Human cardiac tissue samples were obtained from patients with MI undergoing heart transplantation at Nanjing First Hospital. Inclusion criteria: adult patients (≥ 18 years) undergoing orthotopic heart transplantation; diagnosis of end-stage ischemic cardiomyopathy secondary to previous myocardial infarction or coronary artery disease; availability of left ventricular myocardial tissue for analysis. Exclusion criteria: non-ischemic cardiomyopathy; active infection or systemic inflammatory disease; malignant tumors; autoimmune diseases; incomplete clinical information. Regions of the left ventricle exhibiting marked wall thinning and obvious white fibrosis were defined as the infarct zone, areas 1-2 cm adjacent to the infarct were defined as the border zone, and regions approximately 3 cm away without apparent wall thinning or white fibrosis were defined as the remote zone. Immediately after surgical excision, tissue specimens were dissected into small pieces (1 cm × 1 cm × 0.5 cm) according to the anatomical location and pathological lesion, fixed in 10% neutral-buffered formalin, dehydrated, embedded in paraffin, and stored as formalin-fixed paraffin-embedded (FFPE) blocks at room temperature. For histological analysis, paired myocardial tissue samples were obtained from both the infarct border zone (MI sample) and the remote non-infarcted zone (normal tissues) of each explanted heart. The study was conducted in accordance with the Declaration of Helsinki (revised in 2013) and was approved by the Ethics Committee of Nanjing First Hospital, Nanjing Medical University (04/04/2019, KY20190404-030KS-01). Prior to study participation, written informed consent was obtained from every patient.

Homozygous CARD10-deficient (Card10^-/-) mice with a C57BL/6 genetic background were kindly supplied by the Lin Xin Laboratory, Tsinghua University. WT mice of the same genetic background were purchased from GemPharmatech (Nanjing, China). Experimental MI was in 8-week-old male C57BL/6 mice using an established surgical procedure. Animals were randomly assigned to the indicated groups. No formal sample size calculation was performed. Sample sizes were selected based on previous studies and our laboratory experience. No animals or samples were excluded unless death occurred before completion of the experimental protocol. Following induction of anesthesia with isoflurane (5%, 1 L/min), the animals underwent left thoracotomy for ligation of the left anterior descending coronary artery (LAD) under microscopic guidance. Mice in the sham group underwent an identical surgical procedure except for LAD ligation. Following surgery, mice received postoperative analgesia with buprenorphine (0.05-0.1 mg/kg, subcutaneously) and were monitored daily for signs of pain or distress. Animals were monitored daily for body weight, wound healing, activity, and general health throughout the study. The study was approved by the Experimental Animal Ethics Committee of Nanjing First Hospital, Nanjing Medical University (DWSY-24180857). When euthanasia was required, mice were subjected to isoflurane inhalation (5%, 5 L/min) followed by exposure to an overdose of CO₂ until death was confirmed. A total of 88 mice were enrolled in the study. One mouse died from causes unrelated to the experimental procedures, and the remaining 87 mice were included in the study. All analyses, such as histological, immunofluorescence, western blot, and so on, were performed by investigators blinded to the treatment groups.

When testing heart tissue, whole heart lysis was used. Lysis of tissue and cell samples was performed on ice using Radioimmunoprecipitation Assay buffer (RIPA) buffer supplemented with protease and phosphatase inhibitor cocktail (20109ES05, YEASEN, China), and protein concentrations were quantified using a bicinchoninic acid (BCA) assay kit (KGP902, KeyGEN Biotech, China). Equal amounts of protein were separated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking with 5% bovine serum albumin (BSA) prepared in Phosphate-Buffered Saline (PBS) for 1 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies diluted in 3% BSA. Following antibodies were used: p-histone H2A.X (1:1000, 2557, Cell Signaling Technology, USA), CARD10 (1:1000, ab137383, Abcam, UK); p16 (1:1000, ab51243, Abcam, UK); glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:5000, 8884, Cell Signaling Technology, USA); protein kinase B (AKT) (1:1000, 9272, Cell Signaling Technology, USA); p-AKT (1:1000, 4060, Cell Signaling Technology, USA); phosphoinositide 3-kinase (PI3K) (1:1000, 4292, Cell Signaling Technology, USA); and p-PI3K (1:1000, 17366, Cell Signaling Technology, USA). Following three Tris-Buffered Saline with Tween 20 (TBST) washes, membranes were exposed to Horseradish Peroxidase (HRP)-conjugated secondary antibodies prepared in 1% BSA for 1 h at room temperature. Anti-rabbit IgG (1:8000, #7074, CST, USA) or anti-mouse IgG (1:8000, #91196, CST, USA) was selected according to the primary antibody used. Protein bands were developed using Immobilon® UltraPlus Western HRP Substrate (WBULP-100ML, Millipore, USA) and subsequently detected with an automatic chemiluminescence imaging analysis system (3300 Mini, Clinx Science Instruments, China). Densitometric analysis of protein bands was performed using NIH ImageJ software (NIH, USA).

