A novel S100A-TLR3-IFIT3 signaling axis promotes cardiomyocyte apoptosis during myocardial infarction

A novel S100A-TLR3-IFIT3 signaling axis promotes cardiomyocyte apoptosis during myocardial infarction | 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 Article A novel S100A-TLR3-IFIT3 signaling axis promotes cardiomyocyte apoptosis during myocardial infarction shubai Liu, cheng chen, Haoqiang Chen, zhicheng He, Yuanzhi Chen, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8733111/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Ischemic cardiomyopathy is associated with myocardial injury and increased mortality. During myocardial infarction (MI), necrotic cardiomyocytes release damage-associated molecular patterns (DAMPs) that trigger inflammatory responses, yet the specific cardiac alarmins and downstream mechanisms driving cardiomyocyte apoptosis remain unclear. In this study, we identified endogenous S100 protein isoforms as key cardiac alarmins released during myocardial ischemic injury and elucidated their role in activating IFIT3 overexpression through innate immune signaling pathways. Mining patient biopsy expression data and verifying a rat model of left anterior descending (LAD) artery occlusion, we validated IFIT3 overexpression in both human and rodent cardiac tissues during acute myocardial infarction (AMI). To investigate the functional role of IFIT3, we employed CRISPR-Cas9 gene-editing technology to knock out IFIT3 in AC-16 human cardiac cells and developed a continuous oxygen-glucose deprivation/reperfusion (OGD/R) model to mimic MI at the cellular level. IFIT3 knockout significantly inhibited apoptosis induced by OGD/R and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), as detected by Annexin V-FITC/PI double staining. Mechanistically, we utilized Type I interferons, TLR agonists, and STING agonists to dissect the dominant DAMP signaling pathway, revealing that S100 proteins activate IFIT3 overexpression through the TLR3/TICAM1/IRF3 pathway, thereby promoting cardiomyocyte apoptosis. This research establishes a novel S100-TLR3-IFIT3 signaling axis in the pathophysiology of ischemic cardiomyopathy, providing new mechanistic insights and potential therapeutic targets for myocardial ischemic injury. Health sciences/Diseases/Cardiovascular diseases/Cardiomyopathies Biological sciences/Immunology/Cell death and immune response Health sciences/Diseases/Immunological disorders Myocardial ischemic injury TLR3 S100 proteins DAMPs IFIT3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Insufficient blood supply to the myocardium results in injury and death of heart muscle tissue, a condition known as ischemic cardiomyopathy (ISCM), which can arise from both acute and chronic myocardial infarction (MI)[1]. ISCM is the most prevalent cause of mortality [2, 3]. During the MI, debris from necrotic cardiac myocytes activates damage-associated molecular patterns (DAMPs) via pattern recognition receptors (PRRs) within the innate immune system, thereby initiating an inflammatory response [4]. The S100 protein family comprises 21 isoforms characterized by small proteins that contain calcium-binding motifs, including S100A1 through S100A16, S100G, S100P, and S100B [5]. These proteins exhibit both positive and negative roles in cardiovascular diseases. Elevated serum levels of S100A have been identified as a highly sensitive biomarker for acute myocardial injury [6]. Among the isoforms, S100A1 is the most prevalent in cardiomyocytes and is passively released from ischemic cardiomyocytes during acute MI, functioning as a cardiac alarmin [7, 8]. S100A8/9 has been shown to predict the prognosis of patients with MI and is implicated in the regulation of both cardiovascular diseases [9], [10] and autoimmune myocarditis [11, 12]. Calprotectin (S100A8/A9) and S100A1, recognized as DAMPs, trigger inflammatory responses by binding to Toll-like receptor 4 (TLR-4) and initiate a signaling cascade in MI [8, 13, 14]. Additionally, S100A9 plays a crucial role in the maturation of TLR3-containing early endosomes into late endosomes, which subsequently fuse with lysosomes to form an endo-lysosomal compartment. Upon sensing its agonist/PAMP, TLR3 is activated by recruitment of the adaptor protein TRIF and the assembly of key signaling complexes, which subsequently activate IRF3 and NF- κ B [15]. The regulatory factors of interferons (IFNs) modulate the expression of specific genes in the heart that encode pro-inflammatory cytokines and other IFNs [4]. These genes include interferon-stimulated genes (ISGs) and the IFIT (IFN-induced proteins with Tetratricopeptide Repeat (TPR) motifs) family proteins [16]. Activation of IFIT family members by IFNs initiates various signal transduction pathways, which play a critical role in regulating both cell-intrinsic and cell-extrinsic immune responses. This modulation is essential for minimizing immune-mediated damage and cardiac remodeling [17]. The IFIT2/3 genes are highly expressed in patients with cardiovascular disease and MI, and their expression levels are indicative of cardiac function impairment in MI patients[18]. Additionally, IFIT2/3 were associated with the activation of mediators such as of IRF3 and NF- κ Β [19, 20]. Overexpression of IFIT2 causes caspase-3 activation and plasma membrane asymmetry disruption, leading to apoptotic cell death [20]. Furthermore, IFIT2 overexpression induces apoptosis by modulating the balance between pro-apoptotic and anti-apoptotic Bcl-2 family proteins, thereby altering mitochondrial membrane permeability [21]. Inhibition IFIT2 degradation results in perinuclear aggregation and promotes apoptosis [22]. IFIT2/3 is overexpressed in ISCM patients' cardiac tissues and rat MI models. Notably, the IFIT2/3 level is significantly reduced in the Metoprolol -treated group compared to the MI LAD group, which correlates with a decrease in apoptotic cells [23]. Moreover, overexpression of IFIT3 exacerbates cardiomyocyte apoptosis and ventricular damage in MI, consequently impairing cardiac function. However, the mechanism linking endogenous S100A family protein release to IFIT3 activation during MI remains unclear. This study aims to investigate the potential mechanisms connecting the release of endogenous S100A family proteins and the activation of IFIT3 overexpression via the TLRs-STING signaling pathway, which may induce cardiomyocyte apoptosis during the progression of MI. Builds on our previous work [23], a rat myocardial ischemia model and immunofluorescence staining were employed to confirm the upregulation of IFIT3 in cardiomyocytes under ischemic conditions. Multiple cell lines were utilized for this investigation. Additionally, the study employed an in vitro cellular model of oxygen-glucose deprivation/reperfusion (OGD/R) to simulate the MI process and to explore the mechanisms contributing to cardiac damage and dysfunction. Results The overexpression of IFIT3 is functionally with cardiomyocyte myocardial injury. Our previous study revealed a significant upregulation of IFIT3 in the cardiac tissues of patients with myocardial ischemia ( Fig. 1 a )[23] . To investigate the functional role and regulatory mechanism of IFIT3 in myocardial ischemic injury, we employed a rat model of acute myocardial ischemia. Model induction was successfully confirmed 24 hours post-ischemia by assessing cardiac function through small-animal Doppler echocardiography ( Fig. 1 b ) . Echocardiographic results demonstrated a significant decrease in left ventricular ejection fraction (LVEF) and fractional shortening (FS) at the 24-hour time point ( Fig. 1 c-d ) . Additionally, we observed impaired motion of the anterior wall ( Fig. 1 e-g ) and ventricular dilation ( Fig. 1 h ). In contrast, the motion of the posterior wall remained largely unaffected (Fig. S1 a, b) . Collectively, these findings confirm the successful establishment of the acute myocardial ischemia model in rats. Infarct size was further quantified by Masson's trichrome staining of cardiac cross-sections, which revealed that the infarcted area accounted for approximately 40% of the left ventricle ( Fig. 1 i ) . Notably, IFIT3 expression was significantly upregulated in the infarcted region of rat hearts compared with the control group ( Fig. 1 j, S1c ) . Furthermore, immunofluorescence analysis identified markedly elevated IFIT3 levels in a subset of cardiomyocytes located specifically within the border zone of the myocardial infarction ( Fig. 1 k ) . IFIT3 Deficiency Protects Cardiomyocytes from OGD-Induced Apoptosis. To assess the role of IFIT3 in cardiomyocyte responses to ischemic damage, an AC-16 cell exposed to oxygen-glucose deprivation (OGD) model was utilized to investigate the role of IFIT3, which simulate ischemic conditions. Following 24 hours of OGD exposure, AC-16 cells displayed notable morphological changes, transitioning from an elongated spindle shape to a circular morphology ( Fig. 2 a ) . Western blot analysis indicated a significant increase in IFIT3 levels after 2 hours of OGD ( Fig. 2 b, S2a ) , coinciding with the expression of Cleaved-Caspase3/Caspase3 ( Fig. 2 b, S2b-d ) . The CRISPR/Cas9 system was employed to knockout IFIT3 in AC-16 cells to assess its functional impact on cardiomyocyte apoptosis induced by OGD. The complete elimination of IFIT3 in the IFIT3-deficient cells (AC-16) was verified using Western blot analysis, and the morphological alterations of the cells were minimal ( Fig. 2 c ) . A whole transcriptomic profiling was conducted to investigate the primary affected functions and pathways between IFIT3 KO and wild-type AC-16 cells. After normalization and filtering, a total of 960 significantly altered genes were identified, comprising 258 up-regulated and 702 down-regulated genes (IFIT3 KO vs control, fold change (FC) > 2.0 or FC < 0.50, Fig. 2 d). The top 20 Gene Ontology (GO) enrichment analyses were summarized, encompassing molecular functions, cellular components, and biological processes. Notably, enriched processes included immune system processes and responses to stimulus, etc (Fig. 2 e). In particular, the hallmark of apoptosis was emphasized in the Gene Set Enrichment Analysis (GSEA) comparing IFIT3 KO cells to control cells, prompting further investigation (Fig. 2 f). The accompanying heatmap illustrated 13 genes significantly down-regulated in IFIT3 KO cells involved in the hallmark of apoptosis, including BNIP3, BCL2L11, etc (Fig. 2 g). Under resting conditions, IFIT3 knockout did not enhance cardiomyocyte apoptosis. A trend toward an attenuated apoptotic rate was observed in IFIT3-knockout AC16 cells relative to wild-type controls after 2 hours of OGD (Fig. S2 e) . In contrast, after 4 and 24 hours of OGD, the apoptotic ratio of IFIT3-knockout AC-16 cells was significantly reduced ( Fig. 2 h, i ) . Additionally, 999 genes were identified as significantly altered when comparing IFIT3 KO and wild-type AC-16 cells after 2 hours of OGD (345 up-regulated and 654 down-regulated genes in IFIT3 KO, FC > 2.0 or FC < 0.50,) ( Fig. S2 f ). The hallmark of apoptosis was notably highlighted as being inhibited in IFIT3 knockout cells ( Fig. S2 g ). These findings demonstrated that the IFIT3 KO significantly mitigates AC-16 cardiomyocyte apoptosis induced by persistent OGD. IFIT3 knockout confers cardio-protection against H/R injury via preserving mitochondrial membrane potential and suppressing apoptosis. After two hours of OGD followed by three hours of reoxygenation, the different stages of apoptotic ratio (early, late, and total) of wild-type AC-16 cells significantly increased to approximately 20%. In contrast, IFIT3 knockout markedly reduced the apoptotic ratio ( Fig. 3 a ) . When the duration of OGD was extended to four and eight hours, followed by three hours of reoxygenation, the apoptotic ratio in IFIT3 knockout AC-16 cells consistently decreased and remained significantly lower than that observed in wild-type cells ( Fig. 3 b, c ) . These results suggest that IFIT3 deficiency substantially mitigates cardiomyocyte apoptosis induced by ischemia-reperfusion in cardiac myocytes. To investigate the potential regulatory interactions between IFIT3 and mitochondrial signaling, CCCP was employed to induce apoptosis in AC-16 cardiomyocytes by disrupting membrane potential. Notably, IFIT3 knockout significantly attenuated CCCP-induced cardiomyocyte apoptosis following treatment with concentrations of 2.5 µM and 5 µM over a 24-hour period. The apoptotic ratios at different stages—early, late, and total— were significantly lower in IFIT3 knockout AC-16 cells compared to wild-type AC-16 cells (Fig.S3a, b) . It is suggested that IFIT3 plays a detrimental role in the regulation of cardiomyocyte apoptosis through modulation of the mitochondrial membrane potential. Following the induction of OGD, the JC-10 assay was utilized to evaluate mitochondrial membrane potential. The expression of IFIT3 was significantly maintained in AC-16 cells after a 2-hour OGD exposure. Under baseline conditions, the mitochondrial membrane potential in IFIT3-deficient AC-16 cells exhibited minimal variation (Fig. 3 d, JC-10, red color ). However, after 2-hour OGD, the IFIT3 knockout group demonstrated a higher mitochondrial membrane potential, indicated by a weaker green signal, compared to the wild-type group ( Fig. 3 d, JC-10, green color) . These findings suggested that the absence of IFIT3 in cardiomyocytes may facilitate the preservation of mitochondrial membrane potential during ischemic conditions, potentially conferring identifiable anti-apoptotic properties. Furthermore, the study examined the levels of two critical apoptosis markers, BCL2 and BAX, in the context of IFIT3 knockout. IFIT3 knockout markedly altered the expression of key apoptotic regulators, characterized by increased BCL2 and decreased BAX levels ( Fig. 3 e-g ) , thereby significantly elevating the BCL2/BAX ratio ( Fig. 3 h ) . These findings imply that IFIT3 knockout enhance mitochondrial membrane potential and elevates cellular anti-apoptotic capacity by increasing BCL2 expression and decreasing BAX expression. IFIT3 Overexpression Activates the Extrinsic Apoptotic Pathway To further investigate the mechanism by which IFIT3 regulates apoptosis, we transiently overexpressed IFIT3 in AC16 cardiomyocytes ( Fig. 4 a ) via plasmid transfection. Following 48 hours of IFIT3 overexpression, cells were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) for 2 or 4 hours, followed by 3 hours of reoxygenation. The results showed that IFIT3 overexpression significantly increased the ratio of cleaved caspase-3 to caspase-3, indicating the initiation of the apoptotic program. Concurrently, we detected significant activation of cleaved caspase-8, whereas the expression levels of caspase-8, caspase-9, and cleaved caspase-9 showed no significant changes ( Fig. 4 b S 4 a-c ) . These findings suggest that IFIT3 overexpression likely activates the extrinsic apoptotic pathway. To further validate the pro-apoptotic effect of IFIT3, we performed an immunofluorescence assay. After 72 hours of IFIT3 overexpression combined with OGD/R (4h/3h) induction, a significant increase in cardiomyocyte apoptosis was observed ( Fig. 4 c ) , confirming that IFIT3 overexpression potently induces cell death. Alternative signaling activated IFIT3 overexpression in cardiomyocytes during myocardial ischemic injury The IFIT3 gene was initially identified as being induced by interferons, so three types of human type I interferons (IFN α-1b, IFN α-2a, and IFN α-2b) were employed to stimulate IFIT3 expression in myocardial cells (AC-16 cells) over a duration of 24 hours. This experiment yielded a significant increase in IFIT3 expression ( Fig. S5a ). Additionally, varying doses of IFN α-1b and IFN α-2a were tested on mouse cardiomyocytes (HL-1 cells), resulting in a notable stimulation of IFIT3 overexpression ( Fig.S5b ), whereas IFN α-2b exhibited minimal effect on IFIT3 expression( Fig.S5c ). This discrepancy might be attributed to the differences in the protein sequences of IFN α-2b between human and mouse species. Furthermore, treatment with type I interferons (IFN α-1b, IFN α-2a, and IFN α-2b) significantly stimulated IFIT3 overexpression in HEK293 cells ( Fig.S5d ). These results affirm that interferons can induce the IFIT3 production in both cardiomyocytes and non-cardiomyocyte cell types. Notably, the IFIT3 expression was significantly elevated in AC-16 cells after 2 hours of OGD, while transcriptome analysis revealed no detectable expression of IFN α1, IFN α2, or IFN γ mRNA ( Fig.S5e ). This observation indicates that IFIT3 overexpression during OGD may occur via an alternative signaling pathway that operates independently of interferon regulation. Activated TLRs and IRF3 signaling to induce IFIT3 overexpression during myocardial ischemic injury TLRs (Toll-like receptors), which serve as pattern recognition receptors, play a crucial role in mediating inflammatory responses to DAMP during myocardial ischemia and hypoxia. To investigate the TLRs signaling pathways that may be activated by IFIT3 overexpression, the mRNA expression levels of several key signaling regulators