Changes Of HtrA2/Omi In Myocardium Of Aged Rats And The Potential Significance

Background:Aging is the sum of all the changes that take place in the body with the passage of time which ultimately lead to functional impairment and death.Aging might be defined as an increased vulnerability to internal and external stress-a gradual inability to respond to physiological challenges.Data from numerous animal researches and clinical studies support that aging is a major independent risk factor in the markedly enhanced risk for cardiovascular diseases especially coronary heart disease.Compared with young adult,the aging heart is more susceptible to ischemia/reperfusion,has a larger infarct size as well as worse prognosis and thrombolytic therapy.However,the mechanism responsible for this age-related pathologic change remains largely elusive.A recent study showed that there was a significant loss of cardiomyocytes during aging,both in animals and human beings.For example,from 17 to 90, the cardiomyocytes lost almost one third because of aging alone;and a healthy,70-year-old male individual has lost nearly 30%of the myocytes population of the left and right ventricles as a result of aging alone.The cardiomyocytes play a key role in cardiac function as a basic unit.That implicated that the cardiomyocytes loss along with aging was one of the most important factors lead to cardiac dysfunction and higher disease susceptibility.Nowadays,the tendency of the old population is boosting,accompanied with high morbidity and mortality of cardiovascular diseases.Therefore,to reveal the mechanisms for age-associated cardiac structural and functional remodeling and develop novel therapies to prevent or delay cardiovascular changes associated with aging and to reduce the risk for age-associated cardiovascular diseases.The main cause of organ cell loss is cell death.Two most common forms of cell death are necrosis and apoptosis.Many studies suggest that both apoptosis and necrosis are involved in the heart of aging mice.But as time flies,the apoptotic cell death increase,but not necrosis.So we get a conclusion that the apoptosis play the major role in aging cardiomyocytes loss.Apoptosis is an active programmed cell death characterized by a series of stereotypic morphological and biochemical features.Therefore,it's easier to inhibit apoptosis than to prohibit necrosis.Under the physiological conditions,apoptosis is manipulated by the balance between pro-apoptotic and anti-apoptotic proteins,and these proteins also are manipulated by the balance of some upstream regulator proteins.As a result of complicated signal transduction of apoptosis,many particular cell types(such as cardiac cell) couldn't maintain the balance exactly over some special cell stress(such as aging and ischemia/reperfusion).Therefore,to determine the crucial pairs of pro-/anti- apoptosis signals is the most significant steps to solve aging problem,cardiac dysfunction,and ischemic heart disease.XIAP(X chromosome-linked Inhibitor of Apoptosis Protein,XIAP) is the best-characterized and most potent Caspase inhibitor.It plays a crucial role by binding with Caspase-3 and Caspase-9.However,when apoptosis stimuli,Smac/DIABLO which is called the second mitochondria-derived activator of Caspases(Smac) or direct inhibitor of apoptosis-binding protein with low PI(DIABLO) and HtrA2/Omi are released from the mitochondria.Binding Smac/DIABLO or HtrA2/Omi to XIAP disrupts the binding of XIAP to Caspases and relieves the inhibition of apoptosis.Compared to Smac/DIABLO,AIF or cytochrome c,HtrA2/Omi is a nuclear encoded mitochondrial serine protease that not only interacts with XIAP by binding activity and promotes Caspase-dependent apoptosis,but also cleavage XIAP by its serine protease activity and promotes Caspase-independent apoptosis.Recent study has demonstrated that overexpression of HtrA2/Omi markedly increases apoptosis.Treatment with small interference RNA(siRNA) of HtrA2/Omi has shown to be more resistant to apoptosis induced by X-rays.A growing number of studies from animal and clinic experiment have been reported that maybe aging is associated with more cardiomyocytes loss,which results from cell apoptosis,and then leads to cardiac dysfunction.But the mechanism of aging heart soaring loss remains to be ecucidated.Moreover,the