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

Nuclear respiratory factor-1 promotes CFLAR transcription in H9C2 cardiomyocytes, protecting them against hypoxia-induced apoptosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Nuclear respiratory factor-1 promotes CFLAR transcription in H9C2 cardiomyocytes, protecting them against hypoxia-induced apoptosis Hui Li, Yunxia Ma, Junliang Li, Siyu Hou, Hui Song, Yazhou Zhu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6499729/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jun, 2025 Read the published version in Molecular Biology Reports → Version 1 posted 6 You are reading this latest preprint version Abstract 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. NRF-1 CFLAR hypoxia H9C2 cardiomyocytes apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Heart Failure (HF) occurs at the end stage of various heart diseases. It is characterized by high morbidity, mortality, diminished functional capacity, poor quality of life, and a substantial economic and emotional burden for society and the healthcare system [ 1 ] . With the increase in the aging population, fast-paced lifestyles, and rising work-related stress worldwide, the prevalence of HF increases annually [ 2 ] . The burden posed by HF on global healthcare costs is a growing concern. Although medications can alleviate the symptoms, and delay the progression of HF, their prolonged use may lead to drug resistance and side effects [ 3 , 4 ] . Currently, heart transplantation is the most effective treatment for HF; however, its availability is limited by donor shortage and the risk of transplant rejection [ 5 , 6 ] . Gene therapy, powered by the advancements in gene-editing and -delivery technologies, holds promise to become a novel treatment method for HF. Gene therapy provides long-term therapeutic benefits with minimal side effects [ 7 – 9 ] . However, due to the challenges associated with identifying suitable target genes, gene therapy for HF is still in the early stages of development. The pathogenesis of HF is complex, but numerous studies have reported that ischemia- and hypoxia-induced cardiomyocyte apoptosis play critical roles in the extensive loss of cardiomyocytes, ultimately leading to HF [ 10 , 11 ] . Therefore, elucidating the molecular mechanisms underlying cardiomyocyte apoptosis in HF and identifying effective therapeutic target genes are essential for advancing the clinical application of gene therapy in HF. Previous studies have highlighted that nuclear respiratory factor-1 (NRF-1) plays a protective role against apoptosis in H9C2 cardiomyocytes under both chemical hypoxia conditions induced by CoCl 2 and physical hypoxia with 1% O 2 [ 12 – 14 ] . NRF-1 is a key transcription factor that regulates cellular energy metabolism, mitochondrial biogenesis, oxidative stress responses, cell growth, autophagy, and unfolded protein responses [ 15 – 17 ] . We also found that in H9C2 cells, under conditions of hypoxia, NRF-1 enhances cellular energy metabolism, stabilizes the mitochondrial membrane potential, inhibited reactive oxygen species (ROS) production, promotes mitophagy, and inhibits apoptosis via the death receptor and mitochondrial pathways [ 12 , 18 ] . Thus, NRF-1 shows promise as a therapeutic target for the treatment of HF. Despite these findings, the mechanisms whereby NRF-1 inhibits apoptosis in cardiomyocytes remain unclear. Caspase 8 and FADD-like apoptosis regulator (CFLAR), also known as FLIP (FADD-like IL-1beta-converting enzyme-inhibitory protein), is a key anti-apoptotic molecule [ 19 ] . Although CFLAR mRNA has multiple splice variants, it primarily exists in two protein forms: the long isoform (CFLAR L ) and the short isoform (CFLAR S ) [ 20 ] . CFLAR inhibits Caspase-8 activation by preventing its dimerization in the death-inducing signaling complex (DISC), thereby modulating apoptosis via death receptor pathways [ 19 , 21 ] . Conversely, activated Caspase-8 cleaves Bid, a proapoptotic molecule in the cytoplasm, resulting in tBid formation. The -COOH terminus of tBid translocates to the outer mitochondrial membrane and promotes Bax and Bad dimerization, which, in turn, initiates the mitochondrial apoptotic pathway [ 22 ] . Our previous research revealed that both overexpression and knockdown of NRF-1 alter the gene and protein expression of CFLAR [ 12 ] . Moreover, our previous unpublished data obtained using CUT&Tag indicated that the promoter region of Cflar gene contains an NRF-1 transcription factor-binding site (TFBS), suggesting that CFLAR may be one of the target molecules regulated by NRF-1 to protect H9C2 cells from hypoxia-induced apoptosis. However, the roles of NRF-1 in CFLAR transcription and the mechanisms underlying its role remain unclear. In the present study, we investigated the interaction between NRF-1 and CFLAR in H9C2 cells under hypoxic conditions and analyzed their regulatory effects on cardiomyocyte apoptosis. To the best of our knowledge, our study is the first to demonstrate that NRF-1 specifically targets and regulates CFLAR transcription; our findings will provide new insights into potential therapeutic strategies for HF. 2. Materials and Methods 2.1 Cell culture and hypoxia treatment The H9C2 and HEK 293 T cells were originally obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China) and have since maintained in our laboratory. The NRF-1 overexpressing H9C2 cell line (H9C2 + pCDH-NRF-1), the NRF-1 knockdown H9C2 cell line (H9C2 + sh-NRF-1), and their corresponding control cell line (H9C2 + pCDH-vector and H9C2 + sh-vector) generated following the methodologies previously reported by our group [ 12 , 13 ] . All cell lines were cultured in DMEM with high glucose (SH30243.01; HyClone), supplemented with 10% fetal bovine serum (900-108; Gemini Bio) and 1% penicillin-streptomycin (P1400; Solarbio), under standard conditions of 5% CO 2 , 90% humidity, and a temperature of 37°C. To create a hypoxic environment in vitro, the cells were transferred to a tri-gas incubator (Model 3131; Thermo Fisher Scientific) set to contain 1% O 2 , 5% CO 2 and 94% N 2 . 2.2 Predict TFBS of NRF-1 on Cflar gene The specific DNA sequence bound by the NRF-1 protein was identified through CUT&Tag sequencing. GIV software was utilized to analyze the results to locate peak sequences of the Cflar gene. The peak regions were extended by 200 bp in both upstream and downstream directions to extract the corresponding DNA sequences. Subsequently, the Motifs Genome.pl tool from HOMER software was employed to predict motifs within these sequences, which were subsequently matched against existing motif data in the JASPAR database to identify NRF-1 TFBS on Cflar gene. 2.3 ChIP and qPCR Chromatin immunoprecipitation References EZ CHIP Kit 22 Assays (17–371, Millipore) was used to detect the NRF-1 binding in the promoter region of Cflar gene. Following the manufacturer’s protocol, proteins were cross-linked to DNA by incubating 1 × 10 7 cells in 1% formaldehyde for 10 min at 37°C. The cell pellets were resuspended with 1 ml SDS Lysis Buffer which contains 5 µl Protease Inhibitor Cocktail II. To fragment the chromatin to 200–1000 bp, ultrasound it ten times (20 s pulse, 50 s rest) with an Ultrasonic Processor (SCIENTZ-950E, SCIENTZ). The ultrasound effect was detected using 1.5% agarose gel electrophoresis. Immunocomplexes were isolated from the remaining lysates using 1 µg of NRF-1 antibody. After elution, cross-linking removal, and purification, DNA was obtained and stored at -20℃. qPCR was performed on a 7500 system (Thermo, USA) using primers for the Cflar promoter: 5’-GTACGTGACTTGAGCGGTGT-3’(F), 5’-CTGCAGTCCTCCGTG CC-3’(R). The production size is 177 bp, including the predicted NRF-1 binding site. The result was also analyzed by 1.5% agarose gel electrophoresis. 2.4 Luciferase reporter assay The Cflar promoter sequence, spanning from − 1,000 to + 401 relative to the proposed transcriptional start site (TSS) of Cflar exon 1 (NC_086027.1), was cloned and subsequently substituted for the promoter region of firefly luciferase in the psiCHECK 2-Basic vector (Promega, USA). Based on the predicted binding sites of NRF-1, four additional reporter plasmids were constructed, each containing mutations at different positions (Fig. 1 ). 293T cells were seeded in 24-well plates (5×10 4 cells/well), and subsequently transfected with 50 ng of luciferase reporter plasmids and 50 ng of pCDH-NRF-1 plasmids. After 48 h of transfection, measuring the activity of luciferase by a dual-luciferase reporter assay kit (E2920, Promega). According to the manufacture’s protocols, removed the cell culture medium in the 24-well plate and added 500 µl detection working solution to each well, the cells were fully lysed for 15 min. Then, 100 µl of lysate was transferred into a light-tight whiteboard 96-well plate (avoiding bubbles) to detect firefly fluorescence. Subsequently, 50 µl of Stop reaction solution was added to each well and gently mixed while protecting from light for an additional 10 minutes to terminate firefly fluorescence. The fluorescence emitted by sea kidney was detected. The results were presented as the ratio of sea kidney fluorescence to firefly fluorescence for each hole, and the values of each experimental group were normalized and analyzed. 2.5 siRNA of CFLAR in NRF-1 overexpressing H9C2 cells The target sequences for CFLAR siRNA and Oligo sequences of negative control (siRNA-NC) were synthesized from Genepharma (Beijing, China). The two most effective siRNA and siRNA-NC sequences are as follows: siRNA-CFLAR-1 (F: 5’-CCUCCUGGAUUGUUUAAGUTT-3’; R: 5’-ACUUAAACAAUCCAGGAGGTT-3’); siRNA-CFLAR-2 (F: 5’GAGCCAGUGUGUGGAAUAUTT-3’; R: 5’-AUAU UCCACACACUGGCUCTT-3’); siRNA-NC (F: 5’-UUCUUCGAACGUGUCACGU TT-3’; R: 5’-ACGUGACACGUUCGGAGAATT-3’). The two siRNA and siRNA-NC were transfected into H9C2 + pCDH-NRF-1 cell line respectively using Lipofectamine 2000 (11668-027, Invitrogen) according to the manufacturer’s protocols. After 72 h of transfection, CFLAR protein expression levels were assessed, and follow-up hypoxia experiments were performed. 