Inhibit of the cGAS-STING-STAT1 pathway protects heart from the Doxorubicin-induced cardiotoxicity

Inhibit of the cGAS-STING-STAT1 pathway protects heart from the Doxorubicin-induced cardiotoxicity | 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 Inhibit of the cGAS-STING-STAT1 pathway protects heart from the Doxorubicin-induced cardiotoxicity Ning Hou, Xun YUAN, Wenqi Tian, Yuan Qin, Ruchao Jiang, Xianneng Lu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4253972/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 Doxorubicin (DOX) is a common clinical chemotherapeutic drug. However, DOX-induced cardiotoxicity (DIC) limits the wide and long-term clinical use to treat cancers. This study aims to dissect the mechanism in which DNA damage-triggered micronucleus (MN) formation activates the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-STAT1 pathway in cardiac fibroblasts during DIC. C57BL/6J mice were intravenously injected with 10 mg/kg of DOX to establish an acute DOX-induced cardiac injury mouse model. Meanwhile, C57BL/6J mice were intraperitoneally injected with STING inhibitor C-176 (10 mg/kg/week) or intravenously injected with STING siRNA (10 nM/week) prior to DOX (5 mg/kg/week) intravenous injection for 4 weeks to establish a chronic DIC mouse model. After 1 week of Dox injection, mice were harvested for further analysis. Measurements included echocardiography, immunohistochemical analyses, Masson and Sirius Red staining, and Western blots. Here, we showed that the cGAS-STING-STAT1 pathway was activated in cardiac fibroblasts during DIC. The STING inhibition by C-176 or the STING knockdown via siRNA in DOX-induced chronic cardiotoxicity mouse heart attenuated the DOX-induced cardiac dysfunction, cardiac fibrosis, and the inflammatory response. Mechanistically, we also demonstrated that the DOX-induced DNA damage-triggered MN formation impaired the nuclear stability, initiating the activation of the cGAS-STING-STAT1 pathway in cardiac fibroblasts during DIC. Our study illustrated that the activation of the cGAS-STING-STAT1 pathway initiated by DOX-induced DNA damage and MN formation stimulated proinflammatory responses in cardiac fibroblasts, thus promoting myocardial fibrosis during DIC. Health sciences/Diseases/Cardiovascular diseases Biological sciences/Cell biology Doxorubicin Cardiotoxicity STING MN Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Doxorubicin (DOX) is a classic anthracycline chemotherapeutic agent, which is widely used in clinical cancer therapy. Despite the numerous new chemotherapeutic drugs researched and developed in recent years, DOX is still commonly used in therapy for a wide range of cancers, including solid tumors and hematological malignancies, such as breast, stomach, and liver cancers and leukemia 1 , 2 . However, the side effects of DOX limit its clinical application. Among many kinds of adverse reactions, cardiotoxicity is one of the most serious dose-dependent toxic effect, which could lead to dose-dependent accumulative cardiac dysfunction. DOX-induced cardiotoxicity (DIC) has been raised to describe this chemotherapy-related cardiac dysfunction (CTRCD) 3 – 5 . Most studies focused on the cardiomyocyte injury in DIC. Increasing evidence shows that cardiac fibroblast dysfunction is involved in DIC. The potential mechanisms include disrupted DNA damage responses (DDRs) 6 , apoptosis 7 , mitophagy 8 and mitochondrial dysfunction 9 . Therefore, dissecting the molecular mechanisms of cardiac fibroblasts in DIC is necessary. DOX exerts an anticancer effect mainly through activating DSBs and inhibiting topoisomerase II (Top II) activity, which is related to DNA replication. DOX binds DNA and Top II to form a ternary Top II-DOX-DNA cleavage complex, which could induce DNA double-strand breaks (DSBs) and then trigger cell death 10 , 11 . DDRs are initiated when DSBs form to inhibit the cell cycle and repair DNA. If the DNA damage is not repaired, cells undergo cellular senescence 12 . Micronucleus (MN) is a traditional biomarker of DNA damage and chromosome instability 13 , 14 . MN is generated from the fragile nuclear envelope triggered by DSBs or the nucleoplasmic bridge formation induced during mitosis 15 . A recent study showed that MN involves dilated cardiomyopathy with arrhythmias and cardiac fibrosis 16 . Recent researchers also indicated that the formation of MN is mainly caused by mitotic errors and DNA damage 17 , 18 . Meanwhile, MN was detected in cardiomyocytes and heart tissue with BAG3 deletion and cardiomyocytes treated with MG132 19 . However, the mechanism responsible for MN formation in DIC remains poorly understood. Cyclic GMP-AMP synthase (cGAS) is a nuclear acid sensor, which could be activated by exogenous and endogenous DNA and RNA, such as microbial pathogens, mitochondrial DNA, and MN 20 . The activation of cGAS recruits GTP and ATP to form 2′3′-cGAMP isomer and then initiates its downstream signaling effector, stimulator of interferon genes (STING), which employs and phosphorylates TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) 21 , 22 . cGAS-STING is an important signaling pathway in the innate immune system. Recently, the cGAS-STING pathway had been explored in cardiovascular diseases, such as the myocardial ischemic diseases 23 , pressure overload-induced heart failure 24 , myocardial infarction 25 – 27 and DIC 28 . When cGAS-STING is activated, the inflammation-related type-I interferon (IFN) response is triggered, which is the classic downstream pathway. Therefore, cGAS-STING is generally believed to be closely related to inflammation response 22 . Signal transducer and activator of transcription 1 (STAT1) is an important protein during many responses 29 . When STAT1 is activated, it would be phosphorylated and polymerized to form dimers, then translocated to nucleus. p-STAT1 in the nucleus exerts effects to mediate cell inflammation 30 . However, the molecular mechanism of the cGAS-STING-STAT1 pathway triggered by MN in DIC remains inadequately understood. Here, we provide the evidence that MN formation triggers the cGAS-STING-STAT1 pathway in cardiac fibroblasts during DIC. Further analysis indicates that the STING inhibition by STING inhibitor and the STING down-regulation by siRNA attenuate the DOX-induced cardiac dysfunction by inhibiting the STING-STAT1 pathway in cardiac fibroblasts during DIC. Our results suggest that the cGAS-STING-STAT1 pathway could be the potential therapeutic target to treat DIC. Results DOX induced cardiac fibrosis and cardiac dysfunction initiated by DNA damage in heart Cardiac dysfunction is an indicator of DIC during DOX-involved chemotherapy regimen 31 . To define the cardiotoxicity of DOX, we recorded the cardiac function by echocardiography. Our animal study involved intravenous administration of DOX (10 mg/kg). The intravenous route was selected because it is more clinically relevant and elicits less injury compared with other approaches. Echocardiography was performed before DOX administration and 72 h after DOX or NS treatment. The schematic illustrates the timeline of the DOX administration and echocardiography (Fig. 1 a). The results of the echocardiography M-mode of the mouse heart short-axis section showed that the mouse cardiac systolic function decreased after DOX (10 mg/kg) intravenous treatment for 72 h, and ejection fraction (EF) and fractional shortening (FS) exhibited significant reduction (Fig. 1 b c). Previous studies have detected cardiac fibrosis in DOX-induced cardiomyopathy 32 . To evaluate for changes in cardiac fibrosis in DIC, cardiac morphological changes were first analyzed. The Masson staining and Sirius Red staining graphs of heart tissue paraffin sections depict that mouse myocardial fibrosis exhibited after DOX intravenous treatment for 72 h (Fig. 1 d e). Because DDR was initiated when DSBs formed to inhibit the cell cycle and repair DNA, we next assessed the level of DSBs by detecting the expression of the phosphorylated H2AX (g-H2AX) at Ser139, which is a DNA damage marker. After DOX (10 mg/kg) treatment for 8, 16, 24, 48, and 72 h, the γ-H2A.X expression first increased significantly 8h after DOX administration and then decreased (Fig. 1 f). Immunofluorescence staining of g-H2AX in mouse heart tissues confirmed the DNA damage. The intensity of γ-H2A.X positive signals was significantly stronger than that of the NS group (Fig. 1 g). Our results proved that DOX-induced cardiac dysfunction and fibrosis with elevated cardiac DNA damage. MN formation was detected in cardiac fibroblasts after DOX treatment DDR is a multi-step, dynamic process. Accumulated DNA damage limits the cell function, leading to genomic instability, if the damage is persistent. DNA damage in the nucleus results in the leakage of the nucleus DNA, causing the formation of MN. To determine whether MN is formed in DIC caused by accumulated DNA damage, we isolated cardiomyocytes and cardiac fibroblasts to identify the specific effects of DOX on cardiomyocytes or cardiac fibroblasts. After isolation and culture, 1 µM DOX was used to treat cardiomyocytes and cardiac fibroblasts for 2, 4, 8, 16, and 24 h. γ-H2A.X was detected by Western blot and immunofluorescence. In cardiomyocytes, the expression level of γ-H2A.X increased after DOX treatment (Fig. 2 a), and the immunofluorescence results are in accordance with the Western blot results (Fig. 2 b). Although we detected DNA damage in both kinds of cells after DOX treatment, we identified some differences between them. DOX-induced cardiac fibroblasts accumulated DNA damage, as indicated by DAPI staining (Fig. 2 c, d). Cardiac fibroblasts with DOX treatment showed significant MN formation; with the prolongation of DOX treatment time, the proportion of MN formation increased significantly (Fig. 2 e, f). To explore the effect of MN, we determined whether damaged DNA was contained by them. Cardiac fibroblasts were stained with γ-H2AX, DAPI, and vimentin, and the confocal graphs display that MN was colocalized with γ-H2A.X (Fig. 2 g). These results indicated that DOX induced cardiac fibroblast MN formation, in which the MN stain was positive for the DNA damage marker, g-H2AX. cGAS-STING pathway was activated in DIC Studies have demonstrated that MN sensed by cGAS leads to the activation of the cGAS-STING pathway triggered by the accumulation of DNA damage. We further tested the cGAS-STING pathway in the hearts of the DOX-treated mice. Our results indicated that DOX triggered MN formation in cardiac fibroblasts, and the elevated MN formation activated the cGAS-STING pathway in the hearts (Fig. 3 a, b, c). That is, DOX induced genomic instability of cardiac fibroblasts, causing cytoplasmic DNA leakage and cGAS-STING pathway activation. To test the direct effects of DOX on cardiac fibroblasts, cardiac fibroblasts were treated with 1 µM DOX for 2, 4, 8, 16, and 24 h. cGAS-STING pathway proteins were detected by Western blot. cGAS expression increased in a short time after DOX treatment and then decreased significantly. The phosphor-TBK1 level was reduced, whereas phosphor-STING and phosphor-IRF3 gradually increased (Fig. 3 d, e). To clarify whether