Necroptosis is a key contributor to impaired cardiac repair following myocardial infarction

Figures

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  Fig. 1. Endothelial ADAM17 knockdown worsens post-MI survival due to increased LV rupture. A Schematic of tamoxifen (TMX)-induced ADAM17 knockdown in Adam17f/f/Cdh5-CreERT2 (Adam17EC-KD) mice. Mice received Tamoxifen (i.p., 5 days) at 10 weeks of age, followed by LAD-ligation (or sham) at 12 weeks of age. Kaplan–Meier analysis for post-MI survival (B) and LV rupture (C) in Adam17f/f and Adam17EC-KD mice post-MI (Log-rank test). D Infarct size measurements at 3 and 7 days post-MI (n = 7–12/group). E Representative Masson’s trichrome-stained heart sections post-sham or MI from each genotype. UMAP plot (F) and cell distribution (G) of major cardiac cell populations identified by snRNA-seq from sham, peri-infarct, and infarct regions of Adam17f/f and Adam17EC-KD hearts. CM cardiomyocyte, FB fibroblast, EC endothelial cell, Mac macrophages, Neu neutrophils, BC B cells, TC cytotoxic T cells, SMC smooth muscle cells, PC pericytes. H Heatmap of representative genes for the indicated cellular functions (z-score) across sham, infarct, and peri-infarct myocardium. Genes are grouped by the indicated functional categories. Ad17 = Adam17, Inf = infarct, Peri = Peri-infarct. Averaged data are presented…

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  Fig. 2. Endothelial ADAM17 deficiency modulates immune cell recruitment and enhances NETosis following MI. Representative immunofluorescence staining and quantification of neutrophils (LY6G, red) (A), cytotoxic T cells (CD8a, red) (B), and macrophages (CD68, red) (C) in the infarct (Inf), peri-infarct (Peri), and non-infarct (non) regions of Adam17f/f and Adam17EC-KD hearts at post-MI day 1 (top row) and day 3 (bottom row). Nuclei counterstained by DAPI (blue). D Representative images of Neutrophil Extracellular Traps (NETs) by co-staining for myeloperoxidase (MPO, green) and citrullinated histone H3 (CitH3, red), at 1 day post-MI. Corresponding surface intensity plots and quantification of MPO/CitH3 colocalization are shown for each genotype. Quantification represents averaged values from 8 to 10 sections/heart, and 4 hearts/group/genotype. Averaged data are presented as mean ± SEM. Statistical analysis was performed using two-way ANOVA with a Bonferroni post hoc test. *p < 0.05 vs. Adam17f/f.

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  Fig. 3. Loss of ADAM17 compromises endothelial cell adhesion, but immune cell depletion does not reduce Post-MI LV rupture incidence. A Representative immunoblot and protein quantification of adhesion molecules (ICAM1, VCAM1, JAM-A, CXCR3) and cytotoxic protein granzyme B (GZMB) in infarct (Inf), peri-infarct (Peri), and non-infarct (Non) regions at 3 days post-MI and sham heart tissue in Adam17f/f and Adam17EC-KD mice. n = 4–6 hearts/group/genotype. B Representative immunoblot and protein quantification of adhesion molecules (ICAM1, VCAM1, JAM-A, CXCR3) in human coronary endothelial cells (EC), with intact Adam17 levels (scrambled siRNA, ECWT) or siRNA-mediated Adam17 knockdown (ECAd17-KD), under normoxia (21% O₂) or hypoxia (1% O₂) conditions (n = 3 independent EC cultures, total of 6 wells/group). C Schematic illustration of antibody administration timeline for depletion of neutrophils (anti-LY6G) or cytotoxic CD8+ T cells (anti-CD8a) in Adam17EC-KD mice post-MI. The colored circles show the days when the antibodies were injected. D Kaplan–Meier post-MI survival curves for Adam17EC-KD mice following neutrophil (green) or cytotoxic T-cell depletion (purple) compared to no treatment…

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  Fig. 4. Endothelial ADAM17 deficiency impairs endothelial cell number and VEGFR2 signaling after MI. A Representative CD31 immunofluorescence (red) and DAPI nuclear staining (blue) in sham, regions of Adam17f/f and Adam17EC-KD hearts at 3 days post-MI or Sham, and corresponding quantification (n = 3–6 hearts/group; 8–10 sections per heart). B Representative micro-CT reconstructions of coronary vasculature and averaged quantification of vessel volume relative to LV volume in sham and post-MI day 3 or Sham hearts (Red=coronaries in anterior LV wall, blue=other coronaries). C Representative immunoblot and quantification for phosphorylated and total levels of VEGFR and AKT1/2 post-MI/ sham in indicated groups genotype (n = 4–6/group). D Co-culture of human coronary endothelial cells with intact Adam17 (scrambled siRNA, ECWT) or siRNA-mediated Adam17 knockdown (ECAd17-KD) with wildtype cardiac myofibroblasts (intact Adam17 expression) under normoxia or hypoxia (±TGF-β to simulate the pro-fibrotic and hypoxic infarct environment). Number of branches and total length of tubular structures formed by ECs were quantified (n = 4 independent experiments, 3 wells/experiment). E Representative…