2.6 CFLAR overexpression in NRF-1 knockdown H9C2 cells To differentiate from the green fluorescence of the H9C2 + sh-NRF-1 and H9C2 + sh-vector cell lines, we constructed a CFLAR (NM_001033864.4) overexpression using the pLVX-IRES-mCherry lentiviral vector (BR023; Fenghui Biotech), which emits red fluorescence. Cells transfected with the empty vector (pLVX-vector) served as the control group. Virus packaging and cell infection were performed as previously described [ 12 ] . After puromycin selection, flow cytometry was used to isolate stably transfected cells based on red and green double-labeled fluorescence, ensuring a positive cell rate of over 95% in each experiment. Overexpression efficiency was evaluated by western blot analysis. The transfected cell lines were designated as sh-NRF-1 + pLVX-CFLAR, sh-NRF-1 + pLVX-vector, sh-vector + pLVX-CFLAR, and sh-vector + pLVX-vector for subsequent experiments. 2.7 Cell growth and death monitored by Bio-Tek multi-mode imaging system The same number of cells from each group were seeded in a 12-well plate. Hochest33342 was used to detect live cell numbers and PI was used to detect dead cell numbers. Following the instructions, the Hochest33342/PI dual staining reagent (KGA212, Keygen Biotech) was added to the culture medium and mixed before being distributed into each well. The cells were cultured in a Bio-Tek multi-mode cell imaging system under hypoxic condition (1% O2, 5% CO2, 94% N2) at 37°C using Gen5 software. Blue and red fluorescence of each well was measured every 30 minutes for 24 hours, and the results were collected for subsequent analysis. 2.8 TUNEL assay After 12 h of hypoxia treatment, cells from each group were fixed with 4% formaldehyde. Cell apoptosis was assessed using a TUNEL assay kit (C1082; Beyotime) in accordance with the manufacturer's protocol. Hematoxylin was used to stain the nuclei. The cells were photographed with a microscope (EVOS FL) and the apoptosis rate was determined by calculating the ratio of TUNEL-positive cells to the total cells counted in each field. Randomly selected three fields from each group to calculate an average. 2.9 Caspase − 8, -9 and − 3 activity Colorimetric assay kits for Caspase-8, -9 and − 3 (KGA 202, KGA 302, and KGA 402; KeyGEN, China) were employed to evaluate the activities of Caspase-8, -9 and − 3. Following a 12-hour hypoxia treatment, cells were lysed using the lysis buffer provided in the kit to extract protein. Equal volumes of protein (50 µl) were combined with the reaction buffer (50 µl) and incubated at 37°C in the dark for1 h. Absorbance was subsequently measured using a SkanIt@ Software microplate reader set to a wavelength of 405 nm. The activities of Caspase-8, -9 and − 3 are presented as fold changes compared with control values. 2.10 Western blot analysis After cells been exposed to hypoxia for 12 h, total proteins were extracted using a whole cell lysis assay kit (KGP250, KeyGEN). Equivalent protein quantities (30 µg) were subjected to 12% SDS-PAGE and subsequently transferred onto a nitrocellulose membrane (HATF00010; Millipore). Blocked the membrane with 5% BSA in TBST for 2 h. Incubated the membranes overnight at 4°C with the corresponding primary antibodies: NRF-1 (ab175932, Abcam), CFLAR (DF7010, Affinity), Caspase-8 (AP0358, Bioworld), Bid (GTX110568, GenTex), Bcl-2 (AF6139, Affinity), Bax (ab32503, Abcam) and β-actin (ab49900, Abcam). Washed with TBST for 5 min three times, then blotted the membrane using the appropriate HRP-conjugated secondary antibodies (A21020/A21010, Abbkine) for 1 h. Immunoreactive bands were visualized using a chemiluminescence kit (KGP1121, KeyGEN) and quantified with the ChemiDoc™ Touch imaging system (Bio-Rad, USA). The expression levels of each protein were compared to β-actin, and the results were presented as the fold change relative to the control group. 2.11 Statistical analysis All experiments were conducted independently at least three times. The results were presented as the mean ± standard deviation (SD). Statistical difference was analyzed by SPSS version 23.0 using one-way analysis of variance (ANOVA), and Fisher’s least significant difference (LSD) test was used to compare the significant difference between groups. The differences were indicated statistically significant with P < 0.05. 3. Results 3.1 NRF-1 binds to the Cflar promoter and transcriptionally regulates its expression To investigate whether CFLAR is transcriptionally regulated by NRF-1 in response to hypoxia, we analyzed the binding sites of NRF-1 on the Cflar gene using GIA software and predicted the binding sequence at + 243/+257 (Fig. 1 A). ChIP-PCR analysis confirmed that NRF-1 binding to the Cflar promoter in H9C2 cells (Fig. 1 B). The Dual-luciferase reporter assay demonstrated that NRF-1 directly enhances the activity of the Cflar promoter. However, it was found that the functional binding site for NRF-1 is located upstream of our predicted position rather than at the anticipated location (Fig. 1 C). Collectively, these data indicate that NRF-1 binds to the Cflar promoter and transcriptionally regulates its expression. 3.2 CFLAR siRNA reverses the effects of NRF-1 overexpression on cell growth and death in hypoxic H9C2 cells To confirm that CFLAR is a functional target of NRF-1 in protecting H9C2 cells against hypoxia-induced apoptosis, siRNA of CFLAR was used in NRF-1 overexpressed H9C2 cells. The results obtained from Bio-Tek multi-mode cell imaging system indicate that, as the duration of hypoxia increases, the live cell index initially slightly increased but then gradually decreased (Fig. 2 A), while the dead cell index gradually rose (Fig. 2 B). When exposed to hypoxia for 12 h (Fig. 2 C), compared to the pCDH-vector group, the live cell index of the pCDH-NRF-1 group is higher, and the dead cells index is lower. But in the pCDH-NRF-1 + siRNA-Cflar-1 group and pCDH-NRF-1 + siRNA-Cflar-2 group, the live cell index was lower and the dead cells index is higher, compared to the pCDH-NRF-1 group and pCDH-NRF-1 + siRNA-NC group. The videos are shown in the supplementary material. These results indicated that siRNA of CFLAR can reverse the effects of NRF-1 overexpression on cell growth and death in hypoxic H9C2 cells. The videos are shown in the supplementary material. 3.3 CFLAR siRNA mitigates the protect effects of NRF-1 overexpression on cell apoptosis in hypoxic H9C2 cells via the death receptor pathway To clarify the interaction between NRF-1 and CFLAR, as well as their regulatory pathways involved in apoptosis within hypoxic H9C2 cells, we evaluated the apoptosis rate, activities of Caspase-8, -9, and − 3, and levels of apoptosis-related proteins. As shown in Fig. 3 A, after 12 hours of hypoxia, the pCDH-NRF-1 group exhibited a significantly lower apoptosis rate than the pCDH-vector group. Conversely, both pCDH-NRF-1 + siRNA-Cflar-1 and pCDH-NRF-1 + siRNA-Cflar-2 groups exhibited showed markedly higher apoptosis rates compared to the pCDH-NRF-1 + siRNA-NC group. CFLAR siRNA increased Caspase-8 activity in both pCDH-NRF-1 and pCDH-vector cells (Fig. 3 B). The activity of Caspase-9 in the pCDH-NRF-1 + siRNA-Cflar-2 group was higher than that in the pCDH-vector group. No significant difference was observed in Caspase-3 activity. As shown in Fig. 3 C, CFLAR siRNA significantly reduced the protein levels of CFLAR L and CFLARs in both pCDH-NRF-1 and pCDH-vector cells, while increasing cleaved Caspase-8 and tBid levels. However, it did not affect NRF-1, Bit, Bcl-2, or Bax levels. These results indicate that in hypoxic H9C2 cells, CFLAR siRNA can reverse the protective effects of NRF-1 overexpression on apoptosis through the death receptor pathway, but not via the mitochondrial pathway. 3.4 CFLAR overexpression can alleviate cell apoptosis induced by NRF-1 shRNA knockdown in hypoxic H9C2 cells via the death receptor pathway Next, we investigated the effect of CFLAR overexpression on apoptosis in H9C2 cells with NRF-1 shRNA knockdown after 12 hours of hypoxia. The apoptosis rate was lower in the sh-NRF-1 + pLVX-CFLAR group compared to the sh-NRF-1 + pLVX-vector group (Fig. 4 A). In both sh-NRF-1 and sh-vector cells, CFLAR overexpression reduced Caspase-8 activity (Fig. 4 B). The activities of Caspase-9 and Caspase-3 were significantly decreased by CFLAR overexpression in sh-vector cells but not in sh-NRF-1 cells. Additionally, CFLAR overexpression increased the protein levels of CFLAR L and CFLARs, while decreasing the protein levels of cleaved Caspase-8 and tBid in both sh-NRF-1 cells and sh-vector cells (Fig. 4 C). However, no significant difference was shown in the protein levels of NRF-1, Bid, Bcl-2, and Bax. These data demonstrated that CFLAR overexpression can alleviate cell apoptosis induced by NRF-1 shRNA knockdown in hypoxic H9C2 cells via the death receptor pathway rather than the mitochondrial pathway. 