MN activates cGAS nucleic acid sensor, cardiac fibroblasts were stained with DAPI and cGAS. The confocal photos show that MN was colocalized with cGAS signals (Fig. 3 f). We also verified this pathway activation in mouse heart tissue. Different groups of C57BL/6 mice were intravenously injected with 10 mg/kg of DOX for 8, 16, 24, 48, and 72 h, and cGAS-STING pathway proteins were detected by Western blot. Most proteins in this pathway increased at the timepoint of DOX treatment for 16 h (Fig. 3 a, b). Cardiac fibroblasts were isolated and stained via immunofluorescence, and the colocalization of MN and cGAS signals was observed using a confocal microscope (Fig. 3 f). Our results demonstrated that the cGAS-STING pathway connected the DOX-induced cardiac fibroblast MN formation by sensing cytoplasmic DNA because of nuclear DNA damage. STING inhibition decreased the p-STAT1 expression and NF-κB nucleus accumulation in DOX-induced cardiac fibroblasts To clarify whether the cGAS-STING pathway participates in DOX-induced cardiac structure injury and dysfunction, the STING inhibitor C-176 and knock-down by STING shRNA were used to block this pathway. Our results revealed that the cGAS-STING pathway involved in DIC was triggered by DNA damage and MN formation in cardiac fibroblasts. We then explored the function of this pathway in inflammation given that the cardiac fibroblasts initiated cardiac dysfunction. In cultured cardiac fibroblasts, the STING inhibition by C-176 prevented the DOX-induced increases in p-STING and p-IRF3 expression in cardiac fibroblasts (Fig. 4 a, b). Meanwhile, C-176 reduced the DOX-induced nuclear NF-κB accumulation, leading to reduced cardiac fibroblast inflammation (Fig. 4 c). These results demonstrated that C-176 suppressed cardiac inflammation through the inhibition of STING. The STING inhibition in turn suppressed the phosphorylation of STAT1. Our previous data has demonstrated that C-176 decreased the nuclear NF-κB accumulation in DOX-induced cardiac fibroblasts. Previous study has illustrated that the STING inhibition suppressed the STAT1 phosphorylation in an LPS-induced ALI mouse model 33 . To reveal the molecular mechanism of how STING inhibition affected DIC, we first analyzed the STAT1 phosphorylation in cardiac fibroblasts after DOX treatment. The results showed that the phosphorylation of STAT1 was highly increased after DOX treatment (Fig. 5 a, b). On the contrary, our data revealed that the STING inhibition by C-176 highly suppressed the phosphorylation of STAT1, which is a transcription factor strongly associated with the activation of STING (Fig. 5 a, b, c, d). These results suggested that STING inhibition suppressed the DOX-induced STAT1 phosphorylation in cardiac fibroblasts. To further identify whether the STING knockdown by shRNA was related to p-STAT1 expression in nucleu, we tested the p-STAT1 expression in cardiac fibroblasts treated by DOX. STING knock-down by STING shRNA decreased the level of p-STAT1 to STAT1 in DOX treated cardiac fibroblasts (Fig. 6 a, b). The immunofluorescence results indicate that the STING knock-down by STING siRNA attenuate the p-STAT1 nucleu expression in DOX-induced p-STAT1 nucleu accumulation (Fig. 6 c). STING inhibition by C-176 protected the heart from DIC To explore the role of the cGAS-STING signaling pathway in DIC, we inhibited STING by C-176 administration in a DOX-induced chronic cardiac mouse model (Fig. 7 a). Our results revealed that the chronic DOX intravenous administration did not affect the mouse heart weight-to-body weight ratio (Fig. 7 b, c, d, e). Then, we evaluated the cardiac structure and function by echocardiography. The inhibition of STING by C-176 attenuated the DOX-induced cardiac dysfunction, as proven by the increased EF and FS (Fig. 7 f, g). Moreover, we found that the STING inhibition by C-176 decreased the DOX-induced cardiac fibrosis (Masson staining and Sirus Red staining were performed at the completion of the DOX challenge) (Fig. 7 h, i). Considering that the STING inhibition decreased the p-STAT1 nuclear accumulation in cardiac fibroblasts, we evaluated the p-STAT1 protein expression in these mice. As shown in (Fig. 7 j, k), the STING inhibition by C-176 significantly decreased the p-STAT1 protein expression in the DOX-induced cardiac heart tissue. A cytoplasmic and nuclear protein isolation protocol was implemented on the mouse heart tissue to further identify the p-STAT1 expression in the nucleus. The STING inhibition by C-176 decreased the p-STAT1 nuclear expression in the heart tissue of the DOX-induced cardiac mouse model (Fig. 7 l, m). Because p-STAT1 is an important transcriptional factor for regulating inflammation, we detected the IL-1 expression by immunohistochemical staining. The STING inhibition by C-176 decreased the cardiac IL-1 expression in the DOX-induced chronic mouse model (Fig. 7 n, o). These results indicated that C-176 attenuated the DOX-induced cardiac dysfunction by inhibiting the cGAS-STING pathway, which decreased the DOX-induced cardiac local inflammation by downregulating p-STAT1 in cardiac fibroblasts. STING knockdown by STING siRNA protected the heart from DIC To further explore the role of the cGAS-STING signaling pathway in DIC, we knock-down the STING by shRNA administration in a DOX-induced chronic cardiac mouse model (Fig. 8 a). We observed that the chronic DOX intravenous administration did not affect the mouse heart weight-to-body weight ratio (Fig. 8 b, c, d, e). Then, we evaluated the cardiac structure and function by echocardiography. The inhibition of STING by shRNA attenuated the DOX-induced long-term cardiac dysfunction, as proven by the increased EF and FS (Fig. 8 f, g). Moreover, we found that the STING knock-down by STING shRNA decreased the DOX-induced cardiac fibrosis (Masson staining and Sirus Red staining were performed at the completion of the DOX challenge) (Fig. 8 h), suggesting that STING down-regulation attenuate DOX-induced cardiac fibrosis. To further determine whether STING knock-down by shRNA resulted in down-regulation of p-STAT1, we detected the p-STAT1 expression in heart tissue. As shown in (Fig. 8 i), the STING knock-down by STING shRNA significantly decreased the p-STAT1 protein expression in the DOX-induced cardiac heart tissue. The STING knock-down by STING shRNA decreased the p-STAT1 nuclear expression in the heart tissue of the DOX-induced cardiac mouse model (Fig. 8 j). Because p-STAT1 is an important transcriptional factor for regulating inflammation, we detected the IL-1 expression by immunohistochemical staining. The STING knock-down by STING shRNA decreased the cardiac IL-1 expression in the DOX-induced chronic mouse model (Fig. 8 k). These results indicated that STING knock-down by STING shRNA attenuated the DIC by inhibiting the cGAS-STING pathway, which decreased the DOX-induced cardiac local inflammation by downregulating p-STAT1 in cardiac fibroblasts. Discussion CTRCD has become prevalent in cancer treatment given the high survival rates of administration of chemotherapeutic agents 4 . However, type 1 CTRCD initiated by DOX leads to inversible cardiac dysfunction 34 . Therefore, verification of the biological mechanisms is critical to prevent type 1 CTRCD. DOX is the major characteristic agent to induce type 1 CTRCD 35 . Our study revealed that DOX activated the cGAS-STING-STAT1 pathway in cardiac fibroblasts. STING inhibitor C-176 and STING down-regulation by STING siRNA attenuated in cardiac fibroblasts during DIC by inhibiting STING-STAT1 pathway. The cardiac fibroblast MN formation activated the cGAS-STING pathway, in which improving the p-STAT1 nuclear accumulation in DOX-induced cardiac fibroblasts. The cGAS-STING pathway in cardiac fibroblasts also regulated the NF-κB nuclear accumulation after DOX treatment. It was responsible for inflammatory cytokine production, which initiated fibroblast and cardiac inflammation. The pharmacological inhibition of STING by C-176 and the downregulation of STING by siRNA attenuated DIC by inhibiting the STING-STAT1 pathway. These findings suggest the cGAS-STING-p-STAT1 axis in fibroblasts as a novel, critical factor involved in DIC. Inflammatory mechanisms play an important role in the response of cGAS-STING pathway activation 22 , 36 . In this study, we found that MN formation was elevated after DOX treatment in cardiac fibroblasts. The pharmacological inhibition of STING with C-176 and the downregulation of STING by siRNA significantly alleviated DOX-induced cardiac short- and long-term cardiac dysfunctions. In addition, we demonstrated that the cardiac fibroblast nuclear DNA, which escaped into the cytoplasm under DOX treatment, could promote cardiac fibroblast accumulation of p-STAT1 and NF-κB in the nucleus via the cGAS-STING signaling pathway. The STING inhibition by C-176 and the STING downregulation by siRNA significantly decreased the p-STAT1 and NF-kB nuclear accumulation and alleviated cardiac inflammation (Fig. 4 , 5 , 6 ). STING is an intracellular signaling protein that senses cGAMP upon stimulation, leading to the production of IFNs and other inflammatory mediators 37 . Research on the role of the cGAS-STING pathway in CVDs has grown in recent years 23 , 25 – 28 . However, relatively little is known about the role of the STING- STAT1 pathway in the pathological process of chronic DIC. In this study, we demonstrated the activation of the STING-STAT1 pathway in chronic DIC. Previous work by Wei Luo and colleagues showed that the cGAS-STING pathway was activated in endothelial cells following DOX treatment 28 . They also revealed that the cGAS-STING pathway was upregulated in cardiomyocytes after the treatment 28 . Meanwhile, other researchers showed that the level of the cGAS-STING pathway in cardiomyocytes was activated post-DOX treatment 38 . Our study adds to this body of work by demonstrating that the MN formation triggered by DOX treatment during DIC activated the cGAS-STING pathway in cardiac fibroblasts. Following DOX treatment, this pathway triggered the phosphorylation of STAT1, a key signaling molecule involved in inflammatory responses. These findings suggest that the cGAS-STING pathway plays a critical role in chronic DIC through its activation in cardiac fibroblasts and the subsequent phosphorylation of STAT1. This pathway may represent a novel target for the treatment of chronic DIC and other inflammatory CVDs. C-176, a highly selective small molecular inhibitor of STING, is widely used to block the STING-dependent pathway 39 . Recent studies have shown that C-176 can effectively attenuate DOX-induced cardiac dysfunction 28 . However, the exact molecular mechanisms of STING in cardiac local inflammation in DIC are not fully understood. To further investigate the biological mechanisms of C-176 in attenuating DOX-induced cGAS-STING-STAT1 pathway activation in cardiac fibroblasts, we administered C-176 via intraperitoneal injection in our study. The pharmacological inhibition of STING with C-176 significantly attenuated DIC, thereby increasing the EF and FS in DOX-induced cardiac dysfunction. The inhibition of STING with C-176 also alleviated the increased