The Mechanism Study Of The Cardioprotection Against Ischemia-reperfusion Injury Of PPARγ Agonist Pioglitazone In Rats

Ischemic heart disease is the leading cause of mortality in industrialized countries.Most of the treatments available for myocardial infarction,including thrombolytic therapy,interventional treatment and coronary artery bypass surgery,are toward to restore the coronary flow by releasing the occlusion. However,the reperfusion results in myocardial damage.Ischemia-reperfusion injury obstacles the ischemic myocardial getting most benefits from the reperfusion strategy.Previous studies have shown that various kinds of PPARγligands(including rosiglitazone,troglitazone and pioglitazone et al)can protect the myocardial from ischemia reperfusion(I/R)injury by simulating ischemia preconditioning(IPC).The mechanism of the protection has been studied to some extent.This study employs primary culture rat myocardial cells,freshly isolated cardiac myocytes and adult rats to further investigate the mechanism of the protection against I/R injury of pioglitazone based on previous study.1.Objectivea)To study the role of ATP-sensitive potassium channel in the cadiocyte protection against hypoxia-reoxygenation injury of pioglitazone.b)To observe the effect of pioglitazone on ATP-sensitive potassium currents of freshly isolated ventricular myocytes.c)To investigate the mRNA and protein expression changes of ERK1/2,pERK1/2,p38,p-p38,TGFβ1,COX-2 and HIF-1αduring I/R and the effect of pioglitazone on the expression.2.Methodsa)Primary cultures of cardiac myocytes were prepared from the ventricles of neonatal SD rats.The myocytes were divided into different groups to subject to 0.1,1.0,2.0μM pioglitazone pretreatment.At the same time,PPARγreceptor specific antagonist GW9662,sarcolemmal ATP-sensitive potassium (sarcK_ATP)channel blocker glibenclamide and mitochondrial ATP-sensitive potassium(mitoK_ATP)channel blocker 5-HD were added in different groups. Then hypoxia- reoxygenation injury model were established.The cells stained with Annexin V FITC/PI and Hoechst33258 which was used to detect the apoptosis of cardial myocyte.Western-blot and RT-PCR were employed to detect the expression of ATP-sensitive potassium channel subunits.b)Freshly isolated SD adult rats cardiac myocytes were prepared.The sarcK_ATP channel currents were recorded after the cells subjected to hypoxia.To observe the effect of pioglitazone on the sarcK_ATPchannel currents.c)30 adult SD rats were randomly divided into 5 groups.Vehicle,5,10,20mg·kg^-1·d^-1pioglitazone were lavaged for 7 days.Subsequently the heart I/R model were made in vivo by ligation of the anterior descending branch for 30 min and reperfusion for 30 min.Western-blot and RT-PCR were employed to investigate the expression changes of ERK1/2,pERK1/2,p38, p-p38,TGFβ1,COX-2 and HIF-1αmRNA and protein during I/R and the effect of pioglitazone on the expression.3.Resultsa)Myocardial cells were induced to apoptosis by hypoxia-reoxygenation.The percentage of early apoptotic cells increased from 0.20±0.03%of control group to 12.22±1.45%of hypoxia-reoxygenation group(P＜0.05). Pioglitazone decrased myocardial cells apoptosis(0.1μM group:10.09±0.67 %,1.0μM:9.16±1.47%,2.0μM:8.32±0.89%).There were significantly difference only between the highest dose group and hypoxia-reoxygenation group(P＜0.05).PPARγreceptor specific antagonist GW9662 abolished the protection against apoptosis of 2.0μM pioglitazone(12.46±1.62%,P＜0.05).b)MitoK_ATPchannel blocker 5-HD attenuated the protection against apoptosis of 2.0μM pioglitazone(10.00±0.50%,P＜0.05)and sarcK_ATPchannel blocker glibenclarnide had no effect on the protection.c)Pioglitazone upregulated the expression of mitoK_ATPchannel subunit Kir6.1 both in mRNA and protein level.Pioglitazone had no effect on the mRNA expression of sarcK_ATPchannel subunit Kir6.2.d)K_ATPchannel currents were recorded successfully after the freshly isolated SD adult rats cardiac myocytes subjected to hypoxia in.Pioglitazone had no effect on the K_ATPchannel currents.e)The expression of ERK1/2 and p38 protein of rats myocardium were not change after I/R while the expression of p-ERK1/2 and p-p38 protein increased.The expression of TGFβ1 and COX-2 increased both in mRNA and protein level.The expression of HIF-1αmRNA increased too. Pioglitazone upregulated the expression of p-ERK1/2 and downregulated p-p38 protein while had no effect on the expression of ERK1/2 and p38 protein.Pioglitazone upregulated the expression of TGFβ1 and COX-2 both in mRNA and protein level and the expression of HIF-1αin mRNA level.4.Conclusion:a)Pioglitazone pretreatment can reduce myocardial cells apoptosis induced by hypoxia-reoxygenation via partly activation of PPARγ.b)Pioglitazone exerts cardioprotection against apoptosis via opening mitoK_ATP channel and dose not involve sarcK_ATPchannel.c)Regulating the expression of reperfusion injury salvage kinase(RISK)may have ralitionship with the cardioprotection against I/R of pioglitazone.