Exercise improves doxorubicin-induced cardiotoxicity by regulating mitochondrial quality control through activation of the SIRT1-FOXO1 pathway

Exercise improves doxorubicin-induced cardiotoxicity by regulating mitochondrial quality control through activation of the SIRT1-FOXO1 pathway | 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 Exercise improves doxorubicin-induced cardiotoxicity by regulating mitochondrial quality control through activation of the SIRT1-FOXO1 pathway Jiawei Fang, Qiaohui Ren, Yichao Liu, Cuomu Danzeng, Wenya Qiang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8426076/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Doxorubicin (DOX) is a highly effective broad-spectrum chemotherapeutic agent, yet its clinical application is severely limited by dose-dependent cardiotoxicity, for which preventive strategies are lacking. Exercise has emerged as a promising non-pharmacological intervention; its cardioprotective effects may involve silent information regulator 1 (SIRT1), though the precise mechanisms remain unclear. This study aimed to investigate the effect of exercise intensity on DOX-induced cardiotoxicity through the SIRT1-FOXO1 pathway and mitochondrial homeostasis. Using a murine model of DOX-induced cardiac injury, we implemented exercise regimens of varying intensities. Results demonstrated that moderate-intensity exercise (MIE) significantly attenuated DOX-induced cardiac dysfunction, apoptosis, and oxidative stress, while favorably regulating proteins essential for mitochondrial biogenesis and dynamics.Mechanistically, MIE activated myocardial SIRT1, leading to deacetylation of FOXO1, which enhanced antioxidant defenses and conferred cardioprotection. Notably, low-intensity exercise (LIE) offered only modest benefits, whereas high-intensity exercise (HIE) exacerbated myocardial oxidative stress and injury.Collectively, our findings establish that MIE alleviates DOX-induced cardiotoxicity by activating the SIRT1-FOXO1 pathway and restoring mitochondrial homeostasis, thereby providing a critical experimental foundation for developing tailored exercise regimens to prevent chemotherapy-related cardiac dysfunction.. Health sciences/Cardiology Biological sciences/Cell biology Biological sciences/Drug discovery Biological sciences/Physiology Cardiotoxicity Doxorubicin Mitochondrial quality control Exercise intensity SIRT1-FOXO1 pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction Doxorubicin (DOX) has become a vital treatment for specific cancers, such as certain childhood leukemias and breast cancer, due to its broad-spectrum and highly effective anticancer properties 1 , 2 . However, its inherent dose-dependent toxicity can affect multiple organs, including the heart, liver, kidneys, and gonads, thereby severely limiting the drug's clinical application potential 3 , 4 . DOX-induced cardiotoxicity is due to persistent cardiomyocyte damage, which may ultimately lead to congestive heart failure. Currently, the mechanism by which DOX affects cardiac function remains incompletely elucidated; however, it is known to be driven by multiple factors, including mitochondrial dysfunction, oxidative stress, apoptosis, and impaired autophagy flux 5 . Dexrazoxane is currently the only drug approved by the FDA for mitigating DOX-induced cardiotoxicity. However, its clinical application is subject to strict limitations due to the potential for reduced tumor sensitivity and an increased risk of bone marrow suppression 6 . In this context, exploring safe and effective non-pharmacological intervention strategies holds significant clinical significance for preventing or mitigating DOX-related cardiac injury and enhancing treatment safety for patients. Silent Information Regulator 1 (SIRT1) is an NAD+-dependent deacetylase that participates in critical physiological processes, such as cellular senescence, oxidative stress, and energy metabolism, by regulating multiple key targets 7 , 8 . SIRT1 is primarily localized within the cell nucleus, but under specific conditions, it can translocate to the cytoplasm, thereby regulating its downstream target, forkhead box protein O1 (FOXO1). Mitochondrial quality control (MQC) is a crucial mechanism for maintaining the integrity and function of the intracellular mitochondrial network, primarily involving processes such as mitochondrial biogenesis, dynamics (fusion and fission), and mitochondrial autophagy 9 . The SIRT1-FOXO1 pathway plays a central regulatory role in maintaining MQC by coordinating the