level of p-STAT1 and the cardiac local inflammation in vitro and in vivo. Another significant finding from this study was that STING played a mediating role in the inflammatory response through the activation of the NF-κB signaling pathways within cardiac fibroblasts. STING mediated the nuclear accumulation of NF-κB following immune responses. We speculated that STING may influence the immune response in cardiac fibroblasts following DOX treatment through the activation of NF-κB pathways. Our results also indicated that the phosphorylation of STAT1 (p-STAT1) was increased after DOX treatment, which was suppressed by C-176 and STING siRNA administration. The downregulation of p-STAT1 was associated with a reduction in fibrosis following DOX treatment. Recent studies have suggested that targeting the cGAS-STING pathway may represent a therapeutic strategy for mitigating cardiac dysfunction following ischemic heart diseases 23 , chemotherapeutic-induced cardiotoxicity 28 , or myocardial infarction 27 . cGAMP binds to the endoplasmic reticulum, promoting STING dimerization. Subsequently, STING recruits and initiates the IRF3-dependent innate immune response, leading to the production of interferons and an NF-κB-dependent inflammatory response 40 . This condition results in the creation of proinflammatory factors, including TNF-a and IL-1. Our results indicated that the activation of p-STAT1 was dependent on the increased expression of p-STING in cardiac fibroblasts following DOX treatment. The inhibition of the STING downregulated the STING-STAT1 pathway, thereby attenuating DIC. Therefore, the cGAS-STING-STAT1 pathway triggered by DNA damage-induced MN formation in cardiac fibroblasts during DIC may represent the potential therapeutic target to treat DIC. Conclusion Our study revealed that the cGAS-STING-STAT1 pathway played an important role in DIC by stimulating the MN in cardiac fibroblasts. Furthermore, the inhibition of STING mitigated DOX-induced cardiac dysfunction. These findings suggest that the cGAS-STING-STAT1 pathway triggered by MN may serve as a potential therapeutic target for the prevention of CTRCD. Materials and Methods Animal study design All animal experiments were conducted following the guidelines of animal welfare and approved by the Animal Care and Use Committee of Guangzhou Medical University (GY2023-108). In the first acute experiment, mice aged 6–8 weeks were purchased from GemPharmatech Co., Ltd., China and raised at the Guangzhou Medical University Laboratory Animal Center. The mice were randomly divided into saline and DOX groups. The DOX group was injected intravenously with 10 mg/kg of DOX (S1280, Selleck, China). Different groups of mice were harvested at 8, 16, 24, 48, and 72 h after injection. The saline group was injected intravenously with the same dosage of saline. In the second chronic STING inhibitor C-176 experiment, C57BL/6 male mice aged 6–8 weeks were randomly divided into Saline + Corn Oil, Saline + C-176, DOX + Corn Oil, and DOX + C-176 groups. Mice were injected intravenously with 5 mg/kg of saline or DOX once a week and injected intraperitoneally with 10 mg/kg of corn oil or C-176 (S6575, Selleck, China) dissolved in DMSO and diluted in corn oil at the same time. After 4 weeks, the treatment was stopped for 1 week. In the third chronic Sting1 siRNA experiment, Sting1 siRNA (5′-GGAGCCGAAGACTGTACAT-3′) was purchased from Guangzhou RiboBio Co., Ltd. C57BL/6 male mice aged 6–8 weeks were randomly divided into NC + NS, NC + DOX, and Sting1 siRNA + DOX groups. Mice were injected intravenously with 5 mg/kg of NS or DOX once a week and injected intravenously with 10 nmol NC or Sting1 siRNA twice a week before DOX treatment. Echocardiography Before treating with DOX, mice were subjected to transthoracic echocardiography (Vevo 2100 Imaging System, VisualSonics, Canada) to obtain baseline data. Then, every week before injecting, mice were also subjected to echocardiography to obtain cardiac function data. The body temperature of mice was maintained at 36.9°C–37.3°C, while the heart rate was maintained at 550–650 bpm. Echocardiographic M-mode tracing records were used to measure cardiac structure, and at least five cardiac cycles were utilized to calculate the left ventricular EF (LVEF) and the left ventricular FS (LVFS). The measurement application was Vevo Lab. Histological and immunofluorescence analyses One week after the DOX treatment for 4 weeks, mice were sacrificed, and hearts were fixed with 4% paraformaldehyde for 24 h at 4°C, dehydrated with a gradient concentration of ethanol, embedded with paraffin, and sectioned. Tissue sections were stained with Masson’s trichrome immunohistochemical reagent and Picro-Sirius Red Solution to analyze cardiac fibrosis. To observe inflammatory cell infiltration, immunohistochemistry analysis was performed on the above paraffin sections with IL-1β antibody (11E5; Santa Cruz Biotechnology; Cat#sc-52012; 1:100). Mice were harvested at the end point of the experiment, and hearts were fixed with 4% paraformaldehyde for 24 h at 4°C, dehydrated in 30% sucrose solution for 24 h at 4°C, embedded with OCT, and sectioned to acquire 5 µm frozen sections for immunofluorescence analysis. The above frozen sections were blocked and permeabilized for 45 min in a blocking buffer, comprising 1% bovine serum albumin and 0.3% Triton X-100 in PBS. Slides were incubated with primary antibodies diluted in the blocking buffer for 12 h at 4°C and with secondary antibodies diluted in the blocking buffer for 45 min at 37°C. A DAPI staining solution (Beyotime; Cat#C1006) was used for staining for 10 min at room temperature. The following antibodies were applied: anti-phosphor-Histone H2A.X (Ser139) rabbit antibody (20E3; Cell Signaling Technology; Cat#9718; 1:100), anti-cGAS rabbit antibody (D3O8O; Cell Signaling Technology; Cat#31659; 1:100), anti-phosphor-STAT1 (Ser727) rabbit antibody (SN67-04; HUABIO; Cat#ET1611-20; 1:100), anti-NF-κB rabbit antibody (D14E12; Cell Signaling Technology; Cat#8242T; 1:100), goat anti-rabbit IgG DyLight 488 (Abbkine; Cat#A23220; 1:200), goat anti-mouse IgG DyLight 488 (Abbkine; Cat#A23210; 1:200), goat anti-rabbit IgG DyLight 594 (Abbkine; Cat#A23420; 1:200), and goat anti-mouse IgG DyLight 594 (Abbkine; Cat#23410; 1:200). Western blot assay Heart tissue and cells were collected with RIPA lysis buffer (Thermo Fisher Scientific; Cat#89901) containing protease and phosphatase inhibitor (Thermo Fisher Scientific; Cat#78442), fully ground, and vortexed for complete total protein lysis. The protein was separated on 8–15% SDS-polyacrylamide gels by electrophoresis and transferred to 0.45 µm PVDF membranes. After blocking with 5% nonfat milk diluted in TBST for 45 min at room temperature, the membranes were incubated with primary antibodies diluted in a blocking buffer for 12 h at 4°C and then incubated with an HRP-conjugated secondary antibody diluted in a blocking buffer for 45 min at room temperature. SuperSignal West Pico PLUS (Thermo Fisher Scientific; Cat#34580) was used to detect by Amersham Imager 600. The following antibodies were used: anti-phosphor-Histone H2A.X (Ser139) rabbit antibody (20E3; Cell Signaling Technology; Cat#9718; 1:1000), anti-Histone H2A.X rabbit antibody (D17A3; Cell Signaling Technology; Cat#7631; 1:1000), anti-cGAS rabbit antibody (D3O8O; Cell Signaling Technology; Cat#31659; 1:1000), anti-cGAS rabbit antibody (HUABIO; Cat#HA500023; 1:1000), anti-phosphor-TMEM173/STING (Ser366) rabbit antibody (Affinity Biosciences; Cat#AF7416; 1:1000), anti-STING rabbit antibody (D2P2F; Cell Signaling Technology; Cat#13647; 1:1000), anti-phosphor-TBK1/NAK (Ser172) rabbit antibody (D52C2; Cell Signaling Technology; Cat#5483; 1:1000), anti-TBK1/NAK rabbit antibody (E813G; Cell Signaling Technology; Cat#38066; 1:1000), anti-phosphor-IRF3 (Ser396) rabbit antibody (D6O1M; Cell Signaling Technology; Cat#29047; 1:1000), anti-IRF3 rabbit antibody (D83B9; Cell Signaling Technology; Cat#4302; 1:1000), anti-phosphor-STAT1 (Ser727) rabbit antibody (SN67-04; HUABIO; Cat#ET1611-20; 1:1000), and anti-STAT1 rabbit antibody (HUABIO; Cat#ET1606-39; 1:1000). Nucleocytoplasmic separation Heart tissue was ground with a grinding rod in CER Ⅰ reagent in a nucleocytoplasmic separation kit (Thermo Fisher Scientific; Cat#78833), then CER Ⅱ was added in tubes. After vortex and centrifugation, the supernatant was aspirated, and cytoplasm protein was extracted and collected. The precipitate was washed in PBS, and NER reagent was added into tubes. After vortex and centrifugation, nucleus protein could be extracted. Afterward, the protein expression levels could be detected by Western blot. Cell isolation, culture, and stimulation Cardiac fibroblasts were isolated by digesting neonatal SD rat ventricular tissue with Trypsin without EDTA (Thermo Fisher Scientific; Cat#15050065) and type II collagenase (MP Biomed; Cat#100502), filtrated through a 70 µm cell strainer, and purified by differential anchoring velocity. Primary cardiac fibroblasts were resuspended in high glucose DMEM with 10% FBS for 24 h and passaged to the second generation. DOX powder was dissolved in sterile water and diluted to 1 mM. The STING inhibitor C-176 was dissolved in DMSO and diluted to 1, 2, and 4 mM. When the cell density reached 70% of the dish, cells in the logarithmic growth phase were treated with 1 µM DOX for 2, 4, 8, 16, and 24 h. To inhibit the pathway, 2 µM DOX and 1, 2, and 4 µM STING inhibitor C-176 were used for different groups of cardiac fibroblasts; two control groups for DMSO and 4 µM C-176 were also set up to control the influence of dissolvent. When the cell density of cardiac fibroblasts reached 50% of the dish, Sting1 siRNA (F: 5′-CAACAGUGUCUAUGAACUUTT-3′; R: 5′-AAGUUCAUAGACACUGUUGTT-3′) was transfected into cardiac fibroblasts by transfection reagent RNA iMAX (Thermo Fisher Scientific; Cat#13778030). After transfecting in Opti-MEM (Gibco; Cat#31985070) without FBS for 6 h, cells were continuously cultured in a high glucose DMEM culture medium with 10% FBS for 16 h and then treated with 1 µM DOX for 8 h. The above cells were collected in RIPA, and the cell crawling slides were fixed with 4% PFA and then kept at − 20°C. Statistical analysis All data are expressed as mean ± standard error of the mean (mean ± SEM). Data were analyzed with GraphPad Prism 9.0 software (GraphPad, San Diego, CA). The student t test (2 tailed) was performed to compare 2 groups. If SD un-equation with Welch’s correction. One-way ANOVA followed by the Tukey post hoc test was used to compare multiple groups. A value of P < 0.05 was considered significant. Declarations Competing Interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding This work was supported in part by the open research funds from the Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People's Hospital (grant No. 202201-203), the Guangzhou Science and Technology Bureau Project (grant No. G24151045), Guangzhou Medical University Research and Innovation Ability Enhancement Project (grant No. 02-410-2405004, 02-410-2302358XM). Author Contributions XY, WJ and NH were involved in designed and guarantors of the project. XY, WT and YQ were involved to the performance of the experiments, data analysis and wrote the manuscript. RJ, GZ, XL, YL and QZ participated in data analysis and interpretation. XY, WJ and NH supervised the work and revised the manuscript. ACKNOWLEDGEMENTS This work was supported in part by Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People's Hospital (grant No. 202201-203), the Guangzhou Science and Technology Bureau Project (grant No. G24151045), Guangzhou Medical University Research and Innovation Ability Enhancement Project (grant No. 02-410-2405004, 02-410-2302358XM). We are grateful to Prof. Jiandong Luo at the Guangzhou Medical University for his kind guidance during the research. We are also very grateful to our colleagues at the Department of Pharmacology in Guangzhou Medical University for their kind guidance during the use of some experimental instruments. References Carvalho, C., Santos, R.X., Cardoso, S., Correia, S., Oliveira, P.J., Santos, M.S., et al.: Doxorubicin: the good, the bad and the ugly effect. Curr. Med. Chem. 16 , 3267–3285 (2009) Songbo, M., Lang, H., Xinyong, C., Bin, X., Ping, Z., Liang, S.: Oxidative stress injury in doxorubicin-induced cardiotoxicity. Toxicol. 