The Pro-survival Effects And Mechanism Of Propofol Postconditioning Against Myocardial Ischemia/reperfusion Injury

Background:Ischemic preconditioning (IPC) was first coined by Murry to describe a phenomenon whereby brief episodes of ischemia/reperfusion (I/R) could attenuate myocardial damage, but its clinical application has been disappointing due to the unpredictability of ischemia. Ischemic postconditioning (I-postC) performed after ischemia has solved the problem of unpredictability. Furthermore, pharmacological postconditioning, which only requires a drug given as an adjunctive intervention during early reperfusion, can also achieve cardioprotection and is easier to implement.Recent evidences indicate that the volatile anesthetic isoflurane reduces myocardial injury when administered at the early phase of reperfusion. Whether the intravenous anesthetic propofol (2.6-diisopropylphenol) when given at the onset of reperfusion is also capable of producing cardioprotective effects or not is still unclear. Some researches have demonstrated that propofol preconditioning could protect cardiomyocytes through scavenging free radicals, enhancing antioxidant capacity and inhibiting membrane calcium channels. However, subsequent studies indicated that pro-survival signal transduction pathways played an important role in cardioprotection, which stimulated cellular endogenous anti-injury capacity to protect themselves. The present study was designed to determine whether P-PostC is protective against I/R injury and examine our hypothesis of the mechanism using a hypoxia/reoxygenation (H/R) model of cardiomyocytes in vitro.Section 1 The establishment and evaluation of hypoxia/reoxygenation model with primary cultured cardiomyocytesObjective:To discuss the methods of separating,purifying and fostering cardiac muscle cells and the hypoxia/reoxygenation (H/R) model of neonatal rat cardiomyocytes in vitro.Methods:Hearts were obtained from 1-day-old rats and dissociated with enzyme solution. Bromodeoxyuridine was added in the medium to inhibit growth of fibroblasts. Four days of cardiomyocytes were serum-starved overnight. For hypoxia, the culture media was replaced by low glucose and serum free DMEM balanced in 5%CO2/95%N2 and cardiomyocytes were then exposed to hypoxia for 12 hours in a hypoxic incubator or a hypoxic chamber. For reoxygenation, the solution was changed to NCS-DMEM under normal atmosphere for 4 h.Result:Myocardial cells were confluent and spontaneously beating at the frequency of 90-150 beats/min. Trypan blue test confirmed that the survival rate of cardiomyocytes was 95.5%. The rate of specificα-sarcomeric actin positive cells was over 95% visualized by immunofluorescence staining. Cell shrinkage and reduction of frequency and activity of cardiomyocyte spontaneous contractions were observed in hypoxia/reoxygenation cardiomyocytes. The viabilities of cardiomyocytes in the all the H/R groups were reduced in a time-dependent manner. In hypoxia for 3 hours, 6 hours and reoxygenation for 4 hours group, the damages of cardiomyocytes were not obvious. But in hypoxia for more than 12 hours group, the necrotic cells were floating in the medium.Conclusion:The primary cultured cardiomyocytes in our experiment were of high viability and high purity. The established hypoxia/reoxygenation model of cardiomyocytes was simple and reliable.Section 2 Propofol postconditioning protects against cardiomyocyte apoptosis in hypoxia/reoxygenation injury by suppressing NF-κB translocation via ERK MAPK pathwayObjective:Propofol postconditioning (P-PostC) has cardioprotective effects against I/R injury of the heart, which is associated with inhibition of mitochondrial permeability transition pore (MPTP) opening. But few studies have been conducted to investigate the effect of propofol on the ERK pathway and to determine whether it could provide postconditioning to ischemic myocardial injury as well. We hypothesized that P-PostC exerts cytoprotective action through the activation and regulation of NF-κB via ERK MAPK signal transduction pathway in myocytes. The purpose of this study was to determine that propofol postconditioning is cardioprotective and to determine the underlying molecular mechanism. Methods:Primary cultured neonatal rat cardiomyocytes were exposed to 12 h of hypoxia followed by 4 h of reoxygenation (H/R) and postconditioned by different concentrations of propofol at the onset of reperfusion with and without specific inhibitors of mitogen-activated protein kinases (MAPKs). Cell apoptosis and the generation of intracellular reactive oxygen species (ROS) were measured using FACScalibur flow cytometric analysis ERK1/2 phosphorylation and nuclear factor-kappaB (NF-κB) translocation were determined by western blot and immunofluorescence, respectively.Results:Propofol postconditioning enhanced cell viability and reduced apoptosis compared with H/R only cardiomyocytes. Meanwhile, the present study examined the involvement of ROS in the effects of P-PostC in cardiomyocyte exposure to H/R. Fluorescence intensity was measured after postconditioning with increasing concentrations of propofol on cardiomyocytes exposed to H/R. P-PostC reduced mean DCF fluorescence markedly in a concentration-dependent