| BackgroundAs populations age in middle- and low-income countries over the next 25 years, the proportion of deaths due to noncommunicable diseases will rise significantly. Globally, deaths from cancer will increase from 7.4 million in 2004 to 11.8 million in 2030, and deaths from cardiovascular diseases will rise from 17.1 million to 23.4 million in the same period ( world health statistics 2008 ).Coronary heart disease is the leading cause of death worldwide, and 3.8 million men and 3.4 million women die of the disease each year. After an acute myocardial infarction, early and successful myocardial reperfusion with the use of thrombolytic therapy or primary percutaneous coronary intervention ( PCI ) or emergency coronary artery bypass grafting ( CABG ) is the most effective strategy for reducing the size of a myocardial infarct and improving the clinical outcome. The process of restoring blood flow to the ischemic myocardium, however, can induce injury. This phenomenon, termed myocardial reperfusion injury, can paradoxically reduce the beneficial effects of myocardial reperfusion. The injury culminates in the death of cardiac myocytes that were viable immediately before myocardial reperfusion. This form of myocardial injury, which by itself can induce cardiomyocyte death and increase infarct size ,may in part explain why, despite optimal myocardial reperfusion, the rate of death after an acute myocardial infarction approaches 10%, and the incidence of cardiac failure after an acute myocardial infarction is almost 25%.Studies in animal models of acute myocardial infarction suggest that lethal reperfusion injury accounts for up to 50% of the final size of a myocardial infarct, and in these models a number of strategies have been shown to ameliorate lethal reperfusion injury. Yet, the translation of these beneficial effects into the clinical setting has been disappointing. But in 2003, Prof Zhao Zhiqing et al. showed that after a 45-minute episode of sustained myocardial ischemia, the interruption of myocardial reperfusion with three 30-second cycles of myocardial ischemia and reperfusion could reduce the myocardial infarct size significantly. They named this form of cardioprotection ischemic postconditioning. Shortly after that,Staat and Laskey respectively used ischemic postconditioning in patients with acute myocardial infarction who are undergoing PCI with a protocol that has reduced myocardial infarct size by 36% and improved myocardial reperfusion.Although the precise mechanism of ischemic postconditioning–induced protection is not fully understood, but the procedure has been shown to target the important mediators of lethal reperfusion injury by reducing oxidative stress, decreasing intracellular Ca2 + overload, improving endothelial function, attenuating apoptotic cardiomyocyte death, reducing neutrophil accumulation, and delaying the restoration of neutral pH. Furthermore, ischemic postconditioning activates the reperfusion injury salvage kinase (RISK) pathway, which include the phosphatidylinositol-3-kinase-Akt pathway ( PI3K-Akt ) and the extracellular signal-regulating kinases ( ERK ) pathway. Recently, several studies have suggested that phosphorylation of glycogen synthase kinase-3beta (GSK-3β) at Ser9 enhances myocardial tolerance during ischemia- reperfusion injury.In the majority of studies, ischemic postconditioning (IPost) has been demonstrated to protect adult rodent hearts from myocardium ischemia- reperfusion (I/R) injury in vitro, and has implicated the involvement of several candidate signaling pathways such as PI3-K/Akt, ERK1/2, and GSK-3βin cardioprotective signaling and pro-cell survival mechanisms. Recently, some researches have found that the cardioprotection of IPost is lost in aged mice. However, little is known with regards to these signaling mechanisms during advanced age.Aim(1) to determine whether ischemic postconditioning applied at the beginning of reperfusion could protect adult hearts in vivo from ischemia reperfusion injury .(2) to evaluate the hypothesis that ischemic postconditioning could protect ischemia/reperfusion aged hearts in vivo. And to compare the cardioprotection effects of ischemic postconditioning between aged and adult heats.(3) to investigate whether IPost's cardioprotection is dependent on the activation of PI3-K/Akt and GSK-3β, and if activation of PI3-K/Akt or GSK-3βmay be a novel therapeutic strategy for reducing myocardial I/R injury. Methods1. Male Sprague Dawley (SD) rats (adult, 3-4 months aged, 16-18 months) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and experienced left thoracotomy. a 6-0 silk suture was placed under the left anterior descending coronary artery (LAD), and ends of the suture were threaded through a segment of polyethylene tube to form a snare for the reversible LAD occlusion. Rats were subiected to 30 min myocardial ischemia and 3 h reperfusion.2. Needle electrodes were placed subcutaneously on the limbs, and an electrocardiogram was continuously recorded.the right carotid artery was cannulated with a 24-gauge angiocath connected to a fluid-filled pressure transducer for monitoring Hemodynamic properties. The left external jugular vein was cannulated for drug administration.3. The rats were assigned to one of the following groups based upon the intervention performed (n = 8 per adult group, n = 6 per aged group): (1) I/Radult and I/Raged: received no other intervention either before or after LAD occlusion (2) IPostadult and IPostaged: received ischemic postconditioning induced by four cycles of 10 seconds of ischemia and 10 seconds of reperfusion at the beginning of reperfusion (3) IPost+LYadult and IPost+LYaged: received ischemic postconditioning in the presence of the selective PI3K inhibitor, LY294002 (0.3 mg/kg) administered via the left external jugular vein immediately at the beginning of reperfusion (4) Vehicleadult and Vehicleaged: received ischemic postconditioning in the presence of an equal volume of vehicle, 0.02% dimethyl sulfoxide (DMSO) without LY294002 at the beginning of reperfusion (5) Shamadult and Shamaged:p lacement of the silk suture under the LAD and no other intervention. 