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Immunol. 17 , 1142–1149 (2016) Additional Declarations (Not answered) Supplementary Files SupplementaryFilesOriginalWesternBlotFinal.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Scheme of the DOX challenge in mice and time points of functional assessments and heart harvest. b. Echocardiography M-mode of the short-axis section motion patterns of the left ventricles of the hearts of mice treated with DOX for 72 h. c. EF and FS statistic graphs (n=4/ group. d. Masson staining graphs of the heart paraffin sections of mice treated with NS or DOX for 72 h (n=5-10/group). e. Sirius Red staining of heart paraffin sections and polarized light photography. f. Western blot detection of DNA damage marker γ-H2A.X expression in DOX treated for 8, 16, 24, 48, and 72 h (n=4/group). g. γ-H2A.X immunofluorescence staining and statistic graphs of heart tissue frozen sections of mice treated with NS and DOX for 8 h, where red stands for α-actinin, green for γ-H2A.X, and blue for DAPI, 10-15 images acquired from each mice (n=5-6/group). *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 \u003cem\u003evs.\u003c/em\u003eNS.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4253972/v1/e351aedfb901870c86cc71d7.png"},{"id":57677684,"identity":"64f874d2-14d3-4493-8859-cd6c06bf5e38","added_by":"auto","created_at":"2024-06-04 08:10:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4040893,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDOX induced DNA damage and promoted MN formation in cardiac fibroblasts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea. Detection of γ-H2A.X expression of cardiomyocytes treated with DOX by Western blot and statistical analysis (n=5/group). b. Immunofluorescence staining of γ-H2A.X of cardiomyocytes treated with DOX for 8 and 24 h, where red stands for α-actinin, blue for DAPI, and green for γ-H2A.X. c. Detection of γ-H2A.X expression of cardiac fibroblasts treated with DOX by Western blot and statistical analysis (n=5/group). d. Immunofluorescence staining of γ-H2A.X of cardiac fibroblasts treated with DOX for 8 and 24 h, where red stands for vimentin, blue for DAPI, and green for γ-H2A.X. e. MN comparison representative immunofluorescence graphs of rat cardiomyocytes and cardiac fibroblasts treated with DOX for 8 h, where green stands for α-actinin and vimentin, blue for DAPI, and white for DAPI grey-scale images. f. Statistic diagram of the percentage of MN in cardiac fibroblasts treated with DOX for different durations, statistical analyze in each group from 17 images (n=17/group). g. Representative immunofluorescence staining images of MN and γ-H2A.X, where red stands for vimentin, green for γ-H2A.X, and white for DAPI grey-scale images. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 \u003cem\u003evs.\u003c/em\u003e CON\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4253972/v1/8ad17157cbd6143f68e1b2f0.png"},{"id":57678132,"identity":"97fc9a9b-a7d6-406e-af8b-ffad42d6c7b5","added_by":"auto","created_at":"2024-06-04 08:18:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2595948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ecGAS-STING pathway was activated by DNA in MN in vivo and in vitro.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea. Representative graphs of the protein expression levels in the cGAS-STING pathway detected by Western blot in mouse heart tissues. b. Statistical analysis graphs of the protein expression levels in the cGAS-STING pathway (n=4/group). c. Overall and enlarged views of the immunofluorescence staining of MN and cGAS in the cardiac fibroblasts of adult mice, where red stands for vimentin, green for cGAS, and white for DAPI grey-scale images. d. Representative graphs of the protein expression levels in the cGAS-STING pathway in neonatal rat cardiac fibroblasts detected by Western blot. e. Statistical analysis graphs of the protein expression levels in the cGAS-STING pathway (n=4/group). f. Overall and enlarged views of the immunofluorescence staining of MN and cGAS in neonatal rat cardiac fibroblasts, where red stands for cGAS, green for vimentin, blue for DAPI, and white for DAPI grey-scale images. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003cem\u003evs.\u003c/em\u003e CON\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4253972/v1/d4451b98b5ade18b3af6541a.png"},{"id":57677687,"identity":"73408f18-044e-4c19-8744-f42c652788e9","added_by":"auto","created_at":"2024-06-04 08:10:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1100939,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSTING inhibitor C-176 could attenuate the inflammation response in cardiac fibroblasts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea. Cardiac fibroblasts treated with 1 μM DOX and 1, 2, and 4 μM STING inhibitor C-176 for 8 h and cGAS-STING pathway proteins detected by Western blot. b. Statistical analysis graphs of the protein expression levels in the cGAS-STING pathway (n=6/group). c. Representative graphs of NF-κB immunofluorescence staining in cardiac fibroblasts treated with DOX and C-176, where red stands for vimentin, green for NF-κB, and blue for DAPI, and statistical analysis chart of the mean nuclear NF-κB fluorescence intensity (n=8/group). *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 \u003cem\u003evs.\u003c/em\u003e CON, †\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ††\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 \u003cem\u003evs.\u003c/em\u003e DOX\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4253972/v1/9d04b3d1e2ff712c4b39f263.png"},{"id":57678573,"identity":"f7c1bafb-e450-48fd-a826-2a5f962ffde1","added_by":"auto","created_at":"2024-06-04 08:26:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1403541,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSTING pathway downstream inflammation-related protein phosphor-STAT1 expression was regulated by STING activation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea. Detection of phosphor-STAT1 (Ser727) and STAT1 expression levels by Western blot in cardiac fibroblasts treated with DOX for different durations (n=4/group). b. Representative graphs of the immunofluorescence staining of phosphor-STAT1 (Ser727) signals in cardiac fibroblasts treated with DOX for 8 and 24 h, where red stands for vimentin, green for phosphor-STAT1, and blue for DAPI, and statistical analysis chart of the mean nuclear phosphor-STAT1 fluorescence intensity (n=5-7/group). C. Detection of phosphor-STAT1 (Ser727) and STAT1 expression levels by Western blot in cardiac fibroblasts treated with DOX and C-176 for 8 h (n=5/group). d. Immunofluorescence staining of phosphor-STAT1(Ser727) in cardiac fibroblasts treated with DOX and 4 μM C-176 for 8 h, where red stands for vimentin, green for phosphor-STAT1, and blue for DAPI, and statistical analysis chart of the mean nuclear phosphor-STAT1 fluorescence intensity (n=6/group). *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003cem\u003evs.\u003c/em\u003eCON, †\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ††\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 \u003cem\u003evs.\u003c/em\u003e DOX\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4253972/v1/2d4439e98de8cb58974e89a5.png"},{"id":57677688,"identity":"b6b6da2d-97f5-4674-93ba-262990668dc0","added_by":"auto","created_at":"2024-06-04 08:10:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1259396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSting1 siRNA could decrease the expression levels of STING and its downstream proteins and attenuate the inflammation response in cardiac fibroblasts.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea. Detection of STING-IRF3-STAT1 pathway protein expression by Western blot in cardiac fibroblasts transfected with Sting1 siRNA and treated with DOX. b. Statistical analysis graphs of STING-IRF3-STAT1 protein expression levels (n=4-5/group). c. Representative and statistical analysis graphs of the immunofluorescence staining of phosphor-STAT1 in cardiac fibroblasts transfected with Sting1 siRNA and treated with DOX, where red stands for vimentin, green for phosphor-STAT1 (Ser727), and blue for DAPI (n=4-8/group). *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05 \u003cem\u003evs.\u003c/em\u003e NC, ††\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 \u003cem\u003evs.\u003c/em\u003e NC+DOX\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4253972/v1/40af241172b1d41c631ec198.png"},{"id":57678134,"identity":"a115199e-68bb-4c88-a628-39f16242e351","added_by":"auto","created_at":"2024-06-04 08:18:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3300409,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibiting STING by C-176 attenuated the cardiac inflammation response by downregulating STAT1 phosphorylation in DOX-treated mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea. Scheme of DOX and C-176 injection into mice. b. Representative photos of the hearts of different mouse groups at week 5. c. Statistic chart of the heart weight of different mouse groups at week 5 (n=15/group). d. Statistic chart of body weight changes of different mouse groups (n=15-16/group). e. Statistic chart of the body weight and the heart weight-to-body weight ratio of mice at week 5 (n=15/group). f. Statistic line chart of the changes in heart LVEF and EF of mice in each group at week 5 detected by echocardiography (n=7-8/group). G. Statistic line chart of the changes in heart LVFS and FS of mice in each group at week 5 detected by echocardiography (n=7-8/group). h. Representative images and statistical analysis of Masson’s trichrome staining of mouse hearts (n=5/group). i. Representative graphs of the Sirius Red staining of mouse heart tissue paraffin sections. j. Western blot detection of heart tissue phosphor-STAT1 (Ser727) expression level in all groups of mice and statistic chart (n=13/group). k. Western blot detection of phosphor-STAT1 (Ser727) in the nucleus and plasm in mouse heart tissue. l. Immunofluorescence staining of phosphor-STAT1(Ser727) in mouse heart tissue, where red stands for α-actinin, green for phosphor-STAT1 (Ser727), and blue for DAPI. m. Statistical graph of the mean intranuclear phosphor-STAT1 (Ser727) fluorescence intensity (n=6/group). n. Immunohistochemical staining of mouse heart tissue paraffin sections with IL-1β. o. Statistical graph of average IL-1β optical density in mouse hearts in each group (n=6/group). *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003cem\u003evs.