manner compared with the H/R group. The cell viability and apoptosis assays showed that the optimal concentration of propofol for P-PostC was 50μM. Alteration in activity of ERK 1/2 was determined by western blotting of total and phosphorylated ERK 1/2 proteins. Propofol postconditioning stimulated expression of phosphor-ERKs. H/R markedly induced p65 nuclear factor-κB (NF-κB) nuclear translocation in cardiomyocytes, while propofol postconditioning significantly suppressed the H/R-primed NF-κB translocation. Moreover, addition of the MAP kinase kinase 1 (MEK1) inhibitor U0126 into cardiomyocytes 30 min before H/R eliminated the cardioprotection of propofol postconditioning.Conclusions:There are three principal findings of these studies. First, the application of P-PostC at the onset of reoxygenation after hypoxia significantly increased cardiomyocyte viability and reduced the proportion of early cell apoptosis. Consistent with these changes, P-PostC decreased the intracellular ROS generation at the optimum propofol concentration. Second, the anti-apoptotic effect of P-PostC in isolated cardiomyocytes was probably related to activation of ERK MAPK and the repression of NF-κB nucleus translocation. And third, the ERK pathway inhibitor U0126 blocked the inhibiting effect of P-PostC on cell apoptosis and NF-κB translocation, suggesting a key role of ERK MAPK in P-PostC. Together, these findings indicate that P-PostC stimulated myocyte endogenous cardioprotection against H/R injury through pro-survival signal transduction pathways. Section 3 Propofol Postconditioning Protects Heart-derived H9c2 Cells from Hypoxia/reoxygenation Injury via Autophagy involving the SAPK/JNK PathwayObjective:There is increasing evidence that the induction of autophagy is associated with ischemia/reperfusion (I/R) injury. The progress of autophagy can be activated in cardiomyocytes during I/R, which may not only lead to programmed cell death, but also have cardioprotection to some extent. Our earlier research found that propofol postconditioning showed protective effects on H/R-induced apoptosis in cardiac cells. However, the mechanism of propofol postconditioning aganst I/R injury has not yet been fully elucidated. We hypothesized that propofol might induce autophagy for the protection of the myocardium. To test this hypothesis, we examined the effects of propofol postconditioning at different doses on the induction of the autophagy, and discuss the potential regulation mechanisms and signaling pathways of autophagy.Methods:The heart-derived H9c2 cells were exposed to 12 h of hypoxia followed by 4 h of reoxygenation (H/R) and postconditioned by different concentrations of propofol at the onset of reperfusion. The activity of cardiomyocytes and cell apoptosis were measured for evaluating the degree of cardiomyocytes H/R injury. The induction of autophagy in H/R and propofol postconditioning treated myocytes was detected by western blot and immunofluorescence. Futhermore, the activation of JNK in autophagy induced by propofol postconditioning was also determined by western blot with or without SP600125, the inhibitor of JNK.Results:Propofol postconditioning markedly stimulated the induction of autophagy in H9c2 cardiac myoblast cells. The induction of autophagy was confirmed by the increased protein expression of LC3-II and the enhanced ratio of LC3-II/LC3-I. The expression of LC3-II was a little enhanced in H/R H9c2 cells, furthermore, propofol postconditioning (12.5-100μM) group significantly increased the expression of LC3-II in a dose-dependent manner compared with H/R cells. The inhibition of autophagy by 3-MA diminished the cardioprotection of propofol postconditioning. Propofol postconditioning mediated autophagic induction was negatively correlated with the induction of apoptosis. when autophagy was highly induced in propofol postconditioning group, the apoptosis was less than the H/R group in which autophagy was activated slightly. When the autophagy was reduced in 3-MA-treated cardiomyocytes, the percentage of apoptosis was increased compared with P-PostC group. Furthermore, we found that propofol postconditioning induced autophagy for the protection of myocardium through the activation of SAPK/JNK survival signal pathways. H/R activated the phosphorylation of JNK and propofol postconditioning further stimulated the phosphorylation of JNK in a dose-dependent manner in H9c2 cell lines. JNK-specific inhibitor could block autophagy induced by propofol postconditioning.Conclusions:In this study, we showed that propofol postconditioning significantly alleviated the cardiomyocytes H/R injury and reduced the percentage of apoptotic cells induced by H/R. These results were consistent with our previous research. Additionally, the results of our study confirmed for the first time that propofol postconditioning induced cell survival through the induction of autophagy in H9c2 cardiac myoblast cells, and the autophagy in part was mediated through the activation of SAPK/JNK survival pathway. Our research not only brings an explanation for the induction of autophagy in response to H/R injury, but also provides a novel mechanism for the endogenous defensive cardioprotection activated by porpofol postconditioning.