aforementioned processes. Specifically, SIRT1-mediated deacetylation of FOXO1 enhances its transcriptional activity on downstream targets such as peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), superoxide dismutase (SOD), and microtubule-associated protein light chain 3 (LC3), thereby promoting mitochondrial biogenesis, reactive oxygen species (ROS) neutralisation, and the clearance of damaged organelles 10 , 11 . Given the pivotal role of the SIRT1-FOXO1 pathway in sustaining MQC, we hypothesize that specific activation of this pathway could effectively ameliorate DOX-induced mitochondrial dysfunction. As a non-pharmacological intervention, exercise plays a significant role in the prevention and management of cardiovascular disease. Its cardioprotective effects have been demonstrated to be closely associated with improvements in MQC, a process involving multifaceted regulatory mechanisms 12 . Exercise promotes mitochondrial biogenesis by activating the PGC-1α signaling pathway, thereby increasing the number and membrane surface area of mitochondria 13 , 14 . Concurrently, exercise enhances endogenous antioxidant defenses, reducing excessive ROS accumulation and mitigating the oxidative damage it induces 15 . These mechanisms act synergistically to maintain mitochondrial homeostasis and safeguard normal cardiac function. Notably, the cardioprotective effects of exercise are influenced by multiple factors, with exercise intensity being a significant factor. Low-intensity swimming effectively enhances skeletal muscle PGC-1α mRNA expression in SD rats 16 . In comparison, MIE produces a more sustained elevation in PGC-1α mRNA expression, which significantly exceeds the transient and limited increase induced by HIE 17 , 18 . Therefore investigating the differential regulatory mechanisms of exercise intensity on MQC and its downstream pathways holds significant importance for elucidating precise protective strategies against cardiovascular disease. This study established a DOX-induced cardiac injury mouse model to systematically examine the cardioprotective effects of exercise and compare the effects of different exercise intensities. The research further focused on the SIRT1-FOXO1 signalling pathway and the MQC mechanism, aiming to elucidate their potential roles and provide a scientific foundation for the precise clinical application of exercise therapy. 2 Result 2.1 Effects of different exercise intensities on DOX-induced cardiac function and structure in mice This study first established a DOX-induced mouse cardiac injury model (Fig. 1 A). Analysis of physiological parameters revealed that DOX treatment significantly reduced mouse heart weight (HW), body weight (BW), and the heart-to-body weight ratio (HW/BW) compared to the control group (Fig. 1 B-E). All exercise interventions effectively prevented DOX-induced decrease in heart weight (Fig. 1 B). Echocardiographic assessment revealed that both LVEF and LVFS were significantly lower in the DOX-treated group compared to the control group. In the exercise intervention group, LIE showed no significant improvement in cardiac function, whereas MIE markedly improved LVEF and LVFS in DOX-treated mice. Conversely, HIE further reduced both LVEF and LVFS (Fig. 1 F). Histopathological analysis further revealed marked ventricular dilatation accompanied by vacuolar degeneration of cardiomyocytes in the DOX-only group. No significant dilatation or vacuolar degeneration was observed in the right ventricles of any exercise intervention group (Fig. 1 G). Meanwhile, H&E staining revealed that single DOX treatment disrupted myocardial tissue architecture, whereas myocardial cells in the exercise groups exhibited a tighter and more orderly arrangement. Masson staining results revealed that DOX treatment significantly increased the area of myocardial fibrosis, while MIE effectively reduced this pathological change. No significant improvement was observed in the LIE and HIE groups (Fig. 1 H-I). 