UPF1 Alleviates Myocardial Ischemia-Reperfusion Injury by Regulating SMURF2-Mediated Ubiquitination Degradation of FOXA2.

Acute myocardial infarction is a common critical disease of cardiovascular system, which is mainly treated by drug thrombolysis or percutaneous coronary intervention. 1) However, the blocked blood vessels can lead to progression of myocardial ischemia/reperfusion injury (MIRI) after reperfusion, manifested as reperfusion arrhythmia and decreased cardiac function. 2) In severe cases, life-threatening ventricular arrhythmias can occur, leading to sudden death. The mechanism of MIRI includes cell death, inflammation, neurohumoral activation and oxidative stress. Apoptosis is an active and programmed cell death. Molecular cardiovascular research results have confirmed that apoptosis is the main form of myocardial cell injury, and the occurrence of this process is closely related to a variety of genes. 3) Therefore, it is of great clinical significance to explore molecular mechanism of cardiomyocyte apoptosis in MIRI to reduce myocardial injury. Protease-activated receptor 4 (PAR4), a pro-apoptotic protein, belongs to the family of G protein-coupled receptors. 4) Inhibiting PAR4 can effectively block mitogen-activated protein kinase signal transduction and liver apoptosis following brain death. 5) Moreover, PAR4 can induce apoptosis in cells of lung cancer. 6) Recent data have proven that cardiomyocyte apoptosis can be triggered by PAR4 stimulation. However, PAR4 deficiency can reduce cardiomyocyte apoptosis following MIRI, thereby alleviating MIRI. 7) Nevertheless, the molecular mechanism of PAR4 in MIRI is still unknown. Forkhead box A2 (FOXA2), acting as a transcription factor, is essential for both endoplasmic reticulum stress and oxidative stress. 8) FOXA2 downregulates ERβ by transcriptionally inhibiting IGF2BP1, thereby suppressing pyroptosis in endometriosis. 9) Additionally, FOXA2 reduces liver cancer cell migration and invasion via transcriptionally inhibiting microRNA-103a-3p. 10) Besides, FOXA2 safeguards cardiomyocytes against necrosis induced by MIRI. 11) Through the prediction of the JASPAR database, we discovered that FOXA2 has the potential to act as a transcription factor for PAR4. Further investigation is required to explore the interaction between FOXA2 and PAR4 in the context of MIRI. Ubiquitination is an important post-translational modification process. In this process, E3 ubiquitin ligases play a vital role by attaching ubiquitin molecules to target proteins, thereby facilitating protein degradation and regulating various cellular activities. 12) SMAD-specific E3 ubiquitin ligase 2 (SMURF2) is an E3 ligase involved in substrate recognition and ubiquitination. Studies have reported that SMURF2 has pro-apoptotic effects in some diseases. For example, in chronic heart failure rats, miR-568 inhibits SMURF2 to mitigate cardiomyocyte apoptosis, oxidative stress, and cardiac dysfunction. 13) Suppression of SMURF2 enhances the proliferation and reduces apoptosis and inflammation in chondrocytes of osteoarthritis. 14) Through the UbiBrowser database, we predicted that SMURF2 is a potential ubiquitinating enzyme of FOXA2. However, there is currently no experimental evidence to demonstrate whether SMURF2 can also mediate the ubiquitination degradation of FOXA2 in MIRI. Up-frameshift 1 (UPF1) is an ATP-dependent RNA helicase that promotes mRNA decay pathways regulated by different RNA-binding proteins. Studies have shown that by promoting the degradation of FOXD1 mRNA, UPF1 mitigates lung ischemia/reperfusion (I/R) injury. 15) Meanwhile, UPF1 has anti-apoptotic effects in related studies such as heart and cancer cell proliferation. For example, Avery et al. found that loss of UPF1 expression represses cell growth and induces apoptosis in Drosophila development. 16) Additionally, knockout of UPF1 inhibits mRNA decay and blocks the transcription of bag3−/−mutants, resulting in severe defects in the heart and skeletal muscle. 17) We found that UPF1 is a potential RNA binding protein of SMURF2 through Starbase database analysis. Based on the findings mentioned above, our hypothesis is that UPF1 plays a role in the degradation of SMURF2 mRNA, leading to a decrease in SMURF2 expression. This, in turn, inhibits SMURF2-mediated ubiquitination degradation of FOXA2, resulting in increased levels of FOXA2. Subsequently, elevated FOXA2 transcriptionally inhibits PAR4, thereby reducing apoptosis and alleviating MIRI. The outcomes of our study may potentially offer theoretical support for developing novel therapeutic approaches for MIRI.