4. Discussion In the present study, we clarified the role of NRF-1 in regulating CFLAR expression, and ultimately, protecting H9C2 cells against hypoxia-induced apoptosis. We identified NRF-1 as a regulator of Cflar gene transcription and observed that CFLAR siRNA mitigated the effects of NRF-1 overexpression on cell growth, death, and apoptosis in hypoxic H9C2 cells. Conversely, overexpression of CFLAR alleviated the apoptosis induced by NRF-1 shRNA knockdown. The regulatory influence of NRF-1 on CFLAR expression primarily affects the death receptor pathway, rather than the mitochondrial pathway. These findings strongly suggest that NRF-1 directly inhibits apoptosis in hypoxia-induced cardiomyocytes. Thus, our study highlights the potential of NRF-1 to serve as a novel therapeutic target for the treatment of HF. The Cut&Tag, ChIP, and luciferase assay results confirmed the presence of a functional NRF-1 transcription factor-binding site in the promoter region of the Cflar gene. However, this binding site does not align with the one predicted by our software analysis. This discrepancy may be attributed to the specificity of H9C2 cells, or DNA-folding characteristics. Since the specific binding sequence was not the primary focus of our study, we did not pursue it. Further exploration of this sequence could provide valuable insights into the underlying mechanisms of NRF-1 regulation. To the best of our knowledge, ours is the first study to provide evidence that NRF-1 directly regulates CFLAR transcription. CFLAR is an anti-apoptotic molecule, plays a crucial role in the regulation of apoptosis [ 19 , 23 ] . Previous research has investigated the role of CFLAR in hypoxic cardiomyocytes, including our own previous study that observed a reduced CFLAR expression and suppressed apoptosis when cardiomyocytes were subjected to physical hypoxia (1% O 2 ) [ 12 ] . Importantly, we had also found that overexpression of NRF-1 reversed apoptosis by modulating both the death receptor and mitochondrial pathways [ 12 ] . In the present study, we further investigated whether CFLAR serves as a target of NRF-1 in inhibiting hypoxia-induced apoptosis. We used siRNA to reduce CFLAR expression in NRF-1 overexpressing H9C2 cells. Our results indicate that CFLAR siRNA reversed the effects of NRF-1 overexpression on cell growth, death, and apoptosis. However, CFLAR overexpression in hypoxic H9C2 cells counteracted the effects induced by NRF-1 knockdown. These results are aligned with previous research reporting that CFLAR overexpression significantly improves the viability of hypoxia/reoxygenation (H/R) cardiomyocytes, reduces myocardial infarct volume in rats with I/R injury, increases the Bcl-2 levels, and decreases the Caspase-3, -8, and − 9 levels [ 24 ] . Similarly, another study using human uterosacral ligament fibroblasts (hUSLFs) reported that CoCl 2 -induced hypoxia increased hypoxia-inducible factor-1a (HIF-1a) levels, reduced CFLAR expression, and led to apoptosis via the death receptor and mitochondrial pathways [ 25 ] . Since we found that CFLAR does not affect NRF-1 expression, it can be concluded that NRF-1 regulates CFLAR unidirectionally. CFLAR appears to be a target of NRF-1, regulating cleaved Caspase-8 expression in the death receptor pathway and tBid (Fig. 5 ). These findings are consistent with the role of CFLAR in inhibiting Caspase-8 activation and Bid cleavage, as outlined in the introduction. However, it did not markedly impact the activities of Bid, Bcl-2, or Bax in the mitochondrial pathway. This may be due to the level of CFLAR overexpression and interference, the sensitivity of cell lines to hypoxia, and the duration of hypoxia. NRF-1 may also target other molecules that contribute to apoptosis through mitochondrial or alternative pathways; this aspect should be confirmed via further investigations. CFLAR is not only involved in apoptosis but also plays a role in regulating necrosis, inflammation, endoplasmic reticulum (ER) stress, and autophagy, making it a prominent molecule for studying apoptosis and diseases such as cancer [ 26 – 32 ] . Our discovery that NRF-1 regulates CFLAR expression opens new avenues for understanding how NRF-1 modulates these biological processes, particularly in the context of cardiomyocyte apoptosis and HF. By elucidating the molecular mechanism whereby NRF-1 regulates CFLAR expression, this study provides a novel perspective on the potential therapeutic application of NRF-1 in modulating CFLAR-related pathways in various diseases. Despite these important findings, this study has a few limitations. For example, due to time and resource constraints, we could not perform an in-depth analysis of the binding sequence of NRF-1 in the Cflar gene promoter. Furthermore, we only verified the regulatory effect of NRF-1 on CFLAR in the H9C2 cell line; whether this phenomenon is also prevalent in other cell types remains unclear. Future studies using primary cardiac myocytes and in vivo models of myocardial hypoxia in rats or mice would be beneficial in validating these findings in a more physiologically relevant context. Additionally, if we have to consider NRF-1 as a potential therapeutic target for HF, its therapeutic efficacy and safety and the mechanisms underlying its effects should be further explored through in vivo experiments. 5. Conclusion In the present study, we identified NRF-1 as a regulator of CFLAR transcription, which plays a critical role in hypoxia-induced apoptosis in cardiomyocytes. By highlighting CFLAR as a direct target molecule for NRF-1, we provide new insights into the molecular mechanisms that govern the regulation of the death receptor pathway, and ultimately influencing cardiomyocyte survival under hypoxic stress. Thus, our study supports the use of NRF-1 as a potential therapeutic target for HF. Future research should include in vivo studies and explore other target molecules regulated by NRF-1 to fully uncover its therapeutic potential. Declarations Competing Interests The authors declare that there are no financial or non-financial interests related to this work. Funding This work was supported by the Natural Science Foundation of Ningxia Province (No. 2023AAC03159 and 2024AAC03478), Key Research and Development Program of Ningxia Province (No. 2024BEH04100 and No. 2023BEG03052) and Ningxia Medical University Scientific Research Project (No. XT2022002) Author Contribution Hui Li: Writing-original draft, Validation, Conceptualization, Funding acquisition. Yunxia Ma: Writing-original draft, Funding acquisition. Junliang Li: Visualization. Siyu Hou: Methodology, Validation, Data curation. Yunxia Ma: Funding acquisition. Song Hui: Writing – review & editing. Yazhou Zhu: Visualization, Software. Wei Zhao: Project administration, Supervision, Resources. Acknowledgments We thank the members of the Institute of Medical Sciences of Ningxia Hui Autonomous Region for providing excellent technical assistance. Data availability statement Data will be made available on request. Requests for data can be sent to the first author: Hui Li, e-mail: [email protected] . References Savarese G, Becher PM, Lund LH, Seferovic P, Rosano GMC, Coats AJS (2023) Global burden of heart failure: a comprehensive and updated review of epidemiology[J]. Cardiovasc Res 118(17):3272–3287 Spoladore R, Ciampi CM, Ossola P, Sultana A, Spreafico LP, Farina A, Fragasso G (2025) Heart Failure and Osteoporosis: Shared Challenges in the Aging Population[J]. J Cardiovasc Dev Dis, 12(2) Heidenreich P (2024) Heart failure management guidelines: New recommendations and implementation[J]. J Cardiol 83(2):67–73 Bauersachs J (2021) Heart failure drug treatment: the fantastic four[J]. Eur Heart J 42(6):681–683 Coniglio AC, Patel CB, Kittleson M, Schlendorf K, Schroder JN, Devore AD (2022) Innovations in Heart Transplantation: A Review[J]. J Card Fail 28(3):467–476 Awad MA, Shah A, Griffith BP (2022) Current status and outcomes in heart transplantation: a narrative review[J]. Rev Cardiovasc Med 23(1):11 Argirò A, Ding J, Adler E (2023) Gene therapy for heart failure and cardiomyopathies[J]. Rev Esp Cardiol (Engl Ed) 76(12):1042–1054 Korpela H, Järveläinen N, Siimes S, Lampela J, Airaksinen J, Valli K, Turunen M, Pajula J, Nurro J, Ylä-Herttuala S (2021) Gene therapy for ischaemic heart disease and heart failure[J]. J Intern Med 290(3):567–582 Wang S, Li Y, Xu Y, Ma Q, Lin Z, Schlame M, Bezzerides VJ, Strathdee D, Pu WT (2020) AAV Gene Therapy Prevents and Reverses Heart Failure in a Murine Knockout Model of Barth Syndrome[J]. Circ Res 126(8):1024–1039 Teringova E, Tousek P (2017) Apoptosis in ischemic heart disease[J]. J translational Med 15(1):87 Virzì G, Clementi A, Ronco C (2016) Cellular apoptosis in the cardiorenal axis[J]. Heart Fail Rev 21(2):177–189 Li H, Niu N, Yang J, Dong F, Zhang T, Li S, Zhao W (2021) Nuclear respiratory factor 1 protects H9C2 cells against hypoxia-induced apoptosis via the death receptor pathway and mitochondrial pathway[J]. Cell Biol Int 45(8):1784–1796 Niu N, Li Z, Zhu M, Sun H, Yang J, Xu S, Zhao W, Song R (2019) Effects of nuclear respiratory factor1 on apoptosis and mitochondrial dysfunction induced by cobalt chloride in H9C2 cells[J]. Mol Med Rep 19(3):2153–2163 Niu N, Li H, Du X, Wang C, Li J, Yang J, Liu C, Yang S, Zhu Y, Zhao W (2022) Effects of NRF-1 and PGC-1α cooperation on HIF-1α and rat cardiomyocyte apoptosis under hypoxia[J]. Gene 834:146565 Wang D, Zhang J, Lu Y, Luo Q, Zhu L (2016) Nuclear respiratory factor-1 (NRF-1) regulated hypoxia-inducible factor-1alpha (HIF-1alpha) under hypoxia in HEK293T[J]. IUBMB Life 68(9):748–755 Dastghaib S, Shafiee SM, Ramezani F, Ashtari N, Tabasi F, Saffari-Chaleshtori J, Siri M, Vakili O, Igder S, Zamani M, Niknam M, Nasery MM, Kokabi F, Wiechec E, Mostafavi-Pour Z, Mokarram P, Ghavami S (2025) NRF-mediated autophagy and UPR: Exploring new avenues to overcome cancer chemo-resistance[J]. Eur J Pharmacol 988:177210 Perez CM, Felty Q (2022) Molecular basis of the association between transcription regulators nuclear respiratory factor 1 and inhibitor of DNA binding protein 3 and the development of microvascular lesions[J]. Microvasc Res 141:104337 Li J, Li H, Niu N, Zhu Y, Hou S, Zhao W (2025) NRF-1 promotes FUNDC1-mediated mitophagy as a protective mechanism against hypoxia-induced injury in cardiomyocytes[J]. Exp Cell Res 446(1):114472 Yang CY, Lien CI, Tseng YC, Tu YF, Kulczyk AW, Lu YC, Wang YT, Su TW, Hsu LC, Lo YC, Lin SC (2024) Deciphering DED assembly mechanisms in FADD-procaspase-8-cFLIP complexes regulating apoptosis[J]. Nat Commun 15(1):3791 Safa AR (2012) c-FLIP, a master anti-apoptotic regulator[J]. Exp Oncol 34(3):176–184 Hughes MA, Powley IR, Jukes-Jones R, Horn S, Feoktistova M, Fairall L, Schwabe JW, Leverkus M, Cain K, Macfarlane M (2016) Co-operative and Hierarchical Binding of c-FLIP and Caspase-8: A Unified Model Defines How c-FLIP Isoforms Differentially Control Cell Fate[J]. Mol Cell 61(6):834–849 Li H, Zhu H, Xu CJ, Yuan J (1998) Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis[J]. Cell 94(4):491–501 Huang Y, Chi W, Li Y, Zhang C, Li J, Meng F (2025) Morphine Preconditioning Alleviates Ischemia/Reperfusion-induced Caspase-8-dependent Neuronal Apoptosis Through cPKCγ-NF-κB-cFLIP L Pathway[J]. J Neurosurg Anesthesiol 37(1):75–87 Liu D, Wu H, Li YZ, Yang J, Yang J, Ding JW, Zhou G, Zhang J, Wang X, Fan ZX (2021) Cellular FADD-like IL-1β-converting enzyme-inhibitory protein attenuates myocardial ischemia/reperfusion injury via suppressing apoptosis and autophagy simultaneously[J]. Nutr Metab Cardiovasc Dis 31(6):1916–1928 Zhao X, Liu L, Li R, Wei