\u003c/em\u003eNS+Corn Oil, †\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ††\u003cem\u003eP\u003c/em\u003e＜0.01 \u003cem\u003evs.\u003c/em\u003e DOX+Corn Oil\u003c/p\u003e","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-4253972/v1/64695135b7eca493b6bba744.png"},{"id":57677690,"identity":"3ae628f3-3e4e-4e94-b209-4fe0141a5b93","added_by":"auto","created_at":"2024-06-04 08:10:15","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2284813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibiting STING by siRNA attenuated the cardiac inflammation response by downregulating STAT1 phosphorylation in DOX-treated mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea. Scheme of DOX and Sting1 siRNA injection into mice. b. Representative photos of the hearts of different mouse groups at week 5. c. Statistic chart of the heart weight of different mouse groups at week 5 (n=3-5/group). d. Statistic chart of the body weight change of different mouse groups (n=3-5/group). e. Statistic chart of the body weight and heart weight-to-body weight ratio of mice at week 5 (n=3-5/group). f. Statistic line chart of the changes in heart LVEF and EF of mice in each group at week 5 detected by echocardiography (n=3-5/group). g. Statistic line chart of the changes in heart LVFS and FS of mice in each group at week 5 detected by echocardiography (n=3-5/group). h. Representative images and statistical analysis of Masson’s trichrome staining of mouse hearts (n=4) and representative graphs of the Sirius Red staining of mouse heart tissue paraffin sections. i. Western blot detection of the STING and phosphor-STAT1 (Ser727) expression level in heart tissue in all groups of mice and statistic chart (n=3-4/group). j. Immunofluorescence staining and statistical analysis of mouse heart tissue with phosphor-STAT1(Ser727), where red stands for α-actinin, green for phosphor-STAT1 (Ser727), and blue for DAPI (n=8/group). k. Statistical graph of average IL-1β optical density in mouse hearts in each group (n=4/group).Immunohistochemical staining of mouse heart tissue paraffin sections with IL-1β. **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003cem\u003evs.\u003c/em\u003e NC+NS, †\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ††\u003cem\u003eP\u003c/em\u003e＜0.01 \u003cem\u003evs.\u003c/em\u003e NC+DOX\u003c/p\u003e","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-4253972/v1/fbad8d85faffef703d689cb1.png"},{"id":57678577,"identity":"907df1db-62c5-4d28-9906-cacb5b31b1fe","added_by":"auto","created_at":"2024-06-04 08:26:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17655825,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4253972/v1/8ed4bd95-66ee-4a7c-a544-f6a0b30d93c6.pdf"},{"id":57677692,"identity":"2fd6bbdc-1bf6-495e-b3a3-5b4d4e523706","added_by":"auto","created_at":"2024-06-04 08:10:16","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":10995225,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFilesOriginalWesternBlotFinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-4253972/v1/00c63614895d3de6fc2eb124.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Inhibit of the cGAS-STING-STAT1 pathway protects heart from the Doxorubicin-induced cardiotoxicity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDoxorubicin (DOX) is a classic anthracycline chemotherapeutic agent, which is widely used in clinical cancer therapy. Despite the numerous new chemotherapeutic drugs researched and developed in recent years, DOX is still commonly used in therapy for a wide range of cancers, including solid tumors and hematological malignancies, such as breast, stomach, and liver cancers and leukemia\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. However, the side effects of DOX limit its clinical application. Among many kinds of adverse reactions, cardiotoxicity is one of the most serious dose-dependent toxic effect, which could lead to dose-dependent accumulative cardiac dysfunction. DOX-induced cardiotoxicity (DIC) has been raised to describe this chemotherapy-related cardiac dysfunction (CTRCD)\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Most studies focused on the cardiomyocyte injury in DIC. Increasing evidence shows that cardiac fibroblast dysfunction is involved in DIC. The potential mechanisms include disrupted DNA damage responses (DDRs) \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, apoptosis\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, mitophagy\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and mitochondrial dysfunction\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Therefore, dissecting the molecular mechanisms of cardiac fibroblasts in DIC is necessary.\u003c/p\u003e \u003cp\u003eDOX exerts an anticancer effect mainly through activating DSBs and inhibiting topoisomerase II (Top II) activity, which is related to DNA replication. DOX binds DNA and Top II to form a ternary Top II-DOX-DNA cleavage complex, which could induce DNA double-strand breaks (DSBs) and then trigger cell death\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. DDRs are initiated when DSBs form to inhibit the cell cycle and repair DNA. If the DNA damage is not repaired, cells undergo cellular senescence\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Micronucleus (MN) is a traditional biomarker of DNA damage and chromosome instability\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. MN is generated from the fragile nuclear envelope triggered by DSBs or the nucleoplasmic bridge formation induced during mitosis\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. A recent study showed that MN involves dilated cardiomyopathy with arrhythmias and cardiac fibrosis\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Recent researchers also indicated that the formation of MN is mainly caused by mitotic errors and DNA damage\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Meanwhile, MN was detected in cardiomyocytes and heart tissue with BAG3 deletion and cardiomyocytes treated with MG132\u003csup\u003e19\u003c/sup\u003e. However, the mechanism responsible for MN formation in DIC remains poorly understood.\u003c/p\u003e \u003cp\u003eCyclic GMP-AMP synthase (cGAS) is a nuclear acid sensor, which could be activated by exogenous and endogenous DNA and RNA, such as microbial pathogens, mitochondrial DNA, and MN\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The activation of cGAS recruits GTP and ATP to form 2\u0026prime;3\u0026prime;-cGAMP isomer and then initiates its downstream signaling effector, stimulator of interferon genes (STING), which employs and phosphorylates TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ecGAS-STING is an important signaling pathway in the innate immune system. Recently, the cGAS-STING pathway had been explored in cardiovascular diseases, such as the myocardial ischemic diseases\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, pressure overload-induced heart failure\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, myocardial infarction\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and DIC\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. When cGAS-STING is activated, the inflammation-related type-I interferon (IFN) response is triggered, which is the classic downstream pathway. Therefore, cGAS-STING is generally believed to be closely related to inflammation response\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Signal transducer and activator of transcription 1 (STAT1) is an important protein during many responses\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. When STAT1 is activated, it would be phosphorylated and polymerized to form dimers, then translocated to nucleus. p-STAT1 in the nucleus exerts effects to mediate cell inflammation\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. However, the molecular mechanism of the cGAS-STING-STAT1 pathway triggered by MN in DIC remains inadequately understood. Here, we provide the evidence that MN formation triggers the cGAS-STING-STAT1 pathway in cardiac fibroblasts during DIC. Further analysis indicates that the STING inhibition by STING inhibitor and the STING down-regulation by siRNA attenuate the DOX-induced cardiac dysfunction by inhibiting the STING-STAT1 pathway in cardiac fibroblasts during DIC. Our results suggest that the cGAS-STING-STAT1 pathway could be the potential therapeutic target to treat DIC.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDOX induced cardiac fibrosis and cardiac dysfunction initiated by DNA damage in heart\u003c/h2\u003e \u003cp\u003eCardiac dysfunction is an indicator of DIC during DOX-involved chemotherapy regimen\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. To define the cardiotoxicity of DOX, we recorded the cardiac function by echocardiography. Our animal study involved intravenous administration of DOX (10 mg/kg). The intravenous route was selected because it is more clinically relevant and elicits less injury compared with other approaches. Echocardiography was performed before DOX administration and 72 h after DOX or NS treatment. The schematic illustrates the timeline of the DOX administration and echocardiography (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The results of the echocardiography M-mode of the mouse heart short-axis section showed that the mouse cardiac systolic function decreased after DOX (10 mg/kg) intravenous treatment for 72 h, and ejection fraction (EF) and fractional shortening (FS) exhibited significant reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb c). Previous studies have detected cardiac fibrosis in DOX-induced cardiomyopathy\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. To evaluate for changes in cardiac fibrosis in DIC, cardiac morphological changes were first analyzed. The Masson staining and Sirius Red staining graphs of heart tissue paraffin sections depict that mouse myocardial fibrosis exhibited after DOX intravenous treatment for 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBecause DDR was initiated when DSBs formed to inhibit the cell cycle and repair DNA, we next assessed the level of DSBs by detecting the expression of the phosphorylated H2AX (g-H2AX) at Ser139, which is a DNA damage marker. After DOX (10 mg/kg) treatment for 8, 16, 24, 48, and 72 h, the γ-H2A.X expression first increased significantly 8h after DOX administration and then decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Immunofluorescence staining of g-H2AX in mouse heart tissues confirmed the DNA damage. The intensity of γ-H2A.X positive