were assessed in AC-16 cells following 2 hours of OGD. Notably, the expression level of Myd88, a crucial signaling adaptor of TLR4 activation, remained unchanged after 2 hours of OGD compared to baseline conditions ( Fig. 5 a ) . In contrast, the mRNA level of Ticam1, which serves as the key signaling adaptor of TLR3 activation, showed a significantly upregulated, indicating the activation of TLR3 ( Fig. 5 b ) . Furthermore, the protein expression of TICAM1 was significantly elevated after OGD ranging from 2 to 12 hours ( Fig. 5 c ) , while MyD88 expression was not detected in the Western blot analysis. These findings suggests that TLR3 is activated during the OGD period (0-24h), in contrast to TLR4. The study utilized isoform-specific TLR agonists and STING agonists to validate the inducibility of IFIT3 expression in cardiomyocytes, aiming to replicate the signaling mechanisms associated with myocardial injury. A total of 13 agonists were employed for this investigation. The TLR agonists, namely Pam3CSK4 (TLR1/2), HKLM (TLR2), FLA-ST (TLR5), FSL-1 (TLR6/2), Imiquimod (TLR7), ssRNA 40 (TLR8), ODN 2006 (TLR9), and ODN 2216 (TLR9) induced IFIT3 overexpression in AC-16 cells ( Fig. 5 d, e ) . In contrast, this induction effect was not observed in HL-1 mouse cells under identical treatment conditions. Notably, TLR3 agonists (Poly I:C both HMW and LMW) significantly enhanced IFIT3 production in both AC-16 ( Fig. 5 f ) and HL-1 cells ( Fig. 5 g ) . Additionally, the TLR4 agonist (LPS-EK) also induced IFIT3 production in AC-16 ( Fig. 5 d ) and HL-1 cells ( Fig. 5 h ) , albeit with a weaker effect compared to TLR3 agonists. Furthermore, the STING agonist 2'3-cGAMP elicited a stronger IFIT3 expression than ISD in AC-16 cells ( Fig. 5 e ) , while the same concentration of STING agonist (Poly I:C HMW and LPS) resulted in minimal IFIT3 overexpression in HL-1 cells. These results suggest that certain damage-associated endogenous molecules activate TLR3 or IRF3 to induce IFIT3 overexpression during myocardial ischemia and hypoxia via an IFN regulation-independent pathway. S100 family proteins stimulated IFIT3 overexpression in cardiomyocytes The S100 proteins possess a shared calcium-binding domain characterized by an EF-hand structure and are distributed in multiple cellular compartments ( Fig. 6 a ) . Although S100 family proteins play opposing regulatory roles in the progression of various cardiovascular diseases, limited research has investigated the interaction between S100 family proteins and IFIT3. Treatment with S100 isoforms (A1, A6, A8, A9, and B) at a concentration of 2.0µg/mL for 24 hours resulted in an increase in IFIT3 protein expression in AC-16 cells. Among the S100A isoforms, S100A1 and S100A9 exhibited the most significant inducible effects, whereas S100B induced changes in IFIT3 expression that were not statistically significant ( Fig. 6 b ) . Additionally, treatment with all five S100 isoforms (2.0µg/mL, 24 hours) led to a significant increase in IFIT3 expression in mouse cardiac fibroblasts (MCFs) ( Fig.S6a) . Furthermore, treatment with S100A isoforms (A1, A6, A8, and A9) (2.0µg/mL, 24 hours) significantly enhanced IFIT3 expression in HEK293 cells; however, the changes in IFIT3 expression induced by S100B were not statistically significant ( Fig.S6b ). Afterward, AC-16 cells were stimulated with S100A1 and S100A9 at various time points (2.0 µg/mL for 2, 4, 8, 12, and 24 hours). The expression of the IFIT3 protein was significantly increased starting at 2 hours, peaking after 24 hours of stimulation (Fig. 6 c, d). These results suggest a previously unrecognized association between stimulation by S100 family proteins and the overexpression of IFIT3. S100A1 and S100A9 are the predominant S100 isoforms released from damaged cardiomyocytes during MI [7, 24]. It can be inferred that S100A1 and S100A9 serve as endogenous damage-associated pattern molecules that activate IFIT3 expression during MI. S100A1 and S100A9 activated IFIT3 overexpression via TLR3/TICAM1/IRF3 signaling pathway Our findings indicate that the expression of IFIT3 is independent of interferon (IFN) induction but is instead influenced by the activation of TICAM1, which implicates the TLR3/TICAM1/IRF3 signaling pathway. Treatment with S100A1 and S100A9 resulted in the upregulation of TLR3 and TICAM1 (Fig. 6 e, g), thereby indicating activation of TLR3 (Fig. 6 e-f, S6 c, d, Fig. 6 g-h, S6 i, j, respectively ). Furthermore, IRF3 and p-IRF3 expression increased significantly, suggesting that S100A1 (Fig. 6 e, f, S6 c, d) and S100A9 (Fig. 6 g, h, S6 m, n) induce IFIT3 expression through the TLR3/TICAM1/IRF3 pathway. Furthermore, there was a significant increase in the expression levels of IRF3 and phosphorylated IRF3 (p-IRF3), suggesting that S100A1 ( Fig. 6 e, f ) and S100A9 ( Fig. 6 g, h ) induce IFIT3 expression via the TLR3/TICAM1/IRF3 pathway. In addition, the signaling key regulators TLR4/MyD88 were detected following treatment of S100A1 ( Fig. 6 e, Fig.S6 e) and S100A9 ( Fig. 6 g, Fig.S6 k) . Although the expression of TLR4 did not show significant changes, there was a time-dependent increase in MyD88with. It is hypothesized that TLR3 and TLR4 may interact during stimulation with S100A1 and S100A9 in AC-16 cells, suggesting a potential regulatory role for TLR4. However, this hypothesis remains unconfirmed within this study, necessitating further research to explore this possibility. IFIT3 Knockout abolished cardiomyocyte apoptosis initialized by S100A1 and S100A9 This study aims to investigate whether S100A1 and S100A9 can induce apoptosis in cardiomyocyte. Caspase 3 and Cleaved-Caspase 3 levels were measured in AC-16 cells following 24 hours of stimulation with either S100A1 ( Fig. 7 a, S7a, b ) or S100A9 ( Fig. 7 b, S7c, d ) . The results demonstrated that both S100A1 and S100A9 are capable of inducing apoptosis in cardiomyocyte. The Cleaved-Caspase 3 level increased significantly after 4 hours of S100A1 stimulation and continued to increase until 12 hours ( Fig. 7 a, b ) . In experiments involving S100A9, the Cleaved-Caspase 3 level showed a significant increased after 8 hours of stimulation ( Fig. 7 c, d ) . To investigate the role of IFIT3 in regulating the apoptosis induced by S100A1 and S100A9, the IFIT3 gene was knocked out in AC-16 cells. Subsequently, the expression levels of Caspase 3 and Cleaved-Caspase 3 were measured in response to treatment with S100A1 and S100A9. In wild-type cardiomyocytes, the levels of IFIT3 (Fig.S7e) , CASP3 (Fig.S7f) , and Cleaved-CASP3 (Fig.S7g) increased with prolonged exposure to S100A1. In contrast, S100A1 did not elicit any changes in IFIT3 expression in AC-16 cells with IFIT3 knockout ( Fig. 7 e, f ) . Notably, the CASP3 levels in IFIT3 knockout cardiomyocytes were significantly higher than in wild-type cells under rest conditions. However, with extended exposure, the CASP3 level did not increase in the IFIT3 knockout cells in response to S100A1 ( Fig. 7 e, f ) . Furthermore, the level of activated Cleaved-CASP3 were significantly lower in IFIT3 knockout cells compared to wild-type cells after 24h and 48 hours of S100A1 induction ( Fig. 7 e ) . This result suggests that IFIT3 knockout attenuates the apoptosis induced by S100A1. In the experiment involving S100A9-induced AC-16 cells, the expression of IFIT3 exhibited a progressive increase with prolonged S100A9 induction time ( Fig. 7 g ) . Meanwhile, IFIT3 knockout cardiomyocytes showed higher levels of CASP3 expression compared to wild-type cardiomyocytes (Fig.S7j) . Additionally, the of CASP3 expression was increased with extended S100A9 induction time (Fig. 7Si) . However, the level of Cleaved-CASP3 protein was lower in IFIT3 knockout cardiomyocytes than in wild-type cardiomyocytes, although this difference without statistical significance (( Fig. 7 g, S7j ) . In vivo validation of TLR3/IRF3 pathway activation and apoptosis in the myocardial infarct zone To validate the TLR3/IRF3-IFIT3 signaling axis and apoptotic activation in vivo, cardiac tissues from the infarct area of acute MI rat models were analyzed by Western blot and immunofluorescence staining. Western blot analysis revealed significant upregulation of TLR3, phosphorylated IRF3 (p-IRF3), pro-caspase-3, and cleaved caspase-3 in the MI infarct zone (n = 6, 24h post-MI), accompanied by decreased total IRF3 levels ( Fig. 8 a-c ) . These findings indicate activation of the TLR3/IRF3 signaling pathway and execution of apoptosis in ischemic myocardium. To determine the spatial relationship between IFIT3 expression and cardiomyocyte apoptosis, infarct area sections were co-stained with antibodies against cardiac troponin T (cTnT, green), cleaved caspase-3 (yellow), and IFIT3 (red), with DAPI counterstaining for nuclei (blue). Immunofluorescence analysis revealed marked elevation of cleaved caspase-3 that co-localized with both IFIT3 and cTnT specifically in cardiomyocytes within the border zone of the myocardial infarction ( Fig. 8 d ) . These results demonstrate that MI-induced IFIT3 overexpression coincides with cardiomyocyte apoptosis in the infarct border zone, supporting activation of the TLR3/IRF3/p-IRF3 signaling pathway during myocardial ischemic injury. In summary, we identified multiple S100 protein family isoforms (S100A1/6/8/9/B) as endogenous damage-associated molecular patterns (DAMPs) that drive IFIT3-mediated cardiomyocyte apoptosis during myocardial infarction. Specifically, S100A1 and S100A9 activate the TLR3/TICAM1/IRF3 signaling pathway, leading to IFIT3 overexpression. During myocardial ischemic injury, S100 proteins released from necrotic cardiomyocytes are recognized by TLR3/4 receptors on neighboring viable cardiomyocytes, triggering IFIT3-dependent apoptosis that amplifies tissue damage in the infarct border zone ( Fig. 8 e ) . This paracrine injury mechanism establishes the S100A-TLR3-IFIT3 axis as a critical damage amplification loop in ischemic cardiomyopathy. Materials and Methods Dataset collection and preprocessing As outlined in a prior study[23], the analysis of human transcriptomic data was based on previously published datasets( GSE57338) that complied with ethical standards, including the principles of the Declaration of Helsinki. The transcriptional profiles of 313 patient left ventricle biopsies from patients were analyzed utilizing the Affymetrix Human Gene 1.1 ST Array (platform GPL11532) ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE57338 , accessed on 1 January 2015). The dataset comprised 33,297 probes designed to measure alterations in transcriptional profiles associated with the physiological characteristics of hearts affected by heart failure. In this study, our focus to clarify the gene expression characteristics related to ISCM. The biopsy samples included normal hearts as control (Health, n = 136), and ischemic cardiomyopathy (ISCM, n = 95, supplemental Table 1 ). Each gene expression reading was normalized and transformed using the log2 scale. The expression of the IFIT3 gene in each group was presented as means ± standard error of the mean (SEM). Rat Model of Myocardial Infarction and Echocardiography Adult male Sprague-Dawley rats (200–220 g) were obtained from Hunan SJA Laboratory Animal Co., Ltd (People's Republic of China license No. SCXK (Xiang) 2019-0004). All protocols were approved by the Institutional Animal Care and Use Committee of the Kunming Institute of Botany, Chinese Academy of Sciences, and conducted in accordance with NIH guidelines. After one week of acclimation, rats were randomly assigned to sham-operated (n = 12) or myocardial infarction (MI, n = 15) groups. The MI model was established under isoflurane anesthesia (RWD, R510-22-10, 3% for induction, 1.5% for maintenance). A left thoracotomy was performed to expose the heart, and the left anterior descending coronary artery was permanently ligated. After 24 hours of ischemia, cardiac function was reassessed under anesthesia using high-frequency (20 MHz) transthoracic echocardiography (Vevo 3100, Fujifilm VisualSonics or Mindray M9, Shenzhen Mindray Bio-Medical Electronics). Left ventricular dimensions and systolic function parameters, including ejection fraction and fractional shortening, were measured to validate the model[25, 26]. Following functional assessment, rats were euthanized, and heart tissues were collected for subsequent analysis. Tissue Embedding and Sectioning Hearts harvested from Sprague-Dawley rats were fixed in 4% paraformaldehyde for 24 hours, followed by standard paraffin embedding. Serial sectioning was performed starting from the cardiac apex. Trimming continued until the left ventricular chamber was fully exposed. Subsequently, consecutive 3-µm-thick sections were collected with the following scheme: five consecutive sections were retained. Approximately 50 µm of tissue was discarded (corresponding to ~ 16 sections). This cycle was repeated until a total of 35 sections per heart were obtained. Six distinct anatomical levels spanning from apex to base were systematically preserved for analysis. Masson's Trichrome Staining One section from each of the six anatomical levels (total: 6 sections/heart) was selected for Masson's trichrome staining Masson reagent (Servicebio, Cat#G1006-100ML) following the established protocol. This approach ensured representative sampling of the entire ventricular structure. Stained sections were scanned using a digital slide scanner (Pannoramic Scan) for subsequent quantitative analysis of collagen deposition. Immunofluorescence Staining Immunofluorescence staining was performed on AC-16 cell slides or paraffin-embedded ventricular tissue sections. For each target, three consecutive sections from the third anatomical level (mid-ventricular region, sections 15–20) were processed. After antigen retrieval and blocking, sections were incubated with primary antibodies against (1:500 dilution) overnight at 4°C, followed by appropriate secondary antibodies (1:500 dilution). Nuclei were counterstained with DAPI. All images were acquired using a confocal microscope under consistent exposure settings and quantified using ImageJ software with appropriate thresholding algorithms. Cells culture The mouse cardiomyocyte cell HL-1 [27] was obtained from Sigma (USA) and cultured in DMEM medium, which is a valuable model system to address questions of cardiac biology at the cellular & molecular levels. Human AC-16 cells were purchased from ATCC (CRL-3568) and cultured in DMEM/F-12 medium, which is derived from primary adult ventricular tissue and can be induced to differentiate into mature cardiomyocytes. Mouse cardiac fibroblasts (MCFs; catalog number 340098) were sourced from the Beijing Beina Chuanglian Biotechnology Research Institute and maintained in RPMI 1640 medium. HEK293 cells (CRL1573) were pursued from ATCC and maintained in DMEM medium. All medium were supplemented with 10% fetal bovine serum plus 100 U/ml penicillin/streptomycin (P1400, Solarbio, Beijing, China), and 4mM L-glutamine in 37°C, 5% CO 2 atmosphere. Cells were cultured in a 37°C, 5% CO 2 atmosphere. Generation of IFIT3-Knockout Cells Using CRISPR-Cas9 An IFIT3-knockout cell model was established using the Lenti-CRISPR v2 system[28]. A specific sgRNA (5′-GACACCTAGATGGTAACAACG-3′) was cloned into the vector to target the IFIT3 coding region. The constructed plasmid was amplified in DH5α competent cells and verified by sequencing. For transfection, cells were seeded in 6-well plates until 50–60% confluent. Complexes of plasmid DNA (2–5 µg) and Lipofectamine 2000 (4–10 µL, Lipofectamine™ 2000, 11668500) were prepared in Opti-MEM ((Opti-MEM™, Gibco, 31985070) and added to the cells. After 4 hours, the medium was supplemented with an additional 1 mL of complete medium. At 24 hours post-transfection, selection was initiated with puromycin (0.8–2 µg/mL). Polyclonal knockout cells were obtained after 7–10 days of selection, and IFIT3 depletion was confirmed by Western blot. The resulting cells were used for subsequent experiments at early passages. Overexpression of IFIT3 in AC-16 Cells AC-16 cells were seeded in 6-well plates at a density of 2–4×10⁵ cells per well and transfected with 1–2 µg of pcDNA3.1-IFIT3-FLAG plasmid (encoding NM_001549) using Lipofectamine 3000 reagent (Lipofectamine™ 3000 transfection Kit, L3000015). Cells transfected with empty vector served as the control. After 48 hours, total protein was extracted and subjected to Western blot analysis using an anti-FLAG antibody to confirm overexpression efficiency. Successfully transfected cells were used for subsequent functional experiments. Modeling Cardiomyocyte Injury via Oxygen-Glucose Deprivation/Reperfusion (OGD/R) An OGD/R model [29, 30] was established to simulate ischemia/reperfusion injury and investigate the role of IFIT3 in cardiomyocytes. Cells were cultured in six-well plates until reaching 80–90% confluence. For the OGD/R group, the culture medium was replaced with a balanced salt solution (BSS), and cells were transferred to a sealed hypoxia chamber. The chamber was flushed with 95% N₂/5% CO₂ for 15 minutes until the oxygen indicator turned blue-purple, then sealed and incubated at 37°C for the designated