2.2 Effects of different exercise intensities on DOX-induced myocardial oxidative stress and apoptosis First, we measured ROS levels in myocardial tissue. The results showed that DOX treatment significantly increased myocardial ROS production. In contrast, MIE effectively reduced this effect, suggesting that MIE may exert its action by enhancing the endogenous antioxidant defense system. Although LIE and HIE partially alleviated ROS accumulation, their effects were not significant (Fig. 2A-B), reflecting differences in redox regulation among varying exercise intensities. In terms of energy metabolism, DOX significantly reduced myocardial ATP content, suggesting mitochondrial dysfunction. MIE intervention significantly restored ATP levels, thereby improving mitochondrial oxidative phosphorylation efficiency and energy metabolism balance, whereas the LIE and HIE groups showed no significant improvement (Fig. 2C). To further evaluate apoptosis induced by oxidative damage, we employed TUNEL staining for detection. Results showed that DOX significantly increased the apoptosis rate of cardiomyocytes, while MIE effectively inhibited this process. In contrast, the number of apoptotic cells in the LIE and HIE groups remained significantly higher than that in the control group (Fig. 2D-E), indicating their weaker anti-apoptotic effects. In summary, MIE effectively alleviates DOX-induced myocardial oxidative stress, improves mitochondrial function, and inhibits apoptosis, whereas LIE and HIE did not demonstrate significant protective effects in these aspects. 2.3 Effects of different exercise intensities on DOX-induced MQC imbalance The results in Fig. 3A show that DOX treatment led to an abnormal increase in myocardial mitochondrial number, a fragmented morphology, and the accumulation of autophagic vacuoles. MIE effectively reversed these morphological abnormalities. Although autophagic vacuoles decreased in the LIE and HIE groups, they remained higher than in the control group, suggesting that MIE may promote expected degradation during autophagy. Further Western blot analysis was used to measure the expression of proteins related to mitochondrial dynamics. In the DOX group, mitochondrial fusion proteins MFN1 and MFN2 showed decreased expression, indicating suppression of the fusion process. Both MIE and HIE restored their protein expression to normal levels, whereas LIE did not exhibit a significant reversal effect (Fig. 3B-C). Concurrently, DOX induces increased expression of the fission regulatory protein DRP1, indicating that DOX promotes the fission process, ultimately disrupting the fusion-fission equilibrium and shifting it toward the fission state. Additionally, DOX suppressed the expression of PGC-1α, a key factor in mitochondrial biogenesis, and upregulated the levels of PARK2, an autophagy-related protein. MIE effectively reversed these abnormal protein expressions (Fig. 3D-E). In summary, the results of this study indicate that MIE can effectively improve the MQC imbalance caused by DOX, while LIE and HIE did not demonstrate the same degree of protective effect. 2.4 MIE activates the SIRT1-FOXO1 pathway in DOX-damaged cardiomyocytes This study compared the protein expressions of SIRT1 and FOXO1 in the myocardial tissues of five groups of experimental mice. Western blot results showed that DOX treatment significantly reduced the protein expression levels of SIRT1 and FOXO1 (Fig. 4A-B), suggesting that it may inhibit the activity of the SIRT1-FOXO1 pathway, thereby affecting downstream oxidative stress and metabolic regulatory functions. In the exercise intervention group, MIE effectively reversed DOX-induced suppression of SIRT1 and FOXO1 expression, while HIE also showed some upregulating effects. Conversely, LIE further reduced the expression of both proteins. Further assessment of FOXO1 acetylation levels revealed that DOX significantly increased FOXO1 acetylation, while MIE intervention markedly suppressed this excessive acetylation phenomenon. LIE and HIE showed no significant ameliorative effects. The results indicate that MIE effectively activates the SIRT1-FOXO1 signaling pathway in DOX-damaged cardiomyocytes, whereas LIE and HIE exert limited regulatory effects on this pathway. 3 Discussion As a widely used anthracycline antibiotic, DOX exhibits significant antitumor effects, but its severe cardiotoxicity limits its clinical application 3 . Therefore, identifying strategies that can effectively mitigate DOX-induced cardiac damage has become a significant focus of research in this field. The main findings of this study include: (1) MIE significantly ameliorates DOX-induced cardiac dysfunction and myocardial structural abnormalities; (2) MIE exerts its cardioprotective effects by modulating MQC; (3) The SIRT1-FOXO1 axis is a key pathway through which MIE mitigates DOX-induced cardiotoxicity. Our study confirms that exercise intervention can alleviate DOX-induced cardiotoxicity, with exercise intensity being the key determinant of its efficacy. MIE significantly ameliorates DOX-induced cardiac dysfunction