P38α deficiency alleviates myocardial ischemia/reperfusion injury by stabilizing c‑Myc to inhibit ferroptosis

Figures

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  Fig. 1. Ischemia/reperfusion (I/R) stress elicits p38α activation and ferroptosis in cardiomyocytes. a Single-cell transcriptomic profiling from the Human Protein Atlas (HPA) database reveals expression of p38α (MAPK14) in cardiomyocytes. b Schematic illustration of the in vivo mouse myocardial ischemia/reperfusion (I/R) model and in vitro oxygen–glucose deprivation/reperfusion (OGD/R) model employed to recapitulate cardiac I/R injury. c, d Western blot analysis and quantification of phosphorylated p38α (p-p38α), total p38α, and the ferroptosis marker GPX4 in sham-operated and I/R-treated mouse hearts, showing markedly elevated p-p38α and reduced GPX4 levels with a significant inverse correlation (n = 6). e, f Representative immunofluorescence images and quantitative analysis of cardiac tissue sections co-stained for cTnT (cardiomyocyte marker) with p-p38α or GPX4 (n = 8). g Schematic workflow depicting the isolation and primary culture of neonatal mouse cardiomyocytes (NMCMs) from postnatal day 1–3 mice. h, i Western blot analysis and quantification of p-p38α, total p38α, and the ferroptosis marker GPX4 in control and OGD/R-treated NMCMs, showing markedly elevated p-p38α and reduced…

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  Fig. 2. Genetic ablation of p38α mitigates myocardial I/R injury. a Cell viability assays in HL-1 cardiomyocytes following p38α knockdown with or without OGD/R challenge (n = 4). b Flow cytometry analysis of intracellular ROS levels via DCFH-DA staining in HL-1 cells (n = 4). c Cell viability assays in NMCMs following p38α genetic knockout (p38α-CKO) with or without OGD/R challenge (n = 4). d, e In vivo myocardial I/R injury assessment in wild-type (WT) and p38α-CKO mice. d Hematoxylin–eosin (H&E) staining and quantitative analysis of myocardial lesion area (n = 5); e Evans blue/TTC double-staining and quantification of infarct size relative to area-at-risk (AAR) (n = 5). f Representative B-mode echocardiography images of WT and p38α-CKO mouse hearts at baseline (Sham) and 24 h post-I/R (n = 5). g Quantitative analysis of left ventricular ejection fraction (EF) and fractional shortening (FS) from echocardiography (n = 5). h, i TUNEL staining and quantitative analysis of damaged cells in myocardial tissues from WT and p38α-CKO mice post-I/R (n = 4). j Quantification of serum myocardial injury markers, including creatine kinase (CK-MB), creatine kinase (CK), and lactate dehydrogenase…

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  Fig. 3. p38α deficiency alleviates myocardial I/R injury by inhibiting ferroptosis. a KEGG pathway enrichment analysis of differentially expressed genes (DEGs) in shp38α vs. OGD/R group, with the ferroptosis pathway highlighted in red. b Cell viability was detected by CCK-8 assay in HL-1 cells with or without shp38α transfection, treated with or without erastin (n = 4). c MDA level (n = 4), d LDH release (n = 4), and e GSH content (n = 4) were measured to assess oxidative stress and ferroptosis in HL-1 cells. f, g Flow cytometry analysis of mean Cy3-A fluorescence intensity of Fe2Orange f (n = 6) and FITC-A of C11 BODIPY g (n = 4) to detect intracellular Fe.2+ and lipid peroxidation, respectively. h Western blot analysis of ferroptosis-related proteins (SLC40A1, GPX4, ACSL3, HMOX1) in HL-1 cells, with quantitative results shown in the heatmap (n = 4). i Western blot analysis of ferroptosis-related proteins in myocardial tissues from p38α-CKO mice and WT mice with or without I/R injury, with quantitative heatmap results (n = 4). j Immunofluorescence staining of 4-HNE (green) in myocardial tissues, DAPI (blue) for nuclear staining, and quantitative analysis of mean fluorescence intensity…

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  Fig. 4. p38α interacts with c-Myc to regulate ferroptosis in myocardial I/R injury. a Heatmap of transcription factor expression in HL-1 cells transfected with shp38α or shControl, with or without OGD/R treatment. b qRT-PCR analysis of c-Myc mRNA expression in HL-1 cells (n = 6). c Western blot analysis of S62-phosphorylated c-Myc (S62-p–c-Myc) and total c-Myc protein levels (n = 4). d Quantification of p–c-Myc and c-Myc protein expression from panel c (n = 4). e CCK-8 assay for cell viability in HL-1 cells with shp38α, shc-Myc, or oec-Myc transfection, with or without OGD/R (n = 6). f Flow cytometry analysis of intracellular ROS levels detected by DCFH-DA staining (n = 6). g MS analyzed the interaction proteins of c-Myc in HL-1 cells. h Immunofluorescence co-localization staining of c-Myc (green) and p-p38α (red) in HL-1 cells, with DAPI (blue) for nuclear staining (scale bar: 10 μm). i-j Co-IP assay verifying the direct binding between p-p38α and c-Myc or p-p38α and S62-p–c-Myc in HL-1 cells. k Molecular docking model of p-p38α (cyan) and c-Myc (blue) interaction. l Binding energy contribution of key amino acid residues in the p38α-c-Myc complex. m Schematic diagram of c-Myc protein…