OGD period. Control cells were maintained in DMEM/F-12 medium under normoxic conditions. After OGD, cells were returned to a normal oxygenated incubator (95% air/5% CO₂) with complete medium for 3 hours of reperfusion. Protein lysates were collected at the endpoint for subsequent analysis. RNA Sequencing and Bioinformatic Analysis AC-16 cells were cultured in 6-cm dishes until reaching 70–80% confluence. The experimental group was subjected to 2 hours of oxygen-glucose deprivation (OGD), while the control group was maintained under standard culture conditions. Total RNA was extracted using TRIzol reagent and sent to Shanghai Meijie Biomedical Technology Co., Ltd. for transcriptome sequencing. Differentially expressed genes (DEGs) were identified using a threshold of |log₂FC| > 2 and adjusted p-value (padjust) < 0.05. Gene ontology (GO) and pathway enrichment analyses of DEGs associated with IFIT3 knockout were performed using Metascape[31]. Gene Set Enrichment Analysis (GSEA) was conducted to rank genes based on their correlation with IFIT3 knockout, with gene set permutations performed 1000 times for each analysis. Enriched pathways were classified based on the nominal p-value and normalized enrichment score (NES). Type-I interferons, TLRs agonists, IRF3 agonists and S100 proteins stimulation Toll-like receptor agonists (tlrl-kit1hw, Pam3sk4, HKLM, FSL-1, Poly(I:C) HMW, Poly(I:C) LMW, LPS-EK, FLA-ST, imiquimod, ssRNA40/LyoVec™, ODN2006), ODN2216 [tlrl-2216] and STING agonists (ISD Control/LyoVec [tlrl-isdcc], 2’3-cGAMP [tlrl-nacga23-02]) were bought from Invivogen . The recombinant human S100 family proteins were sourced from Solarbio (S100A1, P02881; S100A6, P00170; S100A8, P00431), S100A9, P02686; S100 Calcium Binding Protein B/S100B, P00663). The cells were restimulated with TLR ligands (TLR1/2/3/4/5/6/7/8/9) including Pam3sk4, HKLM, FSL-1, Poly(I:C) HMW, Poly(I:C) LMW, LPS-EK, FLA-ST, imiquimod, ssRNA40/LyoVec™, ODN2006) or STING agonists (ISD Control/LyoVec, 2’3-cGAMP) at the mentioned concentrations or cultured without stimulation(PBS solution, pH 7.2–7.4) as a negative control. Agonists were added to the media at the determined concentrations and cells were incubated at 37°C for the specified duration. For assessing IFIT3 responses, AC-16, HL-1, and HEK293 cells (2 × 10 5 ) in 1ml complete media were added to each of the duplicate ligand- or medium containing wells and incubated at 37 ◦C for 24 h with 5% CO 2 . Three types of type-Ⅰ interferons and three stimulant dosages were selected to evaluate the dose-dependent effect. Cells were stimulated for TLRs agonists and STING agonists. The concentrations of stimuli were aligned with the manufacturer’s guidelines. After 24 hours, cells were harvested, and total protein was extracted. Mitochondrial Membrane Potential Assay Mitochondrial membrane potential (ΔΨm) was assessed using the JC-10 assay kit (Solarbio, CA1310) according to the manufacturer's instructions. Wild-type and IFIT3-deficient AC-16 cells were seeded in 6-well clear-bottom plates and subjected to oxygen-glucose deprivation/reperfusion (OGD/R). After treatment, cells were washed with Hanks’ solution containing 0.02% Pluronic F-127 and incubated with 500 µL of JC-10 working solution (6.67 µg/mL) for 20 minutes at 37°C. Following dye loading, cells were washed thoroughly, mounted with antifade medium containing DAPI, and imaged under a fluorescence microscope. JC-10 aggregates (red fluorescence) and monomers (green fluorescence) were detected at excitation/emissionex wavelengths of 540/590 nm and 490/530 nm, respectively[32]. CCCP-Induced Mitochondrial Depolarization and Apoptosis Carbonyl cyanide-3-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, disrupts mitochondrial membrane potential by facilitating proton leakage across the inner membrane, leading to loss of transmembrane potential and induction of apoptosis[33]. To examine the role of IFIT3 in mitochondrial-dependent apoptosis, AC-16 cells at 70–80% confluence were treated with CCCP (0, 2.5, or 5 µM, Solarbio, Beijing, C6700) for 24 hours. Cells were then harvested and analyzed for apoptosis using Annexin V-FITC/PI staining and flow cytometry. Assessment of Apoptosis by Flow Cytometry Cell apoptosis was quantified using an Annexin V-FITC/PI apoptosis detection kit (Solarbio, CA1020). Briefly, harvested cells were washed with cold PBS and resuspended in 1× binding buffer. The cell suspension was incubated with 5 µL Annexin V-FITC for 5 minutes at room temperature in the dark, followed by addition of 5 µL propidium iodide (PI). Samples were analyzed within 1 hour on a BD FACSCalibur flow cytometry system. Fluorescence signals were collected in the FL1 channel for Annexin V-FITC and FL2 channel for PI. Data were processed using FlowJo software, and apoptotic populations were distinguished as follows: early apoptotic (Annexin V-FITC+/PI−) and late apoptotic (Annexin V-FITC+/PI+). Western blot Cells were gently washed three times with phosphate-buffered saline (PBS) and subsequently lysed in RIPA lysis buffer (Beyotime Biotechnology, China) after treated with drugs or control conditions. Protein concentration of cell extracts was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, A23225) the, which facilitated the adjustment of protein concentration for subsequent experiments. Proteins were isolated using SDS-PAGE on a 10%-15% gel at a loading concentration of 20 µg per well, followed by wet-transfer to polyvinylidene difluoride (PVDF, 0.22µm) membranes (Millipore, USA). The membranes were blocked at room temperature for one hour with 5% skim milk dissolved in TBST solution (FD bioscience, China), and incubated overnight at 4°C with a specific primary antibody. The membranes were washed three times in TBS-T solution for 5 minutes each time and subsequently incubated with either HRP-labeled Goat Anti-Rabbit IgG(H + L) (1:5000, Beyotime Biotechnology, China, A0208) or HRP-labeled Goat Anti-Mouse IgG(H + L) (1:5000, Beyotime Biotechnology, China, A0216) secondary antibodies at room temperature for one hour. Immunoreactive proteins were visualized according to the manufacturer's instructions before exposure of PVDF membranes using Tanon 5200 (Tanon, China) using an enhanced chemiluminescence kit (NCM Biotech, P10100). The standard methods were using the following antibodies: IFIT3 Polyclonal Antibody (Thermo Fisher Scientific, PA5-22230, 1:3000), β-Actin Rabbit mAb (High Dilution) (ABclonal, AC026, 1:20 000), Anti-GAPDH rabbit polyclonal (Sangon Biotech, D110016, 1: 5000); Anti-TRIF (TICAM1) (Abcam, ab302562,1:2000), TLR3 Rabbit Polyclonal Antibody (Beyotime, AF8184, 1:1500), TLR4 Rabbit pAb (ABclonal, A5258, 1:1500), Anti-MyD88 (Abcam, ab133739, 1:2000), Phospho-IRF-3 (Ser396) (D6O1M) Rabbit (Cell Signaling Technology, 29047, 1:1000), IRF3 Rabbit Monoclonal Antibody (Beyotime, AF2485, 1:1500); Caspase-3 Antibody (Cell Signaling Technology, # 9662S, 1:1500), Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb (Cell Signaling Technology, #94530, 1:1500). Statistical Analysis Data are presented as means ± standard error of the mean (SEM). Differences in experimental values were assessed using the student's t-test, conducted with Prism software (GraphPad Software 9.0, Inc., San Diego, CA, USA). A p-value of less than 0.05 was considered statistically significant (*: p <0.05; **: p <0.01; ***: p <0.001). Discussion In this study, we employed a systematic research strategy to elucidate the mechanism underlying IFIT3 overexpression and its pro-apoptotic effects in cardiomyocytes during myocardial infarction (MI), extending our previous research [23]. Initially, we validated IFIT3 overexpression in cardiac biopsies from ischemic patients’ expression profiles (dataset reference) and LAD-induced MI rat models. Using the immortalized human cardiac myocyte cell line AC-16 combined with CRISPR-Cas9 gene editing and oxygen-glucose deprivation (OGD) modeling, we demonstrated that IFIT3 knockout significantly attenuates cardiomyocyte apoptosis under both resting and persistent OGD conditions, which minimizes interference from cytokines released by other cardiac cell types, such as fibroblasts and monocytes, etc. Critically, we identified S100A1 and S100A9 as endogenous cardiac alarmins that activate IFIT3 overexpression through the TLR3/TICAM1/IRF3 signaling pathway, thereby promoting cardiomyocyte apoptosis during MI. In the heart, TLR2, TLR3, TLR4, and TLR5 represent the predominant TLR isoforms [34], with TLR4 and TLR2 being the most extensively studied in the context of myocardial injury. TLR signaling operates through two major pathways: the myeloid differentiation factor 88 (MyD88)-dependent pathway (primarily activating NF-κB and inflammatory cytokines) and the TRIF/TICAM1-dependent pathway (activated by TLR3 and TLR4, leading to IRF3 and type I interferon production). The MyD88-dependent pathway is primarily responsible for activating nuclear factor- κ B (NF- κ B), a major inflammatory transcription factor, which further stimulates the production of inflammatory cytokines. In contrast, the TRIF-dependent pathway can be triggered by TLR3 and TLR4, leading to the activation of interferon regulatory factor 3 (IRF3) and NF- κ B, and consequently results in the production of type I interferon and inflammatory cytokines [35]. TLR3, an intracellular subtype predominantly expressed by cardiomyocytes [36], exhibits upregulated expression and signaling that contribute to persistent autophagy following MI, which in turn promotes heart failure and increases lethality. Enhanced TLR4 activation and elevated expression of proinflammatory mediators downstream of TLR4 signaling have been observed in circulating leukocytes from humans with acute MI [37], contributing to the development of heart failure [38]. Additionally, cardiac TLR4 expression increases following both acute MI [39] and chronic HF[40, 41]. While TLR4 agonist stimulated significant IFIT3 overexpression than other TLR isoforms (TLR1/2/6/7/9) in AC-16 ( Fig. 5 d ) and HL-1 cells ( Fig. 5 h ) , TLR3 agonists induced more strong response of IFIT3 overexpression in AC-16 ( Fig. 5 f ) and HL-1 cells ( Fig. 5 g ) . Furthermore, the key regulator of TLR3 signaling, TRIF (TICAM1), exhibited significantly higher expression after OGD ( Fig. 5 b, c ) . This finding suggests that the TLR3/TRIF pathway, rather than the TLR4/MyD88 pathway, is preferentially activated during ischemic injury in cardiomyocytes. Supporting this, both TLR3 and TLR4 agonists, as well as STING agonists, induced IFIT3 overexpression in AC-16 cells, with TLR3 agonists eliciting particularly robust responses ( Fig. 5 d, e ) . These results indicate that endogenous DAMPs activate the TLR3/IRF3 axis to drive IFIT3 overexpression during myocardial ischemia through an IFN-independent pathway. This mechanism aligns with previous observations that Mice with genetic disrupting or deficiency of TLR4 [42], MyD88 [43], or TLR3[44] reduces infarct size following ischemia/reperfusion and ameliorates pathological remodeling after non-reperfused MI [39, 43, 45], while sustained TLR3 signaling promotes maladaptive responses including apoptosis, inflammation, fibrosis, and oxidative stress. Necrotic cardiac myocytes release a diverse array of endogenous DAMPs, including S100A1, S100A8/A9 and S100β, which activate TLR3/4 and subsequently upregulate key regulatory effectors in both infarcted and remote myocardium following MI [8, 24]. S100A1 is the most abundant S100 isoforms in cardiomyocytes and is passively released from damaged cardiomyocytes during MI, resulting in a dramatically increased serum levels in acute MI patients. Calprotectin (S100A8/A9) serves as an endogenous agonist of TLR4[11] whose expression is elevated in infarcted myocardium and patient blood, contributing to cardiomyocyte death through mitochondrial dysfunction mediated by TLR4 in the ischemic/re-perfused heart. S100A9 overexpression exacerbates MI/R injury, whereas genetic deletion or pharmacological blockade of S100A9 provides protection against such MI in murine models [15]. In the present study, our results indicated that S100A isoforms (S100A1/6/8/9) (2.0µg/mL, 24 hours) treatment significantly induced IFIT3 overexpression in AC-16 cells, HEK293 and MCF cells. Notably, S100A1 and S100A9 exhibited the most potent effects of in AC-16 cells. Mechanistically, S100A1/9 induced IFIT3 overexpression through the TLR3/TICAM1/IRF3 signaling pathway and promoted apoptosis in AC-16 cells, while IFIT3 knockout significantly attenuated S100A1/9-induced apoptosis. Furthermore, to validate our proposed S100A-TLR3-IFIT3 signaling mechanism in vivo, we examined key pathway components in cardiac tissues from acute MI rat models. Western blot analysis of infarct zone tissues (n = 6, 24h post-MI) demonstrated significant increases in TLR3, phosphorylated IRF3 (p-IRF3), and both pro-caspase-3 and cleaved caspase-3 expression, while total IRF3 levels decreased ( Fig. 8 a-c ) . The elevation of p-IRF3 relative to total IRF3 indicates robust activation of IRF3-dependent transcription, consistent with TLR3/TICAM1 pathway engagement. Concurrently, the increased cleaved caspase-3/pro-caspase-3 ratio confirms execution of the apoptotic cascade in ischemic myocardium. To establish the cellular localization of IFIT3 expression relative to apoptotic cardiomyocytes, we performed triple immunofluorescence staining of infarct sections with cardiac troponin T (cTnT, cardiomyocyte marker; green), cleaved caspase-3 (apoptosis marker; yellow), and IFIT3 (red), with DAPI nuclear counterstaining (blue). Confocal microscopy revealed prominent co-localization of IFIT3, cleaved caspase-3, and cTnT specifically in cardiomyocytes within the infarct border zone (Fig. 8 D), demonstrating that IFIT3 overexpression occurs in apoptotic cardiomyocytes at the interface between viable and necrotic tissue. This spatial pattern suggests that IFIT3-expressing cardiomyocytes in the border zone are undergoing apoptosis mediated by TLR3/IRF3 signaling, likely in response to DAMPs released from adjacent necrotic cells. Collectively, these in vivo findings validate that MI activates the TLR3/IRF3/p-IRF3 signaling pathway, driving IFIT3 overexpression and subsequent caspase-3-mediated apoptosis in border zone cardiomyocytes. In this study, our results establish that S100A1/9 proteins released from damaged cells during myocardial injury are recognized as DAMPs by neighboring cardiomyocytes, triggering aberrant IFIT3 overexpression that amplifies cardiomyocyte apoptosis and exacerbates myocardial damage ( Fig. 8 e ) . The most innovative point of this study is to identify the S100A1/9-TLR3-IFIT3 signaling axis as a critical amplification loop in myocardial ischemic injury, wherein damaged cardiomyocytes release alarmins that activate pro-apoptotic pathways in neighboring cells, propagating tissue damage. This pathway represents a promising therapeutic target for MI intervention. Pharmacological inhibition of S100A1/9, TLR3 antagonism, or IFIT3 suppression could potentially interrupt this damage amplification cascade and preserve myocardial function following ischemic injury. These molecular insights advance our understanding of the pathophysiology underlying ischemic cardiomyopathy progression and provide a mechanistic foundation for developing targeted therapies. Several limitations warrant consideration in this study. First, the majority of experiments were conducted using immortalized human (AC-16) or mouse (HL-1) cardiomyocyte cell lines, which, despite being derived from primary adult ventricular tissue and serving as valuable model systems for investigating cardiac functions at cellular and molecular levels, require validation in clinical patient samples to strengthen reliability. Second, while IFIT3 knockdown was achieved using CRISPR-Cas9 gene editing and validated by Western blot, future studies should confirm these findings in cardiomyocytes derived from IFIT3 knockout animals. Third, although IFIT2 has been extensively studied in MI contexts and likely contributes to cardiomyocyte injury, this study focused specifically on IFIT3 and did not examine IFIT2 knockout effects, which represents an important area for future investigation. In summary, this study elucidates a novel molecular mechanism whereby endogenous S100A1/9 alarmins interact with TLR3/4 receptors to drive inducible IFIT3 overexpression and subsequent cardiomyocyte apoptosis during myocardial infarction ( Fig. 8 e ) . These findings reveal the critical role of IFIT3 in propagating myocardial injury and identify the S100A1/9-TLR3-IFIT3 signaling pathway as a potential target for clinical intervention in MI, contributing to a more comprehensive understanding of ischemic cardiomyopathy pathophysiology and offering new therapeutic avenues for treatment. Declarations Ethics approval and consent to participate The animal study was approved by the institutional review board of KIB (KIB-R-018). Not applicable for clinical study requirement. Consent for publication All authors have agreed to submit and be published. Availability of data and material Supplementary materials can be found can be found online. Competing interests None. Funding This work was supported by the following funding sources: To Dr. Shubai Liu: A grant for outstanding talent from abroad from the Chinese Academy of Sciences; Startup Support Funding (E0241211H1); and grants from the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences (Y8677211K1, Y8690211Z1). To Dr. Cheng Chen: The Yunnan Provincial Department of Science and Technology - Kunming Medical University Joint Special Project on Applied Basic Research (Grant No. 202501AY070001-223), the Opening Foundation of The First People's Hospital of Yunnan Province (2023YJZX-YX001) and the Yunnan Provincial Clinical Medical Center Research Project (Grant No. 2024YNLCYXZX0180). Additional support was received from "The Pilot Project for Clinical Collaboration of Traditional Chinese and Western Medicine in Major and Complicated Diseases in Yunnan Province: Chronic Heart Failure." All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Author’s Contributions Designed the overall project study: S.L., W.X., and Y.H.; Collected data, performed data analysis, and drafted the manuscript: C.C., Z.H; performed the animal experiments and collected the data: C.C., Z.H., Y.C.; performed the animal experiments and data analysis: H.Q.C. and H.Z.; interpreted and summarized the results: S.L., W.X., C.C. HQ.C. and Y.H.; wrote and revised the manuscript: C.C., S.L. H.Z., W.X., and Y.H.; all authors have read and approved the final version of manuscript. Acknowledgements The authors gratefully acknowledge the Center for Clinical Medicine Research of The First People's Hospital of Yunnan Province and the Affiliated Hospital of Kunming University of Science and Technology (Kunming 650032, China) for providing the experimental platform and technical support. We extend our sincere thanks to Professor Yalian Sa and Ms. Chengcheng Huang for their invaluable assistance with the experiments. We also wish to thank the College of Chinese Traditional Medicine at Yunnan University of Traditional Chinese Medicine (Kunming 650200, China) for their support. Special gratitude is extended to Professor Yunshu Ma and Professor Lili Cui for their expert assistance with animal experiments and for generously sharing laboratory instrumentation. Availability of data and materials The main figures or supporting data have included all related data and materials in this manuscript. References Prabhu, S.D. and N.G. Frangogiannis, The Biological Basis for Cardiac Repair After Myocardial Infarction: From Inflammation to Fibrosis. Circ Res, 2016. 