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  Fig. 5. Single-nucleus RNA sequencing reveals impaired angiogenesis, reduced EndMT, and enhanced endothelial cell death signatures in Adam17EC-KD hearts post-MI. A UMAP plot of endothelial subclusters (EC_1: naïve, EC_2: proliferative/angiogenic, EC_3: EndMT, EC_4: stressed) with proportional distribution across conditions. B Heatmap of differentially expressed genes defining each EC subcluster. C Heatmaps of genes in ECs corresponding to angiogenesis, adhesion signatures, EndMT, and (early) cell death/stress signatures in indicated groups of either genotype. D Feature plots of representative EndMT (Col1α1, Snai1) and necroptosis-related (Mlkl, Ripk3) genes in peri-infarct and infarct ECs. E CellChat analysis of endothelial cell subclusters (EC_1 to EC_4) with major cardiac cells (CM cardiomyocyte, FB fibroblast, Mac macrophage, Neu neutrophil, BC B cell, TC cytotoxic T cell, and PC pericyte). Ad17 = Adam17, Inf = infarct, peri = peri-infarct.

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  Fig. 6. Endothelial ADAM17 deficiency impairs scar stabilization and endothelial-to-mesenchymal transition (EndMT). A Total, soluble, and insoluble collagen content (hydroxyproline assay) in sham or 3 days post-MI in the indicated groups. B Representative immunoblot and protein quantification of extracellular matrix protein, fibronectin, and related signaling molecules in Adam17f/f and Adam17EC-KD hearts at 3 days post-MI (n = 4–6 hearts/group). C Representative immunofluorescence staining for αSMA (red, marker of fibroblast-like characteristics); VE-Cadherin (green, EC protein) and DAPI (blue, Nuclear staining) in human coronary endothelial cells, with intact (ECWT) or Adam17-knockdown (ECAd17-KD) in control or pro-fibrotic/hypoxia conditions (similar to infarct environment) to induce EndMT. D Representative immunoblot and protein quantification in ECWT and ECAd17-KD in control or pro-fibrotic/hypoxic conditions (n = 3 independent experiments). SM22α smooth muscle protein (transgelin), αSMA alpha smooth muscle actin, LOX lysyl oxidase. Averaged data are presented as mean ± SEM. Statistical comparisons were performed using two-way ANOVA with a Bonferroni post hoc test. *p < 0.05.

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  Fig. 7. Endothelial ADAM17 deficiency promotes necroptotic cell death and suppresses apoptotic signaling following MI. A Representative immunofluorescent co-staining and quantification of dead/dying cells (Propidium Iodide, PI, red; DAPI, blue) in infarct (Inf), peri-infarct (Peri), and non-infarct (Non) regions at 3days post-MI (n = 4 hearts/group/genotype). B Representative immunoblot and protein quantification for pro-apoptosis (Caspase 3) and anti-apoptosis (Caspase 8) in indicated groups (n = 4–6 hearts/group/genotype). C Representative immunoblot and protein quantification for necroptosis-related proteins: Mixed Lineage Kinase Domain-Like (MLKL), receptor-interacting serine/threonine kinases (RIPK1, RIPK3), Tumor necrosis factor receptors (TNFR1, TNFR2), and Tumor necrosis factor-alpha (TNFα) at 3 days post-MI or sham in each genotype (n = 4–6 hearts/group/genotype). Averaged data are presented as mean ± SEM. Statistical analyses were performed using two-way ANOVA with a Bonferroni post hoc test. *p < 0.05 vs. Adam17f/f. Ad17f/f = Adam17f/f and Ad17EC-KD = Adam17EC-KD.

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  Fig. 8. Endothelial ADAM17 loss reduces TNFR1 shedding and heightens necroptosis, while necroptosis inhibition prevents post-MI rupture and restores the endothelial cell population in Adam17EC-KD mice. A Representative immunofluorescence images and quantification of propidium iodide (PI, red) and DAPI (blue) staining in endothelial cells (ECWT and ECAd17-KD) cultured under normoxia or hypoxia (n = 3 independent experiments; 6–8 fields per condition). B Representative immunoblots and protein quantification for Mixed Lineage Kinase Domain Like (MLKL and pMLKL), and Tumor necrosis factor receptor 1 (TNFR1) and receptor 2 (TNFR2), soluble (in conditioned media) and membrane-bound (in cell lysate), in ECs with intact Adam17 (ECWT) or with Adam17 knockdown (ECAd17-KD). C Schematic of treatment of mice with a necroptosis inhibitor, Necrostatin-1 (Nec-1), and post-MI survival curve showing that pharmacological inhibition of necroptosis improved post-MI survival in Adam17EC-KD mice by preventing LV rupture. D CD31 (red) immunostaining at day 7 post-MI in Nec-1–treated mice shows reversal of the drastic decrease in coronary density in Adam17EC-KD-MI compared to Adam17f/f-MI hearts in the infarct,…