Nuclear respiratory factor-1 promotes CFLAR transcription in H9C2 cardiomyocytes, protecting them against hypoxia-induced apoptosis

Background Inhibition of hypoxia-induced apoptosis in cardiomyocytes is crucial for heart failure treatment. Previous research suggests that nuclear respiratory factor-1 (NRF-1) protects hypoxic cardiomyocytes against apoptosis. In the present study, we hypothesized that NRF-1 regulates the expression of Caspase 8 and FADD-like apoptosis regulator (CFLAR) and thus contributes to the regulation of apoptosis in hypoxic cardiomyocytes. Methods and Results Chromatin immunoprecipitation (ChIP) and Dual-Glo luciferase assays confirmed that NRF-1 binds to the Cflar gene promoter and regulates its transcriptional activity. Furthermore, the interactions between NRF-1 and CFLAR and their effects on H9C2 cardiomyocytes apoptosis were tested under hypoxic conditions. Using the BioTek imaging system, we showed that CFLAR siRNA reversed the effects of NRF-1 overexpression on cell growth and death; CFLAR siRNA markedly increased the apoptosis rates and the Caspase-3 and Caspase-8 activities in NRF-1-overexpressing cells. Conversely, in NRF-1-knockdown cells, CFLAR overexpression suppressed hypoxia-induced apoptosis. Western blot analysis showed that NRF-1-mediated regulation of CFLAR expression primarily influences the protein levels of cleaved Caspase-8 and tBid, without any significant differences in Bid, Bcl-2, and Bax expression. Conclusions we demonstrated that NRF-1 directly regulates CFLAR expression, thereby inhibiting the death receptor pathway, and ultimately, protects H9C2 cardiomyocytes from hypoxia-induced apoptosis. Our findings will provide new insights into the molecular mechanisms underlying the protective role of NRF-1 and support its potential to serve as a therapeutic target for ameliorating heart failure.