Molecular Targets Of Ischemic Postconditioning For The Regulation Of Cardiomyocyte Apoptosis

BackgroundStimulation of endogenous protective mechanism and increase of the abilityagainst injury are the definitive therapy to salvage myocardium followingischemia/reperfusion injury. Ischemic postconditioning is one of the strongestendogenous protective mechanisms injury. It, defined as brief periods of reperfusionalternating with re-occlusion applied during the very early minutes of reperfusion, iscapable of protecting myocardium from ischemia/reperfusion injury. However, themechanism by which ischemic postconditioning inhibits myocardium injury remainslargely unknown.Ischemia/reperfusion stimulates cardiomyocyte apoptosis by triggeringmitochondrial apoptotic pathway. Ischemic postconditioning inhibites cardiomyocyteapoptosis induced by ischemia/reperfusion. Consequently, it leads us to considerwhether ischemic postconditioning protects cardiomyocytes fromischemia/reperfusion injury through regulating mitochondrial apoptotic pathway.PUMA (p53upregulated modulator of apoptosis) is a target gene of p53. It is locatedin mitochondria and exerts pro-apoptotic function. ARC (apoptosis repressor withcaspase recruitment domain) is the first anti-apoptotic protein so far identified to behighly and specifically expressed in the cardiac tissue. It has been demonstrated thatPUMA and ARC play pivotal roles in controlling mitochondrial apoptotic pathway.Based on the similar regulatory pathways of apoptosis between ischemicpostconditioning and PUMA/ARC, we speculate that ischemic postconditioningtargets PUMA and ARC to inhibit cardiomyocyte injury.ObjectiveIn cultured neonatal rat cardiomyocytes subjected to hypoxic postconditioning,we observe the relation between the PUMA and ARC expression and the inhibitoryeffect of hypoxic postconditioning on mitochondrial apoptotic pathway. The aim ofthe study is to prove that hypoxic postconditioning inhibits mitochondrial apoptoticpathway by targeting ARC and PUMA, consequently protecting cardiomyocytes from apoptosis.Methods1. Establishment of hypoxia/reoxygenation and hypoxic postconditioning modelsin vitro:(1) Hypoxia/reoxygenation model: the cultured neonatal ratcardiomyocytes were placed into a hypoxic incubator with95%N2-5%CO2at37C. After3h hypoxia, the cells were transferred to a normoxic incubator for3h reoxygenation.(2) Hypoxic postconditioning model: cardiomyocytes wereplaced into a hypoxic incubator with95%N2-5%CO2at37C. After3h hypoxia,the celles were transferred to a normoxic incubator for5min and then returnedback to the hypoxic incubator for additional5min. The cycle of hypoxicpostconditioning was repeated three times at the onset of3h of reoxygenation.2. Analysis of cardiomyocyte apoptosis: Cells were labelled with annexin V-FITCand propidium iodide for25min. The samples were analysed by flow cytometry.3. Cell death assessment: Cell death was determined by Trypan Blue exclusion andthe numbers of Trypan Blue-positive and-negative cells were counted on ahaemocytometer.4. Detection of the expression of PUMA and ARC proteins: The total proteins ofcardiomyocytes were extracted. And Western blot was employed to detect theexpression of ARC and PUMA proteins.5. The expression of cytochrome c and Bax in mitochondria and cytosol: Cytosolicand mitochondrial fractions were isolated. The expression of cytochrome c andBax in mitochondria and cytosol was observed by Western blot.6. Caspase-3activity assay: Caspase-3activity was measured using thecolorimetric activity assay. Absorbance at405nm was measured with amicroplate autoreader.7. Detection of mitochondrial membrane potential: Cardiomyocytes were labeledby MitoProbeTM JC-1. Mitochondrial membrane potential was analyzed byflow cytometry with488nm excitation.8. Image analysis, data management and statistics analysis: Integrated opticaldensity of mRNA and protein bands were performed with software ImagePro-Plus (version4.11). Experimental data were represented by mean±standard deviation (x±s). SPSS13.0statistics software was introduced. Bonferroni's testwas employed to analyze statistical differences between every two-sampleamong several groups. P<0.05was considered statistically significant.Results1. Hypoxic postconditioning inhibits cardiomyocyte apoptosis by down-regulatingPUMA(1) The role of PUMA in hypoxia/reoxygenation-induced cardiomyocyte apoptosis:Hypoxia/reoxygenation up-regulated PUMA mRNA and its protein levels(P<0.001compared to control). Concomitantly, cardiomyocyte apoptosis anddeath were increased under hypoxia/reoxygenation. In contrast, Inhibition ofendogenous PUMA expression by using its siRNA decreased significantlycardiomyocyte apoptosis and death induced by hypoxia/reoxygenation(P<0.001).