and myocardial structural abnormalities, while LIE yields limited benefits. HIE did not demonstrate any improvement in cardiac function. This finding aligns with existing research on the critical role of exercise intensity. Notably, while long-term regular exercise has been proven to exert cardiovascular protective effects through multiple mechanisms 19 and even improve the prognosis of heart failure patients 20 , exercise intensity remains the core factor determining both its protective efficacy and safety. In particular, HIE may induce myocardial ischemia by substantially increasing myocardial oxygen consumption. Simultaneously, it enhances oxygen metabolism, leading to excessive ROS production and lactic acid accumulation, which in turn triggers oxidative damage and apoptosis 21 , ultimately resulting in myocardial structural injury 22 . The findings of this study further reinforce the understanding that in the DOX-induced cardiotoxicity model, MIE demonstrated the most beneficial effects in alleviating oxidative stress and reducing apoptosis, confirming the critical role of exercise intensity. In summary, the alleviation of DOX-induced cardiotoxicity through exercise intervention is related to exercise intensity, and MIE is considered an intervention strategy with greater cardioprotective potential. Another finding of this study is that MIE improves DOX-induced cardiotoxicity by regulating MQC. Mitochondrial dysfunction is one of the core mechanisms underlying DOX-induced cardiotoxicity. Studies have demonstrated that DOX exerts its antitumor effects through sustained activation of the Hippo pathway, while simultaneously inducing significant myocardial mitochondrial damage. This mechanism constitutes a key basis for its adverse cardiac effects 23 . This study observed a decrease in myocardial ATP synthesis capacity, disruption of mitochondrial ultrastructure, and dysregulation of key MQC proteins such as PGC-1α, MFN1, MFN2, and DRP1 following DOX treatment, consistent with previous research reports 24 – 26 . Notably, we observed downregulation of mitochondrial fusion proteins MFN1/2 and upregulation of fission protein DRP1 following DOX treatment, indicating impaired mitochondrial fusion and excessive activation of fission. This imbalance leads to the fragmentation of the mitochondrial network and induces respiratory chain uncoupling, ultimately impairing the efficiency of energy metabolism 27 . These findings suggest that DOX disrupts mitochondrial dynamics by promoting fission and inhibiting fusion. MIE effectively reverses these abnormal protein expressions, restoring the structural and functional integrity of mitochondria, suggesting that its cardioprotective effects are closely associated with reestablishing mitochondrial dynamic equilibrium. In summary, MIE ultimately disrupts the vicious cycle between DOX-induced mitochondrial dysfunction and myocardial injury by regulating the MQC balance. This study further clarifies that the activation of the SIRT1-FOXO1 pathway plays a core regulatory role in the process of MIE improving the cardiotoxicity of DOX. Experimental results indicate that DOX treatment significantly suppresses SIRT1 expression and increases FOXO1 acetylation levels, whereas MIE effectively reverses these changes, restoring pathway activity. Further mechanistic studies indicate that MIE promotes mitochondrial biogenesis mediated by PGC-1α, mitochondrial fusion involving MFN1/2, and PARK2-associated autophagic clearance by activating the SIRT1-FOXO1 pathway, thereby synergistically restoring mitochondrial functional integrity. The findings are highly consistent with previous studies, indicating that SIRT1, as a key regulator of energy metabolism and oxidative stress, is crucial for maintaining MQC 28 . For instance, Yao et al. found that SIRT1 regulates mitophagy to promote the clearance of damaged mitochondria 29 . Mitochondria are highly dynamic organelles, and the dynamic equilibrium between mitochondrial fusion and fission is a crucial mechanism for maintaining mitochondrial homeostasis 30 , 31 . Ding M et al. demonstrated that SIRT1 activates PGC-1α and inhibits DRP1-mediated excessive fission 32 . Furthermore, SIRT1 also modulates the PGC-1α/PDK2/PARL axis to promote mitochondrial fusion 33 . These studies collectively demonstrate that SIRT1 plays multiple roles in coordinating mitochondrial homeostasis. Our research further confirms that MIE reverses DOX-induced myocardial MQC imbalance by activating the upstream SIRT1-FOXO1 pathway, which