Septin4 participates in hypoxia-induced cardiomyocytes injury
Given that no study has evaluated the effect of hypoxia-induced cardiomyocytes injury on Septin4 expression so far, we subjected H9c2 cells to hypoxia treatment for 0, 6, 12 and 24 hours to establish hypoxia‐induced injury. Successful establishment of the hypoxic model was confirmed by the observation of an obviously decreased cells viability (Additional file 1 Supplemental Fig. 1c) and a significantly increased cells apoptosis rate (Additional file 1 Supplemental Fig. 1d, e). By western blot analysis, we found obviously increased expression levels of Septin4, HIF-1α and cleaved caspase3 with the prolonging of hypoxic time (Additional file 1 Supplemental Fig. 1a, b). Thus, Septin4 may play a role in the hypoxia-induced injury model, which was discussed in the following experiments on overexpression and knockdown of Septin4 in H9c2 cells.
Cell viability assay and flow cytometry analysis showed that overexpression of Septin4 significantly aggravated hypoxia-induced H9c2 apoptosis (Fig. 1c and Fig. 1d, e), while knockdown of Septin4 by using the identified most efficacious Septin4 siRNA sequence (Fig. 1f) significantly alleviated hypoxia-induced H9c2 apoptosis (Fig. 1h and Fig. 1i, j). In addition, overexpression of Septin4 significantly increased hypoxia-induced H9c2 cells apoptosis maker cleaved caspase3 protein expression (Fig. 1a, b), while knockdown of Septin4 significantly decreased cleaved caspase3 (Fig. 1f, g), and HIF-1α showed an opposite trend.
Taken as a whole, these results demonstrated that Septin4 played a negative role in cardiomyocytes survival under hypoxia.
Septin4 participates in hypoxia-induced cardiomyocytes injury by down-regulating expression levels of HIF-1α
The increase in HIF-1α expression levels by hypoxia induces HIF-1 activity, which is quite important in protecting cardiomyocytes from ischemic heart disease [22-24]. We next explored the role of Septin4 in the expression changes of HIF-1α in H9c2 cells under hypoxia. First, we found that the expression levels of both Septin4 and HIF-1α increased with the prolonging hypoxic time (Fig. 1a, b). Second, we evaluated the effect of Septin4 on HIF-1α in hypoxic H9c2 cells and found that the expression levels of HIF-1α significantly increased from hypoxic H9c2 cells with stably silenced of Septin4, while markedly decreased after the overexpression of Septin4 in H9c2 cells on the basis of stably silenced of Septin4 (Fig. 2a, b).
At the same time, we observed an opposite expression trend of the apoptosis maker cleaved caspase3 compared to that of HIF-1α in each group (Fig. 2a, b). In addition, cell viability assay (Fig. 2c) and flow cytometry analysis (Fig. 2d, e) showed that the hypoxia-induced apoptosis was relieved with stably silenced of Septin4, while was aggravated with overexpression of Septin4 on the basis of stably silenced of Septin4.
These findings suggested that Septin4 was involved in hypoxia-induced cardiomyocytes injury by down-regulating HIF-1α.
Septin4 interacts with HIF-1α mainly through its GTPase domain
To explore the mechanism of Septin4 in cardiomyocytes injury, we investigated whether Septin4 and HIF-1α actually interact, which then affected the injury to H9c2 cells. First, results of endogenous co-immunoprecipitation demonstrated that Septin4 is a novel protein interacting with HIF-1α (Fig. 3a, b). Next, we found that under hypoxic stimulation, the binding effect of Septin4 and HIF-1α was enhanced (Fig. 3c, d). These findings suggested that Septin4 participated in hypoxia-induced cardiomyocytes injury by interacting with HIF-1α.