The Committee on Ethical Use of Animals at the First Affiliated Hospital of Jiamusi University approved the animal care protocols and experimental procedures (202407). A total of 20 Sprague Dawley (SD) rats (6–8 weeks old, weighing 200–220 g) were obtained from Hunan Silai Kejingda Experiment Animal Company (Hunan, China). The rats were housed under controlled conditions, including a constant room temperature, a 12/12-hour light/dark cycle, and provided with a standard rodent diet. Animals were grouped as follows (n=5 rats/group): Sham group, I/R group, I/R + overexpression (oe)-negative control (NC) group, I/R + oe-UPF1 group. The oe-UPF1 or oe- NC adenoviruses were purchased from Genechem (Shanghai, China). Seven days before operation, the oe-UPF1 or oe-NC adenoviruses were injected into the rats through the right common carotid artery. 18) Myocardial I/R (MI/R) model was constructed in rats as previously described. 19) Anesthesia was induced in rats through intraperitoneal injection of 1% pentobarbital sodium (60 mg/kg). Subsequently, mechanical ventilation was administered using an animal ventilator following endotracheal intubation. Myocardial ischemia was induced by temporarily ligating the left anterior descending coronary artery using a slipknot for a duration of 30 minutes. Subsequently, a 24-hour period of myocardial reperfusion followed. Sham groups underwent the same surgical procedure without artery ligation. Successful model induction is indicated by the appearance of a white color in the left ventricular apex and anterior wall. Rat cardiomyocytes H9C2 cells, obtained from Chinese Academy of Science Cell Bank (Shanghai, China), were cultured in Dulbecco's Modified Eagle's Medium (DMEM) medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin (Sigma, St. Louis, MO, USA) at 37˚C with 5% CO 2 . For hypoxia/reoxygenation (H/R) treatment, cells were incubated in glucose-free DMEM and placed in a hypoxic incubator (5% CO 2 , 94% N 2 , 1% O 2 ) for 18 hours, followed by 8 hours of reoxygenation (95% O 2 , 5% CO 2 ). Control cells were cultured under normal conditions. For MG132 treatment, cells were treated with MG132 (40 µM, Sigma) for 1.5 hours. The siRNA against SMURF2, as well as siRNA negative control, were obtained from RiboBio (Guangzhou, China). Cells were plated in a 6-well plate. For transfection in a six-well plate format, 100 pM of siRNA and 6 μL of Lipofectamine 3,000 reagent (Invitrogen) was added on top of cells at 60% confluence in each well following manufacturer's instructions for 48 hours. Gene-Pharma (Shanghai, China) supplied lentivirus containing oe-FOXA2, oe-PAR4, oe-UPF1, oe-SMURF2, and oe-NC. For infection, cells were exposed to lentivirus particles (10 9 TU/mL) in culture medium. After 24 hours, infected culture medium was discarded. Total RNA was extracted from cells utilizing TRIzol reagent (Invitrogen) and was reverse-transcribed utilizing a PrimeScript RT reagent Kit (Takara, Kyoto, Japan). Next, SYBR (Thermo Fisher Scientific, Carlsbad, CA, USA) was employed for quantitative real-time PCR on Applied Biosystems Prism 7500. Primer sequences were as follows: UPF1 F: 5′-TGCGGTACGAGGATGCTTAC-3′, R: 5′-CTAAATCCTCATTACCAGAGTCAGT-3′; SMURF2 F: 5′-GCCTGACAGTACTCTGTGCAA-3′, R: 5′-ACGCTGATCGTGACGGAATC-3′; FOXA2 F: 5′-TGCACTCGGCTTCCAGTATG-3′, R: 5′-CTGGCGTTCATGTTGCTCAC-3′; PAR4 F: 5′-ATGCTCGGGTTCAGCATCAG-3′, R: 5′-GGGAAGCCTCGTGGATTAGG-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) F: 5′-GCAAGTTCAACGGCACAG-3′, R: 5′-GCCAGTAGACTCCACGACAT-3′. GAPDH was used as reference gene. Data was analyzed with 2 −∆∆Ct method. Protein extracts of cells and tissues were prepared with RIPA lysis buffer (Beyotime, Shanghai, China). Protein concentrations were assessed with BCA assay (Pierce, Rockford, IL, USA). We subjected