regulates mitochondrial biogenesis, fusion-fission, and autophagy processes. The innovation of this study lies in systematically elucidating the role of different exercise intensities in regulating MQC through the SIRT1-FOXO1 pathway in DOX-induced cardiotoxicity, confirming MIE as the optimal intervention intensity. This discovery not only deepens our understanding of the protective mechanisms of exercise on the heart but also provides a theoretical basis and potential intervention targets for clinically preventing and improving chemotherapy-related cardiac dysfunction. However, it is worth noting that our study still has some limitations. First, the SIRT1-FOXO1 signaling pathway may not be the sole mechanism through which exercise affects mitochondria; exercise may also exert effects via other signaling pathways, which warrants further investigation. Second, we subjected mice to endurance training at varying intensities, but the effects of different intensities of resistance training remain unknown. Future studies could incorporate resistance training at different intensities and combine it with more comprehensive exercise physiology metrics to provide more refined exercise protocols. Despite these limitations, our findings still suggest potential therapeutic strategies for DOX-induced cardiotoxicity. 4 Conclusion In summary, our findings indicate that MIE significantly ameliorates DOX-induced cardiotoxicity, with a protective effect superior to that of LIE and HIE. Mechanistically, MIE effectively alleviates DOX-induced MQC imbalance by activating the SIRT1-FOXO1 signaling pathway. These findings support the beneficial role of exercise in mitigating DOX-related cardiac toxicity and provide a basis for optimizing exercise protocols for patients undergoing anthracycline-based chemotherapy. 5 Materials and methods 5.1 Animals and treatments Eight-week-old C57BL/6 male mice were purchased from the Animal Experiment Center of Xi'an Jiaotong University. They were housed in an SPF environment (temperature: 20–25°C, humidity: 50%) with free access to water and food. One week of adaptive feeding preceded the experiment, followed by random assignment to five groups: control group (Ctrl), doxorubicin model group (DOX), doxorubicin + low-intensity exercise group (DOX + LIE), doxorubicin + moderate-intensity exercise group (DOX + MIE), and doxorubicin + high-intensity exercise group (DOX + HIE). The intervention period for all mice was 4 weeks. Control group mice received weekly intraperitoneal injections of saline (5 mg/kg). In comparison, mice in the other groups received intraperitoneal injections of DOX at the same dose (5 mg/kg) 34 . Echocardiography was used to assess the success of modeling. All experimental procedures were approved by the Experimental Center of Shaanxi Provincial People's Hospital. 5.2 Exercise protocols The FT-201 small animal treadmill was used with electrical stimulation parameters set at 25 V, 0.34 mA, and 2 Hz 35 . The exercise capacity testing protocol was as follows: initial speed, 8.5 m/min; incline, 0°; duration, 9 min. Then, increase the speed to 10 m/min, incline to 5°, and maintain for 3 minutes. Subsequently, increase speed by 2.5 m/min every 3 min (maximum 40 m/min) and incline by 5° every 9 min (maximum 15°). Fatigue Criteria (meet any one of the criteria): Remaining on the grid for ≥ 10 seconds; Time spent on grid > 50%; Transitioning from normal posture to prone position with hind limbs trailing for > 30 seconds. Record speed, slope, and total running distance during fatigue episodes. Based on test results, mice were assigned to different exercise intensity groups (LIE: 55–60% Vmax; MIE: 75% Vmax; HIE: 90% Vmax). They exercised 5 days a week: 40 minutes/day during weeks 1–2, 50 minutes/day during weeks 3–4, and 60 minutes/day during weeks 5–8. Control mice received routine care without exercise intervention. All mice were deeply anesthetized with 5% isoflurane(26675-46-7, MCE) 24 hours after the conclusion of the experiment until they lost the righting reflex and responded to toe pinch. Euthanasia was then promptly performed by cervical dislocation. Cardiac tissue samples were collected for subsequent analysis. 5.3 Echocardiography Cardiac structure and function were assessed using echocardiography (Vevo 3100) prior to exercise intervention and 8 weeks after exercise. Parameters measured: Left ventricular end-diastolic diameter (LVIDd), Left ventricular end-systolic diameter (LVIDs), Left ventricular systolic posterior wall thickness (LVPWs), Left ventricular diastolic posterior wall thickness (LVPWd), Ejection fraction (EF%), Fractional shortening (FS%). 