causes and mechanisms responsible for susceptibility to ischemia/reperfusion of old animals are not clear.XIAP,which is regard as an endogenenous mitochondria anti-apoptosis protein,whether is assosicated with these mechanisms or not.If so, to investigate which other endogenenous proteins could affect XIAP.Is it Smac/DIABLO,or HtrA2/Omi,or both of them? Whether the influence can reveal the more cardiomyocytes loss and cardiac dysfunction of aging and whether the influence could enhance susceptibility to ischemia/reperfusion injury have not been previously demonstrated.Therefore,we do the research as follows. SECTION ONE Upregulation of HtrA2/Omi Associated with Enhanced Myocardial Apoptosis of Aging HeartObjective:1.To demonstrate whether cardiac function and apoptosis index are changed in aging heart;2.To investigate whether anti-apoptotic XIAP and pro-apoptotic HtrA2/Omi, Smac/DIABLO expression have the effect on the aging heart.Methods:(1) Male Sprague-Dawley adult rats(4-6months,n=39) and aging rats(22-24months,n=63) were randomly divided into four groups:①Normal Young Group(n=36):A polyethylene catheter connected with a pressure transducer was inserted into the left ventricular cavity through the right carotid artery, and cardial fuction were measured;②Normal Aging Group(n=36):A polyethylene catheter connected with a pressure transducer was inserted into the left ventricular cavity through the fight carotid artery, and cardial fuction were measured;③Noraml Aging Group+ucf-101(n=6):Administrate ucf-101(1.5μmol/kg) via intraperitoneal injection,and then a polyethylene catheter connected with a pressure transducer was inserted into the left ventricular cavity through the right carotid artery, and cardial fuction were measured;④Noraml Aging Group+DMSO(n=6):Administrate DMSO(1.5μmol/kg) via intraperitoneal injection and then a polyethylene catheter connected with a pressure transducer was inserted into the left ventricular cavity through the right carotid artery, and cardial fuction were measured. (2) After inserting polyethylene catheter connected with a pressure transducer into the left ventricular cavity through the right carotid artery,we detected heart rate(HR),left ventricular pressure(LVDP),dP/dt_max,and cardiac index(CI) and other indices;(3) Myocardial apoptosis was analyzed by TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining and Caspase-3 activity assay;(4) The protein expression level of XIAP,HtrA2/Omi and Smac/DIABLO were determined by Western-blot;(5) The gene expression level of XIAP,HtrA2/Omi and Smac/DIABLO were determined by Real-Time PCR.Results:1.The cardiac function is aggravated in aged rat1.1 Cardiac systolic indexCI(Cardiac Index) is one of the indices which reflect the systolic cardiac function.CI in old group(39.23±1.62s^-1) decreased notably as compared with that in young group(CI: 46.82±3.176s^-1)(P＜0.05)(Figure1,2);1.2 Cardiac diastolic indicesThe diastolic cardiac function indices in old group(LVDP:0.16±0.17kPa,LVEDP: 1.25±0.35kPa) were increased significantly as compared to those in the young group (LVDP:-1.16±0.35kPa,LVEDP:-0.70±0.43kPa)(P＜0.01)(Figure3,4).Data from the cardiac function indicated that both systolic and diastolic cardiac functions were aggravated in aged hearts.2.Increased cell apoptosis in myocardium of old rats2.1 TUNEL stainingMyocardial apoptosis of young and old groups was determined by highly sensitive TUNEL staining.Compared to the percent of TUNEL-positive myocytes of young group (0.86±0.29%),the apoptotic index in the old group was increased significantly(2.86±0.43%, P＜0.05)(Figure5,6). 2.2 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 to the Caspase-3 activity ratio of young group(1.00±0.028) the old group was enhanced significantly (2.21±0.327,P＜0.01)(Figure 7).3.Expression of HtrA2/Omi,XIAP and Smac/DIABLO in myocardium of aged rats3.1 The expression XIAP was diminished in the myocardium of aged ratThe protein content of XIAP was measured by western-blot.The content of XIAP in the old group was decreased significantly as compared with that in the young group(P＜0.05) (Figure 8);The mRNA content of XIAP was measured by Real-Time PCR.The content of XIAP in the old group was decreased significantly as compared with that in the young group(P＜0.01) (Figure9,10).3.2 The expression Smac/DIABLO was