X, Luan W, Liu P, Zhao J (2018) Hypoxia-Inducible Factor 1-α (HIF-1α) Induces Apoptosis of Human Uterosacral Ligament Fibroblasts Through the Death Receptor and Mitochondrial Pathways[J]. Med Sci Monit 24:8722–8733 Zheng H, Zhang Y, Zhan Y, Liu S, Lu J, Wen Q, Fan S (2019) Expression of DR5 and c–FLIP proteins as novel prognostic biomarkers for non–small cell lung cancer patients treated with surgical resection and chemotherapy[J]. Oncol Rep 42(6):2363–2370 Xiaohong W, Jun Z, Hongmei G, Fan Q (2019) CFLAR is a critical regulator of cerebral ischaemia-reperfusion injury through regulating inflammation and endoplasmic reticulum (ER) stress[J]. Biomed Pharmacother 117:109155 Xu D, Wang B, Chen PP, Wang YZ, Miao NJ, Yin F, Cheng Q, Zhou ZL, Xie HY, Zhou L, Liu J, Wang XX, Xue H, Zhang W, Lu LM (2019) c-Myc promotes tubular cell apoptosis in ischemia-reperfusion-induced renal injury by negatively regulating c-FLIP and enhancing FasL/Fas-mediated apoptosis pathway[J]. Acta Pharmacol Sin 40(8):1058–1066 Luebke T, Schwarz L, Beer YY, Schumann S, Misterek M, Sander FE, Plaza-Sirvent C, Schmitz I (2019) c-FLIP and CD95 signaling are essential for survival of renal cell carcinoma[J]. Cell Death Dis 10(6):384 Zhang S, Li N, Wu S, Xie T, Chen Q, Wu J, Zeng S, Zhu L, Bai S, Zha H, Tian W, Wu N, Zou X, Fang S, Luo C, Shi M, Sun C, Shu Y, Luo H (2024) c-FLIP facilitates ZIKV infection by mediating caspase-8/3-dependent apoptosis[J]. PLoS Pathog 20(7):e1012408 Ye C, Jiang W, Hu T, Liang J, Chen Y (2024) The Regulatory Impact of CFLAR Methylation Modification on Liver Lipid Metabolism[J]. Int J Mol Sci, 25(14) Mora-Molina R, Stöhr D, Rehm M, López-Rivas A (2022) cFLIP downregulation is an early event required for endoplasmic reticulum stress-induced apoptosis in tumor cells[J]. Cell Death Dis 13(2):111 Additional Declarations No competing interests reported. Supplementary Files CompositevideoFig.2.mp4 Cite Share Download PDF Status: Published Journal Publication published 06 Jun, 2025 Read the published version in Molecular Biology Reports → Version 1 posted Reviews received at journal 28 Apr, 2025 Reviewers agreed at journal 24 Apr, 2025 Reviewers invited by journal 24 Apr, 2025 Editor assigned by journal 23 Apr, 2025 Submission checks completed at journal 23 Apr, 2025 First submitted to journal 21 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6499729","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":447661413,"identity":"cf2992c4-8ef6-4ac8-b210-4c1d17aa8c04","order_by":0,"name":"Hui Li","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Li","suffix":""},{"id":447661415,"identity":"52486906-9912-4b87-ac98-9a1ff0a484d9","order_by":1,"name":"Yunxia Ma","email":"","orcid":"","institution":"The First People's Hospital of Yinchuan, Ningxia Hui Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Yunxia","middleName":"","lastName":"Ma","suffix":""},{"id":447661416,"identity":"bb9a9602-f44a-49e4-b709-04e6d1665f55","order_by":2,"name":"Junliang Li","email":"","orcid":"","institution":"People's Hospital of Ningxia Hui Autonomous Region, Ningxia Hui Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Junliang","middleName":"","lastName":"Li","suffix":""},{"id":447661418,"identity":"460e4cb3-bd37-4cac-a7e3-86974a46711d","order_by":3,"name":"Siyu Hou","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Siyu","middleName":"","lastName":"Hou","suffix":""},{"id":447661420,"identity":"2a2fe132-872d-4238-90db-046946330458","order_by":4,"name":"Hui Song","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Song","suffix":""},{"id":447661422,"identity":"504d97a7-f655-4f67-afaa-548389dcb7da","order_by":5,"name":"Yazhou Zhu","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yazhou","middleName":"","lastName":"Zhu","suffix":""},{"id":447661424,"identity":"54466ebe-4a4e-4e14-9d34-65c9881351da","order_by":6,"name":"Wei Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYHACxgcfDP7JsbG3HyBaC7PhjIIDxnw8ZxKI1sImzPPhQOI8CQcD4tTLz0hgY+YxuJPeJsGQwPCjYhthLYxALQ/nGDzLbZNuPMDYc+Y2YS3M0vnfDd4YMOe2yRxIYGZsI0ILm3QCmwSPAXM6m0SCAXFaeIBaJHkMDicQr0VC/gEwkA3SDNuAgXyQKL/I9xwARuUfG3n59vaDD35UEKEFBRwgUf0oGAWjYBSMAlwAADvWOLpSj3KTAAAAAElFTkSuQmCC","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":true,"prefix":"","firstName":"Wei","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2025-04-22 03:08:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6499729/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6499729/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11033-025-10636-7","type":"published","date":"2025-06-06T15:57:13+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82125158,"identity":"10018bb4-074c-4e57-bec3-ecaf6ce1c5e9","added_by":"auto","created_at":"2025-05-07 03:45:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":310062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNRF-1 binds to the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCflar\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e promoter and transcriptionally regulates its expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Peaks where NRF-1 binds to \u003cem\u003eCflar\u003c/em\u003e promoter identified by GIA software, along with predicted NRF-1 binding site; B. ChIP assay showing the interaction between NRF-1 and the \u003cem\u003eCflar\u003c/em\u003e promoter. The left image displays PCR results from gel electrophoresis, while the right shows qPCR amplification and melting curves; C. The effect of NRF-1 on regulating the activity of \u003cem\u003eCflar \u003c/em\u003epromoter fragments, either in its wild-type (WT) or mutated forms (MUT) at potential NRF-1 binding sites, was assayed using a dual-luciferase reporter system. Data presented as mean ± SD. ** indicates 0.001 ≤ \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01; N.S indicates no significant difference.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6499729/v1/6d30ddd0d3287a7fbfcb068d.png"},{"id":82122487,"identity":"d08db27b-06f5-43d6-bfec-46e1ed4d9421","added_by":"auto","created_at":"2025-05-07 03:29:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":876089,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of CFLAR siRNA on cell growth and death in NRF-1 overexpressed H9C2 cells in hypoxia.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Normalized live cell index; B. Normalized dead cell index; C. The photographs of cells from each group (bar = \u0026nbsp;1000 μm); Blue indicates live cell nuclei stained with Hochest33342; Red represents dead cells stained with PI. The videos are shown in the supplementary material.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6499729/v1/966b823d551fdfd02f842b2a.png"},{"id":82122490,"identity":"ffcdad8f-ff65-49f4-bb5d-68224f058a6f","added_by":"auto","created_at":"2025-05-07 03:29:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":785574,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of CFLAR siRNA on cell apoptosis in NRF-1 overexpressed H9C2 cells after 12 h of hypoxia.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. The apoptosis rate assessed by TUNEL assay (bar = 100 μm); B. Activity of Caspases-8, -9 and -3; C. Relative protein expression levels of NRF-1, CFLAR\u003csub\u003eL\u003c/sub\u003e, CFLAR\u003csub\u003eS\u003c/sub\u003e, NRF-1, Caspase-8, Bid, tBid, Bcl-2 and Bax; * indicates 0.01 ≤ \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** indicates 0.001\u003cem\u003e ≤ P\u003c/em\u003e \u0026lt; 0.01, *** indicates\u003cem\u003e P \u003c/em\u003e\u0026lt; 0.001; N.S indicates no significant difference.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6499729/v1/169615e4e4dad26d41dbc066.png"},{"id":82122489,"identity":"aba5126a-449f-4137-bcd8-64de5c698a25","added_by":"auto","created_at":"2025-05-07 03:29:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":234801,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of CFLAR overexpression on cell apoptosis in H9C2 cells with NRF-1 knockdown after 12 h of hypoxia.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. The apoptosis rate assessed by TUNEL assay (bar = 100 μm); B. Activity of Caspases-8, -9 and -3; C. Relative protein expression levels of NRF-1, CFLAR\u003csub\u003eL\u003c/sub\u003e, CFLAR\u003csub\u003eS\u003c/sub\u003e, NRF-1, Caspase-8, Bid, tBid, Bcl-2 and Bax; * indicates 0.01 ≤ \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** indicates 0.001\u003cem\u003e ≤ P\u003c/em\u003e \u0026lt; 0.01, *** indicates\u003cem\u003e P \u003c/em\u003e\u0026lt; 0.001; N.S indicates no significant difference.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6499729/v1/f48613043957343a1c651df4.png"},{"id":82122503,"identity":"be5cf388-8631-4694-af63-04101bc49225","added_by":"auto","created_at":"2025-05-07 03:29:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":301580,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA summary diagram illustrated that\u003c/strong\u003e \u003cstrong\u003eNRF-1 inhibits apoptosis of hypoxic cardiomyocytes by regulating CFLAR via the death receptor pathway.