5.4 Histological analysis The heart specimens were fixed in 4% paraformaldehyde, followed by dehydration with graded ethanol, clearing with xylene, and embedding in paraffin. Sections were dewaxed with xylene and hydrated with graded ethanol solutions, then stained using haematoxylin and eosin (H&E), Masson's trichrome, and TUNEL staining kits. Stained sections were embedded in neutral resin, examined under an optical microscope, and imaged using a digital pathology slide scanner (LG-S80). Scan and view using Saiviewer-1.0.9 software. Quantify Masson-stained sections with Aipathwell software. Count TUNEL-positive nuclei under an upright fluorescence microscope (Nikon, Japan). 5.5 ATP content detection Place fresh tissue in pre-chilled lysis buffer and homogenize. Centrifuge for 15 minutes, collect the supernatant, and determine the total protein concentration in the supernatant using the Bradford method. Dilute the ATP assay reagent. Add equal volumes of supernatant and diluted reagent to a 96-well plate. Record the emission intensity at 560 nm using a chemiluminescence detector. Normalize the relative ATP levels to the protein content of each sample. 5.6 Reactive oxygen species detection Thaw frozen sections at room temperature and blot dry. Add autofluorescence quencher for 5 minutes, then rinse under running water. Add ROS staining solution (D7008, Sigma, Shanghai) dropwise and incubate at 37°C in a dark incubator for 30 minutes. Cells were counterstained with DAPI for nuclei and sealed with an anti-fluorescence quenching mounting medium. Images were acquired using an upright fluorescence microscope (Nikon, Japan). Aipathwell software was used for quantitative analysis of fluorescence intensity per unit area. 5.7 Transmission electron microscope Isolate fresh mouse left ventricular heart tissue samples (1 mm³), fix with 2% osmium tetroxide and 1% uranyl acid, rinse with ultrapure water, dehydrate with ethanol, permeate and embed in Eponate 12 resin, prepare sections, and perform double staining with uranin and lead citrate. Analyze under a transmission electron microscope. 5.8 Western blot analysis Cardiac tissue was lysed in pre-chilled RIPA lysis buffer. After centrifugation, the supernatant was collected. Protein concentration was determined using the BCA assay kit (Servicebio, Wuhan, China). An equal volume of protein was mixed with loading buffer, denatured, and transferred to a membrane. Incubate at room temperature for 1 hour, then incubate overnight at 4°C with primary antibodies: anti-SIRT1 (1:1000), anti-FOXO1 (1:1000), anti-Ac-FOXO1 (1:1000), anti-MFN1 (1:1000), anti-MFN2 (1:1000), anti-PGC-1α (1:1000), anti-PARK2 (1:1000), anti-DRP1 (1:1000), and anti-GAPDH. HRP-labeled secondary antibody (1:3000, Servicebio, Wuhan) was incubated at room temperature for 1 hour. ECL chemiluminescence was developed, and images were captured using an SCG-W3000 chemiluminescence reader. Image Quant TL software was used to calculate the gray values of the bands. 5.9 Statistical analysis All data are expressed as mean ± SD. The normality of data distribution was assessed using the Shapiro-Wilk test (α = 0.05), and homogeneity of variance among groups was confirmed by Levene's test. Comparisons of protein expression levels among different exercise intensity groups were performed using one-way analysis of variance (ANOVA). When statistically significant differences existed between groups (P < 0.05), post hoc multiple comparisons were conducted using the Least Significant Difference (LSD) test to identify specific groups exhibiting differences. Statistical analysis was performed using Graphpad Prism 10.0, with a significance level set at α = 0.05. Declarations Ethics statement The animal study was approved by the Ethics Committee of Shaanxi Provincial People's Hospital, with ethics approval number SPPH-LLBG-06-3.1. The study was conducted in accordance with the local legislation and institutional requirements. This study is reported in accordance with the ARRIVE guidelines. Funding The authors declare that financial support was received for the research, authorship, and/or publication of this article. This work was thankfully supported by the Shaanxi Provincial People's Hospital Talent Support Program (2021BJ-04) and Shaanxi Provincial Key Industry Innovation Chain Project (2023-ZDLSF-21). 