Cardioprotection And Mechanisms Of Postconditioning On Hypothermic Preservation In Isolated Rat Hearts

Background and AimAlleviation of reperfusion injury followed the hypothermic preservation is a pivotal factor to ensure the success in heart transplantation. It is more significant to improve the cardiac function efficiently in the patients who underwent heart transplant.The ischemic preconditioning phenomenon was introduced in 1986 by Murry, who found that several brief episodes of ischemia-reperfusion prior to a prolonged ischemic insult significantly decreased the myocardial injury. But it is unfeasible of appling ischemic preconditioning on donor heart during heart transplant.In 2003, Vinten-Johansen and his colleagues reported that brief episodes of myocardial ischemia and reperfusion employed during the beginning of reperfusion immediately after a prolonged ischemic insult have the similar protection as that of the ischemic preconditioning. This phenomenon was termed as "ischemic postconditioning". Compared with the ischemic preconditioning, the ischemic postconditioning is more feasible in clinical conditions such as heart transplant.Mitochondrial ATP-sensitive potassium (mitoK_ATP) channel has been reported to play a predominant role in diversified preconditioning-induced cardioprotection. Selective mitoK_ATP opener diazoxide pharmacologically preconditioning could closely mimic the salutary effects of classical ischemic preconditioning, resulting in decrease in infarct size and enhancement of post-ischemic cardiac contractile recovery. 5-HD, a selective mitochondrial ATP-sensitive potassium channel (mitoK_ATP) inhibitor, could abolish the amelioration of contractile function provided by DE. Some studies have shown that supplementation of diazoxide in Celsior cardioplegia solution, could enhancemyocardial protection by opening mitoK_ATP channel during long-term hypothermic preservation. So we hypothesized that the ischemic postconditioning may have the similiar cardiac protection on hearts after long-term hypothermic preservation. The objective of this study was to investigate whether ischemic postconditioning could contribute the cardioprotective effect to isolated rat hearts after long-term hypothermic preservation.In the present study, we used a model of hypothermic preservation of rat hearts in vitro to investigate the effect of ischemic postconditioning on rat hearts after 3 or 8 hours of hypothermic preservation respectively and to explore the underlying mechanisms. The main point of this study is to make clear the relationship of mitoK_ATP channel and ischemic postconditioning in the hypothermic preserved rat myocardium.Methods1. Establishment of hypothermic preserved myocardium modelImmediately after stunning and cervical dislocation, the male Sprague-Dawley rat hearts were rapidly excised and mounted on a Langendorff apparatus for perfusion at 37℃ with Krebs-Henseleit buffer at constant pressure (76 mmHg). The left atrium was opened, and a fluid filled latex balloon was inserted into the left ventricle, the balloon was connected via a short plastic tube to a pressure transducer and computer. After stabilization for approximately 30 minutes of perfusion, all hearts were perfused with cold Celsior cardioplegic solution to induce cardiac arrest and then removed from the Langendorff apparatus and preserved in Celsior cardioplegic solutions at 4℃ for 3 or 8 hours followed by 60 minutes reperfusion. During reperfusion, the balloon volume was set to the value as given at stabilization. Hemodynamic parameters (left ventricular end-diastolic pressure, left ventricular developed pressure, coronary flow, heart rate, rate-pressure product and maximal rise/fall rate of ventricular pressure) were monitored.2. Measurement of LDH in the coronary effluentTo assess myocardial injury, leakage of lactate dehydrogenase (LDH) during reperfusion was measured in the collected coronary effluent.3. Evaluation of the cardiac arrhythmiaThe cardiac arrhythmia score during the second 10min of reperfusion was evaluated on the basis of the ECG.4. Measurement of the myocardium water contentAfter 60 minutes of reperfusion, the ventricles were weighted. Wet weight was measured after both of the ventricles were incised and the excess fluid was blotted. Dry weight was measured after drying for 48 hours at 80℃. Water content was formulated as follow: [(wet weight - dry weight)/wet weight] ×100.5. Evaluation of the myocardial viabilityMyocardial viability was evaluated by measurement of formazan, a product of 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide, which is proportional to myocardial viability.6. Observation of myocardial ultrastructure by transmission electron microscopeAfter the experiment, ultrastructure of myocardium that subjected to an 8 hours of preservation followed by 60-min reperfusion were observed by transmission electron microscope (Philips Tecnai 10).7. Determination of Fas/FasL proteins by immunohistochemical methodExpression of Fas and FasL proteins was detected by immunohistochemical method. For quantitative analysis, SABC-positive cells were counted and approximately 1000 cells in random fields were scored for determination of the percentage of positive cells.8. Measurement of mPTP openingMitochondria were incubated with 200 μmol/L of CaCl₂, and then the absorbance at 520nm was measured spectrophotometrically, which indicate the opening of mitochondrial permeability transition pore (mPTP).Results1. Effect of ischemic postconditioning on rat hearts suffered from 3h of hypothermic preservation(1) After hypothermic preservation for 3 hours, ischemic postconditioning (3 cycles of 30s of reperfusion and 30s of ischemia) enhanced the recovery of left ventricular diastolic and systolic function compared to control group, and augmented the coronary flow and decreased in the cardiac arrhythmia score during reperfusion.(2) The cardiac protective effects of ischemic postconditioning was attenuated by a selective mitoK_ATP blocker 5-hydroxydecanoate (100 μM).2. Effect of postconditioning on rat hearts suffered from 8h of hypothermic preservation(1) After hypothermic preservation for 8 hours, ischemic postconditioning enhanced the recovery of left ventricular diastolic and systolic function compared to control group, augmented the recovery of coronary flow, myocardial viability and reduced myocardium water content, decreased in the leakage of myocardial enzymes and the cardiac arrhythmia score during reperfusion.(2) Postconditioning induced by 30μM of DE have the similar cardiac protective effects as that of ischemic preconditioning, both of which can be attenuated by 5-hydroxydecanoate.(3) CaCl2 (200μmol/L) reduced the absorbance of cardiac mitochondria at 520nm in control group, which indicated that the mPTP was opened in rat heart suffered from 8h of cold preservation. Ischemic and DE postconditionings could antagonist the CaCl2 (200μmol/L) induced decrease of absorbance in 8h of hypothermic preservated hearts, both of which were abolished by 5-HD.(4) After hypothermic preserved for 8h, the expression of Fas and FasL proteins in rat hearts increased. Compared with control group, DE and ischemic postconditionings reduced the expression of Fas and FasL proteins in rat hearts suffered from 8h of hypothermic preservation (P<0.01), both of which were abrogated by 5-HD.ConclusionsIschemic postconditioning could alleviate the injury in rat hearts suffered from long-term hypothermic preservation. The protection of ischemic postconditioning might be due to the opening of mitoK_ATP channel, which might be involved in the maintenance of mitochondrial integrity, the reduced expression of Fas/FasL proteins and the inhibition of mPTP opening.