119 (1): p. 91-112. Safiri, S., et al., Burden of ischemic heart disease and its attributable risk factors in 204 countries and territories, 1990-2019. Eur J Prev Cardiol, 2022. 29 (2): p. 420-431. 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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-8733111","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":584332824,"identity":"661c3c20-be00-49ee-8fed-8ff5e4fbb467","order_by":0,"name":"shubai Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYDACCQY2hgcGDHIg9oEHRGtJMGAwBmtJIF4LA0NiA4hDlBb52T1mDxIKDqfPDzv8EGiLnZxuAwEtBnfOmBskGBzO3Xg7zQCoJdnY7AAhLRI5ZhJgLbMTQFoOJG4jpEV+BkRLuuHs9A/EaWG4AdGSIC+dQ6QtBjfSyoF+STfcIJ1TcCDBgAi/yM9I3vbgwx9refnZ6Zs/fKiwkyOoBWEdWKUBscrB1jWQonoUjIJRMApGFAAAXPZFsQNpm1MAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-8929-4209","institution":"kunming institute of botany, chinese academic of science","correspondingAuthor":true,"prefix":"","firstName":"shubai","middleName":"","lastName":"Liu","suffix":""},{"id":584332825,"identity":"6cdb01be-f34f-4676-97ce-9c5d7bc43691","order_by":1,"name":"cheng chen","email":"","orcid":"","institution":"kunming institute of botany, chinese academic of science","correspondingAuthor":false,"prefix":"","firstName":"cheng","middleName":"","lastName":"chen","suffix":""},{"id":584332826,"identity":"d9179559-7d43-4f81-b1b9-519a0b5abfd4","order_by":2,"name":"Haoqiang Chen","email":"","orcid":"","institution":"The First People's Hospital of Yunnan Province","correspondingAuthor":false,"prefix":"","firstName":"Haoqiang","middleName":"","lastName":"Chen","suffix":""},{"id":584332827,"identity":"8e43e305-a076-426e-9c43-32bf1b59d4e3","order_by":3,"name":"zhicheng He","email":"","orcid":"https://orcid.org/0000-0002-0254-6520","institution":"","correspondingAuthor":false,"prefix":"","firstName":"zhicheng","middleName":"","lastName":"He","suffix":""},{"id":584332828,"identity":"f04ad757-8d50-48b7-b097-ad9980689c4e","order_by":4,"name":"Yuanzhi Chen","email":"","orcid":"","institution":"Anqing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yuanzhi","middleName":"","lastName":"Chen","suffix":""},{"id":584332829,"identity":"86fe78c7-22fc-4436-8bb8-65d6f820e7b3","order_by":5,"name":"Hong Zhang","email":"","orcid":"","institution":"The First People’s Hospital of Yunnan Province","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Zhang","suffix":""},{"id":584332830,"identity":"15a08fe0-fd03-4868-9509-6ee140b30411","order_by":6,"name":"Wenyong Xiong","email":"","orcid":"https://orcid.org/0000-0001-9174-3667","institution":"Yunnan University","correspondingAuthor":false,"prefix":"","firstName":"Wenyong","middleName":"","lastName":"Xiong","suffix":""},{"id":584332831,"identity":"d5fe4997-94a8-4327-80da-265a2bc7f071","order_by":7,"name":"Yingying He","email":"","orcid":"https://orcid.org/0000-0003-4537-4463","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yingying","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2026-01-29 15:28:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8733111/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8733111/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104403667,"identity":"ae24c787-bca6-407b-a601-f44d11c92455","added_by":"auto","created_at":"2026-03-11 12:18:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":918677,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of IFIT3 was significantly elevated in the cardiac tissue of MI patients and rat model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e The mRNA expression level of \u003cem\u003eIfit3\u003c/em\u003e in the left ventricular. The results were represented as mean±SEM, unpaired t-test, compared to the healthy control group (Health Control, n=136; ISCM patients, n=95). \u003cstrong\u003eb.\u003c/strong\u003e Echocardiographic assessment of cardiac function in rats. Methods for measuring cardiac function (right panel). \u003cstrong\u003ec.\u003c/strong\u003e EF, left ventricle (LV) ejection fraction; \u003cstrong\u003ed.\u003c/strong\u003e FS, LV fractional shortening; \u003cstrong\u003ee, \u003c/strong\u003eIVSd, interventricular septal thickness in diastole; \u003cstrong\u003ef. \u003c/strong\u003eLVIDd, LV internal dimension-diastole; \u003cstrong\u003eg.\u003c/strong\u003e IVSs, interventricular septal thickness in systole; \u003cstrong\u003eh.\u003c/strong\u003e LVIDs, LV internal diameter in systole. Sham group: n=12; ISCM 24h group: n=15. \u003cstrong\u003e\u0026nbsp;i.\u003c/strong\u003e Masson staining of rat heart sections. Paraffin sections, 3 μm thick. The heart sections were arranged from left to right in an order from the apex to the remote myocardium. The proportion of the infarcted area in the left ventricle (right panel). Data were represented as mean ± SEM, unpaired t-test, n=3, compared to the sham group.\u003cstrong\u003e j.\u003c/strong\u003e IFIT3 expression in the infarcted area of the left ventricle of rats. \u003cstrong\u003ek.\u003c/strong\u003e Immunofluorescence of rat heart sections. The primary antibody Ctn-t-FITC (Protech, Cardiac Troponin T Monoclonal Antibody CoraLite® Plus 488, CL488-68300), IFIT3 (Thermo Fisher Scientific, PA5-22230), and the secondary antibody CoraLite594-conjugated Goat Anti-Rabbit IgG (H+L) (Protech, SA00013-4) were all diluted at a ratio of 1:500. The primary antibody was incubated overnight at 4 degrees Celsius, and the secondary antibody was incubated at room temperature for 2 hours. Statistical results of fluorescence intensity (right panel). Data were represented as mean±SEM, unpaired t-test, compared to the sham group, n=3.\u003c/p\u003e","description":"","filename":"Binder11.png","url":"https://assets-eu.researchsquare.com/files/rs-8733111/v1/21f377dbb14918655f408460.png"},{"id":104403830,"identity":"ebafd8e5-f32a-43c7-81b5-b142ce5e7b56","added_by":"auto","created_at":"2026-03-11 12:19:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":377117,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional and pathway enrichment analyses, and apoptosis analyses of the IFIT3 knockout in cardiomyocytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Cellular phenotypes of cardiomyocytes induced by OGD 24h. After OGD 24h, cardiomyocytes (AC-16) were atrophy and with cell membrane wrinkled, the volume were decreased and the shape were change to flat. \u003cstrong\u003eb.\u003c/strong\u003e Continuous OGD induces increased expression of IFIT3 and apoptosis in cardiomyocytes.\u003cstrong\u003e c. \u003c/strong\u003eThe diagram illustrates of CRISPR-Cas9 knockout of IFIT3 in cardiomyocytes AC-16. \u003cstrong\u003ed. \u003c/strong\u003eSignificant changes in gene expression were observed in AC-16 cells following IFIT3 knockout. Upregulated genes are indicated in red, downregulated genes in blue, and genes exhibiting no statistical difference are represented in gray (n=3). \u003cstrong\u003ee.\u003c/strong\u003e Representative the top 20 Gene Ontology (GO) process annotations were enriched. \u003cstrong\u003ef.\u003c/strong\u003e The Hallmark apoptosis was enriched as determined by GSEA. \u003cstrong\u003eg\u003c/strong\u003e. Heat maps displayed the significantly altered genes involved in the positive regulation of the apoptosis process. \u003cstrong\u003eh.\u003c/strong\u003e IFIT3 KO significantly reduced cardiomyocyte apoptosis induced by persistent OGD for both 4 hours and 24 hours. Apoptosis induced by persistent oxygen glucose deprivation for 4 hours was assessed using flow cytometry. \u003cstrong\u003ei. \u003c/strong\u003eApoptosis induced by persistent oxygen glucose deprivation for 24 hours was detected by flow cytometry. Compared to the wild-type group (WT), the knockout of IFIT3 did not induce apoptosis in normal AC-16 cells. All results were analyzed using a t-test or ANOVA test, data are expressed as mean ± SEM, compared to the wild-type, n=3.\u003c/p\u003e","description":"","filename":"Binder12.png","url":"https://assets-eu.researchsquare.com/files/rs-8733111/v1/571e8ab87d3545759c450b98.png"},{"id":104404872,"identity":"67092f74-9a4e-42a5-93bd-fcd58af9dd41","added_by":"auto","created_at":"2026-03-11 12:21:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":471078,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIFIT3 KO reduces cardiomyocyte apoptosis induced by oxygen glucose deprivation and reperfusion (OGD/R)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea-c.\u003c/strong\u003e Flow cytometry analysis revealed a significant reduction in cardiomyocyte apoptosis in the IFIT3 knockout group compared to the wild-type (WT) control group following OGD for 2 hours \u003cstrong\u003e[a]\u003c/strong\u003e, 4 hours\u003cstrong\u003e [b]\u003c/strong\u003e, and 8 hours \u003cstrong\u003e[c]\u003c/strong\u003e, as well as after a 3-hour reperfusion period. All results were used t-test and expressed mean ± SEM, compared with the wild-type, n=3. Flow cytometry analysis revealed a significant reduction in cardiomyocyte apoptosis in the IFIT3 knockout (KO) group compared to the wild-type (WT) control group following oxygen-glucose deprivation (OGD) for 2 hours (\u003cstrong\u003e[a]\u003c/strong\u003e), 4 hours \u003cstrong\u003e([b])\u003c/strong\u003e, and 8 hours \u003cstrong\u003e([c])\u003c/strong\u003e, as well as after a 3-hour reperfusion period. \u003cstrong\u003ed.\u003c/strong\u003eMitochondrial membrane potential was assessed using the JC-10 fluorescent mitochondrial marker after 2 hours of hypoxic glucose deprivation, as described in the Methods section. A decrease in mitochondrial membrane potential is indicated by green fluorescence, while high mitochondrial membrane potential is indicated by red fluorescence (left panel). The ratio of red to green fluorescence intensity was analyzed (right panel, n=3). \u003cstrong\u003ee.\u003c/strong\u003e Western blot analysis determined the expression levels of BCL2 \u003cstrong\u003e(f)\u003c/strong\u003e and Bax \u003cstrong\u003e(g)\u003c/strong\u003ein AC-16 cells following IFIT3 knockout. The quantification of BCL2 expression (WT: 1.000 ± 0.0187, KO. \u003cem\u003eIfit3\u003c/em\u003e: 2.168 ± 0.350) (\u003cstrong\u003ef\u003c/strong\u003e) and Bax (WT: 1.899 ± 0.174, KO. \u003cem\u003eIfit3\u003c/em\u003e: 1.000± 0.0177) (\u003cstrong\u003eg\u003c/strong\u003e). (\u003cstrong\u003eh\u003c/strong\u003e). The ratio of BCL2/Bax. All results were analyzed using a t-test and expressed mean±SEM, compared with the wild-type, n=4.\u003c/p\u003e","description":"","filename":"Binder13.png","url":"https://assets-eu.researchsquare.com/files/rs-8733111/v1/1dd4b2e059b58d85afe864cf.png"},{"id":104403705,"identity":"3072d31a-694b-4f9d-adc8-0bf234879dcb","added_by":"auto","created_at":"2026-03-11 12:18:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":468212,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIFIT3 overexpression exacerbates cardiomyocyte apoptosis through the extrinsic pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eWestern blot analysis confirming IFIT3 protein expression levels 48 hours after transfection with an IFIT3-overexpressing plasmid. Cells were seeded in 6-well plates at a density of 2 × 10⁵ cells per well and transfected with 2 µg of plasmid per well. B. Western blot analysis of key apoptosis-related proteins following IFIT3 overexpression and subsequent OGD/R injury. Protein levels of caspase-9, cleaved caspase-9, caspase-8, cleaved caspase-8, caspase-3, and cleaved caspase-3 were assessed after 48 hours of IFIT3 overexpression, followed by OGD/R challenges of 2h/3h and 4h/3h, respectively. \u003cstrong\u003ec.\u003c/strong\u003e Representative immunofluorescence images showing co-localization of IFIT3 (red) and cleaved caspase-3 (green) in cardiomyocytes under the indicated conditions. Nuclei were counterstained with DAPI (blue). The primary antibody IFIT3 (Thermo Fisher Scientific, PA5-22230), cleaved caspase3 (Protech, 68773-1-Ig) and the secondary antibody CoraLite594-conjugated Goat Anti-Rabbit IgG (H+L) (Protech, SA00013-4), FITC conjugated Goat Anti-mouse IgG (H+L) (A0428) were all diluted at a ratio of 1:500. The primary antibody was incubated overnight at 4 degrees Celsius, and the secondary antibody was incubated at room temperature for 2 hours, n=3.\u003c/p\u003e","description":"","filename":"Binder14.png","url":"https://assets-eu.researchsquare.com/files/rs-8733111/v1/781ebd5bdcf311c7dc091d05.png"},{"id":104177792,"identity":"30668d95-73c8-4232-9267-09c1a3fcae82","added_by":"auto","created_at":"2026-03-08 16:49:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":125912,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTLRs signaling pathways activated in AC-16 during OGD.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA quantitative whole transcriptome analysis was conducted to assess the gene expression of MyD88 \u003cstrong\u003e(a)\u003c/strong\u003e, TICAM1\u003cstrong\u003e(b) \u003c/strong\u003ein AC-16 cells subjected to OGD for 2 hours. WB was utilized to detect the expression of TICAM1 in AC-16 cells after OGD deprivation at various time points: 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours \u003cstrong\u003e(c)\u003c/strong\u003e. Quantitative analysis was performed to evaluate the expression of IFIT3 in AC-16 cells induced by TLR agonists\u003cstrong\u003e (d, e, f)\u003c/strong\u003e, as well as in HL-1 cells following stimulation with TLR3/4 agonists\u003cstrong\u003e (g, h)\u003c/strong\u003e. Statistical analyses were carried out using a t-test, and results were expressed mean ± SEM, * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, compared with the control group (Ctrl), n\u0026gt;3.\u003c/p\u003e","description":"","filename":"Binder15.png","url":"https://assets-eu.researchsquare.com/files/rs-8733111/v1/5b87128befee1412b5ddd458.png"},{"id":104404021,"identity":"4443c486-361e-47d0-8c26-6ec5b8743148","added_by":"auto","created_at":"2026-03-11 12:19:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":267101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS100 family proteins induced IFIT3 overexpression in cardiomyocytes by \u0026nbsp;TLR3/4 signaling pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Subcellular location of S100 in Cardiomyocytes. \u003cstrong\u003eb.\u003c/strong\u003e Western Blot analysis detected IFIT3 expression in AC-16 cells following a 24-hour induction with S100 family proteins. The induced concentrations of S100A1, S100A6, S100A8, S100A9 and S100B were each 2µg/ml. \u003cstrong\u003ec\u0026amp;d.