signals was significantly stronger than that of the NS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). Our results proved that DOX-induced cardiac dysfunction and fibrosis with elevated cardiac DNA damage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMN formation was detected in cardiac fibroblasts after DOX treatment\u003c/h2\u003e \u003cp\u003eDDR is a multi-step, dynamic process. Accumulated DNA damage limits the cell function, leading to genomic instability, if the damage is persistent. DNA damage in the nucleus results in the leakage of the nucleus DNA, causing the formation of MN. To determine whether MN is formed in DIC caused by accumulated DNA damage, we isolated cardiomyocytes and cardiac fibroblasts to identify the specific effects of DOX on cardiomyocytes or cardiac fibroblasts. After isolation and culture, 1 \u0026micro;M DOX was used to treat cardiomyocytes and cardiac fibroblasts for 2, 4, 8, 16, and 24 h. γ-H2A.X was detected by Western blot and immunofluorescence. In cardiomyocytes, the expression level of γ-H2A.X increased after DOX treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), and the immunofluorescence results are in accordance with the Western blot results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Although we detected DNA damage in both kinds of cells after DOX treatment, we identified some differences between them. DOX-induced cardiac fibroblasts accumulated DNA damage, as indicated by DAPI staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d). Cardiac fibroblasts with DOX treatment showed significant MN formation; with the prolongation of DOX treatment time, the proportion of MN formation increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, f). To explore the effect of MN, we determined whether damaged DNA was contained by them. Cardiac fibroblasts were stained with γ-H2AX, DAPI, and vimentin, and the confocal graphs display that MN was colocalized with γ-H2A.X (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). These results indicated that DOX induced cardiac fibroblast MN formation, in which the MN stain was positive for the DNA damage marker, g-H2AX.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ecGAS-STING pathway was activated in DIC\u003c/h2\u003e \u003cp\u003eStudies have demonstrated that MN sensed by cGAS leads to the activation of the cGAS-STING pathway triggered by the accumulation of DNA damage. We further tested the cGAS-STING pathway in the hearts of the DOX-treated mice. Our results indicated that DOX triggered MN formation in cardiac fibroblasts, and the elevated MN formation activated the cGAS-STING pathway in the hearts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b, c). That is, DOX induced genomic instability of cardiac fibroblasts, causing cytoplasmic DNA leakage and cGAS-STING pathway activation. To test the direct effects of DOX on cardiac fibroblasts, cardiac fibroblasts were treated with 1 \u0026micro;M DOX for 2, 4, 8, 16, and 24 h. cGAS-STING pathway proteins were detected by Western blot. cGAS expression increased in a short time after DOX treatment and then decreased significantly. The phosphor-TBK1 level was reduced, whereas phosphor-STING and phosphor-IRF3 gradually increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, e). To clarify whether MN activates cGAS nucleic acid sensor, cardiac fibroblasts were stained with DAPI and cGAS. The confocal photos show that MN was colocalized with cGAS signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). We also verified this pathway activation in mouse heart tissue. Different groups of C57BL/6 mice were intravenously injected with 10 mg/kg of DOX for 8, 16, 24, 48, and 72 h, and cGAS-STING pathway proteins were detected by Western blot. Most proteins in this pathway increased at the timepoint of DOX treatment for 16 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Cardiac fibroblasts were isolated and stained via immunofluorescence, and the colocalization of MN and cGAS signals was observed using a confocal microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). Our results demonstrated that the cGAS-STING pathway connected the DOX-induced cardiac fibroblast MN formation by sensing cytoplasmic DNA because of nuclear DNA damage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSTING inhibition decreased the p-STAT1 expression and NF-κB nucleus accumulation in DOX-induced cardiac fibroblasts\u003c/h2\u003e \u003cp\u003eTo clarify whether the cGAS-STING pathway participates in DOX-induced cardiac structure injury and dysfunction, the STING inhibitor C-176 and knock-down by STING shRNA were used to block this pathway. Our results revealed that the cGAS-STING pathway involved in DIC was triggered by DNA damage and MN formation in cardiac fibroblasts. We then explored the function of this pathway in inflammation given that the cardiac fibroblasts initiated cardiac dysfunction. In cultured cardiac fibroblasts, the STING inhibition by C-176 prevented the DOX-induced increases in p-STING and p-IRF3 expression in cardiac fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Meanwhile, C-176 reduced the DOX-induced nuclear NF-κB accumulation, leading to reduced cardiac fibroblast inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). These results demonstrated that C-176 suppressed cardiac inflammation through the inhibition of STING.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe STING inhibition in turn suppressed the phosphorylation of STAT1. Our previous data has demonstrated that C-176 decreased the nuclear NF-κB accumulation in DOX-induced cardiac fibroblasts. Previous study has illustrated that the STING inhibition suppressed the STAT1 phosphorylation in an LPS-induced ALI mouse model\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. To reveal the molecular mechanism of how STING inhibition affected DIC, we first analyzed the STAT1 phosphorylation in cardiac fibroblasts after DOX treatment. The results showed that the phosphorylation of STAT1 was highly increased after DOX treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). On the contrary, our data revealed that the STING inhibition by C-176 highly suppressed the phosphorylation of STAT1, which is a transcription factor strongly associated with the activation of STING (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b, c, d). These results suggested that STING inhibition suppressed the DOX-induced STAT1 phosphorylation in cardiac fibroblasts.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further identify whether the STING knockdown by shRNA was related to p-STAT1 expression in nucleu, we tested the p-STAT1 expression in cardiac fibroblasts treated by DOX. STING knock-down by STING shRNA decreased the level of p-STAT1 to STAT1 in DOX treated cardiac fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). The immunofluorescence results indicate that the STING knock-down by STING siRNA attenuate the p-STAT1 nucleu expression in DOX-induced p-STAT1 nucleu accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSTING inhibition by C-176 protected the heart from DIC\u003c/h2\u003e \u003cp\u003eTo explore the role of the cGAS-STING signaling pathway in DIC, we inhibited STING by C-176 administration in a DOX-induced chronic cardiac mouse model (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Our results revealed that the chronic DOX intravenous administration did not affect the mouse heart weight-to-body weight ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, c, d, e). Then, we evaluated the cardiac structure and function by echocardiography. The inhibition of STING by C-176 attenuated the DOX-induced cardiac dysfunction, as proven by the increased EF and FS (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef, g). Moreover, we found that the STING inhibition by C-176 decreased the DOX-induced cardiac fibrosis (Masson staining and Sirus Red staining were performed at the completion of the DOX challenge) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh, i). Considering that the STING inhibition decreased the p-STAT1 nuclear accumulation in cardiac fibroblasts, we evaluated the p-STAT1 protein expression in these mice. As shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ej, k), the STING inhibition by C-176 significantly decreased the p-STAT1 protein expression in the DOX-induced cardiac heart tissue. A cytoplasmic and nuclear protein isolation protocol was implemented on the mouse heart tissue to further identify the p-STAT1 expression in the nucleus. The STING inhibition by C-176 decreased the p-STAT1 nuclear expression in the heart tissue of the DOX-induced cardiac mouse model (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003el, m). Because p-STAT1 is an important transcriptional factor for regulating inflammation, we detected the IL-1 expression by immunohistochemical staining. The STING inhibition by C-176 decreased the cardiac IL-1 expression in the DOX-induced chronic mouse model (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003en, o). These results indicated that C-176 attenuated the DOX-induced cardiac dysfunction by inhibiting the cGAS-STING pathway, which decreased the DOX-induced cardiac local inflammation by downregulating p-STAT1 in cardiac fibroblasts.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSTING knockdown by STING siRNA protected the heart from DIC\u003c/h2\u003e \u003cp\u003eTo further explore the role of the cGAS-STING signaling pathway in DIC, we knock-down the STING by shRNA administration in a DOX-induced chronic cardiac mouse model (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). We observed that the chronic DOX intravenous administration did not affect the mouse heart weight-to-body weight ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb, c, d, e). Then, we evaluated the cardiac structure and function by echocardiography. The inhibition of STING by shRNA attenuated the DOX-induced long-term cardiac dysfunction, as proven by the increased EF and FS (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ef, g). Moreover, we found that the STING knock-down by STING shRNA decreased the DOX-induced cardiac fibrosis (Masson staining and Sirus Red staining were performed at the completion of the DOX challenge) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eh), suggesting that STING down-regulation attenuate DOX-induced cardiac fibrosis. To further determine whether STING knock-down by shRNA resulted in down-regulation of p-STAT1, we detected the p-STAT1 expression in heart tissue. As shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ei), the STING knock-down by STING shRNA significantly decreased the p-STAT1 protein expression in the DOX-induced cardiac heart tissue. The STING knock-down by STING shRNA decreased the p-STAT1 nuclear expression in the heart tissue of the DOX-induced cardiac mouse model (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ej). Because p-STAT1 is an important transcriptional factor for regulating inflammation, we detected the IL-1 expression by immunohistochemical staining. The STING knock-down by STING shRNA decreased the cardiac IL-1 expression in the DOX-induced chronic mouse model (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ek). These results indicated that STING knock-down by STING shRNA attenuated the DIC by inhibiting the cGAS-STING pathway, which decreased the DOX-induced cardiac local inflammation by downregulating p-STAT1 in cardiac fibroblasts.