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6499729/v1/7f70875fbb71bf203bf4e33c.png"},{"id":84242507,"identity":"fc434974-b5eb-4f0a-8da1-c04277fe816b","added_by":"auto","created_at":"2025-06-09 16:08:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3571245,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6499729/v1/7e8620a6-35fe-4a10-8641-04fff698b386.pdf"},{"id":82122508,"identity":"c39021d5-dd7d-45de-ac89-5de0b7b1978d","added_by":"auto","created_at":"2025-05-07 03:29:20","extension":"mp4","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":5099247,"visible":true,"origin":"","legend":"","description":"","filename":"CompositevideoFig.2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6499729/v1/707ca86e6f59cbf3caa7b168.mp4"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nuclear respiratory factor-1 promotes CFLAR transcription in H9C2 cardiomyocytes, protecting them against hypoxia-induced apoptosis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHeart Failure (HF) occurs at the end stage of various heart diseases. It is characterized by high morbidity, mortality, diminished functional capacity, poor quality of life, and a substantial economic and emotional burden for society and the healthcare system \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. With the increase in the aging population, fast-paced lifestyles, and rising work-related stress worldwide, the prevalence of HF increases annually \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. The burden posed by HF on global healthcare costs is a growing concern. Although medications can alleviate the symptoms, and delay the progression of HF, their prolonged use may lead to drug resistance and side effects \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Currently, heart transplantation is the most effective treatment for HF; however, its availability is limited by donor shortage and the risk of transplant rejection \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Gene therapy, powered by the advancements in gene-editing and -delivery technologies, holds promise to become a novel treatment method for HF. Gene therapy provides long-term therapeutic benefits with minimal side effects \u003csup\u003e[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. However, due to the challenges associated with identifying suitable target genes, gene therapy for HF is still in the early stages of development. The pathogenesis of HF is complex, but numerous studies have reported that ischemia- and hypoxia-induced cardiomyocyte apoptosis play critical roles in the extensive loss of cardiomyocytes, ultimately leading to HF \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Therefore, elucidating the molecular mechanisms underlying cardiomyocyte apoptosis in HF and identifying effective therapeutic target genes are essential for advancing the clinical application of gene therapy in HF.\u003c/p\u003e \u003cp\u003ePrevious studies have highlighted that nuclear respiratory factor-1 (NRF-1) plays a protective role against apoptosis in H9C2 cardiomyocytes under both chemical hypoxia conditions induced by CoCl\u003csub\u003e2\u003c/sub\u003e and physical hypoxia with 1% O\u003csub\u003e2\u003c/sub\u003e \u003csup\u003e[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. NRF-1 is a key transcription factor that regulates cellular energy metabolism, mitochondrial biogenesis, oxidative stress responses, cell growth, autophagy, and unfolded protein responses \u003csup\u003e[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. We also found that in H9C2 cells, under conditions of hypoxia, NRF-1 enhances cellular energy metabolism, stabilizes the mitochondrial membrane potential, inhibited reactive oxygen species (ROS) production, promotes mitophagy, and inhibits apoptosis via the death receptor and mitochondrial pathways \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Thus, NRF-1 shows promise as a therapeutic target for the treatment of HF. Despite these findings, the mechanisms whereby NRF-1 inhibits apoptosis in cardiomyocytes remain unclear.\u003c/p\u003e \u003cp\u003eCaspase 8 and FADD-like apoptosis regulator (CFLAR), also known as FLIP (FADD-like IL-1beta-converting enzyme-inhibitory protein), is a key anti-apoptotic molecule \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Although CFLAR mRNA has multiple splice variants, it primarily exists in two protein forms: the long isoform (CFLAR\u003csub\u003eL\u003c/sub\u003e) and the short isoform (CFLAR\u003csub\u003eS\u003c/sub\u003e) \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. CFLAR inhibits Caspase-8 activation by preventing its dimerization in the death-inducing signaling complex (DISC), thereby modulating apoptosis via death receptor pathways \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Conversely, activated Caspase-8 cleaves Bid, a proapoptotic molecule in the cytoplasm, resulting in tBid formation. The -COOH terminus of tBid translocates to the outer mitochondrial membrane and promotes Bax and Bad dimerization, which, in turn, initiates the mitochondrial apoptotic pathway \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Our previous research revealed that both overexpression and knockdown of NRF-1 alter the gene and protein expression of CFLAR \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Moreover, our previous unpublished data obtained using CUT\u0026amp;Tag indicated that the promoter region of \u003cem\u003eCflar\u003c/em\u003e gene contains an NRF-1 transcription factor-binding site (TFBS), suggesting that CFLAR may be one of the target molecules regulated by NRF-1 to protect H9C2 cells from hypoxia-induced apoptosis. However, the roles of NRF-1 in CFLAR transcription and the mechanisms underlying its role remain unclear.\u003c/p\u003e \u003cp\u003eIn the present study, we investigated the interaction between NRF-1 and CFLAR in H9C2 cells under hypoxic conditions and analyzed their regulatory effects on cardiomyocyte apoptosis. To the best of our knowledge, our study is the first to demonstrate that NRF-1 specifically targets and regulates CFLAR transcription; our findings will provide new insights into potential therapeutic strategies for HF.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell culture and hypoxia treatment\u003c/h2\u003e \u003cp\u003eThe H9C2 and HEK 293 T cells were originally obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China) and have since maintained in our laboratory. The NRF-1 overexpressing H9C2 cell line (H9C2\u0026thinsp;+\u0026thinsp;pCDH-NRF-1), the NRF-1 knockdown H9C2 cell line (H9C2\u0026thinsp;+\u0026thinsp;sh-NRF-1), and their corresponding control cell line (H9C2\u0026thinsp;+\u0026thinsp;pCDH-vector and H9C2\u0026thinsp;+\u0026thinsp;sh-vector) generated following the methodologies previously reported by our group \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. All cell lines were cultured in DMEM with high glucose (SH30243.01; HyClone), supplemented with 10% fetal bovine serum (900-108; Gemini Bio) and 1% penicillin-streptomycin (P1400; Solarbio), under standard conditions of 5% CO\u003csub\u003e2\u003c/sub\u003e, 90% humidity, and a temperature of 37\u0026deg;C.\u003c/p\u003e \u003cp\u003eTo create a hypoxic environment in vitro, the cells were transferred to a tri-gas incubator (Model 3131; Thermo Fisher Scientific) set to contain 1% O\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e and 94% N\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Predict TFBS of NRF-1 on \u003cem\u003eCflar\u003c/em\u003e gene\u003c/h2\u003e \u003cp\u003eThe specific DNA sequence bound by the NRF-1 protein was identified through CUT\u0026amp;Tag sequencing. GIV software was utilized to analyze the results to locate peak sequences of the \u003cem\u003eCflar\u003c/em\u003e gene. The peak regions were extended by 200 bp in both upstream and downstream directions to extract the corresponding DNA sequences. Subsequently, the Motifs Genome.pl tool from HOMER software was employed to predict motifs within these sequences, which were subsequently matched against existing motif data in the JASPAR database to identify NRF-1 TFBS on \u003cem\u003eCflar\u003c/em\u003e gene.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 ChIP and qPCR\u003c/h2\u003e \u003cp\u003eChromatin immunoprecipitation References EZ CHIP Kit 22 Assays (17\u0026ndash;371, Millipore) was used to detect the NRF-1 binding in the promoter region of \u003cem\u003eCflar\u003c/em\u003e gene. Following the manufacturer\u0026rsquo;s protocol, proteins were cross-linked to DNA by incubating 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells in 1% formaldehyde for 10 min at 37\u0026deg;C. The cell pellets were resuspended with 1 ml SDS Lysis Buffer which contains 5 \u0026micro;l Protease Inhibitor Cocktail II. To fragment the chromatin to 200\u0026ndash;1000 bp, ultrasound it ten times (20 s pulse, 50 s rest) with an Ultrasonic Processor (SCIENTZ-950E, SCIENTZ). The ultrasound effect was detected using 1.5% agarose gel electrophoresis. Immunocomplexes were isolated from the remaining lysates using 1 \u0026micro;g of NRF-1 antibody. After elution, cross-linking removal, and purification, DNA was obtained and stored at -20℃.\u003c/p\u003e \u003cp\u003eqPCR was performed on a 7500 system (Thermo, USA) using primers for the Cflar promoter: 5\u0026rsquo;-GTACGTGACTTGAGCGGTGT-3\u0026rsquo;(F), 5\u0026rsquo;-CTGCAGTCCTCCGTG CC-3\u0026rsquo;(R). The production size is 177 bp, including the predicted NRF-1 binding site. The result was also analyzed by 1.5% agarose gel electrophoresis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Luciferase reporter assay\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eCflar\u003c/em\u003e promoter sequence, spanning from \u0026minus;\u0026thinsp;1,000 to +\u0026thinsp;401 relative to the proposed transcriptional start site (TSS) of \u003cem\u003eCflar\u003c/em\u003e exon 1 (NC_086027.1), was cloned and subsequently substituted for the promoter region of firefly luciferase in the psiCHECK 2-Basic vector (Promega, USA). Based on the predicted binding sites of NRF-1, four additional reporter plasmids were constructed, each containing mutations at different positions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e293T cells were seeded in 24-well plates (5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well), and subsequently transfected with 50 ng of luciferase reporter plasmids and 50 ng of pCDH-NRF-1 plasmids. After 48 h of transfection, measuring the activity of luciferase by a dual-luciferase reporter assay kit (E2920, Promega). According to the manufacture\u0026rsquo;s protocols, removed the cell culture medium in the 24-well plate and added 500 \u0026micro;l detection working solution to each well, the cells were fully lysed for 15 min. Then, 100 \u0026micro;l of lysate was transferred into a light-tight whiteboard 96-well plate (avoiding bubbles) to detect firefly fluorescence. Subsequently, 50 \u0026micro;l of Stop reaction solution was added to each well and gently mixed while protecting from light for an additional 10 minutes to terminate firefly fluorescence. The fluorescence emitted by sea kidney was detected. The results were presented as the ratio of sea kidney fluorescence to firefly fluorescence for each hole, and the values of each experimental