\u003c/strong\u003e Western Blot analysis examined the time course of IFIT3 induction in AC-16 cells following stimulation with S100A1 (2 µg/ml, \u003cstrong\u003eC\u003c/strong\u003e) and S100A9 (2 µg/ml, \u003cstrong\u003ed\u003c/strong\u003e). All results were analyzed using a t-test and expressed mean ± SEM, with comparisons made against the control group (Ctrl), n\u0026gt;4. \u003cstrong\u003ee\u0026amp;g.\u003c/strong\u003e TLR3/4 signaling pathway activation in AC-16 cells following the induction of S100A1 and S100A9 (2.0 µg/mL, for 2 to 24 hours).\u003c/p\u003e","description":"","filename":"Binder16.png","url":"https://assets-eu.researchsquare.com/files/rs-8733111/v1/246e2e586c62ecafd4001bc5.png"},{"id":104177799,"identity":"3a986ee9-67ae-4a16-84ab-45ed4d7cb763","added_by":"auto","created_at":"2026-03-08 16:49:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":151322,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS100A1 and S100A9 stimulated IFIT3 overexpression and induced AC-16 cells apoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea-f.\u003c/strong\u003e Western blot analysis was conducted to quantitatively assess the expression levels of CASP3 and Cleaved-CASP3 in AC-16 cells treated with S100A1 (2.0 µg /mL, panels \u003cstrong\u003ea, b\u003c/strong\u003e) or S100A9 (2.0 µg/mL, panels \u003cstrong\u003ec, d\u003c/strong\u003e). \u003cstrong\u003ee-g. \u003c/strong\u003eThe effect of IFIT3 knockout on the expression of CASP3 and Cleaved-CASP3 induced by S100A1 (panels \u003cstrong\u003ee, f\u003c/strong\u003e) and S100A9 (panels \u003cstrong\u003eg-h\u003c/strong\u003e) was evaluated. Statistical analyses were performed using a t-test, and results were expressed as mean ± SEM (n=3 or 4). Significance was determined at * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003e p \u003c/em\u003e\u0026lt; 0.01, ****\u003cem\u003e p \u003c/em\u003e\u0026lt; 0.0001 compared with the control group (Ctrl).\u003c/p\u003e","description":"","filename":"Binder17.png","url":"https://assets-eu.researchsquare.com/files/rs-8733111/v1/c8a8b38ab3e5e0c2207145f0.png"},{"id":104177800,"identity":"f98a5dd2-ed43-4101-a5cd-dc9968c5ff5c","added_by":"auto","created_at":"2026-03-08 16:49:52","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":575607,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular profiling of the ISCM-induced signaling pathway and co-localization of IFIT3 with apoptosis in the infarcted rat heart.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eWestern blot analysis of key signaling proteins (TLR3, IRF3, p-IRF3) and apoptosis executor (Caspase-3, Cleaved caspase-3) in the left ventricular infarct area following 24 hours of sustained ischemia. Data was performed from n=6 biologically independent animals per group (Sham vs. ISCM 24h). \u003cstrong\u003eb. \u003c/strong\u003eThe ratio of p-IRF3/IRF3. \u003cstrong\u003ec. \u003c/strong\u003eThe ratio of Cleaved caspase3/ caspase3. \u003cstrong\u003ed\u003c/strong\u003e. Representative immunofluorescence staining of heart paraffin sections for cardiac Troponin T (cTn-T, green), Cleaved caspase-3 (yellow), IFIT3 (red) and nuclei were counterstained with DAPI (blue). Images are representative of findings from n=3 animals per group. \u003cstrong\u003ee\u003c/strong\u003e. Hypothesis the mechanism of S100A1/A9 as DAMPs regulating IFIT3 expression and inducing apoptosis in myocardial ischemic injury. In the context of myocardial ischemic injury, the S100 A1/9 protein released from damaged or necrotic cells may serve as DAMPs, inducing apoptosis in adjacent cardiomyocytes through the activation of TLR3, which exacerbates myocardial injury. DAMPs refer to Damage/Danger Associated Molecular Patterns, while PRRs denote Pattern recognition receptors.\u003c/p\u003e","description":"","filename":"Binder18.png","url":"https://assets-eu.researchsquare.com/files/rs-8733111/v1/3bca5c7454e537a2b76f1df6.png"},{"id":104783961,"identity":"6eeec8c0-3578-4229-b56c-3835fb8d1360","added_by":"auto","created_at":"2026-03-17 08:04:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5419013,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8733111/v1/1fb50b10-a002-4572-94cf-6dc3dca2156a.pdf"},{"id":104779491,"identity":"d09fa3bd-e01e-499b-b06d-db2210cec410","added_by":"auto","created_at":"2026-03-17 07:40:59","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3021176,"visible":true,"origin":"","legend":"Supplemental material - figures","description":"","filename":"SupplementalFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8733111/v1/6598c68bc0c72368b36cee52.pdf"},{"id":104177795,"identity":"3b5a62fc-e51d-4590-afc7-b8712cbb5ca5","added_by":"auto","created_at":"2026-03-08 16:49:52","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":40437,"visible":true,"origin":"","legend":"Supplemental material","description":"","filename":"Supplementalfigleg.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8733111/v1/090ea306fa46e2935f0e6e18.pdf"}],"financialInterests":"There is no duality of interest","formattedTitle":"A novel S100A-TLR3-IFIT3 signaling axis promotes cardiomyocyte apoptosis during myocardial infarction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInsufficient blood supply to the myocardium results in injury and death of heart muscle tissue, a condition known as ischemic cardiomyopathy (ISCM), which can arise from both acute and chronic myocardial infarction (MI)[1]. ISCM is the most prevalent cause of mortality [2, 3]. During the MI, debris from necrotic cardiac myocytes activates damage-associated molecular patterns (DAMPs) via pattern recognition receptors (PRRs) within the innate immune system, thereby initiating an inflammatory response [4].\u003c/p\u003e \u003cp\u003eThe S100 protein family comprises 21 isoforms characterized by small proteins that contain calcium-binding motifs, including S100A1 through S100A16, S100G, S100P, and S100B [5]. These proteins exhibit both positive and negative roles in cardiovascular diseases. Elevated serum levels of S100A have been identified as a highly sensitive biomarker for acute myocardial injury [6]. Among the isoforms, S100A1 is the most prevalent in cardiomyocytes and is passively released from ischemic cardiomyocytes during acute MI, functioning as a cardiac alarmin [7, 8]. S100A8/9 has been shown to predict the prognosis of patients with MI and is implicated in the regulation of both cardiovascular diseases [9], [10] and autoimmune myocarditis [11, 12]. Calprotectin (S100A8/A9) and S100A1, recognized as DAMPs, trigger inflammatory responses by binding to Toll-like receptor 4 (TLR-4) and initiate a signaling cascade in MI [8, 13, 14]. Additionally, S100A9 plays a crucial role in the maturation of TLR3-containing early endosomes into late endosomes, which subsequently fuse with lysosomes to form an endo-lysosomal compartment. Upon sensing its agonist/PAMP, TLR3 is activated by recruitment of the adaptor protein TRIF and the assembly of key signaling complexes, which subsequently activate IRF3 and NF-\u003cem\u003eκ\u003c/em\u003eB [15].\u003c/p\u003e \u003cp\u003eThe regulatory factors of interferons (IFNs) modulate the expression of specific genes in the heart that encode pro-inflammatory cytokines and other IFNs [4]. These genes include interferon-stimulated genes (ISGs) and the IFIT (IFN-induced proteins with Tetratricopeptide Repeat (TPR) motifs) family proteins [16]. Activation of IFIT family members by IFNs initiates various signal transduction pathways, which play a critical role in regulating both cell-intrinsic and cell-extrinsic immune responses. This modulation is essential for minimizing immune-mediated damage and cardiac remodeling [17]. The IFIT2/3 genes are highly expressed in patients with cardiovascular disease and MI, and their expression levels are indicative of cardiac function impairment in MI patients[18]. Additionally, IFIT2/3 were associated with the activation of mediators such as of IRF3 and NF-\u003cem\u003eκ\u003c/em\u003eΒ [19, 20]. Overexpression of IFIT2 causes caspase-3 activation and plasma membrane asymmetry disruption, leading to apoptotic cell death [20]. Furthermore, IFIT2 overexpression induces apoptosis by modulating the balance between pro-apoptotic and anti-apoptotic Bcl-2 family proteins, thereby altering mitochondrial membrane permeability [21]. Inhibition IFIT2 degradation results in perinuclear aggregation and promotes apoptosis [22]. IFIT2/3 is overexpressed in ISCM patients' cardiac tissues and rat MI models. Notably, the IFIT2/3 level is significantly reduced in the Metoprolol -treated group compared to the MI LAD group, which correlates with a decrease in apoptotic cells [23]. Moreover, overexpression of IFIT3 exacerbates cardiomyocyte apoptosis and ventricular damage in MI, consequently impairing cardiac function. However, the mechanism linking endogenous S100A family protein release to IFIT3 activation during MI remains unclear.\u003c/p\u003e \u003cp\u003eThis study aims to investigate the potential mechanisms connecting the release of endogenous S100A family proteins and the activation of IFIT3 overexpression via the TLRs-STING signaling pathway, which may induce cardiomyocyte apoptosis during the progression of MI. Builds on our previous work [23], a rat myocardial ischemia model and immunofluorescence staining were employed to confirm the upregulation of IFIT3 in cardiomyocytes under ischemic conditions. Multiple cell lines were utilized for this investigation. Additionally, the study employed an in vitro cellular model of oxygen-glucose deprivation/reperfusion (OGD/R) to simulate the MI process and to explore the mechanisms contributing to cardiac damage and dysfunction.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eThe overexpression of IFIT3 is functionally with cardiomyocyte myocardial injury.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur previous study revealed a significant upregulation of IFIT3 in the cardiac tissues of patients with myocardial ischemia \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u003cb\u003e)[23]\u003c/b\u003e. To investigate the functional role and regulatory mechanism of IFIT3 in myocardial ischemic injury, we employed a rat model of acute myocardial ischemia. Model induction was successfully confirmed 24 hours post-ischemia by assessing cardiac function through small-animal Doppler echocardiography \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Echocardiographic results demonstrated a significant decrease in left ventricular ejection fraction (LVEF) and fractional shortening (FS) at the 24-hour time point \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-d\u003cb\u003e)\u003c/b\u003e. Additionally, we observed impaired motion of the anterior wall \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-g\u003cb\u003e)\u003c/b\u003e and ventricular dilation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh\u003cb\u003e).\u003c/b\u003e In contrast, the motion of the posterior wall remained largely unaffected \u003cb\u003e(Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea, b)\u003c/b\u003e. Collectively, these findings confirm the successful establishment of the acute myocardial ischemia model in rats. Infarct size was further quantified by Masson's trichrome staining of cardiac cross-sections, which revealed that the infarcted area accounted for approximately 40% of the left ventricle \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei\u003cb\u003e)\u003c/b\u003e. Notably, IFIT3 expression was significantly upregulated in the infarcted region of rat hearts compared with the control group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej, S1c\u003cb\u003e)\u003c/b\u003e. Furthermore, immunofluorescence analysis identified markedly elevated IFIT3 levels in a subset of cardiomyocytes located specifically within the border zone of the myocardial infarction \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIFIT3 Deficiency Protects Cardiomyocytes from OGD-Induced Apoptosis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo assess the role of IFIT3 in cardiomyocyte responses to ischemic damage, an AC-16 cell exposed to oxygen-glucose deprivation (OGD) model was utilized to investigate the role of IFIT3, which simulate ischemic conditions. Following 24 hours of OGD exposure, AC-16 cells displayed notable morphological changes, transitioning from an elongated spindle shape to a circular morphology \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Western blot analysis indicated a significant increase in IFIT3 levels after 2 hours of OGD \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, S2a\u003cb\u003e)\u003c/b\u003e, coinciding with the expression of Cleaved-Caspase3/Caspase3 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, S2b-d\u003cb\u003e)\u003c/b\u003e. The CRISPR/Cas9 system was employed to knockout IFIT3 in AC-16 cells to assess its functional impact on cardiomyocyte apoptosis induced by OGD. The complete elimination of IFIT3 in the IFIT3-deficient cells (AC-16) was verified using Western blot analysis, and the morphological alterations of the cells were minimal \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e. A whole transcriptomic profiling was conducted to investigate the primary affected functions and pathways between IFIT3 KO and wild-type AC-16 cells. After normalization and filtering, a total of 960 significantly altered genes were identified, comprising 258 up-regulated and 702 down-regulated genes (IFIT3 KO vs control, fold change (FC)\u0026thinsp;\u0026gt;\u0026thinsp;2.0 or FC\u0026thinsp;\u0026lt;\u0026thinsp;0.50, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The top 20 Gene Ontology (GO) enrichment analyses were summarized, encompassing molecular functions, cellular components, and biological processes. Notably, enriched processes included immune system processes and responses to stimulus, etc (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). In particular, the hallmark of apoptosis was emphasized in the Gene Set Enrichment Analysis (GSEA) comparing IFIT3 KO cells to control cells, prompting further investigation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). The accompanying heatmap illustrated 13 genes significantly down-regulated in IFIT3 KO cells involved in the hallmark of apoptosis, including BNIP3, BCL2L11, etc (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Under resting conditions, IFIT3 knockout did not enhance cardiomyocyte apoptosis. A trend toward an attenuated apoptotic rate was observed in IFIT3-knockout AC16 cells relative to wild-type controls after 2 hours of OGD \u003cb\u003e(Fig.\u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ee)\u003c/b\u003e. In contrast, after 4 and 24 hours of OGD, the apoptotic ratio of IFIT3-knockout AC-16 cells was significantly reduced \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh, i\u003cb\u003e)\u003c/b\u003e. Additionally, 999 genes were identified as significantly altered when comparing IFIT3 KO and wild-type AC-16 cells after 2 hours of OGD (345 up-regulated and 654 down-regulated genes in IFIT3 KO, FC\u0026thinsp;\u0026gt;\u0026thinsp;2.0 or FC\u0026thinsp;\u0026lt;\u0026thinsp;0.50,) (\u003cb\u003eFig.\u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ef\u003c/b\u003e). The hallmark of apoptosis was notably highlighted as being inhibited in IFIT3 knockout cells (\u003cb\u003eFig.\u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eg\u003c/b\u003e). These findings demonstrated that the IFIT3 KO significantly mitigates AC-16 cardiomyocyte apoptosis induced by persistent OGD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIFIT3 knockout confers cardio-protection against H/R injury via preserving mitochondrial membrane potential and suppressing apoptosis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter two hours of OGD followed by three hours of reoxygenation, the different stages of apoptotic ratio (early, late, and total) of wild-type AC-16 cells significantly increased to approximately 20%. In contrast, IFIT3 knockout markedly reduced the apoptotic ratio \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. When the duration of OGD was extended to four and eight hours, followed by three hours of reoxygenation, the apoptotic ratio in IFIT3 knockout AC-16 cells consistently decreased and remained significantly lower than that observed in wild-type cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, c\u003cb\u003e)\u003c/b\u003e. These results suggest that IFIT3 deficiency substantially mitigates cardiomyocyte apoptosis induced by ischemia-reperfusion in cardiac myocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the potential regulatory interactions between IFIT3 and mitochondrial signaling, CCCP was employed to induce apoptosis in AC-16 cardiomyocytes by disrupting membrane potential. Notably, IFIT3 knockout significantly attenuated CCCP-induced cardiomyocyte apoptosis following treatment with concentrations of 2.5 \u0026micro;M and 5 \u0026micro;M over a 24-hour period. The apoptotic ratios at different stages\u0026mdash;early, late, and total\u0026mdash; were significantly lower in IFIT3 knockout AC-16 cells compared to wild-type AC-16 cells \u003cb\u003e(Fig.S3a, b)\u003c/b\u003e. It is suggested that IFIT3 plays a detrimental role in the regulation of cardiomyocyte apoptosis through modulation of the mitochondrial membrane potential.