After fixation in 4% formalin for 24 h, cardiac tissues were processed through graded ethanol dehydration, infiltrated with paraffin and sectioned into 4-μm-thick slices. Histological examination was performed using hematoxylin and eosin (HE) and Masson’s trichrome staining for evaluation of cardiac structural alterations and collagen deposition using light microscopy.

2,3,5-triphenyltetrazolium chloride (TTC) staining was used to determine the extent of tissue infarct areas. Fresh tissue samples were frozen at -80 °C for 10 min and subsequently sliced into 1 mm sections. The tissue samples were then immersed in 1% TTC (T8877-5G, MilliporeSigma, USA) in saline at 37 °C for 15 min and observed under a light microscope.

For immunofluorescence (IF) staining, tissue samples embedded in Optimal Cutting Temperature (OCT) compound were sectioned at a thickness of 5 μm. The tissue sections were sequentially fixed with 4% paraformaldehyde, permeabilized using 0.5% Triton X-100, and blocked with goat serum (ZLI-9022, Beijing Zhongshan Biotechnology, China) before overnight incubation with primary antibodies at 4 °C. Cultured cells underwent the same staining procedure, with the exception of OCT embedding and cryosectioning. Following antibodies were used: CARD10 (1:100, ab137383, Abcam, UK); alpha-smooth muscle actin (α-SMA) (1:100, A5228, Sigma, USA); von Willebrand factor (VWF) (1:100, ab11713, Abcam, UK); troponin I (1:100, ab47003, Abcam, UK); p16 (1:1000, ab51243, Abcam, UK); vimentin (1:100, 5741, CST, USA). Samples were washed and further incubated with Alexa Fluor^TM 488 chicken anti-rabbit IgG (H+L) (1:5000, A21441, Invitrogen, USA) or F(ab’)2-Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor^TM 546 (1:5000, 11018, Invitrogen, USA) for 1 h at room temperature. Cell nuclei were visualized by Hoechst (B-2261, Sigma, USA) staining. Images were captured using a fluorescence microscope (ZEISS SCOPE.A1, Germany).

For Immunohistochemistry (IHC) staining, tissue samples were embedded in 4% paraformaldehyde and sliced into 4-μm-thick sections. Following xylene deparaffinization and stepwise ethanol rehydration, paraffin sections underwent heat-induced antigen retrieval in citrate buffer (pH 6.0). Endogenous peroxidase was quenched by treatment with 3% hydrogen peroxide before nonspecific binding sites were blocked with normal serum. The sections were subsequently incubated with the appropriate primary antibodies against CARD10 (1:100; ab137383, Abcam, Cambridge, UK) overnight at 4 °C. After three rinses with PBS, the sections were incubated with an HRP-labeled polymer detection reagent (KIT-5004, MXB Biotechnologies, Fuzhou, China). Immunostaining was developed using a 3,3’-diaminobenzidine (DAB) detection kit (ZLI-9018, Beijing Zhongshan Biotechnology Co., Ltd., Beijing, China), followed by hematoxylin nuclear counterstaining. Representative images were acquired using a light microscope.

Cardiac function was assessed four weeks after MI induction by echocardiography. Ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter at end-diastole (LVIDd), and left ventricular internal diameter at end-systole (LVIDs) were measured with a Vevo2100 micro-ultrasound system equipped with a 30 MHz transducer (Visual-Sonics, Toronto, ON).