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCTRCD has become prevalent in cancer treatment given the high survival rates of administration of chemotherapeutic agents\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, type 1 CTRCD initiated by DOX leads to inversible cardiac dysfunction\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Therefore, verification of the biological mechanisms is critical to prevent type 1 CTRCD. DOX is the major characteristic agent to induce type 1 CTRCD\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Our study revealed that DOX activated the cGAS-STING-STAT1 pathway in cardiac fibroblasts. STING inhibitor C-176 and STING down-regulation by STING siRNA attenuated in cardiac fibroblasts during DIC by inhibiting STING-STAT1 pathway. The cardiac fibroblast MN formation activated the cGAS-STING pathway, in which improving the p-STAT1 nuclear accumulation in DOX-induced cardiac fibroblasts. The cGAS-STING pathway in cardiac fibroblasts also regulated the NF-κB nuclear accumulation after DOX treatment. It was responsible for inflammatory cytokine production, which initiated fibroblast and cardiac inflammation. The pharmacological inhibition of STING by C-176 and the downregulation of STING by siRNA attenuated DIC by inhibiting the STING-STAT1 pathway. These findings suggest the cGAS-STING-p-STAT1 axis in fibroblasts as a novel, critical factor involved in DIC.\u003c/p\u003e \u003cp\u003eInflammatory mechanisms play an important role in the response of cGAS-STING pathway activation\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In this study, we found that MN formation was elevated after DOX treatment in cardiac fibroblasts. The pharmacological inhibition of STING with C-176 and the downregulation of STING by siRNA significantly alleviated DOX-induced cardiac short- and long-term cardiac dysfunctions. In addition, we demonstrated that the cardiac fibroblast nuclear DNA, which escaped into the cytoplasm under DOX treatment, could promote cardiac fibroblast accumulation of p-STAT1 and NF-κB in the nucleus via the cGAS-STING signaling pathway. The STING inhibition by C-176 and the STING downregulation by siRNA significantly decreased the p-STAT1 and NF-kB nuclear accumulation and alleviated cardiac inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e,\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSTING is an intracellular signaling protein that senses cGAMP upon stimulation, leading to the production of IFNs and other inflammatory mediators\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Research on the role of the cGAS-STING pathway in CVDs has grown in recent years \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. However, relatively little is known about the role of the STING- STAT1 pathway in the pathological process of chronic DIC. In this study, we demonstrated the activation of the STING-STAT1 pathway in chronic DIC. Previous work by Wei Luo and colleagues showed that the cGAS-STING pathway was activated in endothelial cells following DOX treatment\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. They also revealed that the cGAS-STING pathway was upregulated in cardiomyocytes after the treatment\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Meanwhile, other researchers showed that the level of the cGAS-STING pathway in cardiomyocytes was activated post-DOX treatment\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Our study adds to this body of work by demonstrating that the MN formation triggered by DOX treatment during DIC activated the cGAS-STING pathway in cardiac fibroblasts. Following DOX treatment, this pathway triggered the phosphorylation of STAT1, a key signaling molecule involved in inflammatory responses. These findings suggest that the cGAS-STING pathway plays a critical role in chronic DIC through its activation in cardiac fibroblasts and the subsequent phosphorylation of STAT1. This pathway may represent a novel target for the treatment of chronic DIC and other inflammatory CVDs.\u003c/p\u003e \u003cp\u003eC-176, a highly selective small molecular inhibitor of STING, is widely used to block the STING-dependent pathway\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Recent studies have shown that C-176 can effectively attenuate DOX-induced cardiac dysfunction\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. However, the exact molecular mechanisms of STING in cardiac local inflammation in DIC are not fully understood. To further investigate the biological mechanisms of C-176 in attenuating DOX-induced cGAS-STING-STAT1 pathway activation in cardiac fibroblasts, we administered C-176 via intraperitoneal injection in our study. The pharmacological inhibition of STING with C-176 significantly attenuated DIC, thereby increasing the EF and FS in DOX-induced cardiac dysfunction. The inhibition of STING with C-176 also alleviated the increased level of p-STAT1 and the cardiac local inflammation in vitro and in vivo.\u003c/p\u003e \u003cp\u003eAnother significant finding from this study was that STING played a mediating role in the inflammatory response through the activation of the NF-κB signaling pathways within cardiac fibroblasts. STING mediated the nuclear accumulation of NF-κB following immune responses. We speculated that STING may influence the immune response in cardiac fibroblasts following DOX treatment through the activation of NF-κB pathways. Our results also indicated that the phosphorylation of STAT1 (p-STAT1) was increased after DOX treatment, which was suppressed by C-176 and STING siRNA administration. The downregulation of p-STAT1 was associated with a reduction in fibrosis following DOX treatment.\u003c/p\u003e \u003cp\u003eRecent studies have suggested that targeting the cGAS-STING pathway may represent a therapeutic strategy for mitigating cardiac dysfunction following ischemic heart diseases\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, chemotherapeutic-induced cardiotoxicity\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, or myocardial infarction\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. cGAMP binds to the endoplasmic reticulum, promoting STING dimerization. Subsequently, STING recruits and initiates the IRF3-dependent innate immune response, leading to the production of interferons and an NF-κB-dependent inflammatory response\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. This condition results in the creation of proinflammatory factors, including TNF-a and IL-1. Our results indicated that the activation of p-STAT1 was dependent on the increased expression of p-STING in cardiac fibroblasts following DOX treatment. The inhibition of the STING downregulated the STING-STAT1 pathway, thereby attenuating DIC. Therefore, the cGAS-STING-STAT1 pathway triggered by DNA damage-induced MN formation in cardiac fibroblasts during DIC may represent the potential therapeutic target to treat DIC.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study revealed that the cGAS-STING-STAT1 pathway played an important role in DIC by stimulating the MN in cardiac fibroblasts. Furthermore, the inhibition of STING mitigated DOX-induced cardiac dysfunction. These findings suggest that the cGAS-STING-STAT1 pathway triggered by MN may serve as a potential therapeutic target for the prevention of CTRCD.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnimal study design\u003c/h2\u003e \u003cp\u003e All animal experiments were conducted following the guidelines of animal welfare and approved by the Animal Care and Use Committee of Guangzhou Medical University (GY2023-108).\u003c/p\u003e \u003cp\u003eIn the first acute experiment, mice aged 6\u0026ndash;8 weeks were purchased from GemPharmatech Co., Ltd., China and raised at the Guangzhou Medical University Laboratory Animal Center. The mice were randomly divided into saline and DOX groups. The DOX group was injected intravenously with 10 mg/kg of DOX (S1280, Selleck, China). Different groups of mice were harvested at 8, 16, 24, 48, and 72 h after injection. The saline group was injected intravenously with the same dosage of saline.\u003c/p\u003e \u003cp\u003eIn the second chronic STING inhibitor C-176 experiment, C57BL/6 male mice aged 6\u0026ndash;8 weeks were randomly divided into Saline\u0026thinsp;+\u0026thinsp;Corn Oil, Saline\u0026thinsp;+\u0026thinsp;C-176, DOX\u0026thinsp;+\u0026thinsp;Corn Oil, and DOX\u0026thinsp;+\u0026thinsp;C-176 groups. Mice were injected intravenously with 5 mg/kg of saline or DOX once a week and injected intraperitoneally with 10 mg/kg of corn oil or C-176 (S6575, Selleck, China) dissolved in DMSO and diluted in corn oil at the same time. After 4 weeks, the treatment was stopped for 1 week.\u003c/p\u003e \u003cp\u003eIn the third chronic Sting1 siRNA experiment, Sting1 siRNA (5\u0026prime;-GGAGCCGAAGACTGTACAT-3\u0026prime;) was purchased from Guangzhou RiboBio Co., Ltd. C57BL/6 male mice aged 6\u0026ndash;8 weeks were randomly divided into NC\u0026thinsp;+\u0026thinsp;NS, NC\u0026thinsp;+\u0026thinsp;DOX, and Sting1 siRNA\u0026thinsp;+\u0026thinsp;DOX groups. Mice were injected intravenously with 5 mg/kg of NS or DOX once a week and injected intravenously with 10 nmol NC or Sting1 siRNA twice a week before DOX treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEchocardiography\u003c/h2\u003e \u003cp\u003eBefore treating with DOX, mice were subjected to transthoracic echocardiography (Vevo 2100 Imaging System, VisualSonics, Canada) to obtain baseline data. Then, every week before injecting, mice were also subjected to echocardiography to obtain cardiac function data. The body temperature of mice was maintained at 36.9\u0026deg;C\u0026ndash;37.3\u0026deg;C, while the heart rate was maintained at 550\u0026ndash;650 bpm. Echocardiographic M-mode tracing records were used to measure cardiac structure, and at least five cardiac cycles were utilized to calculate the left ventricular EF (LVEF) and the left ventricular FS (LVFS). The measurement application was Vevo Lab.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHistological and immunofluorescence analyses\u003c/h2\u003e \u003cp\u003eOne week after the DOX treatment for 4 weeks, mice were sacrificed, and hearts were fixed with 4% paraformaldehyde for 24 h at 4\u0026deg;C, dehydrated with a gradient concentration of ethanol, embedded with paraffin, and sectioned. Tissue sections were stained with Masson\u0026rsquo;s trichrome immunohistochemical reagent and Picro-Sirius Red Solution to analyze cardiac fibrosis. To observe inflammatory cell infiltration, immunohistochemistry analysis was performed on the above paraffin sections with IL-1β antibody (11E5; Santa Cruz Biotechnology; Cat#sc-52012; 1:100).