\u003c/p\u003e \u003cp\u003eFollowing the induction of OGD, the JC-10 assay was utilized to evaluate mitochondrial membrane potential. The expression of IFIT3 was significantly maintained in AC-16 cells after a 2-hour OGD exposure. Under baseline conditions, the mitochondrial membrane potential in IFIT3-deficient AC-16 cells exhibited minimal variation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, \u003cb\u003eJC-10, red color\u003c/b\u003e). However, after 2-hour OGD, the IFIT3 knockout group demonstrated a higher mitochondrial membrane potential, indicated by a weaker green signal, compared to the wild-type group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, \u003cb\u003eJC-10, green color)\u003c/b\u003e. These findings suggested that the absence of IFIT3 in cardiomyocytes may facilitate the preservation of mitochondrial membrane potential during ischemic conditions, potentially conferring identifiable anti-apoptotic properties.\u003c/p\u003e \u003cp\u003eFurthermore, the study examined the levels of two critical apoptosis markers, BCL2 and BAX, in the context of IFIT3 knockout. IFIT3 knockout markedly altered the expression of key apoptotic regulators, characterized by increased BCL2 and decreased BAX levels \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-g\u003cb\u003e)\u003c/b\u003e, thereby significantly elevating the BCL2/BAX ratio \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh\u003cb\u003e)\u003c/b\u003e. These findings imply that IFIT3 knockout enhance mitochondrial membrane potential and elevates cellular anti-apoptotic capacity by increasing BCL2 expression and decreasing BAX expression.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIFIT3 Overexpression Activates the Extrinsic Apoptotic Pathway\u003c/h2\u003e \u003cp\u003eTo further investigate the mechanism by which IFIT3 regulates apoptosis, we transiently overexpressed IFIT3 in AC16 cardiomyocytes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e via plasmid transfection. Following 48 hours of IFIT3 overexpression, cells were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) for 2 or 4 hours, followed by 3 hours of reoxygenation. The results showed that IFIT3 overexpression significantly increased the ratio of cleaved caspase-3 to caspase-3, indicating the initiation of the apoptotic program. Concurrently, we detected significant activation of cleaved caspase-8, whereas the expression levels of caspase-8, caspase-9, and cleaved caspase-9 showed no significant changes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb S\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c\u003cb\u003e)\u003c/b\u003e. These findings suggest that IFIT3 overexpression likely activates the extrinsic apoptotic pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further validate the pro-apoptotic effect of IFIT3, we performed an immunofluorescence assay. After 72 hours of IFIT3 overexpression combined with OGD/R (4h/3h) induction, a significant increase in cardiomyocyte apoptosis was observed \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e, confirming that IFIT3 overexpression potently induces cell death.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAlternative signaling activated IFIT3 overexpression in cardiomyocytes during myocardial ischemic injury\u003c/h3\u003e\n\u003cp\u003eThe IFIT3 gene was initially identified as being induced by interferons, so three types of human type I interferons (IFN α-1b, IFN α-2a, and IFN α-2b) were employed to stimulate IFIT3 expression in myocardial cells (AC-16 cells) over a duration of 24 hours. This experiment yielded a significant increase in IFIT3 expression (\u003cb\u003eFig. S5a\u003c/b\u003e). Additionally, varying doses of IFN α-1b and IFN α-2a were tested on mouse cardiomyocytes (HL-1 cells), resulting in a notable stimulation of IFIT3 overexpression (\u003cb\u003eFig.S5b\u003c/b\u003e), whereas IFN α-2b exhibited minimal effect on IFIT3 expression(\u003cb\u003eFig.S5c\u003c/b\u003e ). This discrepancy might be attributed to the differences in the protein sequences of IFN α-2b between human and mouse species. Furthermore, treatment with type I interferons (IFN α-1b, IFN α-2a, and IFN α-2b) significantly stimulated IFIT3 overexpression in HEK293 cells (\u003cb\u003eFig.S5d\u003c/b\u003e). These results affirm that interferons can induce the IFIT3 production in both cardiomyocytes and non-cardiomyocyte cell types. Notably, the IFIT3 expression was significantly elevated in AC-16 cells after 2 hours of OGD, while transcriptome analysis revealed no detectable expression of IFN α1, IFN α2, or IFN γ mRNA (\u003cb\u003eFig.S5e\u003c/b\u003e). This observation indicates that IFIT3 overexpression during OGD may occur via an alternative signaling pathway that operates independently of interferon regulation.\u003c/p\u003e\n\u003ch3\u003eActivated TLRs and IRF3 signaling to induce IFIT3 overexpression during myocardial ischemic injury\u003c/h3\u003e\n\u003cp\u003eTLRs (Toll-like receptors), which serve as pattern recognition receptors, play a crucial role in mediating inflammatory responses to DAMP during myocardial ischemia and hypoxia. To investigate the TLRs signaling pathways that may be activated by IFIT3 overexpression, the mRNA expression levels of several key signaling regulators were assessed in AC-16 cells following 2 hours of OGD. Notably, the expression level of Myd88, a crucial signaling adaptor of TLR4 activation, remained unchanged after 2 hours of OGD compared to baseline conditions \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. In contrast, the mRNA level of Ticam1, which serves as the key signaling adaptor of TLR3 activation, showed a significantly upregulated, indicating the activation of TLR3 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Furthermore, the protein expression of TICAM1 was significantly elevated after OGD ranging from 2 to 12 hours \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e, while MyD88 expression was not detected in the Western blot analysis. These findings suggests that TLR3 is activated during the OGD period (0-24h), in contrast to TLR4.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe study utilized isoform-specific TLR agonists and STING agonists to validate the inducibility of IFIT3 expression in cardiomyocytes, aiming to replicate the signaling mechanisms associated with myocardial injury. A total of 13 agonists were employed for this investigation. The TLR agonists, namely Pam3CSK4 (TLR1/2), HKLM (TLR2), FLA-ST (TLR5), FSL-1 (TLR6/2), Imiquimod (TLR7), ssRNA 40 (TLR8), ODN 2006 (TLR9), and ODN 2216 (TLR9) induced IFIT3 overexpression in AC-16 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, e\u003cb\u003e)\u003c/b\u003e. In contrast, this induction effect was not observed in HL-1 mouse cells under identical treatment conditions. Notably, TLR3 agonists (Poly I:C both HMW and LMW) significantly enhanced IFIT3 production in both AC-16 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e and HL-1 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e. Additionally, the TLR4 agonist (LPS-EK) also induced IFIT3 production in AC-16 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e and HL-1 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh\u003cb\u003e)\u003c/b\u003e, albeit with a weaker effect compared to TLR3 agonists. Furthermore, the STING agonist 2'3-cGAMP elicited a stronger IFIT3 expression than ISD in AC-16 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e, while the same concentration of STING agonist (Poly I:C HMW and LPS) resulted in minimal IFIT3 overexpression in HL-1 cells. These results suggest that certain damage-associated endogenous molecules activate TLR3 or IRF3 to induce IFIT3 overexpression during myocardial ischemia and hypoxia via an IFN regulation-independent pathway.\u003c/p\u003e\n\u003ch3\u003eS100 family proteins stimulated IFIT3 overexpression in cardiomyocytes\u003c/h3\u003e\n\u003cp\u003eThe S100 proteins possess a shared calcium-binding domain characterized by an EF-hand structure and are distributed in multiple cellular compartments \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Although S100 family proteins play opposing regulatory roles in the progression of various cardiovascular diseases, limited research has investigated the interaction between S100 family proteins and IFIT3. Treatment with S100 isoforms (A1, A6, A8, A9, and B) at a concentration of 2.0\u0026micro;g/mL for 24 hours resulted in an increase in IFIT3 protein expression in AC-16 cells. Among the S100A isoforms, S100A1 and S100A9 exhibited the most significant inducible effects, whereas S100B induced changes in IFIT3 expression that were not statistically significant \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Additionally, treatment with all five S100 isoforms (2.0\u0026micro;g/mL, 24 hours) led to a significant increase in IFIT3 expression in mouse cardiac fibroblasts (MCFs) (\u003cb\u003eFig.S6a)\u003c/b\u003e. Furthermore, treatment with S100A isoforms (A1, A6, A8, and A9) (2.0\u0026micro;g/mL, 24 hours) significantly enhanced IFIT3 expression in HEK293 cells; however, the changes in IFIT3 expression induced by S100B were not statistically significant (\u003cb\u003eFig.S6b\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfterward, AC-16 cells were stimulated with S100A1 and S100A9 at various time points (2.0 \u0026micro;g/mL for 2, 4, 8, 12, and 24 hours). The expression of the IFIT3 protein was significantly increased starting at 2 hours, peaking after 24 hours of stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, d). These results suggest a previously unrecognized association between stimulation by S100 family proteins and the overexpression of IFIT3. S100A1 and S100A9 are the predominant S100 isoforms released from damaged cardiomyocytes during MI [7, 24]. It can be inferred that S100A1 and S100A9 serve as endogenous damage-associated pattern molecules that activate IFIT3 expression during MI.\u003c/p\u003e\n\u003ch3\u003eS100A1 and S100A9 activated IFIT3 overexpression via TLR3/TICAM1/IRF3 signaling pathway\u003c/h3\u003e\n\u003cp\u003eOur findings indicate that the expression of IFIT3 is independent of interferon (IFN) induction but is instead influenced by the activation of TICAM1, which implicates the TLR3/TICAM1/IRF3 signaling pathway. Treatment with S100A1 and S100A9 resulted in the upregulation of TLR3 and TICAM1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, g), thereby indicating activation of TLR3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee-f, S6 c, d, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg-h, S6 i, j, \u003cb\u003erespectively\u003c/b\u003e). Furthermore, IRF3 and p-IRF3 expression increased significantly, suggesting that S100A1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, f, S6 c, d) and S100A9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, h, S6 m, n) induce IFIT3 expression through the TLR3/TICAM1/IRF3 pathway. Furthermore, there was a significant increase in the expression levels of IRF3 and phosphorylated IRF3 (p-IRF3), suggesting that S100A1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, f\u003cb\u003e)\u003c/b\u003e and S100A9 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, h\u003cb\u003e)\u003c/b\u003e induce IFIT3 expression via the TLR3/TICAM1/IRF3 pathway.\u003c/p\u003e \u003cp\u003eIn addition, the signaling key regulators TLR4/MyD88 were detected following treatment of S100A1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, \u003cb\u003eFig.S6 e)\u003c/b\u003e and S100A9 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, \u003cb\u003eFig.S6 k)\u003c/b\u003e. Although the expression of TLR4 did not show significant changes, there was a time-dependent increase in MyD88with. It is hypothesized that TLR3 and TLR4 may interact during stimulation with S100A1 and S100A9 in AC-16 cells, suggesting a potential regulatory role for TLR4. However, this hypothesis remains unconfirmed within this study, necessitating further research to explore this possibility.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIFIT3 Knockout abolished cardiomyocyte apoptosis initialized by S100A1 and S100A9\u003c/h2\u003e \u003cp\u003eThis study aims to investigate whether S100A1 and S100A9 can induce apoptosis in cardiomyocyte. Caspase 3 and Cleaved-Caspase 3 levels were measured in AC-16 cells following 24 hours of stimulation with either S100A1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, S7a, b\u003cb\u003e)\u003c/b\u003e or S100A9 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, S7c, d\u003cb\u003e)\u003c/b\u003e. The results demonstrated that both S100A1 and S100A9 are capable of inducing apoptosis in cardiomyocyte. The Cleaved-Caspase 3 level increased significantly after 4 hours of S100A1 stimulation and continued to increase until 12 hours \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b\u003cb\u003e)\u003c/b\u003e. In experiments involving S100A9, the Cleaved-Caspase 3 level showed a significant increased after 8 hours of stimulation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, d\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the role of IFIT3 in regulating the apoptosis induced by S100A1 and S100A9, the IFIT3 gene was knocked out in AC-16 cells. Subsequently, the expression levels of Caspase 3 and Cleaved-Caspase 3 were measured in response to treatment with S100A1 and S100A9. In wild-type cardiomyocytes, the levels of IFIT3\u003cb\u003e(Fig.S7e)\u003c/b\u003e, CASP3\u003cb\u003e(Fig.S7f)\u003c/b\u003e, and Cleaved-CASP3 \u003cb\u003e(Fig.S7g)\u003c/b\u003e increased with prolonged exposure to S100A1. In contrast, S100A1 did not elicit any changes in IFIT3 expression in AC-16 cells with IFIT3 knockout \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee, f\u003cb\u003e)\u003c/b\u003e. Notably, the CASP3 levels in IFIT3 knockout cardiomyocytes were significantly higher than in wild-type cells under rest conditions. However, with extended exposure, the CASP3 level did not increase in the IFIT3 knockout cells in response to S100A1 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee, f\u003cb\u003e)\u003c/b\u003e. Furthermore, the level of activated Cleaved-CASP3 were significantly lower in IFIT3 knockout cells compared to wild-type cells after 24h and 48 hours of S100A1 induction \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. This result suggests that IFIT3 knockout attenuates the apoptosis induced by S100A1.\u003c/p\u003e \u003cp\u003eIn the experiment involving S100A9-induced AC-16 cells, the expression of IFIT3 exhibited a progressive increase with prolonged S100A9 induction time \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e. Meanwhile, IFIT3 knockout cardiomyocytes showed higher levels of CASP3 expression compared to wild-type cardiomyocytes \u003cb\u003e(Fig.S7j)\u003c/b\u003e. Additionally, the of CASP3 expression was increased with extended S100A9 induction time \u003cb\u003e(Fig.\u0026nbsp;7Si)\u003c/b\u003e. However, the level of Cleaved-CASP3 protein was lower in IFIT3 knockout cardiomyocytes than in wild-type cardiomyocytes, although this difference without statistical significance \u003cb\u003e((\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg, S7j\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIn vivo validation of TLR3/IRF3 pathway activation and apoptosis in the myocardial infarct zone\u003c/h3\u003e\n\u003cp\u003eTo validate the TLR3/IRF3-IFIT3 signaling axis and apoptotic activation in vivo, cardiac tissues from the infarct area of acute MI rat models were analyzed by Western blot and immunofluorescence staining. Western blot analysis revealed significant upregulation of TLR3, phosphorylated IRF3 (p-IRF3), pro-caspase-3, and cleaved caspase-3 in the MI infarct zone (n\u0026thinsp;=\u0026thinsp;6, 24h post-MI), accompanied by decreased total IRF3 levels \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea-c\u003cb\u003e)\u003c/b\u003e. These findings indicate activation of the TLR3/IRF3 signaling pathway and execution of apoptosis in ischemic myocardium.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the spatial relationship between IFIT3 expression and cardiomyocyte apoptosis, infarct area sections were co-stained with antibodies against cardiac troponin T (cTnT, green), cleaved caspase-3 (yellow), and IFIT3 (red), with DAPI counterstaining for nuclei (blue). Immunofluorescence analysis revealed marked elevation of cleaved caspase-3 that co-localized with both IFIT3 and cTnT specifically in cardiomyocytes within the border zone of the myocardial infarction \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. These results demonstrate that MI-induced IFIT3 overexpression coincides with cardiomyocyte apoptosis in the infarct border zone, supporting activation of the TLR3/IRF3/p-IRF3 signaling pathway during myocardial ischemic injury.