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  Fig. 5. p-p38α deficiency promotes c-Myc stabilization by inhibiting STUB1-mediated ubiquitination and degradation in myocardial I/R injury. a Western blot analysis of c-Myc protein levels in HL-1 cells with or without shp38α transfection, treated with or without OGD/R and the proteasome inhibitor MG132 (n = 4). b CHX (50 μg/mL) chase assay to detect c-Myc protein stability in HL-1 cells under different treatments (Control, shp38α, OGD/R, shp38α + OGD/R) (n = 4). c Quantitative analysis of remaining c-Myc protein levels from panel b (n = 4). d In vitro ubiquitination assay showing c-Myc ubiquitination levels in HL-1 cells with or without shp38α transfection, with or without OGD/R treatment. e Heatmap of E3 ubiquitin ligase expression in shp38α vs. control HL-1 cells under OGD/R, with STUB1 highlighted as a key candidate. f Western blot and relative quantification of c-Myc protein levels in HL-1 cells with or without shSTUB1 transfection, with or without OGD/R (n = 4). g IP-MS assay confirming the interaction between STUB1 and c-Myc. h Co-IP assay verifying the direct binding between STUB1 and c-Myc in HL-1 cells. i In vitro ubiquitination assay of c-Myc in cells transfected with WT or…

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  Fig. 6. c-Myc transcriptionally inhibits NCOA4 to regulate ferroptosis in myocardial I/R injury. a Heatmap of ferroptosis-related gene expression in shp38α vs. control HL-1 cells under OGD/R, with key ferroptosis regulators (NCOA4, TFRC, ACSL4) highlighted. b qRT-PCR analysis of NCOA4, TFRC, and ACSL4 mRNA expression in HL-1 cells with or without shp38α transfection, with or without OGD/R (n = 6). c qRT-PCR analysis of NCOA4, TFRC, and ACSL4 mRNA expression in HL-1 cells with shc-Myc or oec-Myc transfection, with or without OGD/R (n = 6). d, e Western blot and relative quantification analysis of NCOA4 protein levels in HL-1 cells with shp38α d or shc-Myc/oec-Myc e transfection, with or without OGD/R (n = 4). f Putative c-Myc-binding sites identified in the promoter region of NCOA4 gene. g CUT&RUN-qPCR assays showing binding of c-Myc to NCOA4 promoter (n = 6). h NCOA4 mRNA expression in HL-1 cells treated with shMYC or oeMYC and Act D for 0, 2, 4, 6, and 8 h with or without OGD/R, and was detected by RT‐qPCR (n = 4). i CCK-8 assay for cell viability, and measurement of GSH and MDA levels in HL-1 cells with shc-Myc and/or shNCOA4 transfection, with or without OGD/R (n = 6). j CCK-8 assay…

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  Fig. 7. In vivo validation of the p38α/c-Myc/NCOA4 regulatory axis in NMCMs and AMCMs. a Schematic workflow depicting the isolation of adult mouse cardiomyocytes (AMCMs) and immunofluorescence staining of cTnT (green) in AMCMs. b Co-IP assay confirming the interaction between p-p38α and c-Myc in NMCMs (left) and AMCMs (right) under OGD/R. c Ubiquitination assay of c-Myc in NMCMs and AMCMs with or without p38α knockout and OGD/R. d Co-IP assay verifying the interaction between STUB1 and c-Myc in NMCMs (left) and AMCMs (right), with or without OGD/R. e Western blot and relative quantification analysis of c-Myc protein levels in NMCMs and AMCMs with or without STUB1 knockdown, with or without OGD/R (n = 4). f Western blot and relative quantification analysis of NCOA4 expression in NMCMs and AMCMs with c-Myc knockdown or c-Myc overexpression, with or without OGD/R (n = 4). g CUT&RUN-qPCR assay verifying c-Myc binding to the NCOA4 promoter in NMCMs and AMCMs with or without c-Myc manipulation, with or without OGD/R (n = 6). h Act D assay to detect NCOA4 mRNA stability in NMCMs and AMCMs (n = 4). i, j CCK-8 assay for cell viability, and measurement of MDA levels in NMCMs and AMCMs with p38α…

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  Fig. 8. Pharmacological inhibition of p38α with VX-745 alleviates myocardial I/R injury by suppressing the c-Myc/NCOA4 ferroptosis axis. a Schematic diagram of the experimental protocol for VX-745 administration in mice subjected to myocardial I/R injury. b Serum levels of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN) and creatinine (Cr) in mice with or without VX-745 treatment (n = 6). c Serum levels of creatine kinase isoenzymes (CK-MB) and LDH in I/R-stressed mice with or without VX-745 treatment (n = 6). d Evans blue and TTC double staining of mouse myocardial tissues and quantitative analysis of infarct size (infarction area/AAR, %) (n = 6). e Echocardiographic analysis of left ventricular ejection fraction (EF%) and fractional shortening (FS%) in mice (n = 6). f Levels of Fe 2⁺ and 12-HETE in myocardial tissues (n = 6). g Immunohistochemical staining of 4-HNE in myocardial tissues, with quantitative H-score analysis (scale bar: 50 μm) (n = 6). h Transmission electron microscopy (TEM) analysis of myocardial mitochondrial morphology, with quantitative analysis of mitochondrial cristae number (scale bar: 500 nm) (n = 6). i Western blot and…

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  Fig. 9. Schematic of the mechanisms involving p38α in the regulation of NCOA4-mediated ferroptosis in myocardial I/R injury. Myocardial I/R activates p-p38α, which directly interacts with c-Myc, recruits STUB1 to promote c-Myc ubiquitination and degradation, and de-represses NCOA4 transcription, triggering ferroptosis. P38α deficiency stabilizes c-Myc, suppresses NCOA4, and inhibits I/R-induced ferroptosis, defining the p-p38α/c-Myc/NCOA4 axis as a key regulator of ferroptosis in myocardial I/R injury (Created by Figdraw; ID: OTAIO994bb)