| Background:World Health Organization predicted that ischemic heart disease will be the second threat of health in 2030. Myocardial ischemia induced by coronary artery infarction is one of main fatal causes of ischemic heart disease. Myocardial ischemia initiated a series of pathophysiologic changes, and persistent myocardial ischemia without reperfusion induced cell death, finally, resulting in myocardial dysfunction. It has been suggested that the only available therapy is restoring blood flow of ischemic myocardium as soon as possible, coronary reperfusion, which could reduce cell death and alleviate myocardial dysfunction. However, it has been recognized that although early restoration of blood flow to ischemic myocardium (reperfusion) is necessary to limit ischemic tissue injury, reperfusion itself has deleterious effects on cardiomyocytes that can accelerate and extend tissue injury. Therefore, it is necessary to illustrate the possible mechanisms of reperfusion injury.Myocardial ischemia/reperfusion results in numerous myocardial cell deaths. Two most common forms of cell death are programed cell death and accidental cell death. Programmed cell death characterized by a series of stereotypic morphological and biochemical features. Therefore, it's easier to inhibit programmed cell death than necrosis. It has been demonstrated that cardiomyocyte apoptosis, a special form of programed cell death, plays a pivotal role in myocardial infarction and cardiac dysfuntion induced by myocardial ischemia/reperfusion injury. One of the most widely recognized biochemical features of apoptosis is the activation of a class of aspartate-specific cysteine proteases known as caspases. Cells process multiple caspases, which may work in a cascade fashion. The most potent endogenous inhibitor of caspases is X-linked Inhibitor of Apoptosis Protein (XIAP). Many studies shows that XIAP plays a significant role in cardiomyocyte apoptosis induced by myocardial ischemia/reperfusion, but it has not been well determined that how the dynamic expression of XIAP comes after myocardial ischemia/reperfusion and whether XIAP was involved in myocardial ischemial and reperfusion. If so, the cardiomyocytes play a key role in cardiac function as a basic unit, which implicated that the cardiomyocytes apotosis was one of the most important factors lead to cardiac dysfunction, and how important of XIAP to effect the cardiac function after myocardial ischemia and reperfusion? There, the experiments were designed as follows. Firstly, we detected the time course of XIAP protein and mRNA expression after myocardial ischemia/reperfusion, and then we identified the relationship between XIAP protein expression and cardiac function. Finally, we determined whether XIAP was involved in cardiac dysfunction resulted by myocardial ischemia/ reperfusion with the treatment of Embelin, an XIAP specific inhibitor.Objectives1. To observe the time course of XIAP expression after myocardial ischemia/reperfusion.2. To illustrate whether XIAP was involved in myocardial ischemia/reperfusion injury.Methods1. Male Sprague-Dawley adult rats (4-6 months, n=103) were randomly divided into three groups, including myocardial ischemia/reperfusion group (Left Anterior Descending (LAD) with occlusion), sham group (Left Anterior Descending without occlusion) and drug group.1). Myocardial Ischemia and Reperfusion Group (MI/R Group)①Reperfusion 0 hour Group (ROh):LAD with reversible occlusion,30min ischemia. n=6;②Reperfusion 1 hour Group (Rlh):LAD with reversible occlusion,30min ischemia followed by one hour reperfusion. n=7, one rat was dead caused by ventricular fibrillation in the process of operation, and actually six rats were used in this group;③Reperfusion 2 hour Group (R2h):LAD with reversible occlusion,30min ischemia followed by two hours