\u003c/p\u003e \u003cp\u003eIn summary, we identified multiple S100 protein family isoforms (S100A1/6/8/9/B) as endogenous damage-associated molecular patterns (DAMPs) that drive IFIT3-mediated cardiomyocyte apoptosis during myocardial infarction. Specifically, S100A1 and S100A9 activate the TLR3/TICAM1/IRF3 signaling pathway, leading to IFIT3 overexpression. During myocardial ischemic injury, S100 proteins released from necrotic cardiomyocytes are recognized by TLR3/4 receptors on neighboring viable cardiomyocytes, triggering IFIT3-dependent apoptosis that amplifies tissue damage in the infarct border zone \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. This paracrine injury mechanism establishes the S100A-TLR3-IFIT3 axis as a critical damage amplification loop in ischemic cardiomyopathy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDataset collection and preprocessing\u003c/h2\u003e \u003cp\u003eAs outlined in a prior study[23], the analysis of human transcriptomic data was based on previously published datasets( GSE57338) that complied with ethical standards, including the principles of the Declaration of Helsinki. The transcriptional profiles of 313 patient left ventricle biopsies from patients were analyzed utilizing the Affymetrix Human Gene 1.1 ST Array (platform GPL11532) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE57338\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE57338\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed on 1 January 2015). The dataset comprised 33,297 probes designed to measure alterations in transcriptional profiles associated with the physiological characteristics of hearts affected by heart failure. In this study, our focus to clarify the gene expression characteristics related to ISCM. The biopsy samples included normal hearts as control (Health, n\u0026thinsp;=\u0026thinsp;136), and ischemic cardiomyopathy (ISCM, n\u0026thinsp;=\u0026thinsp;95, \u003cb\u003esupplemental Table\u0026nbsp;1\u003c/b\u003e). Each gene expression reading was normalized and transformed using the log2 scale. The expression of the IFIT3 gene in each group was presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRat Model of Myocardial Infarction and Echocardiography\u003c/h2\u003e \u003cp\u003eAdult male Sprague-Dawley rats (200\u0026ndash;220 g) were obtained from Hunan SJA Laboratory Animal Co., Ltd (People's Republic of China license No. SCXK (Xiang) 2019-0004). All protocols were approved by the Institutional Animal Care and Use Committee of the Kunming Institute of Botany, Chinese Academy of Sciences, and conducted in accordance with NIH guidelines. After one week of acclimation, rats were randomly assigned to sham-operated (n\u0026thinsp;=\u0026thinsp;12) or myocardial infarction (MI, n\u0026thinsp;=\u0026thinsp;15) groups.\u003c/p\u003e \u003cp\u003eThe MI model was established under isoflurane anesthesia (RWD, R510-22-10, 3% for induction, 1.5% for maintenance). A left thoracotomy was performed to expose the heart, and the left anterior descending coronary artery was permanently ligated. After 24 hours of ischemia, cardiac function was reassessed under anesthesia using high-frequency (20 MHz) transthoracic echocardiography (Vevo 3100, Fujifilm VisualSonics or Mindray M9, Shenzhen Mindray Bio-Medical Electronics). Left ventricular dimensions and systolic function parameters, including ejection fraction and fractional shortening, were measured to validate the model[25, 26]. Following functional assessment, rats were euthanized, and heart tissues were collected for subsequent analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTissue Embedding and Sectioning\u003c/h2\u003e \u003cp\u003eHearts harvested from Sprague-Dawley rats were fixed in 4% paraformaldehyde for 24 hours, followed by standard paraffin embedding. Serial sectioning was performed starting from the cardiac apex. Trimming continued until the left ventricular chamber was fully exposed. Subsequently, consecutive 3-\u0026micro;m-thick sections were collected with the following scheme: five consecutive sections were retained. Approximately 50 \u0026micro;m of tissue was discarded (corresponding to ~\u0026thinsp;16 sections). This cycle was repeated until a total of 35 sections per heart were obtained. Six distinct anatomical levels spanning from apex to base were systematically preserved for analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMasson's Trichrome Staining\u003c/h2\u003e \u003cp\u003eOne section from each of the six anatomical levels (total: 6 sections/heart) was selected for Masson's trichrome staining Masson reagent (Servicebio, Cat#G1006-100ML) following the established protocol. This approach ensured representative sampling of the entire ventricular structure. Stained sections were scanned using a digital slide scanner (Pannoramic Scan) for subsequent quantitative analysis of collagen deposition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence Staining\u003c/h2\u003e \u003cp\u003eImmunofluorescence staining was performed on AC-16 cell slides or paraffin-embedded ventricular tissue sections. For each target, three consecutive sections from the third anatomical level (mid-ventricular region, sections 15\u0026ndash;20) were processed. After antigen retrieval and blocking, sections were incubated with primary antibodies against (1:500 dilution) overnight at 4\u0026deg;C, followed by appropriate secondary antibodies (1:500 dilution). Nuclei were counterstained with DAPI. All images were acquired using a confocal microscope under consistent exposure settings and quantified using ImageJ software with appropriate thresholding algorithms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCells culture\u003c/h2\u003e \u003cp\u003eThe mouse cardiomyocyte cell HL-1 [27] was obtained from Sigma (USA) and cultured in DMEM medium, which is a valuable model system to address questions of cardiac biology at the cellular \u0026amp; molecular levels. Human AC-16 cells were purchased from ATCC (CRL-3568) and cultured in DMEM/F-12 medium, which is derived from primary adult ventricular tissue and can be induced to differentiate into mature cardiomyocytes. Mouse cardiac fibroblasts (MCFs; catalog number 340098) were sourced from the Beijing Beina Chuanglian Biotechnology Research Institute and maintained in RPMI 1640 medium. HEK293 cells (CRL1573) were pursued from ATCC and maintained in DMEM medium. All medium were supplemented with 10% fetal bovine serum plus 100 U/ml penicillin/streptomycin (P1400, Solarbio, Beijing, China), and 4mM L-glutamine in 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Cells were cultured in a 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of IFIT3-Knockout Cells Using CRISPR-Cas9\u003c/h2\u003e \u003cp\u003eAn IFIT3-knockout cell model was established using the Lenti-CRISPR v2 system[28]. A specific sgRNA (5\u0026prime;-GACACCTAGATGGTAACAACG-3\u0026prime;) was cloned into the vector to target the IFIT3 coding region. The constructed plasmid was amplified in DH5α competent cells and verified by sequencing. For transfection, cells were seeded in 6-well plates until 50\u0026ndash;60% confluent. Complexes of plasmid DNA (2\u0026ndash;5 \u0026micro;g) and Lipofectamine 2000 (4\u0026ndash;10 \u0026micro;L, Lipofectamine\u0026trade; 2000, 11668500) were prepared in Opti-MEM ((Opti-MEM\u0026trade;, Gibco, 31985070) and added to the cells. After 4 hours, the medium was supplemented with an additional 1 mL of complete medium. At 24 hours post-transfection, selection was initiated with puromycin (0.8\u0026ndash;2 \u0026micro;g/mL). Polyclonal knockout cells were obtained after 7\u0026ndash;10 days of selection, and IFIT3 depletion was confirmed by Western blot. The resulting cells were used for subsequent experiments at early passages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eOverexpression of IFIT3 in AC-16 Cells\u003c/h2\u003e \u003cp\u003eAC-16 cells were seeded in 6-well plates at a density of 2\u0026ndash;4\u0026times;10⁵ cells per well and transfected with 1\u0026ndash;2 \u0026micro;g of pcDNA3.1-IFIT3-FLAG plasmid (encoding NM_001549) using Lipofectamine 3000 reagent (Lipofectamine\u0026trade; 3000 transfection Kit, L3000015). Cells transfected with empty vector served as the control. After 48 hours, total protein was extracted and subjected to Western blot analysis using an anti-FLAG antibody to confirm overexpression efficiency. Successfully transfected cells were used for subsequent functional experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eModeling Cardiomyocyte Injury via Oxygen-Glucose Deprivation/Reperfusion (OGD/R)\u003c/h2\u003e \u003cp\u003eAn OGD/R model [29, 30] was established to simulate ischemia/reperfusion injury and investigate the role of IFIT3 in cardiomyocytes. Cells were cultured in six-well plates until reaching 80\u0026ndash;90% confluence. For the OGD/R group, the culture medium was replaced with a balanced salt solution (BSS), and cells were transferred to a sealed hypoxia chamber. The chamber was flushed with 95% N₂/5% CO₂ for 15 minutes until the oxygen indicator turned blue-purple, then sealed and incubated at 37\u0026deg;C for the designated OGD period. Control cells were maintained in DMEM/F-12 medium under normoxic conditions. After OGD, cells were returned to a normal oxygenated incubator (95% air/5% CO₂) with complete medium for 3 hours of reperfusion. Protein lysates were collected at the endpoint for subsequent analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eRNA Sequencing and Bioinformatic Analysis\u003c/h2\u003e \u003cp\u003eAC-16 cells were cultured in 6-cm dishes until reaching 70\u0026ndash;80% confluence. The experimental group was subjected to 2 hours of oxygen-glucose deprivation (OGD), while the control group was maintained under standard culture conditions. Total RNA was extracted using TRIzol reagent and sent to Shanghai Meijie Biomedical Technology Co., Ltd. for transcriptome sequencing.\u003c/p\u003e \u003cp\u003eDifferentially expressed genes (DEGs) were identified using a threshold of |log₂FC| \u0026gt; 2 and adjusted p-value (padjust)\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Gene ontology (GO) and pathway enrichment analyses of DEGs associated with IFIT3 knockout were performed using Metascape[31]. Gene Set Enrichment Analysis (GSEA) was conducted to rank genes based on their correlation with IFIT3 knockout, with gene set permutations performed 1000 times for each analysis. Enriched pathways were classified based on the nominal p-value and normalized enrichment score (NES).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eType-I interferons, TLRs agonists, IRF3 agonists and S100 proteins stimulation\u003c/h2\u003e \u003cp\u003eToll-like receptor agonists (tlrl-kit1hw, Pam3sk4, HKLM, FSL-1, Poly(I:C) HMW, Poly(I:C) LMW, LPS-EK, FLA-ST, imiquimod, ssRNA40/LyoVec\u0026trade;, ODN2006), ODN2216 [tlrl-2216] and STING agonists (ISD Control/LyoVec [tlrl-isdcc], 2\u0026rsquo;3-cGAMP [tlrl-nacga23-02]) were bought from \u003cem\u003eInvivogen\u003c/em\u003e. The recombinant human S100 family proteins were sourced from Solarbio (S100A1, P02881; S100A6, P00170; S100A8, P00431), S100A9, P02686; S100 Calcium Binding Protein B/S100B, P00663). The cells were restimulated with TLR ligands (TLR1/2/3/4/5/6/7/8/9) including Pam3sk4, HKLM, FSL-1, Poly(I:C) HMW, Poly(I:C) LMW, LPS-EK, FLA-ST, imiquimod, ssRNA40/LyoVec\u0026trade;, ODN2006) or STING agonists (ISD Control/LyoVec, 2\u0026rsquo;3-cGAMP) at the mentioned concentrations or cultured without stimulation(PBS solution, pH 7.2\u0026ndash;7.4) as a negative control. Agonists were added to the media at the determined concentrations and cells were incubated at 37\u0026deg;C for the specified duration. For assessing IFIT3 responses, AC-16, HL-1, and HEK293 cells (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) in 1ml complete media were added to each of the duplicate ligand- or medium containing wells and incubated at 37 ◦C for 24 h with 5% CO\u003csub\u003e2\u003c/sub\u003e. Three types of type-Ⅰ interferons and three stimulant dosages were selected to evaluate the dose-dependent effect. Cells were stimulated for TLRs agonists and STING agonists. The concentrations of stimuli were aligned with the manufacturer\u0026rsquo;s guidelines. After 24 hours, cells were harvested, and total protein was extracted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial Membrane Potential Assay\u003c/h2\u003e \u003cp\u003eMitochondrial membrane potential (ΔΨm) was assessed using the JC-10 assay kit (Solarbio, CA1310) according to the manufacturer's instructions. Wild-type and IFIT3-deficient AC-16 cells were seeded in 6-well clear-bottom plates and subjected to oxygen-glucose deprivation/reperfusion (OGD/R). After treatment, cells were washed with Hanks\u0026rsquo; solution containing 0.02% Pluronic F-127 and incubated with 500 \u0026micro;L of JC-10 working solution (6.67 \u0026micro;g/mL) for 20 minutes at 37\u0026deg;C. Following dye loading, cells were washed thoroughly, mounted with antifade medium containing DAPI, and imaged under a fluorescence microscope. JC-10 aggregates (red fluorescence) and monomers (green fluorescence) were detected at excitation/emissionex wavelengths of 540/590 nm and 490/530 nm, respectively[32].\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eCCCP-Induced Mitochondrial Depolarization and Apoptosis\u003c/h2\u003e \u003cp\u003eCarbonyl cyanide-3-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, disrupts mitochondrial membrane potential by facilitating proton leakage across the inner membrane, leading to loss of transmembrane potential and induction of apoptosis[33]. To examine the role of IFIT3 in mitochondrial-dependent apoptosis, AC-16 cells at 70\u0026ndash;80% confluence were treated with CCCP (0, 2.5, or 5 \u0026micro;M, Solarbio, Beijing, C6700) for 24 hours. Cells were then harvested and analyzed for apoptosis using Annexin V-FITC/PI staining and flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of Apoptosis by Flow Cytometry\u003c/h2\u003e \u003cp\u003eCell apoptosis was quantified using an Annexin V-FITC/PI apoptosis detection kit (Solarbio, CA1020). Briefly, harvested cells were washed with cold PBS and resuspended in 1\u0026times; binding buffer. The cell suspension was incubated with 5 \u0026micro;L Annexin V-FITC for 5 minutes at room temperature in the dark, followed by addition of 5 \u0026micro;L propidium iodide (PI). Samples were analyzed within 1 hour on a BD FACSCalibur flow cytometry system. Fluorescence signals were collected in the FL1 channel for Annexin V-FITC and FL2 channel for PI. Data were processed using FlowJo software, and apoptotic populations were distinguished as follows: early apoptotic (Annexin V-FITC+/PI\u0026minus;) and late apoptotic (Annexin V-FITC+/PI+).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eCells were gently washed three times with phosphate-buffered saline (PBS) and subsequently lysed in RIPA lysis buffer (Beyotime Biotechnology, China) after treated with drugs or control conditions. Protein concentration of cell extracts was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, A23225) the, which facilitated the adjustment of protein concentration for subsequent experiments. Proteins were isolated using SDS-PAGE on a 10%-15% gel at a loading concentration of 20 \u0026micro;g per well, followed by wet-transfer to polyvinylidene difluoride (PVDF, 0.22\u0026micro;m) membranes (Millipore, USA). The membranes were blocked at room temperature for one hour with 5% skim milk dissolved in TBST solution (FD bioscience, China), and incubated overnight at 4\u0026deg;C with a specific primary antibody. The membranes were washed three times in TBS-T solution for 5 minutes each time and subsequently incubated with either HRP-labeled Goat Anti-Rabbit IgG(H\u0026thinsp;+\u0026thinsp;L) (1:5000, Beyotime Biotechnology, China, A0208) or HRP-labeled Goat Anti-Mouse IgG(H\u0026thinsp;+\u0026thinsp;L) (1:5000, Beyotime Biotechnology, China, A0216) secondary antibodies at room temperature for one hour. Immunoreactive proteins were visualized according to the manufacturer's instructions before exposure of PVDF membranes using Tanon 5200 (Tanon, China) using an enhanced chemiluminescence kit (NCM Biotech, P10100). The standard methods were using the following antibodies: IFIT3 Polyclonal Antibody (Thermo Fisher Scientific, PA5-22230, 1:3000), β-Actin Rabbit mAb (High Dilution) (ABclonal, AC026, 1:20 000), Anti-GAPDH rabbit polyclonal (Sangon Biotech, D110016, 1: 5000); Anti-TRIF (TICAM1) (Abcam, ab302562,1:2000), TLR3 Rabbit Polyclonal Antibody (Beyotime, AF8184, 1:1500), TLR4 Rabbit pAb (ABclonal, A5258, 1:1500), Anti-MyD88 (Abcam, ab133739, 1:2000), Phospho-IRF-3 (Ser396) (D6O1M) Rabbit (Cell Signaling Technology, 29047, 1:1000), IRF3 Rabbit Monoclonal Antibody (Beyotime, AF2485, 1:1500); Caspase-3 Antibody (Cell Signaling Technology, # 9662S, 1:1500), Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb (Cell Signaling Technology, #94530, 1:1500).