Gene expression was analyzed by real-time quantitative Polymerase Chain Reaction (qPCR) following an established protocol^[15]. Total RNA was extracted from heart tissues or cells using TRIzol Reagent (15596-026, Invitrogen, USA), and cDNA was generated by Hifair III 1st strand cDNA synthesis superMix (11141ES60, YEASEN, China). qPCR was performed using Hieff UNICON Universal Blue qPCR SYBR Green Master Mix (1184ES08, YEASEN, China). All primer information is summarized in Table 1. The relative Ct (ΔΔCt) was calculated as ΔΔCt = 2^{- (ΔCt MI-ΔCt CONTROL)}, where ΔCt = (Ct_GENE-Ct_GAPDH) for each MI and control conditions;

Primary CFs were isolated from 4-week-old mice as previously described. Briefly, hearts were quickly excised and washed in PBS. After being dissected into fragments of about 1 mm, the tissue was enzymatically digested in Hank’s balanced salt solution (HBSS) supplemented with 0.6 mg/mL collagenase type II (C2-BIOC, Sigma, USA), 0.2 mg/mL collagenase type IV (C4-BIOC, Sigma, USA), and 0.2 mg/mL BSA (BS114-250g, Biosharp, China) for 10 min at 37 °C for enzymatic dissociation. This digestion step was repeated 4-5 times until the tissue fragments became noticeably smaller. Undigested tissue debris was removed by passing the suspension through a 40-μm filter. Erythrocytes were removed using red blood cell lysis buffer (C3702-500 mL, Beyotime, China). The filtrate was centrifuged for 10 min at 1,000 rpm and the resulting pellet was resuspended in DMEM/F12 (11320033, Gibco, USA) supplemented with 1% penicillin-streptomycin (P4333-100ML, Sigma, USA) and 10% fetal bovine serum (A5670502, Gibco, USA). Only cell preparations with > 80% vimentin-positive cells were used for subsequent experiments. All experiments were conducted using cells at passage 2-3.

Human umbilical vein endothelial cells (HUVEC) were kindly provided by the Clinical Medicine Research Center in Nanjing Drum Tower Hospital ^[16]. Cell identity had been routinely authenticated before use. HUVECs were cultured in ECM medium [500 mL of basal medium (1001, Solarbio life science, China), 25 mL of fetal bovine serum (0025, Solarbio life science, China), 5 mL of endothelial cell growth supplement (1052, Solarbio life science, China) and 5 mL of penicillin/streptomycin solution (0503, Solarbio life science, China)] to confluence in 6-well plates. All experiments were conducted using cells at passages 3-5. Cells were scratched with a sterile 10-μL pipette tip, washed with PBS, and incubated in conditioned medium. The conditioned medium was prepared by mixing the supernatant of CF cultures with DMEM/F12 (10% FBS) at a 1:1 ratio. Three areas in each well were randomly selected under a light microscope (IX73, Olympus, Japan). The cell migration rate was calculated as: Cell migration rate = (L₀ - L_t)/L₀*100%, where L₀ represents the scratch width at 0 h and L_t represents the scratch width at 24 h^[17].

HUVECs (3.5 × 10⁵ cells/well) were seeded onto Matrigel (356234, Corning, USA) -coated 96-well plates for tube formation. HUVECs were then incubated with 100 μL of conditioned medium per well at 37 °C for 6 h in humidified atmosphere. The conditioned medium was prepared by mixing the supernatant of CF cultures with ECM medium (same as wound healing assay) at a 1:1 ratio. Images were acquired using a light microscope (IX73, Olympus, Japan) and analyzed with the ImageJ Angiogenesis analyzer plugin^[18].

Mitochondrial membrane potential was assessed using a JC-1 staining kit (C2003S, Beyotime, China). Following PBS washing, CFs were then incubated with JC1 working solution at 37 °C for 20 min. Mitochondrial membrane potential (ΔΨm) was evaluated by calculating the fluorescence intensity ratio of aggregated (red) to monomeric (green) JC-1. JC-1 aggregates were measured at excitation/emission wavelengths of 585/590 nm, and JC-1 monomers were measured at 514/529 nm.