the proteins to 10% acrylamide gels and electrophoretically transferred to PVDF membranes (Millipore, Bedford, MA, USA). After 1 hour of blockade with 5% bovine serum albumin, membranes were incubated with antibodies overnight at 4˚C: UPF1 (#12040, Cell Signaling Technology, CST, Danvers, MA, USA), FOXA2 (#8186T, 1:1,000, CST), PAR4 (#2328, 1:1,000, CST), Bax (ab32503, 1:1,000, Abcam, Cambridge, MA, USA), Cleaved caspase-3 (ab184787, 1:1,000, Abcam), SYVN1 (13473-1-AP, 1:1,000, Proteintech, Rosemont, IL, USA), SMURF2 (18038-1-AP, 1:1,000, Proteintech), WW domain containing E3 ubiquitin protein ligase 2 (WWP2; 12197-1-AP, 1:1,000, Proteintech). After rinsing with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (#7074, 1:1,000, CST) for 1 hour. Bands were developed using enhanced chemiluminescence detection reagent (Bio-Rad, Hercules, CA, USA). Protein expression levels were quantified utilizing Image J (National Institutes of Health, Bethesda, MD, USA) to determine relative quantity. GAPDH (MA5-35235, Invitrogen) was a loading control. Myocardial tissue was promptly frozen at −20°C for approximately 20 minutes to facilitate slicing. Tissue sections were obtained at 1 mm intervals. Subsequently, slices were immersed in a 2% tetrazolium chloride (TTC) solution (Sigma) and incubated at 37°C, shielded from light, for 20 minutes. Afterward, slices were fixed in 4% paraformaldehyde for 24 hours and photographed. Cardiac specimens were preserved using 10% paraformaldehyde, followed by embedding in paraffin and slicing into thin sections of 5 μm thickness. These sections were then placed on slides for hematoxylin-eosin staining (Beyotime) and examined using a microscope (Olympus, Tokyo, Japan). Tissue slices were processed through several stages, including deparaffinization, antigen retrieval, a 30-minute blocking step using goat serum, and overnight incubation at 4°C with a primary antibody specific to UPF1 (#12040, CST. Subsequently, slices were exposed to a biotinylated secondary antibody (65-6140, Invitrogen) and developed utilizing diaminobenzidine for color visualization. Section images were captured using a microscope (Olympus). Deparaffinization of cardiac sections was carried out, followed by a 30-minute treatment with Proteinase K at 37°C. After 2 phosphate-buffered saline (PBS) washes, slides were incubated with a terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling (TUNEL) detection solution (Beyotime) for 1 hour. DAPI staining (Beyotime) was adopted to visualize nucleus. TUNEL-positive cells were observed under a microscope (Olympus). Based on FOXA2 binding sites identified in JASPAR database, the corresponding sequence of PAR4 was amplified and inserted into psiCheck2 control vectors (Promega, Madison, WI, USA) to generate PAR4 wild-type vector (PAR4 promoter wt). Additionally, mutant sequence of PAR4 was introduced in psiCheck2 to create PAR4 mutant-type vector (PAR4 promoter mut). Subsequently, 293T cells were co-treated with the above plasmids along with either oe-FOXA2 or oe-NC. Luciferase activity was assessed utilizing Dual-Luciferase Reporter Assay System (Promega). Magna RNA immunoprecipitation (RIP) assay kits (Millipore) were adopted for RIP assay, following provided protocol. H9C2 cells were lysed and incubated with magnetic beads preconjugated with antibodies anti-UPF1 (#12040, CST) or anti-immunoglobulin G (IgG) (ab182931, Abcam). Immunoprecipitated RNA was analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis. To generate DNA-protein crosslinks, cells were exposed to 4% methanal. Subsequently, sonication was performed to fragment chromatin, followed by immunoprecipitation overnight at 4°C utilizing antibodies specific to FOXA2 (MA5-42678, Invitrogen) or IgG (ab182931, Abcam). The resulting immune complexes