(2) Hypoxic postconditioning inhibits cardiomyocyte apoptosis by regualting PUMAexpression: When cardiomyocytes were postconditioned at the onset ofreoxygenation by brief, repeated cycles of reoxygenation-hypoxia, the levels ofPUMA mRNA and protein were reduced compared with thehypoxia/reoxygenation group. And the number of apoptotic and deadcardiomyocytes was decresed after cardiomyocytes were exposed to hypoxicpostconditioning (P<0.001compared to hypoxia/reoxygenation). However,overexpression of PUMA in cardiomyocytes by infecting adPUMA reducedgreatly the inhibitory effect of hypoxic postconditioning on cell apoptosis anddeath (P<0.001).2. Hypoxic postconditioning up-regulates ARC to inhibit cardiomyocyte apoptosis(1) The role of ARC in cardiomyocyte viability under physiological condition:Transfection of antisense-ARC to cardiomyocytes under physiological conditioninduced an increase in cell apoptosis and death (P<0.05). In contrast, there wasno changes in cell apoptosis and death when cardiomyocytes were transfectedwith scramble-ARC (P>0.05).(2) Involement of ARC in hypoxia/reoxygenation-induced cardiomyocyte apoptosis:Hypoxia/reoxygenation down-regulated the expression of ARC protein, increased cardiomyocyte apoptosis and death (P<0.05). Infection of adARC incardiomyocytes inhibited cell apoptosis and death induced byhypoxia/reoxygenation (P<0.05). However, infection of-gal in cardiomyocyteshad no effect on cell apoptosis and death induced by hypoxia/reoxygenation(P>0.05).(3) ARC is required for the anti-apoptotic effect of hypoxic postconditioning:Hypoxic postconditioning inhibited the down-regulation of ARC protein leveland reduced the number of apoptotic and dead cells induced byhypoxia/reoxygenation (P<0.01). Cells were transfected with ARC antisenseoligodeoxynucleotides or its scrambled forms. And the results showed thattransfection with ARC antisense oligodeoxynucleotides induced a significantincrease in the percentage of apoptotic and death cells under postconditioningcondition (P<0.001, compared with postconditioning+hypoxia/reoxygenation),but not transfection with ARC scramble oligodeoxynucleotides (P>0.05,compared with postconditioning+hypoxia/reoxygenation).3. Studying on the inhibitory effect of hypoxic postconditioning on mitochondrialapoptotic pathway by targeting PUMA and ARC(1) Hypoxic postconditioning targets PUMA and ARC to inhibit the loss ofmitochondrial membrane potential: Hypoxic postconditioning inhibited the lossof mitochondrial membrane potential in cardiomyocytes induced byhypoxia/reoxygenation (P<0.05). However, when the cells were treated byadPUMA or ARC antisense oligodeoxynucleotides, the inhibitory effect ofhypoxic postconditioning disappeared (P<0.01compared with postconditioning).In contrast, treatment of cardiomyocytes with-gal or scramble-ARC had noeffect on the inhibitory role of hypoxic postconditioning in the loss ofmitochondrial membrane potential (P>0.05).(2) The blockage role of hypoxic postconditioning in the translocation of Bax andthe release of cytochrome c by targeting PUMA and ARC:Hypoxia/reoxygenation induced the translocation of Bax from cytosol tomitochondria and the release of cytochrome c from mitochondria to cytosol. Incontrast, hypoxic postconditioning inhibited the translocation of Bax and therelease of cytochrome c induced by hypoxia/reoxygenation. However, when PUMA was overexpressed by adPUMA or endogenous ARC was knocked downby its antisense oligodeoxynucleotides, the inhibitory effect of hypoxicpostconditioning on Bax translocation and cytochrome c release was abolished.Conclusion:1. Hypoxic postconditioning inhibits hypoxia/reoxygenation-inducedcardiomyocyte injury by down-regulation of PUMA expression and up-regulation of ARC expression.2. The cardioprotective effect of hypoxic postconditioning is mediated by PUMAand ARC through inhibiting mitochondrial apoptotic pathway.