group were normalized and analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 siRNA of CFLAR in NRF-1 overexpressing H9C2 cells\u003c/h2\u003e \u003cp\u003eThe target sequences for CFLAR siRNA and Oligo sequences of negative control (siRNA-NC) were synthesized from Genepharma (Beijing, China). The two most effective siRNA and siRNA-NC sequences are as follows: siRNA-CFLAR-1 (F: 5\u0026rsquo;-CCUCCUGGAUUGUUUAAGUTT-3\u0026rsquo;; R: 5\u0026rsquo;-ACUUAAACAAUCCAGGAGGTT-3\u0026rsquo;); siRNA-CFLAR-2 (F: 5\u0026rsquo;GAGCCAGUGUGUGGAAUAUTT-3\u0026rsquo;; R: 5\u0026rsquo;-AUAU UCCACACACUGGCUCTT-3\u0026rsquo;); siRNA-NC (F: 5\u0026rsquo;-UUCUUCGAACGUGUCACGU TT-3\u0026rsquo;; R: 5\u0026rsquo;-ACGUGACACGUUCGGAGAATT-3\u0026rsquo;). The two siRNA and siRNA-NC were transfected into H9C2\u0026thinsp;+\u0026thinsp;pCDH-NRF-1 cell line respectively using Lipofectamine 2000 (11668-027, Invitrogen) according to the manufacturer\u0026rsquo;s protocols. After 72 h of transfection, CFLAR protein expression levels were assessed, and follow-up hypoxia experiments were performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 CFLAR overexpression in NRF-1 knockdown H9C2 cells\u003c/h2\u003e \u003cp\u003eTo differentiate from the green fluorescence of the H9C2\u0026thinsp;+\u0026thinsp;sh-NRF-1 and H9C2\u0026thinsp;+\u0026thinsp;sh-vector cell lines, we constructed a CFLAR (NM_001033864.4) overexpression using the pLVX-IRES-mCherry lentiviral vector (BR023; Fenghui Biotech), which emits red fluorescence. Cells transfected with the empty vector (pLVX-vector) served as the control group. Virus packaging and cell infection were performed as previously described\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. After puromycin selection, flow cytometry was used to isolate stably transfected cells based on red and green double-labeled fluorescence, ensuring a positive cell rate of over 95% in each experiment. Overexpression efficiency was evaluated by western blot analysis.\u003c/p\u003e \u003cp\u003eThe transfected cell lines were designated as sh-NRF-1\u0026thinsp;+\u0026thinsp;pLVX-CFLAR, sh-NRF-1\u0026thinsp;+\u0026thinsp;pLVX-vector, sh-vector\u0026thinsp;+\u0026thinsp;pLVX-CFLAR, and sh-vector\u0026thinsp;+\u0026thinsp;pLVX-vector for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.7 Cell growth and death monitored by Bio-Tek multi-mode imaging system\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe same number of cells from each group were seeded in a 12-well plate. Hochest33342 was used to detect live cell numbers and PI was used to detect dead cell numbers. Following the instructions, the Hochest33342/PI dual staining reagent (KGA212, Keygen Biotech) was added to the culture medium and mixed before being distributed into each well. The cells were cultured in a Bio-Tek multi-mode cell imaging system under hypoxic condition (1% O2, 5% CO2, 94% N2) at 37\u0026deg;C using Gen5 software. Blue and red fluorescence of each well was measured every 30 minutes for 24 hours, and the results were collected for subsequent analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 TUNEL assay\u003c/h2\u003e \u003cp\u003eAfter 12 h of hypoxia treatment, cells from each group were fixed with 4% formaldehyde. Cell apoptosis was assessed using a TUNEL assay kit (C1082; Beyotime) in accordance with the manufacturer's protocol. Hematoxylin was used to stain the nuclei. The cells were photographed with a microscope (EVOS FL) and the apoptosis rate was determined by calculating the ratio of TUNEL-positive cells to the total cells counted in each field. Randomly selected three fields from each group to calculate an average.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Caspase \u0026minus;\u0026thinsp;8, -9 and \u0026minus;\u0026thinsp;3 activity\u003c/h2\u003e \u003cp\u003eColorimetric assay kits for Caspase-8, -9 and \u0026minus;\u0026thinsp;3 (KGA 202, KGA 302, and KGA 402; KeyGEN, China) were employed to evaluate the activities of Caspase-8, -9 and \u0026minus;\u0026thinsp;3. Following a 12-hour hypoxia treatment, cells were lysed using the lysis buffer provided in the kit to extract protein. Equal volumes of protein (50 \u0026micro;l) were combined with the reaction buffer (50 \u0026micro;l) and incubated at 37\u0026deg;C in the dark for1 h. Absorbance was subsequently measured using a SkanIt@ Software microplate reader set to a wavelength of 405 nm. The activities of Caspase-8, -9 and \u0026minus;\u0026thinsp;3 are presented as fold changes compared with control values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Western blot analysis\u003c/h2\u003e \u003cp\u003eAfter cells been exposed to hypoxia for 12 h, total proteins were extracted using a whole cell lysis assay kit (KGP250, KeyGEN). Equivalent protein quantities (30 \u0026micro;g) were subjected to 12% SDS-PAGE and subsequently transferred onto a nitrocellulose membrane (HATF00010; Millipore). Blocked the membrane with 5% BSA in TBST for 2 h. Incubated the membranes overnight at 4\u0026deg;C with the corresponding primary antibodies: NRF-1 (ab175932, Abcam), CFLAR (DF7010, Affinity), Caspase-8 (AP0358, Bioworld), Bid (GTX110568, GenTex), Bcl-2 (AF6139, Affinity), Bax (ab32503, Abcam) and β-actin (ab49900, Abcam). Washed with TBST for 5 min three times, then blotted the membrane using the appropriate HRP-conjugated secondary antibodies (A21020/A21010, Abbkine) for 1 h. Immunoreactive bands were visualized using a chemiluminescence kit (KGP1121, KeyGEN) and quantified with the ChemiDoc\u0026trade; Touch imaging system (Bio-Rad, USA). The expression levels of each protein were compared to β-actin, and the results were presented as the fold change relative to the control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were conducted independently at least three times. The results were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical difference was analyzed by SPSS version 23.0 using one-way analysis of variance (ANOVA), and Fisher\u0026rsquo;s least significant difference (LSD) test was used to compare the significant difference between groups. The differences were indicated statistically significant with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 NRF-1 binds to the \u003cem\u003eCflar\u003c/em\u003e promoter and transcriptionally regulates its expression\u003c/h2\u003e\n \u003cp\u003eTo investigate whether CFLAR is transcriptionally regulated by NRF-1 in response to hypoxia, we analyzed the binding sites of NRF-1 on the \u003cem\u003eCflar\u003c/em\u003e gene using GIA software and predicted the binding sequence at +\u0026thinsp;243/+257 (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). ChIP-PCR analysis confirmed that NRF-1 binding to the \u003cem\u003eCflar\u003c/em\u003e promoter in H9C2 cells (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). The Dual-luciferase reporter assay demonstrated that NRF-1 directly enhances the activity of the \u003cem\u003eCflar\u003c/em\u003e promoter. However, it was found that the functional binding site for NRF-1 is located upstream of our predicted position rather than at the anticipated location (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). Collectively, these data indicate that NRF-1 binds to the \u003cem\u003eCflar\u003c/em\u003e promoter and transcriptionally regulates its expression.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.2 CFLAR siRNA reverses the effects of NRF-1 overexpression on cell growth and death in hypoxic H9C2 cells\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo confirm that CFLAR is a functional target of NRF-1 in protecting H9C2 cells against hypoxia-induced apoptosis, siRNA of CFLAR was used in NRF-1 overexpressed H9C2 cells. The results obtained from Bio-Tek multi-mode cell imaging system indicate that, as the duration of hypoxia increases, the live cell index initially slightly increased but then gradually decreased (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA), while the dead cell index gradually rose (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). When exposed to hypoxia for 12 h (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC), compared to the pCDH-vector group, the live cell index of the pCDH-NRF-1 group is higher, and the dead cells index is lower. But in the pCDH-NRF-1\u0026thinsp;+\u0026thinsp;siRNA-Cflar-1 group and pCDH-NRF-1\u0026thinsp;+\u0026thinsp;siRNA-Cflar-2 group, the live cell index was lower and the dead cells index is higher, compared to the pCDH-NRF-1 group and pCDH-NRF-1\u0026thinsp;+\u0026thinsp;siRNA-NC group. The videos are shown in the supplementary material. These results indicated that siRNA of CFLAR can reverse the effects of NRF-1 overexpression on cell growth and death in hypoxic H9C2 cells. The videos are shown in the supplementary material.\u003c/p\u003e\u003cspan\u003e\u003cstrong\u003e3.3 CFLAR siRNA mitigates the protect effects of NRF-1 overexpression on cell apoptosis in hypoxic H9C2 cells via the death receptor pathway\u003c/strong\u003e\u003cbr\u003e\u003c/span\u003e\n \u003cp\u003eTo clarify the interaction between NRF-1 and CFLAR, as well as their regulatory pathways involved in apoptosis within hypoxic H9C2 cells, we evaluated the apoptosis rate, activities of Caspase-8, -9, and \u0026minus;\u0026thinsp;3, and levels of apoptosis-related proteins. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, after 12 hours of hypoxia, the pCDH-NRF-1 group exhibited a significantly lower apoptosis rate than the pCDH-vector group. Conversely, both pCDH-NRF-1\u0026thinsp;+\u0026thinsp;siRNA-Cflar-1 and pCDH-NRF-1\u0026thinsp;+\u0026thinsp;siRNA-Cflar-2 groups exhibited showed markedly higher apoptosis rates compared to the pCDH-NRF-1\u0026thinsp;+\u0026thinsp;siRNA-NC group. CFLAR siRNA increased Caspase-8 activity in both pCDH-NRF-1 and pCDH-vector cells (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). The activity of Caspase-9 in the pCDH-NRF-1\u0026thinsp;+\u0026thinsp;siRNA-Cflar-2 group was higher than that in the pCDH-vector group. No significant difference was observed in Caspase-3 activity.