Mechanism Of Palmitate Induced The H9c2 Cardiac Cells Apoptosis

Recently, Lipid accumulation in the heart has been observed under conditions of elevated plasma free fatty acids, such as Type 2 diabetes mellitus and is linked with cardiomyocyte apoptosis, left ventricular contractile dysfunction. Fatty acids are a principal source of energy for the heart and the metabolism of fatty acids by the heart has been the subject of detailed reviews. Fatty acids, including palmitate, are present in high levels in the blood of patients following acute myocardial infarction and are responsible for increasing the extent and severity of myocardial injury.Cardiomyocytes are terminally differentiated cells whose inability to reproduce underscores the seriousness of the loss of even a small amount of cardiac muscle cells in the situation of myocardial infarction, which is accompanied by high circulating concentrations of palmitate. The nature of palmitate-induced cardiomyocyte death, however, has not been well characterized. Recently, palmitate-induced apoptosis has been demonstrated in murine hematopoietic cell lines, embryonic chick astrocytes and neuronal cells, pancreatic beta cells and neonatal rat cardiomyocytes. Yet fatty acids have also been implicated in the induction of necrosis. Thus, it is unclear to what extent palmitate-induced cell death should be ascribed to the cascade of caspases, endonucleases and other factors that characterize the genetic program of apoptotic cell death.The mechanisms underlying palmitate-induced cell death are incompletely understood. Palmitate is incorporated into de novo synthesis of ceramide, a lipid-signaling molecule implicated in the induction of apoptosis so that excess palmitate may induce cell death through increased intracellular ceramide concentration. An equally attractive hypothesis, that may be especially relevant for the heart, is that the adverse effect of excess fatty acids is due to other metabolic factors, so called toxic intermediates of fatty acid metabolism.To more completely define the nature and mechanism underlying palmitate-induced cell death, cardiomyocytes were cultured from embryonic rat heart and were treated with palmitate. Part I Effect of Pamitate on H9c2 myocardial cell apoptosis and activity of NADPH oxidaseObjectiveTo investigate the effect of palmitate on apoptosis concentration and time and the activity of NADPH oxidase in the H9c2 myocardial cell.Methods1. To find ideal concentration of palmitate the cardiomyocytes were divided into four groups: control group, 0.2mM palmitate group, 0.5mM palmitate group, 0.8mM palmitate group, and 1.2mM palmitate group. We examined the changes cell viability, cardiomyocyte apoptosis rate, and the activity of caspase-3 in the cardiomyocytes.2. To observe time-effect of cell injury induced by palmitate, the cardiomyocytes were divided into five groups: 0h group, 4h group, 8h group, 16h group, 24h group and 48h. group. Cells were treated according to the instruction manual of AnnexinV-FITC KIT, then to perform FACS to measure the rate of apoptosis.3. To observe the effect of palmitate on the NADPH oxidase subunit p47phox mRNA and protein level.Results1. A dose-dependent relation between the concentration of palmitate and H9c2 cardiomyocyte injury was observed. The H9c2 cardiomyocyte apoptosis rate was increased significantly, while the cellular viability was decreased in the 0.5mM group, this indicated that 0.5mmol/L palmitate was the ideal concentration to induc cardiomyocyte apoptosis.2. A significant time-dependent relation between palmitate and cardiomyocyte injury was observed. H9c2 myocardial cell apoptosis rate was increased greatly after 16h treatment of palmitate .3. p47phox mRNA and protein level was increased significantly campared with the control group after 24h treatment of palmitate.Conclusions1. H9c2 myocardial cell can be induced to injury and apoptosis by palmitate, and appears a significant time-dependent.2. Palmitate induced significantly increase of p47phox mRNA and protein level in H9c2 myocardial cell. Part II The effect of apocynin on the palmitate treated H9c2 cardiomyocytesObjectiveTo investigate whether the NADPH oxidase involved in the process of the palmitate induced H9c2 myocardial cell apoptosis.MethodsThe experiment were divided into four groups: control group, apocynin group, palmitate group, and palmitate+apocynin group. We observed the ROS production using fluorescent probe, the P47phox mRNA was measured by RT-PCR. the Bcl-2,Bax and caspase-3 protein level were measured by western- blot.Results1. The effect of apocynin on the palmitate treated H9c2 cardiomyocytes NADPH oxidase P47phox mRNA and protein level: compared with the control, P47phox mRNA and protein level increased significantly in the palmitate group. (P<0.05). the apocynin can discrease the P47phox mRNA and protein level significantly(P<0.05).2. The effect of apocynin on ROS production in the palmitate treated H9c2 cardiomyocytes: compared with the control,the ROS production significantly increased in palmitate group,apocynin can discrease the ROS production. The positive control is the best power in the ROS production.3. Compared with the control,the caspase-3 activity, caspase-3 and Bax protein level were significantly increased in the palmitate group, and the Bcl-2 protein level was discrease in the palmitate group, apocynin can abatement the above change significantly(P<0.05).ConclusionsPalmitate can increased the NADPH oxidase P47phox mRNA and protein level, and increased the ROS formation in the H9c2 myocardial cell, can led to the decrease of Bcl-2 and increase of Bax protein level, the caspase-3 activity also increased significantly. Apocynin can inhibit these changment. Part III The effect of MAPK and NF-κB on the palmitate treated H9c2 cardiomyocytes apoptosisObjectiveTo investigate the effect of MAPK and NF-κB on the palmitate induced H9c2 myocardial cell apoptosis.MethodsFirst, the experiment were divided into 4 groups: control group, apocynin group, palmitate group and palmitate+apocynin group. The p65, p-p38,p-ERK,p-JNK were measured by western-blotting.Then, the experiment were divided into 5 groups: control group, palmitate group, palmitate + SB203508 group(pal+p38i), palmitate + PD98059 group(pal+ERKi), palmitate + SP600125 group ( pal+JNKi ) . The Bcl-2, Bax were measured by western-blotting. The caspase-3 activity was detected by Enzyme Standardization instrument.Results1. Compared with the control, the P65 in nucleus protein was increased significantly in the palmitate group (P<0.05). the apocynin can discrease the P65 protein level significantly(P<0.05).2. Palmitate can induce the p-p38,p-JNK protein level significantly(P<0.05). apocynin can discrease the p-p38,p-JNK protein level significantly(P<0.05).the p-ERK has no evident changement.3. Palmitate decreased the Bcl-2 protein level and increased Bax significantly(P<0.05). the JNK inhibitor and p38 inhibitor inhibited the above changes. And only the JNK inhibitor had the statistical significanc(eP<0.05). The inhibitor of p38, JNK and ERK decreased the Bax protein level significantly compared with the palmitate group(P<0.05).4. Compared with the control, the caspase-3 activity increased significantly in the pal group, pal+p38i group and pal+JNKi group(sP<0.05). compared with the palmitate group, caspase-3 activity decreased in the pal+p38i group and pal+ERKi group, but the decrease has no statistical significance(P>0.05),and the caspase-3 activity significantly decreased in the pal+JNKi group(P<0.05).ConclusionsThe increase of p47phox mRNA and protein level can induce the NADPH oxidase activity, which can lead to oxidative stress, these maybe the upstream mechanism of palmitate induced H9c2 myocardial cell apoptosis. The NF-κB and p38, JNK MAPK activation may participate the palmitate induced H9c2 myocardial cell apoptosis, and lead to the decrease of Bcl-2 and increase of Bax protein level, and ultimately generate apoptosis.