unchanged in the myocardium of aged ratsThe protein content of Smac/DIABLO was measured by western-blot.The content of Smac/DIABLO in the old group was not significantly different as compared with that in the young group(P＞0.05)(Figure11);The protein content of Smac/DIABLO was measured by Real Time PCR.The content of Smac/DIABLO in the old group was not significantly different as compared with that in the young group(P＞0.05)(Figure12,13).3.3 The expression HtrA2/Omi was enhanced in myocardium of aged rats.The protein content of HtrA2/Omi was measured by western-blot.The content of HtrA2/Omi in the old group was enhanced significantly as compared with that in the young group(P＜0.05)(Figure14);The mRNA content of HtrA2/Omi was measured by Real Time PCR.The content of HtrA2/Omi in the old group was enhanced significantly as compared with that in the young group(P＜0.05)(Figure15,16).4.Inhibition of HtrA2/Omi has a significant cardioprotective effect in reducing cardiac cell apoptosisCaspase-3 is the pivotal protein in the final pathway of cell apoptosis.Myocardial cell apoptosis of ucf-101 and Vehicle groups was quantitatively analyzed by Caspase-3 activity assay. The apoptotic levels of both groups were expressed in a Caspase-3 activity ratio.Compared to the Caspase-3 activity ratio of Vehicle group the ucf-101 group was enhanced significantly (P＜0.05)(Figure17).Conclusion(1) The apoptotic index increased,the cardiac relative mass decreased and the cardiac function was aggravated in the myocardium of aged rats;(2) Enhanced HtrA2/Omi and reduced XIAP in myocardium of aged rats,suggested that HtrA2/Omi increasing caused XIAP degradation Caspases activation,and subsequent apoptosis in aging heart;(3) Therapeutic interventions that inhibit HtrA2/Omi expression may provide an effective method in the delay of aging process in which apoptotic cell death plays an important role. SECTION TWO Upregulation of HtrA2/Omi in Myocardial Ischemia/Reperfusion Injure of Aged RatObjective1.To demonstrate whether cardiac function and apoptosis index are changed in aging heart after ischemia/reperfusion injury.2.To determine the changed content of pro-apoptotic HtrA2/Omi released from mitochondria after ischemia/reperfusion,and to investigate the mechanism of the changes in myocardial ischemia/reperfusion injury of old rats.Methods(1) Male Sprague-Dawley adult rats(4-6months,n=38) and aging rats(22-24months,n=54) were randomly divided into 6 groups:①Young MI/R Group(n=24):Placing a 6-0 silk suture slipknot around the left anterior descending coronary artery.After 30 rain of ischemia,the slipknot was released and the myocardium was reperfused for 3h;②Aging MI/R Group(n=24):Placing a 6-0 silk suture slipknot around the left anterior descending coronary artery.After 30 rain of ischemia,the slipknot was released and the myocardium was reperfused for 3h;③Young sham Group(n=6):Placing a 6-0 silk suture slipknot around the left anterior descending coronary artery,but not tied.The other step is same to Young MI/R group;④Aging sham Group(n=6):Placing a 6-0 silk suture slipknot around the left anterior descending coronary artery,but not tied.The other step is same to Aging MI/R group;⑤Aging MI/R Group+ucf-101(n=6):Administrate ucf-101 via intraperitoneal injection 10min before reperfusion;⑥Aging MI/R Group+DMSO(n=6):Administrate DMSO via intraperitoneal injection 10min before reperfusion; (2) 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;(3) Myocardial apoptosis was analyzed by TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining and Caspase-3 activity assay;(4) At the end of the measurement of cardiac function in vitro,the myocardial infarct size was stained with Even's Blue and TTC and then the area at risk was determined.