4. Blood samples (0.5 mL) were collected at baseline and the end of reperfusion and centrifuged at 2500×g and 4°C for 10 min. The samples were then stored at -70°C until further study. The activity of CK and LDH was analyzed at 25°C using commercially available kits.5. At the end of reperfusion,ischemic and infarct areas were measured by Evans blue and triphenyltetrazolium chloride ( TTC ) staining respectively.6. In separate experiments (n=4 rats per experimental group), phosphorylation of Akt and GSK-3βwere analyzed by Western blotting after 15 min of reperfusion.Results1. Hemodynamic data includes heart rate ( HR ), LVDP and±dP/dtmax (1) HR: At all time points, there were no significant differences among I/Radult, IPostadult, and IPost+LYadult groups, and no significant differences among the I/Raged, IPostaged, and IPost+LYaged groups. At baseline, I 5 min, R 15 min, R 30 min, the HR of I/Raged group were lower than that of I/Radult group. At all time points, there were no significant differences between IPostadult,and IPostaged , between IPost+LYadult and IPost+LYaged group.(2) LVDP: At all time points, there were no significant differences among I/Radult, IPostadult, IPost+LYadult groups, and no significant differences among the I/Raged, IPostaged, IPost+LYaged groups. At points of R 5 min, R1 h, and R 2 h, the LVDP of I/Raged group were lower than that of I/Radult group. At points of R 5 min, R 2 h, the LVDP of IPostaged group were lower than that of IPostadult group. At points of R 2 h and R 3 h, the LVDP of IPost+LYaged group were lower than that of IPost+LYadult group. (3) +dP/dtmax:At R 5 min, R 2 h, and R 3 h, +dP/dtmax of IPostadult were significantly higher than that of I/Radult or IPost+LYadult. But at all time points, there were no significant differences among the I/Raged, IPostaged, IPost+LYaged groups. In the I/Rgroup, there was no significant difference between aged and adult rats. But in the IPost groups, +dP/dtmax of adult rats were significantly higher than that of aged rats during reperfusion except R 1 h. In the IPost+LY groups only at R 30 min +dP/dtmax of adult rats were significantly higher than that of aged rats.(4) -dP/dtmax: At R2 h, R 3 h, -dP/dtmax of IPostadult were significantly higher than that of I/Radult or IPost+LYadult. But at all time points, there were no significant differences among the I/Raged, IPostaged, IPost+LYaged groups. In the I/R and IPost+LY group, only at I 5 min -dP/dtmax of adult rats were significantly lower than that of aged rats. But in the IPost groups, -dP/dtmax of adult rats were significantly higher than that of aged rats at R 5 min and R 15 min.2. The area at risk ( in percentage of the left ventricle ) by LAD occlusion was comparable among six groups ( I/Radult, 52.8±5.2% IPostadult, 52.0±5.0% Ipost + LYadult, 52.2±5.8% I/Raged, 51.3±7.2% IPostaged, 53.8±3.5% IPost+LYaged, 51.1±5.8% ). As expected, the postconditioned hearts developed significantly smaller infarct sizes ( expressed as percentage of the area at risk, IS/AAR ) than I/R hearts (11.9±1.6% in IPostadult vs. 30.2±2.8% in I/Radult, P<0.05 13.7±2.5% in IPostaged vs. 27.5±3.5% in I/Raged, P<0.05 ). The PI3K inhibitor, LY294002, abolished IPost's cardioprotection in aged and adult rats (30.1±2.9% in IPost + LYadult vs. 11.9±1.6% in IPostadult, P<0.05; 33.4±7.0% in IPost + LYaged vs. 13.7±2.5% in IPostaged, P<0.05 ). There were no significant differences between I/Radult and I/Raged, IPostadult and IPostaged, IPost + LYadult and IPost+LYaged. ( all P =NS ). 3. At baseline, the levels of CK in aged groups were lower than that in adult groups, and no differences within aged groups and within adult groups. The levels LDH were no differences between the six groups. Compared with the baseline, CK and LDH release was significantly increased after 3 hours of reperfusion in all I/R, IPost, and IPost+LY groups ( all P < 0.05 vs. baseline ). At 3 h of reperfusion, CK and LDH release in IPost rats was significantly lower than that in I/R groups ( all P< 0.05 vs. I/R groups ). LY294002 prevented the decrease of CK and LDH release in IPost group ( all P<0.05 vs. IPost, not significantly different from I/R groups ).4. There were no differences in total Akt ( t-Akt ) and total GSK-3β( t-GSK-3β) levels between every two groups. But in both adult and aged rat hearts, IPost significantly increased levels of Akt phosphorylation to levels greater than I/R hearts ( p-Akt/t-Akt: 0.64±0.04 in IPostadult vs. 0.40±0.09 in I/Radult, P<0.05 0.63±0.03 in IPostaged vs. 0.39±0.01 in I/Raged, P<0.05 ). The PI3K inhibitor, LY294002, blocked Akt phosphorylation ( p-Akt/t-Akt: 0.34±0.07 in IPost + LYadult vs. 0.64±0.04 in IPostadult, P<0.05 0.35±0.09 in IPost + LYaged vs. 0.63±0.03 in IPostaged ,P<0.05 ). Similarly, IPost significantly increased levels of GSK-3βphosphorylation to levels greater than I/R hearts, and LY294002 attenuated the levels of phosphorylated-GSK-3βin both adult and aged hearts.Conclusion1. Ischemic postconditioning at the onset of reperfusion reduces myocardial infarct size, attenuates the increased levels of CK and LDH, and improve functional preservation after reperfusion in adult SD rats in vivo.2. An infarct sparing effect for IPost in in vivo heart model of aged rats, which unlike the adult rat model, was not associated with functional preservation.3. IPost's cardioprotection in both adult and aged SD rat hearts may be associated with Akt and GSK-3βphosphorylation. |
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