were captured using chromatin immunoprecipitation (ChIP)-grade protein G magnetic beads (Roche, Basel, Switzerland). The immunoprecipitated DNA was subjected to PCR analysis and normalized to input DNA. After treatment, cells were lysed and subjected to an overnight incubation at 4 °C with agarose beads (Roche) conjugated with anti-FOXA2 antibody (#8186T, CST). Following that, beads were rinsed and boiled with SDS loading buffer for further analysis. The ubiquitination level of FOXA2. Levels of FOXA2, SYVN1, SMURF2, and WWP2 were determined utilizing western blot. To assess stability of the FOXA2 protein, cycloheximide (Sigma) was employed. In brief, cell culture medium was supplemented with 125 μg/mL of cycloheximide. At 0, 2, 4, and 8 hours, the protein was extracted from cells. FOXA2 protein level was assessed utilizing Western blot. Actinomycin D (Sigma) was employed to assess stability of SMURF2 mRNA. Briefly, cell culture medium was supplemented with 1 μM actinomycin D. Following incubation for 0, 3, 6, and 9 hours, RNA was extracted from cells. SMURF2 mRNA level was quantified utilizing RT-qPCR. Cell viability was assessed utilizing Cell Counting Kit-8 (CCK-8, Beyotime). H9C2 cells were seeded individually in 96-well plates. After the respective treatments, 10 μL of CCK-8 solution was added to each well and incubated at 37°C for 1 hour. The absorbance at 450 nm was measured with a microplate reader (Thermo Fisher Scientific). To analyze cell apoptosis, an apoptosis assay kit (Annexin V-FITC/propidium iodide [PI], ELabScience, Wuhan, China) was utilized. Following treatment, cells were harvested, washed twice with ice-cold PBS, and suspended in 300 μL of binding buffer. Subsequently, they were incubated in the dark for 10 minutes with 5 μL of Annexin V-FITC and 10 μL of PI. After addition of 200 μL of PBS to each sample, cell apoptosis was assessed utilizing flow cytometry (BD Biosciences, San Diego, CA, USA). Experimental results were shown as mean ± standard deviation of three repeated experiments. Analyses were conducted utilizing SPSS 21.0 (IBM Corp., Armonk, NY, USA). Pairwise comparisons were analyzed with Student t - test, and multigroup comparisons were analyzed with 1-way analysis of variance. For continuous variables that do not follow a normal distribution, we presented them as median (interquartile range) and used non-parametric analysis for comparison. The p<0.05 indicated a statistical difference.

To establish a cell model of MIRI, H9C2 cells were cultured under conditions of H/R. RT-qPCR and Western blot results showed that compared to control group, H/R treatment promoted PAR4 expression ( Figure 1A and B ). Notably, FOXA2 was predicted to be a potential transcription factor of PAR4 by hTFtarget and Cistrome Data Browser database. ChIP assay confirmed the recruitment of FOXA2 to PAR4 promoter region ( Figure 1C ). JASPAR database further predicted binding site of FOXA2 to PAR4 promoter region, and dual luciferase assay verified the transcriptional regulation of FOXA2 on PAR4, suggesting that FOXA2 transcriptionally inhibited PAR4 expression ( Figure 1D ). Next, H9C2 cells were overexpressed with FOXA2 and PAR4, and exposed to H/R treatment. Compared to the blank group, H/R treatment significantly inhibited the expression of FOXA2 while increasing the levels of PAR4. However, FOXA2 was upregulated and PAR4 was downregulated after overexpressing FOXA2, but PAR4 level was increased after PAR4 upregulation ( Figure 1E and F ). CCK8 assay indicated that H/R treatment significantly inhibited cell viability, which was then rescued by FOXA2 upregulation but abolished by reinforced PAR4 ( Figure 1G ). Moreover, flow cytometry showed that apoptosis was increased following H/R treatment, and the introduction of oe-FOXA2 reduced