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Differences in experimental values were assessed using the student's t-test, conducted with Prism software (GraphPad Software 9.0, Inc., San Diego, CA, USA). A \u003cem\u003ep-value\u003c/em\u003e of less than 0.05 was considered statistically significant (*:\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; **:\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; ***:\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we employed a systematic research strategy to elucidate the mechanism underlying IFIT3 overexpression and its pro-apoptotic effects in cardiomyocytes during myocardial infarction (MI), extending our previous research [23]. Initially, we validated IFIT3 overexpression in cardiac biopsies from ischemic patients\u0026rsquo; expression profiles (dataset reference) and LAD-induced MI rat models. Using the immortalized human cardiac myocyte cell line AC-16 combined with CRISPR-Cas9 gene editing and oxygen-glucose deprivation (OGD) modeling, we demonstrated that IFIT3 knockout significantly attenuates cardiomyocyte apoptosis under both resting and persistent OGD conditions, which minimizes interference from cytokines released by other cardiac cell types, such as fibroblasts and monocytes, etc. Critically, we identified S100A1 and S100A9 as endogenous cardiac alarmins that activate IFIT3 overexpression through the TLR3/TICAM1/IRF3 signaling pathway, thereby promoting cardiomyocyte apoptosis during MI.\u003c/p\u003e \u003cp\u003eIn the heart, TLR2, TLR3, TLR4, and TLR5 represent the predominant TLR isoforms [34], with TLR4 and TLR2 being the most extensively studied in the context of myocardial injury. TLR signaling operates through two major pathways: the myeloid differentiation factor 88 (MyD88)-dependent pathway (primarily activating NF-κB and inflammatory cytokines) and the TRIF/TICAM1-dependent pathway (activated by TLR3 and TLR4, leading to IRF3 and type I interferon production). The MyD88-dependent pathway is primarily responsible for activating nuclear factor-\u003cem\u003eκ\u003c/em\u003eB (NF-\u003cem\u003eκ\u003c/em\u003eB), a major inflammatory transcription factor, which further stimulates the production of inflammatory cytokines. In contrast, the TRIF-dependent pathway can be triggered by TLR3 and TLR4, leading to the activation of interferon regulatory factor 3 (IRF3) and NF-\u003cem\u003eκ\u003c/em\u003eB, and consequently results in the production of type I interferon and inflammatory cytokines [35]. TLR3, an intracellular subtype predominantly expressed by cardiomyocytes [36], exhibits upregulated expression and signaling that contribute to persistent autophagy following MI, which in turn promotes heart failure and increases lethality. Enhanced TLR4 activation and elevated expression of proinflammatory mediators downstream of TLR4 signaling have been observed in circulating leukocytes from humans with acute MI [37], contributing to the development of heart failure [38]. Additionally, cardiac TLR4 expression increases following both acute MI [39] and chronic HF[40, 41]. While TLR4 agonist stimulated significant IFIT3 overexpression than other TLR isoforms (TLR1/2/6/7/9) in AC-16 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e and HL-1 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh\u003cb\u003e)\u003c/b\u003e, TLR3 agonists induced more strong response of IFIT3 overexpression in AC-16 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e and HL-1 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e. Furthermore, the key regulator of TLR3 signaling, TRIF (TICAM1), exhibited significantly higher expression after OGD \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, c\u003cb\u003e)\u003c/b\u003e. This finding suggests that the TLR3/TRIF pathway, rather than the TLR4/MyD88 pathway, is preferentially activated during ischemic injury in cardiomyocytes.\u003c/p\u003e \u003cp\u003eSupporting this, both TLR3 and TLR4 agonists, as well as STING agonists, induced IFIT3 overexpression in AC-16 cells, with TLR3 agonists eliciting particularly robust responses \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, e\u003cb\u003e)\u003c/b\u003e. These results indicate that endogenous DAMPs activate the TLR3/IRF3 axis to drive IFIT3 overexpression during myocardial ischemia through an IFN-independent pathway. This mechanism aligns with previous observations that Mice with genetic disrupting or deficiency of TLR4 [42], MyD88 [43], or TLR3[44] reduces infarct size following ischemia/reperfusion and ameliorates pathological remodeling after non-reperfused MI [39, 43, 45], while sustained TLR3 signaling promotes maladaptive responses including apoptosis, inflammation, fibrosis, and oxidative stress.\u003c/p\u003e \u003cp\u003eNecrotic cardiac myocytes release a diverse array of endogenous DAMPs, including S100A1, S100A8/A9 and S100β, which activate TLR3/4 and subsequently upregulate key regulatory effectors in both infarcted and remote myocardium following MI [8, 24]. S100A1 is the most abundant S100 isoforms in cardiomyocytes and is passively released from damaged cardiomyocytes during MI, resulting in a dramatically increased serum levels in acute MI patients. Calprotectin (S100A8/A9) serves as an endogenous agonist of TLR4[11] whose expression is elevated in infarcted myocardium and patient blood, contributing to cardiomyocyte death through mitochondrial dysfunction mediated by TLR4 in the ischemic/re-perfused heart. S100A9 overexpression exacerbates MI/R injury, whereas genetic deletion or pharmacological blockade of S100A9 provides protection against such MI in murine models [15]. In the present study, our results indicated that S100A isoforms (S100A1/6/8/9) (2.0\u0026micro;g/mL, 24 hours) treatment significantly induced IFIT3 overexpression in AC-16 cells, HEK293 and MCF cells. Notably, S100A1 and S100A9 exhibited the most potent effects of in AC-16 cells. Mechanistically, S100A1/9 induced IFIT3 overexpression through the TLR3/TICAM1/IRF3 signaling pathway and promoted apoptosis in AC-16 cells, while IFIT3 knockout significantly attenuated S100A1/9-induced apoptosis.\u003c/p\u003e \u003cp\u003eFurthermore, to validate our proposed S100A-TLR3-IFIT3 signaling mechanism in vivo, we examined key pathway components in cardiac tissues from acute MI rat models. Western blot analysis of infarct zone tissues (n\u0026thinsp;=\u0026thinsp;6, 24h post-MI) demonstrated significant increases in TLR3, phosphorylated IRF3 (p-IRF3), and both pro-caspase-3 and cleaved caspase-3 expression, while total IRF3 levels decreased \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea-c\u003cb\u003e)\u003c/b\u003e. The elevation of p-IRF3 relative to total IRF3 indicates robust activation of IRF3-dependent transcription, consistent with TLR3/TICAM1 pathway engagement. Concurrently, the increased cleaved caspase-3/pro-caspase-3 ratio confirms execution of the apoptotic cascade in ischemic myocardium. To establish the cellular localization of IFIT3 expression relative to apoptotic cardiomyocytes, we performed triple immunofluorescence staining of infarct sections with cardiac troponin T (cTnT, cardiomyocyte marker; green), cleaved caspase-3 (apoptosis marker; yellow), and IFIT3 (red), with DAPI nuclear counterstaining (blue). Confocal microscopy revealed prominent co-localization of IFIT3, cleaved caspase-3, and cTnT specifically in cardiomyocytes within the infarct border zone (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD), demonstrating that IFIT3 overexpression occurs in apoptotic cardiomyocytes at the interface between viable and necrotic tissue. This spatial pattern suggests that IFIT3-expressing cardiomyocytes in the border zone are undergoing apoptosis mediated by TLR3/IRF3 signaling, likely in response to DAMPs released from adjacent necrotic cells. Collectively, these in vivo findings validate that MI activates the TLR3/IRF3/p-IRF3 signaling pathway, driving IFIT3 overexpression and subsequent caspase-3-mediated apoptosis in border zone cardiomyocytes.\u003c/p\u003e \u003cp\u003eIn this study, our results establish that S100A1/9 proteins released from damaged cells during myocardial injury are recognized as DAMPs by neighboring cardiomyocytes, triggering aberrant IFIT3 overexpression that amplifies cardiomyocyte apoptosis and exacerbates myocardial damage \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. The most innovative point of this study is to identify the S100A1/9-TLR3-IFIT3 signaling axis as a critical amplification loop in myocardial ischemic injury, wherein damaged cardiomyocytes release alarmins that activate pro-apoptotic pathways in neighboring cells, propagating tissue damage. This pathway represents a promising therapeutic target for MI intervention. Pharmacological inhibition of S100A1/9, TLR3 antagonism, or IFIT3 suppression could potentially interrupt this damage amplification cascade and preserve myocardial function following ischemic injury. These molecular insights advance our understanding of the pathophysiology underlying ischemic cardiomyopathy progression and provide a mechanistic foundation for developing targeted therapies.\u003c/p\u003e \u003cp\u003eSeveral limitations warrant consideration in this study. First, the majority of experiments were conducted using immortalized human (AC-16) or mouse (HL-1) cardiomyocyte cell lines, which, despite being derived from primary adult ventricular tissue and serving as valuable model systems for investigating cardiac functions at cellular and molecular levels, require validation in clinical patient samples to strengthen reliability. Second, while IFIT3 knockdown was achieved using CRISPR-Cas9 gene editing and validated by Western blot, future studies should confirm these findings in cardiomyocytes derived from IFIT3 knockout animals. Third, although IFIT2 has been extensively studied in MI contexts and likely contributes to cardiomyocyte injury, this study focused specifically on IFIT3 and did not examine IFIT2 knockout effects, which represents an important area for future investigation.\u003c/p\u003e \u003cp\u003eIn summary, this study elucidates a novel molecular mechanism whereby endogenous S100A1/9 alarmins interact with TLR3/4 receptors to drive inducible IFIT3 overexpression and subsequent cardiomyocyte apoptosis during myocardial infarction \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. These findings reveal the critical role of IFIT3 in propagating myocardial injury and identify the S100A1/9-TLR3-IFIT3 signaling pathway as a potential target for clinical intervention in MI, contributing to a more comprehensive understanding of ischemic cardiomyopathy pathophysiology and offering new therapeutic avenues for treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study was approved by the institutional review board of KIB (KIB-R-018). Not applicable for clinical study requirement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have agreed to submit and be published.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary materials can be found can be found online.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the following funding sources:\u003c/p\u003e\n\u003cp\u003eTo Dr. Shubai Liu: A grant for outstanding talent from abroad from the Chinese Academy of Sciences; Startup Support Funding (E0241211H1); and grants from the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences (Y8677211K1, Y8690211Z1).\u003c/p\u003e\n\u003cp\u003eTo Dr. Cheng Chen: The Yunnan Provincial Department of Science and Technology - Kunming Medical University Joint Special Project on Applied Basic Research (Grant No. 202501AY070001-223), the Opening Foundation of The First People\u0026apos;s Hospital of Yunnan Province (2023YJZX-YX001) and the Yunnan Provincial Clinical Medical Center Research Project (Grant No. 2024YNLCYXZX0180).\u003c/p\u003e\n\u003cp\u003eAdditional support was received from \u0026quot;The Pilot Project for Clinical Collaboration of Traditional Chinese and Western Medicine in Major and Complicated Diseases in Yunnan Province: Chronic Heart Failure.\u0026quot;\u003c/p\u003e\n\u003cp\u003eAll other authors have reported that they have no relationships relevant to the contents of this paper to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s Contributions\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDesigned the overall project study: S.L., W.X., and Y.H.; Collected data, performed data analysis, and drafted the manuscript: C.C., Z.H; performed the animal experiments and collected the data: C.C., Z.H., Y.C.; performed the animal experiments and data analysis: H.Q.C. and H.Z.; interpreted and summarized the results: S.L., W.X., C.C. HQ.C. and Y.H.; wrote and revised the manuscript: C.C., S.L. H.Z., W.X., and Y.H.; all authors have read and approved the final version of manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the Center for Clinical Medicine Research of The First People\u0026apos;s Hospital of Yunnan Province and the Affiliated Hospital of Kunming University of Science and Technology (Kunming 650032, China) for providing the experimental platform and technical support. We extend our sincere thanks to Professor Yalian Sa and Ms. Chengcheng Huang for their invaluable assistance with the experiments.\u003c/p\u003e\n\u003cp\u003eWe also wish to thank the College of Chinese Traditional Medicine at Yunnan University of Traditional Chinese Medicine (Kunming 650200, China) for their support. Special gratitude is extended to Professor Yunshu Ma and Professor Lili Cui for their expert assistance with animal experiments and for generously sharing laboratory instrumentation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Availability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe main figures or supporting data have included all related data and materials in this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003ePrabhu, S.D. and N.G. 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H1311-H1318.\u003c/li\u003e\n \u003cli\u003eLu, C., et al., \u003cem\u003eToll-like receptor 3 plays a role in myocardial infarction and ischemia/reperfusion injury.\u003c/em\u003e Biochim Biophys Acta, 2014. \u003cstrong\u003e1842\u003c/strong\u003e(1): p. 22-31.\u003c/li\u003e\n \u003cli\u003eSingh, M.V., et al., \u003cem\u003eMyD88 mediated inflammatory signaling leads to CaMKII oxidation, cardiac hypertrophy and death after myocardial infarction.\u003c/em\u003e J Mol Cell Cardiol, 2012. \u003cstrong\u003e52\u003c/strong\u003e(5): p. 1135-44.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Myocardial ischemic injury, TLR3, S100 proteins, DAMPs, IFIT3","lastPublishedDoi":"10.21203/rs.3.rs-8733111/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8733111/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIschemic cardiomyopathy is associated with myocardial injury and increased mortality. During myocardial infarction (MI), necrotic cardiomyocytes release damage-associated molecular patterns (DAMPs) that trigger inflammatory responses, yet the specific cardiac alarmins and downstream mechanisms driving cardiomyocyte apoptosis remain unclear. In this study, we identified endogenous S100 protein isoforms as key cardiac alarmins released during myocardial ischemic injury and elucidated their role in activating IFIT3 overexpression through innate immune signaling pathways. Mining patient biopsy expression data and verifying a rat model of left anterior descending (LAD) artery occlusion, we validated IFIT3 overexpression in both human and rodent cardiac tissues during acute myocardial infarction (AMI). To investigate the functional role of IFIT3, we employed CRISPR-Cas9 gene-editing technology to knock out IFIT3 in AC-16 human cardiac cells and developed a continuous oxygen-glucose deprivation/reperfusion (OGD/R) model to mimic MI at the cellular level. IFIT3 knockout significantly inhibited apoptosis induced by OGD/R and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), as detected by Annexin V-FITC/PI double staining. Mechanistically, we utilized Type I interferons, TLR agonists, and STING agonists to dissect the dominant DAMP signaling pathway, revealing that S100 proteins activate IFIT3 overexpression through the TLR3/TICAM1/IRF3 pathway, thereby promoting cardiomyocyte apoptosis. This research establishes a novel S100-TLR3-IFIT3 signaling axis in the pathophysiology of ischemic cardiomyopathy, providing new mechanistic insights and potential therapeutic targets for myocardial ischemic injury.\u003c/p\u003e","manuscriptTitle":"A novel S100A-TLR3-IFIT3 signaling axis promotes cardiomyocyte apoptosis during myocardial infarction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-08 16:49:47","doi":"10.21203/rs.3.rs-8733111/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dedcad8a-5ed6-4852-a0e1-ca75a49aa706","owner":[],"postedDate":"March 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":62161314,"name":"Health sciences/Diseases/Cardiovascular diseases/Cardiomyopathies"},{"id":62161315,"name":"Biological sciences/Immunology/Cell death and immune response"},{"id":62161316,"name":"Health sciences/Diseases/Immunological disorders"}],"tags":[],"updatedAt":"2026-03-08T16:49:47+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-08 16:49:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8733111","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8733111","identity":"rs-8733111","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}