Western blot results cardiomyocytes7/27/2023 ![]() One-way ANOVA with Tukey’s multiple comparisons test ( B, C, E, G, J, L, and O) two-way ANOVA with Tukey’s multiple comparisons test ( H, M).Ĭell viability assay and flow cytometry analysis showed that overexpression of Septin4 significantly aggravated hypoxia-induced H9c2 apoptosis (Fig. All data are presented as mean ± SD of three repeated experiments ***<0.001, **<0.01, *<0.05. N, O Flow cytometry analysis of H9c2 cells’ apoptosis rate after transfected with si-Con or si-Septin4 under hypoxia for 24 h. M Cell viability assay of H9c2 cells’ viability after transfected with si-Con or si-Septin4 under hypoxia for 24 h. K, L Western blot analysis of the expression levels of HIF-1α, Septin4, and cleaved caspases3 in H9c2 cells transfected with si-Con or si-Septin4 under hypoxia for 24 h. I, J Flow cytometry analysis of H9c2 cells’ apoptosis rate after transfected with vector empty or Flag-Septin4 under hypoxia for 24 h. H Cell viability assay of H9c2 cells’ viability after transfected with vector empty or Flag-Septin4 under hypoxia for 24 h. F, G Western blot analysis of the expression levels of HIF-1α, Septin4, and cleaved caspases3 in H9c2 cells transfected with vector empty or Flag-Septin4 under hypoxia for 24 h. ![]() ![]() D, E Flow cytometry analysis of H9c2 cells’ apoptosis rate with the prolonging of hypoxic time. C Cell viability assay of H9c2 cells’ viability with the prolonging of hypoxic time. Finally, by reducing HIF-1α levels, Septin4 aggravated the hypoxia-induced cardiomyocytes apoptosis.Ī, B Western blot analysis of the expression levels of Septin4, HIF-1α, and cleaved caspases3 with the prolonging of hypoxic time. The current study found the similar mechanism for the first time in cardiomyocytes that Septin4 can enhance the binding between HIF-1α and the E3 ubiquitin ligase VHL, and then Septin4 reduces the expression levels of the cardio-protective factor HIF-1α through UPS pathway. In fact, previous studies have reported that Septin4 can promote the ubiquitination and degradation of some proteins by enhancing the interaction between these proteins and some E3 ubiquitin ligases. In addition, although it cannot be categorized as any key enzymes in the UPS, Septin4 aggravated the hypoxia-induced cardiomyocytes by reducing HIF-1α levels through the UPS pathway. Here in this study, we found that the cardio-protective factor HIF-1α is a novel interacting protein with Septin4 via Septin4-GTPase domain in hypoxia-induced cardiomyocytes apoptosis. However, the role of Septin4 in hypoxia-induced cardiomyocytes and whether there is another novel substrate for Septin4 in cardiomyocytes are not yet known. ) are also found to be the substrate of Septin4 to promote apoptosis in some tumor cells. Septin4, a protein localized at mitochondrion, is shown to be a proapoptotic protein mainly by promoting the degradation of XIAP, the only IAP protein that can directly inhibit caspases. In addition, researchers have found that the VHL-mediated degradation of HIF-1α can be enhanced by some molecules such as VBP1 and SSAT2. The mechanism has been detailed described that after hydroxylated by prolyl hydroxylase domain enzymes, HIF-1α forms a recognizing site for VHL to bind with, then the binding between VHL and HIF-1α initiate the polyubiquitination and degradation of HIF-1α through UPS pathway. Under normoxia, HIF-1α could hardly be detected in cells, which is due to the existence of VHL, a recognizing subunit of an E3 ubiquitin ligase complex that mediates the degradation of HIF-1α through the UPS (ubiquitination-proteasome system). Under hypoxia, the rapid degradation of HIF-1α in living cells is inhibited, and HIF-1α will subsequently accumulate to a certain level to protect the cardiomyocytes from hypoxia-induced apoptosis mainly by up-regulating the anaerobic process, down-regulating the aerobic process, and restoring the normal delivery of oxygen. HIF-1 plays an important role in mediating cells to adapt to hypoxia, and HIF-1α, the oxygen regulating subunit of HIF-1, is indispensable in this process. Myocardial ischemia can lead to insufficient oxygen supply to the myocardium, which is also called myocardial hypoxia myocardial hypoxia can result in cardiomyocytes apoptosis and necrosis, which can further develop into myocardial infarction and endanger the life of the patient.
0 Comments
Leave a Reply. |