\u003c/p\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC, CFLAR siRNA significantly reduced the protein levels of CFLAR\u003csub\u003eL\u003c/sub\u003e and CFLARs in both pCDH-NRF-1 and pCDH-vector cells, while increasing cleaved Caspase-8 and tBid levels. However, it did not affect NRF-1, Bit, Bcl-2, or Bax levels. These results indicate that in hypoxic H9C2 cells, CFLAR siRNA can reverse the protective effects of NRF-1 overexpression on apoptosis through the death receptor pathway, but not via the mitochondrial pathway.\u003c/p\u003e\u003cspan\u003e\u003cstrong\u003e3.4 CFLAR overexpression can alleviate cell apoptosis induced by NRF-1 shRNA knockdown in hypoxic H9C2 cells via the death receptor pathway\u003c/strong\u003e\u003cbr\u003e\u003c/span\u003e\n \u003cp\u003eNext, we investigated the effect of CFLAR overexpression on apoptosis in H9C2 cells with NRF-1 shRNA knockdown after 12 hours of hypoxia. The apoptosis rate was lower in the sh-NRF-1\u0026thinsp;+\u0026thinsp;pLVX-CFLAR group compared to the sh-NRF-1\u0026thinsp;+\u0026thinsp;pLVX-vector group (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). In both sh-NRF-1 and sh-vector cells, CFLAR overexpression reduced Caspase-8 activity (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). The activities of Caspase-9 and Caspase-3 were significantly decreased by CFLAR overexpression in sh-vector cells but not in sh-NRF-1 cells.\u003c/p\u003e\n \u003cp\u003eAdditionally, CFLAR overexpression increased the protein levels of CFLAR\u003csub\u003eL\u003c/sub\u003e and CFLARs, while decreasing the protein levels of cleaved Caspase-8 and tBid in both sh-NRF-1 cells and sh-vector cells (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). However, no significant difference was shown in the protein levels of NRF-1, Bid, Bcl-2, and Bax. These data demonstrated that CFLAR overexpression can alleviate cell apoptosis induced by NRF-1 shRNA knockdown in hypoxic H9C2 cells via the death receptor pathway rather than the mitochondrial pathway.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn the present study, we clarified the role of NRF-1 in regulating CFLAR expression, and ultimately, protecting H9C2 cells against hypoxia-induced apoptosis. We identified NRF-1 as a regulator of \u003cem\u003eCflar\u003c/em\u003e gene transcription and observed that CFLAR siRNA mitigated the effects of NRF-1 overexpression on cell growth, death, and apoptosis in hypoxic H9C2 cells. Conversely, overexpression of CFLAR alleviated the apoptosis induced by NRF-1 shRNA knockdown. The regulatory influence of NRF-1 on CFLAR expression primarily affects the death receptor pathway, rather than the mitochondrial pathway. These findings strongly suggest that NRF-1 directly inhibits apoptosis in hypoxia-induced cardiomyocytes. Thus, our study highlights the potential of NRF-1 to serve as a novel therapeutic target for the treatment of HF.\u003c/p\u003e \u003cp\u003eThe Cut\u0026amp;Tag, ChIP, and luciferase assay results confirmed the presence of a functional NRF-1 transcription factor-binding site in the promoter region of the \u003cem\u003eCflar\u003c/em\u003e gene. However, this binding site does not align with the one predicted by our software analysis. This discrepancy may be attributed to the specificity of H9C2 cells, or DNA-folding characteristics. Since the specific binding sequence was not the primary focus of our study, we did not pursue it. Further exploration of this sequence could provide valuable insights into the underlying mechanisms of NRF-1 regulation.\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, ours is the first study to provide evidence that NRF-1 directly regulates CFLAR transcription. CFLAR is an anti-apoptotic molecule, plays a crucial role in the regulation of apoptosis \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Previous research has investigated the role of CFLAR in hypoxic cardiomyocytes, including our own previous study that observed a reduced CFLAR expression and suppressed apoptosis when cardiomyocytes were subjected to physical hypoxia (1% O\u003csub\u003e2\u003c/sub\u003e) \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Importantly, we had also found that overexpression of NRF-1 reversed apoptosis by modulating both the death receptor and mitochondrial pathways \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. In the present study, we further investigated whether CFLAR serves as a target of NRF-1 in inhibiting hypoxia-induced apoptosis. We used siRNA to reduce CFLAR expression in NRF-1 overexpressing H9C2 cells. Our results indicate that CFLAR siRNA reversed the effects of NRF-1 overexpression on cell growth, death, and apoptosis. However, CFLAR overexpression in hypoxic H9C2 cells counteracted the effects induced by NRF-1 knockdown. These results are aligned with previous research reporting that CFLAR overexpression significantly improves the viability of hypoxia/reoxygenation (H/R) cardiomyocytes, reduces myocardial infarct volume in rats with I/R injury, increases the Bcl-2 levels, and decreases the Caspase-3, -8, and \u0026minus;\u0026thinsp;9 levels \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Similarly, another study using human uterosacral ligament fibroblasts (hUSLFs) reported that CoCl\u003csub\u003e2\u003c/sub\u003e-induced hypoxia increased hypoxia-inducible factor-1a (HIF-1a) levels, reduced CFLAR expression, and led to apoptosis via the death receptor and mitochondrial pathways \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Since we found that CFLAR does not affect NRF-1 expression, it can be concluded that NRF-1 regulates CFLAR unidirectionally. CFLAR appears to be a target of NRF-1, regulating cleaved Caspase-8 expression in the death receptor pathway and tBid (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These findings are consistent with the role of CFLAR in inhibiting Caspase-8 activation and Bid cleavage, as outlined in the introduction. However, it did not markedly impact the activities of Bid, Bcl-2, or Bax in the mitochondrial pathway. This may be due to the level of CFLAR overexpression and interference, the sensitivity of cell lines to hypoxia, and the duration of hypoxia. NRF-1 may also target other molecules that contribute to apoptosis through mitochondrial or alternative pathways; this aspect should be confirmed via further investigations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCFLAR is not only involved in apoptosis but also plays a role in regulating necrosis, inflammation, endoplasmic reticulum (ER) stress, and autophagy, making it a prominent molecule for studying apoptosis and diseases such as cancer \u003csup\u003e[\u003cspan additionalcitationids=\"CR27 CR28 CR29 CR30 CR31\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Our discovery that NRF-1 regulates CFLAR expression opens new avenues for understanding how NRF-1 modulates these biological processes, particularly in the context of cardiomyocyte apoptosis and HF. By elucidating the molecular mechanism whereby NRF-1 regulates CFLAR expression, this study provides a novel perspective on the potential therapeutic application of NRF-1 in modulating CFLAR-related pathways in various diseases.\u003c/p\u003e \u003cp\u003eDespite these important findings, this study has a few limitations. For example, due to time and resource constraints, we could not perform an in-depth analysis of the binding sequence of NRF-1 in the \u003cem\u003eCflar\u003c/em\u003e gene promoter. Furthermore, we only verified the regulatory effect of NRF-1 on CFLAR in the H9C2 cell line; whether this phenomenon is also prevalent in other cell types remains unclear. Future studies using primary cardiac myocytes and \u003cem\u003ein vivo\u003c/em\u003e models of myocardial hypoxia in rats or mice would be beneficial in validating these findings in a more physiologically relevant context. Additionally, if we have to consider NRF-1 as a potential therapeutic target for HF, its therapeutic efficacy and safety and the mechanisms underlying its effects should be further explored through \u003cem\u003ein vivo\u003c/em\u003e experiments.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn the present study, we identified NRF-1 as a regulator of CFLAR transcription, which plays a critical role in hypoxia-induced apoptosis in cardiomyocytes. By highlighting CFLAR as a direct target molecule for NRF-1, we provide new insights into the molecular mechanisms that govern the regulation of the death receptor pathway, and ultimately influencing cardiomyocyte survival under hypoxic stress. Thus, our study supports the use of NRF-1 as a potential therapeutic target for HF. Future research should include \u003cem\u003ein vivo\u003c/em\u003e studies and explore other target molecules regulated by NRF-1 to fully uncover its therapeutic potential.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that there are no financial or non-financial interests related to this work.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of Ningxia Province (No. 2023AAC03159 and 2024AAC03478), Key Research and Development Program of Ningxia Province (No. 2024BEH04100 and No. 2023BEG03052) and Ningxia Medical University Scientific Research Project (No. XT2022002)\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eHui Li: Writing-original draft, Validation, Conceptualization, Funding acquisition. Yunxia Ma: Writing-original draft, Funding acquisition. Junliang Li: Visualization. Siyu Hou: Methodology, Validation, Data curation. Yunxia Ma: Funding acquisition. Song Hui: Writing \u0026ndash; review \u0026amp; editing. Yazhou Zhu: Visualization, Software. Wei Zhao: Project administration, Supervision, Resources.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe thank the members of the Institute of Medical Sciences of Ningxia Hui Autonomous Region for providing excellent technical assistance.\u003c/p\u003e\n\u003ch2\u003eData availability statement\u003c/h2\u003e\n\u003cp\u003eData will be made available on request. Requests for data can be sent to the first author: Hui Li, e-mail: [email protected].