reperfusion. n=6,;④Reperfusion 3 hour Group (R3h):LAD with reversible occlusion,30min ischemia followed by three hours reperfusion. n=6;⑤Reperfusion 12 hour Group (R12h):LAD with reversible occlusion,30min ischemia followed by twelve hours reperfusion. n=7, one rat was dead caused by unidentified reason in the process of operation, and actually six rats were used in this group;⑥Reperfusion 24 hour Group (R24h):LAD with reversible occlusion,30min ischemia followed by twelve hours reperfusion. n=8, there were two rats were dead caused by ventricular fibrillation in the process of operation, and actually six rats were used in this group; 2). Sham GroupMale Sprague-Dawley adult rats (n=36) were randomly divided into six groups. Each group includes 6 rats. Left Anterior Descending (LAD) without occlusion. The other steps are same to MI/R group;3). Drug Group①MI/R 12hour+Vehicle Group:LAD with reversible occlusion,30min ischemia followed by twelve hours reperfusion. The rats were given 1ml/kg body weight of oil (vehicle) 12 hours before reperfusion. n=12, two rats were dead caused by ventricular fibrillation in the process of operation, and actually ten rats were used in this group.②MI/R 12hour+Embelin Group:LAD with reversible occlusion,30min ischemia followed by twelve hours reperfusion The rats received 20mg/kg body weight embelin in oil 12 hours before reperfusion (500mg/kg). n=15, five rats were dead caused by ventricular fibrillation, pneumonia and tumor in the process of operation, and actually ten rats were used in this group.2. A polyethylene catheter connected with a pressure transducer was inserted into the left ventricular cavity through the right carotid artery, and Left Ventricular Systolic Pressure (LVSP), Left Ventricular Diastolic Pressure(LVDP),+dp/dtmax,-dp/dtmax were measured;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 was determined by Western Blot;5. The mRNA expression level of XIAP was determined by Real time PCR.Results1. The rat model of myocardial ischemia and reperfusion1.1 Time course of cardiomyocyte apoptosis after myocardial ischemia and reperfusion.1.1.2 Caspase-3 activity assayCaspase-3 is the pivotal protein in the final pathway of cell apoptosis. Cardiomyocyte apoptosis was quantitatively analyzed by Caspase-3 activity assay. The apoptotic levels of groups were expressed in a Caspase-3 activity ratio. Compared to the sham group (1.00±0.21), Caspase-3 activity ratio began to increase at 1hour after reperfusion (1.55±0.24, P<0.05), and reach to the peak at 12 hour after reperfusion (1.66±0.21, P<0.05), and return to normal at 24 hour after reperfusion (1.21±0.13, P>0.05). (Figure 4)1.1.2 TUNEL stainingMyocardial apoptosis was determined by highly sensitive TUNEL staining. Compared to Sham group (1.36±0.47%), the percent of TUNEL-positive myocytes and the apoptotic index in MI/R 12 hour group was increased significantly (14.75±1.54%, P<0.05). (Figure5).1.2 Time course of cardiac function after myocardial ischemia and reperfusion.1.2.1 Cardiac systolic indices+dp/dtmax and LVSP are two of indices which reflect the systolic cardiac function. +dp/dtmax began to decrese at 12 hour after reperfusion (404.76±137.94Kpa/s vs. Sham 633.00±59.48Kpa/s, P<0.05), and sustained a lower level to 24 hour after reperfusion (388.71±93.51Kpa/s vs. Sham 653.42±61.41Kpa/s, P<0.05). However, there is no significant difference of LVSP between the two groups. (Figure 6,7)2.2 Cardiac diastolic index-dp/dtmax and LVDP are two of indices which reflect the diastolic cardiac function.