Furthermore, to explore which domain of Septin4 bound with HIF-1α, four truncated plasmids of Septin4 were produced according to their functional domains (Fig. 3e). We then overexpressed full-length Septin4 or various truncated mutants of it in 293T cells for co-immunoprecipitation, the results demonstrated that Septin4 mainly interacted with HIF-1α via its GTPase domain (Fig. 3f).
Septin4 mediates the proteasome degradation of HIF-1α
To further to confirm whether Septin4 affected the expression of HIF-1α, Flag-Septin4 was transfected in H9c2 cells with an increasing amount. We found that the expression of Septin4 increased while the expression of HIF-1α decreased (Fig. 4a, b). What’s more, the knockdown of Septin4 with the three Septin4 siRNA sequences resulted in an obviously decreased expression of Septin4 but a significantly increased expression of HIF-1α (Fig. 4c, d).
The above two findings suggested that Septin4 may mediate the degradation of HIF-1α in some way. Therefore, we investigated whether septin4 affected the stability of HIF-1α by the use of protein synthesis inhibitor CHX to inhibit transcription activity or proteasome inhibitor MG132 to inhibit the proteasome degradation activity.
In the CHX assay, transfection of Flag-Septin4 in H9c2 cells led to an obvious reduction in the expression of endogenous HIF-1α compared to the empty vector group (Fig. 4e, f), while in the MG132 assay with Flag-Septin4 transfected in H9c2 cells, the accumulation of HIF-1α was more faster and marked than the empty vector group (Fig. 4g, h), suggesting that Septin4 reduced the expression of HIF-1α by the proteasome degradation pathway.
Septin4 mediates the polyubiquitination of HIF-1α
Given that HIF-1α commonly degraded by proteasome[25], we then transfected Flag-Septin4 in H9c2 cells whether followed by the treatment of MG132 or not, and found that the interaction between Septin4 and HIF-1α was enhanced by MG132 (Fig. 5a). The results of endogenous co-immunoprecipitation assay also showed a MG132 enhanced interaction between Septin4 and HIF-1α (Fig. 5b).
Furthermore, after co-transfecting Flag-Septin4 and HA-UB in H9c2 cells followed by the treatment of MG132, we found that Septin4 significantly increased HIF-1α ubiquitination levels by co-immunoprecipitation assay (Fig. 5c). What’s more, Septin4 silencing was found to reduce HIF-1α ubiquitination levels in Septin4 knockdown H9c2 cells after overexpression of HA-UB (Fig. 5d). These results suggested that Septin4-mediated degradation of HIF-1α by the proteasome pathway was dependent on polyubiquitination.
Septin4 promotes the ubiquitin-proteasome degradation of HIF-1α by mobilizing von Hippel-Lindau protein (VHL)
The existence of VHL in an E3 ubiquitin ligase complex, which can mediate the rapid proteasome degradation of HIF-1α, makes HIF-1α levels low on normoxia [26, 27]. We next bought HIF-PHDs inhibitor BAY85-3934, which negatively affect the VHL- HIF-1α interaction and then clarified whether Septin4 mediates the ubiquitin-proteasome degradation of HIF-1α via VHL.
First, we evaluated the effect of Septin4 and BAY85-3934 on HIF-1α in hypoxic H9c2 cells and found that the expression levels of HIF-1α significantly decreased under hypoxia after the overexpression of Septin4, while BAY85-3934 can rescue this result (Fig. 6a, b). Second, to explain the phenomenon, we then transfected Flag-Septin4 in H9c2 cells and found that the interaction between HIF-1α and VHL was enhanced (Fig. 6c) while Septin4 silencing was found to reduce this binding effect. (Fig. 6d). Lastly, after co-transfecting Flag-Septin4 and HA-UB in H9c2 cells, we found that BAY85-3934 significantly decreased the ubiquitination levels of HIF-1α (Fig. 6e).
These results confirmed that Septin4 mediated HIF-1α polyubiquitination via VHL and promoted HIF-1α degradation by the proteasome pathway (Fig. 6f).