apoptosis, which was then countered by overexpression of PAR4 ( Figure 1H ). Meanwhile, apoptosis-related proteins Bax and Cleaved caspase-3 were increased after H/R treatment, and FOXA2 overexpression decreased the protein levels of Bax and Cleaved caspase-3, and PAR4 upregulation partially abolished the rescue effect of oe-FOXA2 ( Figure 1I ). Therefore, FOXA2 transcriptionally inhibited PAR4, thereby attenuating H/R-triggered cardiomyocyte apoptosis. DMEM = Dulbecco's Modified Eagle's Medium; FOXA2 = forkhead box A2; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; H/R = hypoxia/reoxygenation; IgG = immunoglobulin G; NC = negative control; oe = overexpression; PAR4 = protease-activated receptor 4; RT-qPCR = reverse transcription quantitative polymerase chain reaction. ** p<0.01, *** p<0.001. H9C2 cells were used for H/R treatment, and treated with proteasome inhibitor MG132. H/R treatment resulted in an increase in SMURF2 level and a decrease in FOXA2 level, and MG132 treatment increased FOXA2 level while having no effect on SMURF2 level ( Figure 2A ). In addition, the level of FOXA2 ubiquitination was increased in H/R group ( Figure 2B ). After treatment of protein synthesis inhibitor cycloheximide, the stability of FOXA2 protein in H/R group was decreased ( Figure 2C ). We next screened for ubiquitinase that may be involved in maintaining FOXA2 stability. We conducted bioinformatics analysis using the BioGRID database and UbiBrowser database, we found that OTU deubiquitinase 3, ring finger protein 4, SMURF1, STIP1 homology and U-box containing protein 1 (STUB1), SYVN1, SMURF2, and WWP2 might be the upstream regulators of FOXA2. Among these, SYVN1, SMURF2, and WWP2 levels were elevated in cells under H/R treatment ( Supplementary Figure 1A ). Next, Co-IP experiments implied that only endogenous SMURF2 interacted with FOXA2 ( Supplementary Figure 1B ). Furthermore, H9C2 cells were transfected with si-NC or si-SMURF2, followed by H/R treatment. FOXA2 protein in SMURF2 knockdown group was increased ( Figure 2D ). Meanwhile, the ubiquitination level of FOXA2 protein in SMURF2 knockdown group was decreased ( Figure 2E ). Besides, the stability of FOXA2 protein was increased in SMURF2 knockdown group ( Figure 2F ). Collectively, these findings suggested that SMURF2 participated in regulating FOXA2 stability by its ubiquitinase activity. FOXA2 = forkhead box A2; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; H/R = hypoxia/reoxygenation; NC = negative control; SMURF2 = SMAD-specific E3 ubiquitin ligase 2. *** p<0.001. UPF1 is a potential RNA binding protein for SMIRF2. Next, we investigated effect of UPF1 on H/R-induced cardiomyocyte apoptosis. H9C2 cells were infected with oe-NC or oe-UPF1, followed by H/R treatment. Compared with the blank group, the expression level of UPF1 was downregulated by H/R treatment, and UPF1 overexpression rescued this downward trend ( Figure 3A and B ). Next, CCK8 assay demonstrated that UPF1 upregulation significantly increased cell viability ( Figure 3C ). Furthermore, apoptosis was inhibited, and levels of Bax and Cleaved caspase-3 were decreased by UPF1 overexpression ( Figure 3D and E ). The data above indicated that overexpressed UPF1 improved H/R-triggered cardiomyocyte apoptosis. GAPDH = glyceraldehyde 3-phosphate dehydrogenase; H/R = hypoxia/reoxygenation; NC = negative control; oe = overexpression; RT-qPCR = reverse transcription quantitative polymerase chain reaction; UPF1 = up-frameshift 1. ** p<0.01, *** p<0.001. To reveal the effect of UPF1 on MIRI in vivo, we established a MI/R rat model. SD rats were subjected to different groups: sham, I/R, I/R + oe-NC, I/R + oe-UPF1. Western blot and immunohistochemistry staining showed that I/R treatment led to a decrease in UPF1 protein in rat heart