Superoxide was measured using MitoSOX staining (M36008, thermofisher, USA). MitoSOX Red reagent was dissolved in anhydrous Dimethyl Sulfoxide (DMSO) and subsequently diluted in HBSS containing calcium and magnesium to obtain a final working concentration of 500 nM. After being washed with PBS, CFs were treated with the MSR working solution at 37 °C for 30 min under light-protected conditions. Fluorescence signals were measured at excitation/emission wavelengths of 396/610 nm.

Transmission electron microscopy (TEM) was used to examine Cellular ultrastructure. Following fixation in 2.5% glutaraldehyde and postfixation with osmium tetroxide, cell samples were dehydrated in a graded ethanol series and infiltrated with epoxy resin. Ultrathin sections were prepared for uranyl acetate and lead citrate staining prior to TEM imaging.

PCR, western blot, and migration assay data are shown as mean ± Standard Error of the Mean (SEM). Comparisons among three or more groups were performed using two-way Analysis of Variance followed by Sidak’s multiple comparisons test. All statistical analyses were conducted using GraphPad Prism 8.02 (GraphPad Software, San Diego, CA, USA). Results were considered statistically significant when P < 0.05.

To investigate the expression and localization of CARD10 following MI, Western blot and IHC staining were performed to compare CARD10 levels between MI and sham-operated mice. CARD10 expression was found elevated in whole heart lysates from MI mice. Expression was found highest at one week after MI induction [Figure 1A and B]. IHC staining of mouse heart tissue one week following MI induction confirmed elevated CARD10 expression [Figure 1C and D]. IF staining showed that CARD10 colocalized with the fibroblast marker α-SMA in a scattered pattern around the border zone of infarcted mouse hearts [Figure 1E]. Additionally, comparable results were found in human heart samples by IHC staining of CARD10 [Figure 1F and G]. Baseline characteristics of the volunteers are in Table 2. These results suggest that CARD10 may contribute to the pathophysiology of MI, potentially through its involvement in CFs.

To investigate the role of CARD10 in MI, MI was induced by LAD coronary artery ligation in Card10^-/- and WT mice. Echocardiography and sample collection for morphological analyses were carried out four weeks after surgery. Heart tissue from Card10^-/- mice exhibited a lower degree of fibrosis compared to heart tissue from WT mice, as assessed by HE and Masson staining [Figure 2A-D]. TTC staining revealed smaller infarct areas in heart tissue from Card10^-/- mice [Figure 2E and F]. Echocardiographic analysis [Figure 2G and H] revealed significantly restored cardiac function in Card10^-/- mice compared to WT mice after MI induction, with higher EF and FS and decreased LVIDd and LVIDs. Reduced heart weights were observed in Card10^-/- mice after MI induction. Furthermore, increased expression of the endothelial cell marker VWF [Figure 2I and J] and the smooth muscle cell marker a-SMA [Figure 2K and L] was found in the cardiac tissue of Card10^-/- mice compared to WT mice after MI induction. This indicates increased angiogenic capacity in mice deficient in CARD10. Collectively, these results of the morphological and functional analyses suggest that CARD10 deficiency attenuates the effects of MI.

Previous studies have suggested that CF senescence may exert beneficial effects following MI^[5]. To assess cellular senescence in our experimental model and the effects of CARD10 deficiency, an SA-β-gal activity assay was performed in heart tissue one week after MI induction. We found an accumulation of SA-β-gal staining in regions adjacent to the infarct area of hearts from Card10^-/- mice that was absent in hearts from WT mice [Figure 3A]. In addition, the expression of senescence markers p16 and phosphorylated histone H2A.X (p-histone H2A.X) was examined using Western blot analysis [Figure 3B and C]. Both markers were expressed at significantly higher levels in Card10^-/- mouse tissue following MI. These results indicate an increased level of senescence in the infarcted hearts of Card10^-/- mice.

