 5.2 Positive correlation between myocardial iNOS expression and NT contentThere was a positive correlation between the expression of iNOS and myocardial NT content in the rats(r=0.600,P＜0.05)which meant the NT content was enhanced with the increase in iNOS expression.The evidence.suggested that the enhanced NT content might be related to the elevated iNOS expression.5.3 The expression eNOS was diminished in the myocardium of aged ratsThe distribution and content of eNOS were determined by IHC and Western-blot.The content of eNOS in the old group(268.4±57.5)was reduced significantly as compared with that in the young group(564.1±52.9)(P＜0.05).5.4 Positive correlation between eNOS expression and the content of S-nitrosylated proteinsThe eNOS expression and the content of S-nitrosylated proteins were positively correlated in the myocardium of the rats(r=0.622,P＜0.05).It was suggested that the decreased content of S-nitrosylated proteins possibly related to the declined eNOS expression in aged heart.Conclusion.(1)The age-related pathologic changes occurred in the aged heart as evidenced by increased apoptotic index,decreased cardiac relative mass and cardiac dysfunction.(2)The dysregulation of RNS occurred in the aged heart evidenced by enhanced S-Nitrosylated proteins and decreased ONOO^- which possibly contributed to the enhanced apoptosis in the aged heart resulting in the cardiac dysfunction.(3)The disorder of NOS evidenced by increased iNOS and declined eNOS in the aged heart possibly resulted in the dysregulation of RNS.(4)Evidence from our study showed that the types of enzymes producing RNS,the toxic production of RNS and its chemical modification played an important role in the aging of myocardium.This evidence suggests that in clinical medicine the administration of intervention,including reducing iNOS,stimulating eNOS activity,removing ONOO^- or supplementing S-nitrosylated proteins,will possibly alleviate aged-associated myocardial injuries.