-dp/dtmax began to decrese at 12 hour after reperfusion (335.00±109.00 Kpa/s vs. Sham 427.83±72.92Kpa/s, P<0.05), and sustained a lower level to 24 hour after reperfusion (-dp/dtmax,353.71±97.21 Kpa/s vs. Sham 459.42±66.22Kpa/s, P<0.05). LVDP began to increase at 12 hour after reperfusion (1.10±0.99 Kpa vs. Sham-1.00±0.77Kpa, P<0.05), and sustained a.high level to 24 hour after reperfusion (0.79±0.50 Kpa vs. Sham-1.18±0.64Kpa, P<0.05). (Figure 8,9) Data from the cardiac function indicated that both systolic and diastolic functions were deteriorated after ischemia and reperfusion, especially at the time of 12 hour and 24 hour.2. Time course of XIAP expression in the heart after myocardial ischemia and reperfusion.2.1 Time course of XIAP protein expression in the heart after myocardial ischemia and reperfusion.The protein content of XIAP in the heart was measured by western-blot. The content of XIAP began to decrease at 3 hour after reperfusion (1.00±0.19 vs. Sham 1.61±0.12. P<0.05), and declined to the bottom at 12 hour after reperfusion (0.65±0.16 vs. Sham 1.42±0.24, P<0.05), and sustained a lower level to 24 hour after reperfusion (0.94±0.16 vs. Sham 1.53±0.21, P<0.05). (Figure 10)2.2 Time course of XIAP mRNA expression in the heart after myocardial ischemia/ reperfusion.The mRNA content of XIAP was measured by Real Time PCR. There is no significant difference of XIAP mRNA level between sham group and MI/R group (P>0.05). (Figure11, 12,13)3. Correlation analysis of XIAP protein expression in the heart with cardiac function after myocardial ischemia and reperfusion.3.1 Positive correlation between. XIAP protein expression with+dp/dtmax after ischemia and reperfusion (n=42, r=0.655, P<0.05), which shows that XIAP protein expression has a close correlation with cardiac systolic function.(Figure 14)3.2.Negative correlation between XIAP protein expression with LVDP after ischemia and reperfusion (n=41, r=-0.528, P<0.05), which shows that XIAP protein expression has a close correlation with cardiac systolic function.(Figure 15)4. Inhibition of XIAP had a significantly effect on increasing cardiomyocyte apoptosis and aggravating cardiac dysfunction after myocardial ischemia and reperfusion4.1 Treatment with specific inhibitor of XIAP, embelin, could increase cardiomyocyte apoptosis after myocardial ischemia and reperfusion.4.1.1 Caspase-3 activity assayCaspase-3 is the pivotal protein in the final pathway of cell apoptosis. Cardiomyocyte apoptosis 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 vehicle group (1.00±0.16), Caspase-3 activity was increased markedly in embelin group(1.49±0.36,P<0.05).(Figurel6)4.1.2 TUNEL stainingMyocardial apoptosis was determined by highly sensitive TUNEL staining. Compared to vehicle group (16.02±1.73%), the percent of TUNEL-positive. myocytes and the apoptotic index in Embelin group was increased significantly (22.97±1.60%. P<0.05). (Figure 17). 4.2 Treatment with specific inhibitor of XIAP, embelin, could aggravate cardiac dysfunction after myocardia ischemia and reperfusion.4.2.1 Cardiac systolic indices+dp/dtmax, LVSP are three of indices which reflect the systolic cardiac function. Compared to the vehicle group (+dp/dtmax,408.63±89.92Kpa/s; LVSP,12.78±1.78 Kpa), the systolic cardiac function decreased markedly in embelin group (+dp/dtmax, 308.13±94.22Kpa/s,P<0.05;LVSP,10.84±1.58Kpa,P<0.05). (Figure18,19)4.2 Cardiac diastolic index-dp/dtmax and LVDP are two of indices which reflect the diastolic cardiac function. Compared to the vehicle group (0.52±1.10Kpa), LVDP was increased significantly in embelin group. (1.64±0.88Kpa P<0.05). However, there was no significant difference of-dp/dtmax between the two groups (Figure20,21)ConclusionThe investigation fistly indicated that lower XIAP protein expression had a close correlation with cardiac dysfunction after myocardial ischemia and reperfusion, and inhibition of XIAP had a significantly aggravated effect on cardiomyocyte apoptosis and cardiac dysfunction, which suggested that the decrease of XIAP was involved in the myocardial ischemia and reperfusion injury. |
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