\u003c/p\u003e \u003cp\u003eMice were harvested at the end point of the experiment, and hearts were fixed with 4% paraformaldehyde for 24 h at 4\u0026deg;C, dehydrated in 30% sucrose solution for 24 h at 4\u0026deg;C, embedded with OCT, and sectioned to acquire 5 \u0026micro;m frozen sections for immunofluorescence analysis. The above frozen sections were blocked and permeabilized for 45 min in a blocking buffer, comprising 1% bovine serum albumin and 0.3% Triton X-100 in PBS. Slides were incubated with primary antibodies diluted in the blocking buffer for 12 h at 4\u0026deg;C and with secondary antibodies diluted in the blocking buffer for 45 min at 37\u0026deg;C. A DAPI staining solution (Beyotime; Cat#C1006) was used for staining for 10 min at room temperature. The following antibodies were applied: anti-phosphor-Histone H2A.X (Ser139) rabbit antibody (20E3; Cell Signaling Technology; Cat#9718; 1:100), anti-cGAS rabbit antibody (D3O8O; Cell Signaling Technology; Cat#31659; 1:100), anti-phosphor-STAT1 (Ser727) rabbit antibody (SN67-04; HUABIO; Cat#ET1611-20; 1:100), anti-NF-κB rabbit antibody (D14E12; Cell Signaling Technology; Cat#8242T; 1:100), goat anti-rabbit IgG DyLight 488 (Abbkine; Cat#A23220; 1:200), goat anti-mouse IgG DyLight 488 (Abbkine; Cat#A23210; 1:200), goat anti-rabbit IgG DyLight 594 (Abbkine; Cat#A23420; 1:200), and goat anti-mouse IgG DyLight 594 (Abbkine; Cat#23410; 1:200).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot assay\u003c/h2\u003e \u003cp\u003eHeart tissue and cells were collected with RIPA lysis buffer (Thermo Fisher Scientific; Cat#89901) containing protease and phosphatase inhibitor (Thermo Fisher Scientific; Cat#78442), fully ground, and vortexed for complete total protein lysis. The protein was separated on 8\u0026ndash;15% SDS-polyacrylamide gels by electrophoresis and transferred to 0.45 \u0026micro;m PVDF membranes. After blocking with 5% nonfat milk diluted in TBST for 45 min at room temperature, the membranes were incubated with primary antibodies diluted in a blocking buffer for 12 h at 4\u0026deg;C and then incubated with an HRP-conjugated secondary antibody diluted in a blocking buffer for 45 min at room temperature. SuperSignal West Pico PLUS (Thermo Fisher Scientific; Cat#34580) was used to detect by Amersham Imager 600. The following antibodies were used: anti-phosphor-Histone H2A.X (Ser139) rabbit antibody (20E3; Cell Signaling Technology; Cat#9718; 1:1000), anti-Histone H2A.X rabbit antibody (D17A3; Cell Signaling Technology; Cat#7631; 1:1000), anti-cGAS rabbit antibody (D3O8O; Cell Signaling Technology; Cat#31659; 1:1000), anti-cGAS rabbit antibody (HUABIO; Cat#HA500023; 1:1000), anti-phosphor-TMEM173/STING (Ser366) rabbit antibody (Affinity Biosciences; Cat#AF7416; 1:1000), anti-STING rabbit antibody (D2P2F; Cell Signaling Technology; Cat#13647; 1:1000), anti-phosphor-TBK1/NAK (Ser172) rabbit antibody (D52C2; Cell Signaling Technology; Cat#5483; 1:1000), anti-TBK1/NAK rabbit antibody (E813G; Cell Signaling Technology; Cat#38066; 1:1000), anti-phosphor-IRF3 (Ser396) rabbit antibody (D6O1M; Cell Signaling Technology; Cat#29047; 1:1000), anti-IRF3 rabbit antibody (D83B9; Cell Signaling Technology; Cat#4302; 1:1000), anti-phosphor-STAT1 (Ser727) rabbit antibody (SN67-04; HUABIO; Cat#ET1611-20; 1:1000), and anti-STAT1 rabbit antibody (HUABIO; Cat#ET1606-39; 1:1000).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eNucleocytoplasmic separation\u003c/h2\u003e \u003cp\u003eHeart tissue was ground with a grinding rod in CER Ⅰ reagent in a nucleocytoplasmic separation kit (Thermo Fisher Scientific; Cat#78833), then CER Ⅱ was added in tubes. After vortex and centrifugation, the supernatant was aspirated, and cytoplasm protein was extracted and collected. The precipitate was washed in PBS, and NER reagent was added into tubes. After vortex and centrifugation, nucleus protein could be extracted. Afterward, the protein expression levels could be detected by Western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCell isolation, culture, and stimulation\u003c/h2\u003e \u003cp\u003eCardiac fibroblasts were isolated by digesting neonatal SD rat ventricular tissue with Trypsin without EDTA (Thermo Fisher Scientific; Cat#15050065) and type II collagenase (MP Biomed; Cat#100502), filtrated through a 70 \u0026micro;m cell strainer, and purified by differential anchoring velocity. Primary cardiac fibroblasts were resuspended in high glucose DMEM with 10% FBS for 24 h and passaged to the second generation.\u003c/p\u003e \u003cp\u003eDOX powder was dissolved in sterile water and diluted to 1 mM. The STING inhibitor C-176 was dissolved in DMSO and diluted to 1, 2, and 4 mM. When the cell density reached 70% of the dish, cells in the logarithmic growth phase were treated with 1 \u0026micro;M DOX for 2, 4, 8, 16, and 24 h. To inhibit the pathway, 2 \u0026micro;M DOX and 1, 2, and 4 \u0026micro;M STING inhibitor C-176 were used for different groups of cardiac fibroblasts; two control groups for DMSO and 4 \u0026micro;M C-176 were also set up to control the influence of dissolvent.\u003c/p\u003e \u003cp\u003eWhen the cell density of cardiac fibroblasts reached 50% of the dish, Sting1 siRNA (F: 5\u0026prime;-CAACAGUGUCUAUGAACUUTT-3\u0026prime;; R: 5\u0026prime;-AAGUUCAUAGACACUGUUGTT-3\u0026prime;) was transfected into cardiac fibroblasts by transfection reagent RNA iMAX (Thermo Fisher Scientific; Cat#13778030). After transfecting in Opti-MEM (Gibco; Cat#31985070) without FBS for 6 h, cells were continuously cultured in a high glucose DMEM culture medium with 10% FBS for 16 h and then treated with 1 \u0026micro;M DOX for 8 h.\u003c/p\u003e \u003cp\u003eThe above cells were collected in RIPA, and the cell crawling slides were fixed with 4% PFA and then kept at \u0026minus;\u0026thinsp;20\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM). Data were analyzed with GraphPad Prism 9.0 software (GraphPad, San Diego, CA). The student \u003cem\u003et\u003c/em\u003e test (2 tailed) was performed to compare 2 groups. If SD un-equation with Welch\u0026rsquo;s correction. One-way ANOVA followed by the Tukey post hoc test was used to compare multiple groups. A value of \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported in part by the open research funds from the Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People's Hospital (grant No. 202201-203), the Guangzhou Science and Technology Bureau Project (grant No. G24151045), Guangzhou Medical University Research and Innovation Ability Enhancement Project (grant No. 02-410-2405004, 02-410-2302358XM).\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eXY, WJ and NH were involved in designed and guarantors of the project. XY, WT and YQ were involved to the performance of the experiments, data analysis and wrote the manuscript. RJ, GZ, XL, YL and QZ participated in data analysis and interpretation. XY, WJ and NH supervised the work and revised the manuscript.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eThis work was supported in part by Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People's Hospital (grant No. 202201-203), the Guangzhou Science and Technology Bureau Project (grant No. G24151045), Guangzhou Medical University Research and Innovation Ability Enhancement Project (grant No. 02-410-2405004, 02-410-2302358XM). We are grateful to Prof. Jiandong Luo at the Guangzhou Medical University for his kind guidance during the research. We are also very grateful to our colleagues at the Department of Pharmacology in Guangzhou Medical University for their kind guidance during the use of some experimental instruments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCarvalho, C., Santos, R.X., Cardoso, S., Correia, S., Oliveira, P.J., Santos, M.S., et al.: Doxorubicin: the good, the bad and the ugly effect. Curr. Med. Chem. \u003cb\u003e16\u003c/b\u003e, 3267\u0026ndash;3285 (2009)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSongbo, M., Lang, H., Xinyong, C., Bin, X., Ping, Z., Liang, S.: Oxidative stress injury in doxorubicin-induced cardiotoxicity. Toxicol. Lett. \u003cb\u003e307\u003c/b\u003e, 41\u0026ndash;48 (2019)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYeh, E.T.: Cardiotoxicity induced by chemotherapy and antibody therapy. Annu. Rev. 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Immunol. \u003cb\u003e17\u003c/b\u003e, 1142\u0026ndash;1149 (2016)\u003c/span\u003e\u003c/li\u003e\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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Doxorubicin, Cardiotoxicity, STING, MN","lastPublishedDoi":"10.21203/rs.3.rs-4253972/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4253972/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDoxorubicin (DOX) is a common clinical chemotherapeutic drug. However, DOX-induced cardiotoxicity (DIC) limits the wide and long-term clinical use to treat cancers. This study aims to dissect the mechanism in which DNA damage-triggered micronucleus (MN) formation activates the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-STAT1 pathway in cardiac fibroblasts during DIC. C57BL/6J mice were intravenously injected with 10 mg/kg of DOX to establish an acute DOX-induced cardiac injury mouse model. Meanwhile, C57BL/6J mice were intraperitoneally injected with STING inhibitor C-176 (10 mg/kg/week) or intravenously injected with STING siRNA (10 nM/week) prior to DOX (5 mg/kg/week) intravenous injection for 4 weeks to establish a chronic DIC mouse model. After 1 week of Dox injection, mice were harvested for further analysis. Measurements included echocardiography, immunohistochemical analyses, Masson and Sirius Red staining, and Western blots. Here, we showed that the cGAS-STING-STAT1 pathway was activated in cardiac fibroblasts during DIC. The STING inhibition by C-176 or the STING knockdown via siRNA in DOX-induced chronic cardiotoxicity mouse heart attenuated the DOX-induced cardiac dysfunction, cardiac fibrosis, and the inflammatory response. Mechanistically, we also demonstrated that the DOX-induced DNA damage-triggered MN formation impaired the nuclear stability, initiating the activation of the cGAS-STING-STAT1 pathway in cardiac fibroblasts during DIC. Our study illustrated that the activation of the cGAS-STING-STAT1 pathway initiated by DOX-induced DNA damage and MN formation stimulated proinflammatory responses in cardiac fibroblasts, thus promoting myocardial fibrosis during DIC.\u003c/p\u003e","manuscriptTitle":"Inhibit of the cGAS-STING-STAT1 pathway protects heart from the Doxorubicin-induced cardiotoxicity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-04 08:10:10","doi":"10.21203/rs.3.rs-4253972/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fbfa17d9-5c2c-4027-b03f-cf65f7344b35","owner":[],"postedDate":"June 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":31303568,"name":"Health sciences/Diseases/Cardiovascular diseases"},{"id":31303569,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2024-06-04T08:10:11+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-04 08:10:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4253972","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4253972","identity":"rs-4253972","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}