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSavarese G, Becher PM, Lund LH, Seferovic P, Rosano GMC, Coats AJS (2023) Global burden of heart failure: a comprehensive and updated review of epidemiology[J]. Cardiovasc Res 118(17):3272\u0026ndash;3287\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpoladore R, Ciampi CM, Ossola P, Sultana A, Spreafico LP, Farina A, Fragasso G (2025) Heart Failure and Osteoporosis: Shared Challenges in the Aging Population[J]. J Cardiovasc Dev Dis, 12(2)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeidenreich P (2024) Heart failure management guidelines: New recommendations and implementation[J]. J Cardiol 83(2):67\u0026ndash;73\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBauersachs J (2021) Heart failure drug treatment: the fantastic four[J]. Eur Heart J 42(6):681\u0026ndash;683\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eConiglio AC, Patel CB, Kittleson M, Schlendorf K, Schroder JN, Devore AD (2022) Innovations in Heart Transplantation: A Review[J]. J Card Fail 28(3):467\u0026ndash;476\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAwad MA, Shah A, Griffith BP (2022) Current status and outcomes in heart transplantation: a narrative review[J]. Rev Cardiovasc Med 23(1):11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArgir\u0026ograve; A, Ding J, Adler E (2023) Gene therapy for heart failure and cardiomyopathies[J]. Rev Esp Cardiol (Engl Ed) 76(12):1042\u0026ndash;1054\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKorpela H, J\u0026auml;rvel\u0026auml;inen N, Siimes S, Lampela J, Airaksinen J, Valli K, Turunen M, Pajula J, Nurro J, Yl\u0026auml;-Herttuala S (2021) Gene therapy for ischaemic heart disease and heart failure[J]. J Intern Med 290(3):567\u0026ndash;582\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S, Li Y, Xu Y, Ma Q, Lin Z, Schlame M, Bezzerides VJ, Strathdee D, Pu WT (2020) AAV Gene Therapy Prevents and Reverses Heart Failure in a Murine Knockout Model of Barth Syndrome[J]. Circ Res 126(8):1024\u0026ndash;1039\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeringova E, Tousek P (2017) Apoptosis in ischemic heart disease[J]. J translational Med 15(1):87\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVirz\u0026igrave; G, Clementi A, Ronco C (2016) Cellular apoptosis in the cardiorenal axis[J]. Heart Fail Rev 21(2):177\u0026ndash;189\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, Niu N, Yang J, Dong F, Zhang T, Li S, Zhao W (2021) Nuclear respiratory factor 1 protects H9C2 cells against hypoxia-induced apoptosis via the death receptor pathway and mitochondrial pathway[J]. Cell Biol Int 45(8):1784\u0026ndash;1796\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiu N, Li Z, Zhu M, Sun H, Yang J, Xu S, Zhao W, Song R (2019) Effects of nuclear respiratory factor1 on apoptosis and mitochondrial dysfunction induced by cobalt chloride in H9C2 cells[J]. Mol Med Rep 19(3):2153\u0026ndash;2163\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiu N, Li H, Du X, Wang C, Li J, Yang J, Liu C, Yang S, Zhu Y, Zhao W (2022) Effects of NRF-1 and PGC-1α cooperation on HIF-1α and rat cardiomyocyte apoptosis under hypoxia[J]. Gene 834:146565\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang D, Zhang J, Lu Y, Luo Q, Zhu L (2016) Nuclear respiratory factor-1 (NRF-1) regulated hypoxia-inducible factor-1alpha (HIF-1alpha) under hypoxia in HEK293T[J]. IUBMB Life 68(9):748\u0026ndash;755\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDastghaib S, Shafiee SM, Ramezani F, Ashtari N, Tabasi F, Saffari-Chaleshtori J, Siri M, Vakili O, Igder S, Zamani M, Niknam M, Nasery MM, Kokabi F, Wiechec E, Mostafavi-Pour Z, Mokarram P, Ghavami S (2025) NRF-mediated autophagy and UPR: Exploring new avenues to overcome cancer chemo-resistance[J]. Eur J Pharmacol 988:177210\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerez CM, Felty Q (2022) Molecular basis of the association between transcription regulators nuclear respiratory factor 1 and inhibitor of DNA binding protein 3 and the development of microvascular lesions[J]. Microvasc Res 141:104337\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Li H, Niu N, Zhu Y, Hou S, Zhao W (2025) NRF-1 promotes FUNDC1-mediated mitophagy as a protective mechanism against hypoxia-induced injury in cardiomyocytes[J]. Exp Cell Res 446(1):114472\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang CY, Lien CI, Tseng YC, Tu YF, Kulczyk AW, Lu YC, Wang YT, Su TW, Hsu LC, Lo YC, Lin SC (2024) Deciphering DED assembly mechanisms in FADD-procaspase-8-cFLIP complexes regulating apoptosis[J]. Nat Commun 15(1):3791\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSafa AR (2012) c-FLIP, a master anti-apoptotic regulator[J]. Exp Oncol 34(3):176\u0026ndash;184\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHughes MA, Powley IR, Jukes-Jones R, Horn S, Feoktistova M, Fairall L, Schwabe JW, Leverkus M, Cain K, Macfarlane M (2016) Co-operative and Hierarchical Binding of c-FLIP and Caspase-8: A Unified Model Defines How c-FLIP Isoforms Differentially Control Cell Fate[J]. Mol Cell 61(6):834\u0026ndash;849\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, Zhu H, Xu CJ, Yuan J (1998) Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis[J]. Cell 94(4):491\u0026ndash;501\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang Y, Chi W, Li Y, Zhang C, Li J, Meng F (2025) Morphine Preconditioning Alleviates Ischemia/Reperfusion-induced Caspase-8-dependent Neuronal Apoptosis Through cPKCγ-NF-κB-cFLIP L Pathway[J]. J Neurosurg Anesthesiol 37(1):75\u0026ndash;87\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu D, Wu H, Li YZ, Yang J, Yang J, Ding JW, Zhou G, Zhang J, Wang X, Fan ZX (2021) Cellular FADD-like IL-1β-converting enzyme-inhibitory protein attenuates myocardial ischemia/reperfusion injury via suppressing apoptosis and autophagy simultaneously[J]. Nutr Metab Cardiovasc Dis 31(6):1916\u0026ndash;1928\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao X, Liu L, Li R, Wei X, Luan W, Liu P, Zhao J (2018) Hypoxia-Inducible Factor 1-α (HIF-1α) Induces Apoptosis of Human Uterosacral Ligament Fibroblasts Through the Death Receptor and Mitochondrial Pathways[J]. Med Sci Monit 24:8722\u0026ndash;8733\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng H, Zhang Y, Zhan Y, Liu S, Lu J, Wen Q, Fan S (2019) Expression of DR5 and c\u0026ndash;FLIP proteins as novel prognostic biomarkers for non\u0026ndash;small cell lung cancer patients treated with surgical resection and chemotherapy[J]. Oncol Rep 42(6):2363\u0026ndash;2370\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiaohong W, Jun Z, Hongmei G, Fan Q (2019) CFLAR is a critical regulator of cerebral ischaemia-reperfusion injury through regulating inflammation and endoplasmic reticulum (ER) stress[J]. Biomed Pharmacother 117:109155\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu D, Wang B, Chen PP, Wang YZ, Miao NJ, Yin F, Cheng Q, Zhou ZL, Xie HY, Zhou L, Liu J, Wang XX, Xue H, Zhang W, Lu LM (2019) c-Myc promotes tubular cell apoptosis in ischemia-reperfusion-induced renal injury by negatively regulating c-FLIP and enhancing FasL/Fas-mediated apoptosis pathway[J]. Acta Pharmacol Sin 40(8):1058\u0026ndash;1066\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuebke T, Schwarz L, Beer YY, Schumann S, Misterek M, Sander FE, Plaza-Sirvent C, Schmitz I (2019) c-FLIP and CD95 signaling are essential for survival of renal cell carcinoma[J]. Cell Death Dis 10(6):384\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang S, Li N, Wu S, Xie T, Chen Q, Wu J, Zeng S, Zhu L, Bai S, Zha H, Tian W, Wu N, Zou X, Fang S, Luo C, Shi M, Sun C, Shu Y, Luo H (2024) c-FLIP facilitates ZIKV infection by mediating caspase-8/3-dependent apoptosis[J]. PLoS Pathog 20(7):e1012408\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYe C, Jiang W, Hu T, Liang J, Chen Y (2024) The Regulatory Impact of CFLAR Methylation Modification on Liver Lipid Metabolism[J]. Int J Mol Sci, 25(14)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMora-Molina R, St\u0026ouml;hr D, Rehm M, L\u0026oacute;pez-Rivas A (2022) cFLIP downregulation is an early event required for endoplasmic reticulum stress-induced apoptosis in tumor cells[J]. Cell Death Dis 13(2):111\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"NRF-1, CFLAR, hypoxia, H9C2 cardiomyocytes, apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-6499729/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6499729/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eInhibition 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.\u003c/p\u003e\u003ch2\u003eMethods and Results\u003c/h2\u003e \u003cp\u003eChromatin immunoprecipitation (ChIP) and Dual-Glo luciferase assays confirmed that NRF-1 binds to the \u003cem\u003eCflar\u003c/em\u003e 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.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003ewe 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.\u003c/p\u003e","manuscriptTitle":"Nuclear respiratory factor-1 promotes CFLAR transcription in H9C2 cardiomyocytes, protecting them against hypoxia-induced apoptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 03:29:15","doi":"10.21203/rs.3.rs-6499729/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-04-28T16:23:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"68272708412546520639669853151597809870","date":"2025-04-24T16:01:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-24T11:10:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-23T08:30:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-23T08:30:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2025-04-22T02:57:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"72aa925d-c619-4f0e-961e-49a7b2be817f","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T16:00:23+00:00","versionOfRecord":{"articleIdentity":"rs-6499729","link":"https://doi.org/10.1007/s11033-025-10636-7","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2025-06-06 15:57:13","publishedOnDateReadable":"June 6th, 2025"},"versionCreatedAt":"2025-05-07 03:29:15","video":"","vorDoi":"10.1007/s11033-025-10636-7","vorDoiUrl":"https://doi.org/10.1007/s11033-025-10636-7","workflowStages":[]},"version":"v1","identity":"rs-6499729","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6499729","identity":"rs-6499729","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}