tissue, while further overexpression of UPF1 increased UPF1 expression ( Figure 4A and B ). Furthermore, TTC staining indicated that I/R treatment resulted in ischemia in rat heart tissue, and overexpressed UPF1 reduced the ischemic area ( Figure 4C ). HE staining revealed that compared with Sham group, I/R group had more serious myocardial injury, while overexpression of UPF1 obviously alleviated it ( Figure 4D ). TUNEL staining discovered that apoptosis in I/R group were higher than that in sham group, and UPF1 upregulation inhibited this upward trend ( Figure 4E ). Finally, the I/R-induced increase in Bax and Cleaved caspase-3 levels was reversed with UPF1 overexpression ( Figure 4F ). I/R treatment increased SMURF2 and PAR4 expression, but decreased FOXA2 expression in rat heart tissue, while UPF1 upregulation overturned these effects ( Figure 4G ). These results implied that reinforced UPF1 prevented myocardial dysfunction caused by I/R in vivo. GAPDH = glyceraldehyde 3-phosphate dehydrogenase; FOXA2 = forkhead box A2; I/R = ischemia/reperfusion; NC = negative control; oe = overexpression; PAR4 = protease-activated receptor 4; SMURF2 = SMAD-specific E3 ubiquitin ligase 2; TTC = tetrazolium chloride; UPF1 = up-frameshift 1. *** p<0.001. StarBase database and POSTAR3 database predicted that UPF1 was a potential RNA binding protein of SMURF2 ( Supplementary Figure 2 ). Based on above findings, we further explored effect of H/R treatment on SMURF2 mRNA stability. SMURF2 mRNA level in H9C2 cells was assessed at 0, 3, 6 and 9 hours after treatment with actinomycin D (1 μM). Stability of SMURF2 mRNA in H/R group was increased ( Figure 5A ). RIP assay verified that there was a binding relationship between UPF1 and SMURF2 mRNA, and this binding relationship was weakened after H/R treatment ( Figure 5B ). Furthermore, H9C2 cells were infected with oe-NC or oe-UPF1, followed by H/R treatment. UPF1 overexpression repressed SMURF2 mRNA and protein expression ( Figure 5C and D ). Notably, UPF1 overexpression reduced the mRNA stability of SMURF2 ( Figure 5E ). Altogether, the above data indicated that UPF1 inhibited SMURF2 expression by decreasing the stability of SMURF2 mRNA. GAPDH = glyceraldehyde 3-phosphate dehydrogenase; H/R = hypoxia/reoxygenation; IgG = immunoglobulin G; NC = negative control; oe = overexpression; RT-qPCR = reverse transcription quantitative polymerase chain reaction; SMURF2 = SMAD-specific E3 ubiquitin ligase 2; UPF1 = up-frameshift 1. ** p<0.01, *** p<0.001. Next, we conducted rescue assays to confirm whether UPF1 inhibited H/R-triggered cardiomyocyte apoptosis through regulating SMURF2, FOXA2 and PAR4 expression. Next, UPF1 and SMURF2 were overexpressed in H9C2 cells, which were then exposed to H/R treatment . Western blot results showed that UPF1 upregulation reduced SMURF2 and PAR4 expression, and increased FOXA2 expression, while overexpressed SMURF2 further increased SMURF2 and PAR4 expression and decreased FOXA2 expression ( Figure 6A ). CCK8 assay demonstrated that reinforced SMURF2 abrogated the promoting effect of UPF1 upregulation on cell viability ( Figure 6B ). Besides, UPF1 overexpression inhibited apoptosis in H/R-induced cells, and SMURF2 overexpression attenuated this inhibitory effect ( Figure 6C ). Additionally, oe-SMURF2 partially rescued the suppressive effects of oe-UPF1 on Bax and Cleaved caspase-3 levels ( Figure 6D ). Altogether, UPF1 suppressed H/R-triggered cardiomyocyte apoptosis through the SMURF2/FOXA2/PAR4 axis. FOXA2 = forkhead box A2; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; H/R = hypoxia/reoxygenation; NC = negative control; oe = overexpression; PAR4 = protease-activated receptor 4; SMURF2 = SMAD-specific E3 ubiquitin ligase 2; UPF1 = up-frameshift 1. * p<0.05, ** p<0.01, *** p<0.001.