To further validate these findings in vitro, primary CFs were isolated from Card10^-/- and WT adult mice and were stimulated with TGF-β (10 ng/mL) for 4 days to establish a senescence model. The Card10^-/- CFs stimulated with TGF-β showed increased SA-β-gal activity compared with WT CFs [Figure 3D]. Consistent with this observation, Western blot analysis demonstrated significantly higher expression of p16 and p-histone H2A.X in the Card10^-/- CFs upon TGF-β stimulation [Figure 3E and F]. IF staining further confirmed an increased number of p16-positive Card10^-/- CFs after TGF-β stimulation [Figure 3G]. These in vitro results are consistent with the in vivo findings, confirming enhanced senescence in Card10^-/- cells, consistent with the in vivo results. Senescent cells are known to develop a SASP, characterized by the release of proinflammatory factors such as Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-α), and Platelet-Derived Growth Factor-AA (PDGF-AA)^[19]. The expression of these factors was assessed by Real-time quantitative polymerase chain reaction (RT-qPCR). Following TGF-β stimulation, Card10^-/- CFs exhibited significantly higher mRNA levels of Il6, Tnf, and Pdgfa compared with WT CFs [Figure 3H]. Collectively, these results indicated that CARD10 deficiency promotes a pronounced senescence phenotype in the heart.

We have previously demonstrated that CARD10 deficiency promotes angiogenesis following MI [Figure 2I-L]. To investigate whether this effect is due to factors released by senescent cells, the effects of CF-derived conditioned medium on migration, proliferation, and angiogenesis of HUVECs were determined. The conditioned medium was prepared by combining the supernatant of Card10^-/- or WT CFs incubated with TGF-β for 4 days with DMEM (10% FBS) at a 1:1 ratio.

HUVECs cultured with Card10^-/- CF-derived conditioned medium exhibited significantly higher migration and proliferation compared with HUVECs cultured with WT CF-derived conditioned medium, as determined by healing [Figure 3I and J] and CCK8 assay [Figure 3K]. Moreover, under the same conditions, HUVECs produced significantly more nodes and junctions as determined in a tube formation assay [Figure 3L-N]. Collectively, these results indicate that senescent fibroblasts promote endothelial cell proliferation and activities that contribute to attenuating the damage associated with MI. This effect may be mediated by secretion of SASP factors, including PDGF-AA.

For the rescue experiment, we used quercetin (10 μM, HY-18085, MedChemExpress, NJ, USA) to anti-senescence for 48 h^[20] to treat CFs. p16 was decreased after quercetin stimulation [Supplementary Figure 1A and B], validating that senescence was alleviated after quercetin stimulation. Then, HUVECs were cultured with CF-derived conditioned medium. The pro-angiogenic effects of Card10^-/- CF-derived conditioned medium on HUVECs were partially diminished [Supplementary Figure 1C-G], implicating that the cardioprotective effects associated with CARD10 deficiency may be mediated by fibroblast senescence.

Mitochondrial dysfunction is a well-established hallmark of cellular senescence. We therefore investigated the contribution of mitochondrial dysfunction to the senescence phenotype induced by CARD10 deficiency in CFs isolated from Card10^-/- and WT adult mice following stimulation with TGF-β. Mitochondrial membrane potential, mtROS production and mitochondrial ultrastructure were assessed by JC-1 staining, MitoSOX Red fluorescence and TEM, respectively. Upon stimulation with TGF-β, Card10^-/- CFs exhibited significantly lower mitochondrial membrane potential compared to WT CFs [Figure 4A and B] but generated significantly more mtROS [Figure 4C and D]. TEM analysis showed mitochondrial ultrastructural abnormalities in WT CFs stimulated with TGF-β that were further aggravated in Card10^-/- CFs [Figure 4E]. These findings indicate that CARD10 deficiency exacerbated mitochondrial damage after TGF-β stimulation.

Given the established role of the PI3K/AKT signaling pathway in both mitochondrial dysfunction and cellular senescence, we next examined its activation status. Notably, phosphorylation of both PI3K and AKT was significantly increased in TGF-β-stimulated Card10^-/- CFs compared to WT CFs [Figure 4F and G], suggesting that the PI3K/AKT pathway may contribute to CARD10-mediated regulation of CF senescence.

This study identifies CARD10 as a novel regulator of cardiac fibroblast senescence and suggests that targeting it may represent a potential strategy to improve cardiac repair after MI. Importantly, given the context-dependent effects of cellular senescence, precise temporal control of senescence modulation will likely be required to maximize beneficial effects while minimizing adverse remodeling.