(5) The protein expression level of XIAP,HtrA2/Omi,Smac/DIABLO from mitochondria and cytosolic extracts in different groups were determined by Western-blot.Results1.Aging increased the susceptibility of myoeardium to ischemia/reperfusion injury1.1 Measurement of Cardiac function1.1.1 Cardiac systolic indexCI(Cardiac Index) is one of the indices which reflect the systolic cardiac function.CI in aging MI/R group(CI:41.68±1.62s^-1) decreased notably as compared with that in young MI/R group(CI:50.034±3.176s^-1,P＜0.05)(Figure21);1.1.2 Cardiac diastolic indicesThe diastolic cardiac function indices in aging MI/R group(LVDP:14.68±0.17kPa, P＜0.05;LVEDP:0.9±0.35kPa) were increased significantly as compared to those in the young MI/R group(LVDP:12.84±0.35kPa,P＜0.05;LVEDP:-0.36±0.43 kPa P＜0.01)Data from the cardiac function indicated that both systolic and diastolic cardiac functions were aggravated in aged hearts after ischemia and reperfusion injury(Figure22,23, 24).1.2 Measurement of infarct sizeThe ratio of area at risk to left ventricular area(AAR/LV):No significant difference was observed among these groups.The myocardial infarct size of aging group was significantly enhanced as compared with young MI/R group(P＜0.05)(Figure25,26); 1.3 Increased cell apoptosis in myocardium of aging rats after ischemia and reperfusion injury1.3.1 TUNEL stainingMyocardial apoptosis of young and aging MI/R groups was determined by highly sensitive TUNEL staining.Compared to the percent of TUNEL-positive myocytes of young MI/R group(14.6±1.7%) the apoptotic index in the old group was increased significantly (19.0±2.1%,P＜0.05)(Figure27,28).1.3.2 Caspase-3 activity assayCaspase-3 is the pivotal protein in the final pathway of cell apoptosis.Myocardial cell apoptosis of young and aging MI/R groups was quantitatively analyzed by Caspase-3 activity assay.The apoptotic levels of both groups were expressed in a Caspase-3 activity ratio. Compared to the Caspase-3 activity ratio of young MI/R group(340+32μmol pNA/mg) the old MI/R group was enhanced significantly(436±35μmol pNA/mg,P＜0.05)(Figure29).2.The enhanced release of HtrA2/Omi,reduced XIAP increased the susceptibility to ischemia/reperfusion injury of aged rats2.1 The expression XIAP was diminished in the myocardium of aging MI/R group.The protein content of XIAP was measured by Western-blot.The content of XIAP in aging MI/R group was decreased significantly as compared with that in the young MI/R group(P＜0.05)(Figure30).2.2 The expression HtrA2/Omi was enhanced in myocardium cytosolic extracts of aging MI/R groupThe protein content of HtrA2/Omi in cytosolic was measured by Western-blot.The content of HtrA2/Omi in aging MI/R group was enhanced significantly as compared with that in the young MI/R group(P＜0.05)(Figure31). 3.Inhibition of HtrA2/Omi decreased the release of HtrA2/Omi to cytosolie and increased the XIAP3.1 The expression HtrA2/Omi was diminished in myocardium cytosolic extracts of aging MI/R group+ucf-101The protein content of HtrA2/Omi in cytosolic was measured by western-blot,ucf-101 reduced significantly the expression of HtrA2/Omi in cytosolic as compared with that in aging MI/R group+Vehicle(P＜0.05)(Figure31).3.2 The expression XIAP was enhanced in myocardium cytosolic extracts of aging MI/R group+ucf-101.The protein content of XIAP was measured by western-blot,ucf-101 increased significantly the expression of XIAP as compared with that in aging MI/R group+Vehicle(P＜0.05)(Figure30).4.Inhibition of HtrA2/Omi can attenuate the aging cardiac function perfectly after ischemia/reperfusion in reducing cardiac cell apoptosis4.1 Caspase-3 activity assayCaspase-3 is the pivotal protein in the final pathway of cell apoptosis.Myocardial cell apoptosis of aging MI/R groups+ucf-101/Vehicle was quantitatively analyzed by Caspase-3 activity assay.The apoptotic levels of both groups were expressed in a Caspase-3 activity ratio. Compared to the Caspase-3 activity ratio of aging MI/R group + Vehicle,aging MI/R group +ucf-101 the old group was decreased significantly(P＜0.05)(Figure32).4.2 Measurement of Cardiac functionThe cardiac function indices in aging MI/R group+Vehicle(LVDP:0.2745±0.15;LVEDP: 0.9750±1.04682;CI:20.8988±8.03547) were not different significantly as compared to those in aging MI/R group +ucf-101(LVDP:4.5000±4.37798,P=0.145;LVEDP:2.825±3.46254, P=0.206;CI:30.3515±12.09784,P=0.214) Through our analyzing,we plan to expend experiment animal quantity to avoid the exaggerated standard diviation(Figure33,34,35). Conclusion(1) Aging increased the susceptibility of myocardium to ischemia/reperfusion injury;(2) Aging enhanced HtrA2/Omi release from mitochondria to cytosolic,and decreased the XIAP expression.When use ucf-101,these results can be counteracted.To some extent,it provided one of novel reasons contributed to the greater susceptibility of aged cardiomyocytes to ischemia/reperfusion injury.