The pathogenesis of MIRI is very complex, mainly involving calcium overload, mitochondrial damage, inflammatory response, oxidative stress and cardiomyocyte apoptosis. Cardiomyocyte apoptosis is the main pathological feature of MIRI, and the degree of apoptosis determines the degree of MIRI. 20) Myocardial ischemia initiates cardiomyocyte apoptosis, and reperfusion further aggravates cardiomyocyte apoptosis, which in turn causes malignant arrhythmia, heart failure and even sudden cardiac death. 21) Therefore, further exploration into underlying mechanism of cardiomyocyte apoptosis is crucial for MIRI treatment. Here, we identified that UPF1 regulated SMURF2-mediated ubiquitination degradation of FOXA2, thus alleviating cardiomyocyte apoptosis after MIRI. FOXA2, a member of the forkhead transcription factor family, exerts transcriptional regulation by binding to the promoter and enhancer regions of target genes. Through the blockade of p38/STAT3 signaling, FOXA2 mitigates lipopolysaccharide-induced pneumonia by suppressing the inflammatory response, oxidative stress, and apoptosis. 22) Besides, by suppressing FOXA2, the circular RNA CNEACR regulates the process of necroptosis in cardiomyocytes. 11) In this study, we found that FOXA2 increased cell viability and inhibited apoptosis in H/R-induced cardiomyocytes. Moreover, we discovered that FOXA2 repressed H/R-stimulated cardiomyocyte apoptosis by suppressing PAR4 transcription, a member of protease activated receptors. In cardiomyocytes, we verified that FOXA2 negatively regulated PAR4 expression by transcriptionally inhibiting PAR4. Notably, deficiency of PAR4 provides cardioprotective effects following acute ischemia-reperfusion injury. The deficiency of PAR4 reduces cardiomyocyte death during ischemia-reperfusion by inhibiting JNK signaling pathway effectors. 23) These findings emphasize the therapeutic potential of the FOXA2/PAR4 axis in promoting cardioprotection against MIRI. SMURF2 is a member of HECT family of E3 ubiquitin ligases and was initially believed to have significant involvement in embryogenesis, maintenance of adult tissue homeostasis, and the development of diverse human diseases. 24) A study showed that suppression of SMURF2 enhances the viability of H9C2 cells stimulated by oxygen-glucose deprivation/reoxygenation (OGD/R) and inhibits apoptosis. 25) In addition, SMURF2 can ubiquitinate and degrade EZH2, leading to activation of Akt/GSK3β signaling. SMURF2 translation suppression by miR-322/503 provides protection against MIRI via modulating EZH2/Akt/GSK3β signaling. 26) Consistent with previous studies, our results revealed that SMURF2 negatively regulated FOXA2 expression in cardiomyocytes by directly targeting FOXA2 and degrading FOXA2. UPF1, an RNA-binding protein, acts as a key protein in pathway of nonsense-mediated mRNA decay. 27) Recent research has provided compelling evidence highlighting the role of UPF1 in the modulation of apoptosis. For example, knockdown of UPF1 enhances apoptosis in colorectal cancer cells. 28) Importantly, recent study has shown that UPF1 mitigates lung I/R injury by facilitating the degradation of FOXD1 mRNA. 15) Here, we found that UPF1 overexpression alleviated H/R-stimulated cardiomyocyte apoptosis and I/R-triggered myocardial dysfunction. UPF1 has also been implicated in the modulation of gastric cancer progression through its role in facilitating the degradation of the long non-coding RNA MALAT1. 29) In this study, we observed the association between UPF1 and SMURF2 mRNA. UPF1 mediated the decay of SMURF2 mRNA and reduces its expression. In cell experiments, we demonstrated that UPF1 inhibited H/R-induced cardiomyocyte apoptosis through SMURF2/FOXA2/PAR4 pathway. Therefore, these results indicated a protective role of UPF1 in MIRI. To conclude, our experiment demonstrated that UPF1 upregulated FOXA2/downregulated PAR4 signaling through suppressing SMURF2 expression, thereby inhibiting cell apoptosis during MIRI. Therefore, the UPF1/SMURF2/FOXA2/PAR4 pathway holds promise in enhancing prognosis of MIRI. However, further clinical research is required to validate findings of this animal study comprehensively.