Research On The Molecular Mechanisms Of FFA-induced H9c2 Cell Apoptosis

Backgroud and ObjectiveMetabolic syndrome (MetS, MS) is a metabolic disorder accompanied by obesity, insulin resistance, type 2 diabetes and dyslipidemia. Patients suffering from MS have higher incidence of cardiovascular disease, compared with healthy people. Free fatty acids (FFAs) were reported to be closely related to MS, and excessive FFA was usually the cause of diverse diseases such as obesity, insulin resistance and type 2 diabetes. Saturated fatty acids (SFA), is considered to be one of the major pathogenic factor. Palmitic acid (PA), a type of SFA, can induce apoptosis in a variety of cells, including in cardiomyocytes. However, the specific molecular mechanisms of PA-induced cardiomyocyte apoptosis remains unknown.β-arrestin 2, belonging to the arrestins family, is not only involved in the desensitization, endocytosis and ubiquitin-mediated degradation of the seven transmembrane receptors (7TMRs), but also acts as a multi-functional scaffold and adapter which participates in various signal pathways. However, the role of β-arresitn 2 in cell apoptosis dependents on its downstream protein, and is also influenced by the cell types and experimental method. Until now, whether or not β-arrestin 2 take part in the PA-induced cardiomyocyte apoptosis is still unreported.The Akt/GSK-3β signaling pathway is widely involved in cell proliferation and apoptosis. It was reported that β-arrestin 2 could recruit c-Src and Akt thereby facilitating the phosphorylation and activation of Akt. Akt then phosphorylates the ninth serine site of GSK-3β and down-regulates its kinase activity which results in the promotion of proliferation and suppression of apoptosis. However, whether the Akt/GSK-3β signaling was involved in PA-induced cell apoptosis is still unknown.To solve the above-mentioned problem, H9c2 cells were imposed to high concentration of PA which imitating the excessive level of FFA in vivo to investigate the molecular mechanisms of FFA-induced cardiomyocytes apoptosis. Our research will provide novel evidence and clinical target for cardiovascular disease therapy.Results1. Palmitic acid induced H9c2 apoptosis in a time and concentration gradient manner.To certificating the infulence of PA on cardiomyocytes, firstly, we performed the SRB experiment and revealed the growth inhibitory effect of PA on H9c2 cells. We then confirmed that PA can induce nuclear condensation and fragmentation in H9c2 apoptosis by use of Hoechst 33258 staining. Finally, western blot and flow cytometry results showed that PA induced H9c2 apoptosis in a time and concentration gradient dependent manner.2. Palmitic acid induced the down-regulation of β-arrestin 2 expression in a time and concentration gradient dependent way. Thus we speculated that β-arrestin 2 may have an inhibitory effect on H9c2 cell apoptosis. However, this conclusion should be further confirmed by additional experiments.To study the function of β-arrestin 2 in PA induced H9c2 apoptosis, we carried out the Western Blot experiment. The results showed that PA may down-regulate the protein level of β-arrestin 2 in a time and concentration gradient dependent fashion. So we speculated that β-arrestin 2 may have an inhibitory effect in PA-induced H9c2 apoptosis. However, overexpression of exogenous P-arrestin 2 promoted PA-induced cell apoptosis. And immunofluorescence experiment revealed the different cellular distribution between exogenous and endogenous β-arrestin 2. Therefore, the role of β-arrestin 2 in PA induced H9c2 apoptosis may be intricate and more experiment need to be done to provide explanation.3. The mRNA level of β-arrestin 2 was not changed in PA induced H9c2 apoptosis, indicating that the down-regulation of the protein may occur during the translation process.The gene expression of eukaryote is complicated and could be modulated in several levels including transcription, post-transcription and translation. The qRT-PCR result showed no obvious changes in the mRNA level of β-arrestin 2. This illustrated that the down-regulation of β-arrestin 2 did not take place in the transcription level, but may happen during the translation process.4. Inhibiting the activity of GSK-3β significantly suppressed PA-induced apoptosis.Western Blot results showed that p-Akt (Ser473) was significantly down-regulated, and the protein level of p-GSK-3p (ser9) was not changed. LiCl, an inhibitor of GSK-3β, significantly suppressed PA induced apoptosis. And interference of GSK-3P by siRNA confirmed this result.Conclusions1. PA induced H9c2 apoptosis and down-regulated p-arrestin 2 protein level in a time and concentration gradient dependent manner. During this progress, the mRNA level of β-arrestin 2 was not changed.2. Inhibition of GSK-3β by its inhibitor LiCl or RNA interference significantly suppressed PA-induced H9c2 apoptosis, indicating the pro-apoptotic effect of GSK-3β.