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12:41:17","extension":"html","order_by":51,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115522,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8426076/v1/eee6e185c223c50780c4db65.html"},{"id":100237226,"identity":"4d4707b1-50e1-4a01-bc37-15503a341e54","added_by":"auto","created_at":"2026-01-14 12:41:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":315037,"visible":true,"origin":"","legend":"\u003cp\u003eMIE significantly alleviated DOX-induced cardiac dysfunction in mice. (\u003cstrong\u003eA\u003c/strong\u003e) Schematic diagram of the animal experimental process. (\u003cstrong\u003eB-D\u003c/strong\u003e) DOX significantly reduced heart weight (HW), body weight (BW), and the heart-to-body weight ratio (HW/BW). Exercise at all intensities effectively reversed DOX-related losses in both HW and HW/BW (n = 6). (\u003cstrong\u003eE\u003c/strong\u003e) Schematic diagrams of mouse hearts in each group (n = 6). (\u003cstrong\u003eF-H\u003c/strong\u003e) Representative echocardiograms, ejection fraction (EF%), and short-axis shortening fraction (FS%) before and after exercise in each group (n = 6). (\u003cstrong\u003eI\u003c/strong\u003e) Representative H\u0026amp;E-stained images (n = 6, scale bars: top = 500 μm, bottom = 50 μm). (\u003cstrong\u003eJ-K\u003c/strong\u003e) Representative Masson-stained cardiac section images and fibrosis scores (n = 6). Data are expressed as a single value, with the mean ± SD. *p \u0026lt; 0.05 indicates significant differences between each intervention group and the control group. #p \u0026lt; 0.05 indicates significant differences between each exercise group and the DOX group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8426076/v1/1e528a2b53bf64b239a65581.png"},{"id":100237199,"identity":"1f25c15c-8b28-47eb-8c39-3842e675171b","added_by":"auto","created_at":"2026-01-14 12:41:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":148837,"visible":true,"origin":"","legend":"\u003cp\u003eMIE mitigated DOX-induced oxidative stress and apoptosis. (\u003cstrong\u003eA-B\u003c/strong\u003e) DHE staining (red) for detecting ROS levels in cardiac tissue (n = 6). (\u003cstrong\u003eC\u003c/strong\u003e) Measurement of ATP content in cardiac tissue (n = 6). (\u003cstrong\u003eD-E\u003c/strong\u003e) TUNEL staining results showing apoptosis levels and quantitative analysis (n = 6). Data are expressed as a single value, with the mean ± SD. *p \u0026lt; 0.05 indicates significant differences between each intervention group and the control group. #p \u0026lt; 0.05 indicates significant differences between each exercise group and the DOX group.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8426076/v1/426a6a0d007772d0b4c5a110.jpg"},{"id":100237257,"identity":"6443cc9d-7174-481b-8539-129542c99b8b","added_by":"auto","created_at":"2026-01-14 12:41:19","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":155502,"visible":true,"origin":"","legend":"\u003cp\u003eMIE restored the DOX-induced imbalance in mitochondrial quality control. (\u003cstrong\u003eA\u003c/strong\u003e) Representative images of mitochondria and autophagic vacuoles under transmission electron microscopy (n = 6). Autophagic vacuoles are marked with red arrows. (\u003cstrong\u003eB-E\u003c/strong\u003e) Western blot detcetion of MFN1, MFN2, and DRP1 protein expression in the hearts from mice (n = 6). (\u003cstrong\u003eF-H\u003c/strong\u003e) Western blot detcetion of PGC-1α and PARK2 protein expression in the hearts from mice (n = 6)(original blots/gels are presented in Supplementary Figure). Data are expressed as a single value, with the mean ± SD. *p \u0026lt; 0.05 indicates significant differences between each intervention group and the control group. #p \u0026lt; 0.05 indicates significant differences between each exercise group and the DOX group.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8426076/v1/ff4c32c86256d8e96ce1194a.jpg"},{"id":100237200,"identity":"3b9d05bf-c91f-4e0d-9e1d-bef62f92253b","added_by":"auto","created_at":"2026-01-14 12:41:05","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":58712,"visible":true,"origin":"","legend":"\u003cp\u003eMIE ameliorates DOX-induced cardiotoxicity via the SIRT1-FOXO1 pathway. (\u003cstrong\u003eA-D\u003c/strong\u003e) Western blot detection of SIRT1, FOXO1, and Ac-FOXO1 protein expression in the hearts from mice. The protein expressions of SIRT1 and FOXO1 in the hearts of mice in the MIE group increased, while the expression of Ac-FOXO1 decreased(original blots/gels are presented in Supplementary Figure). Data are expressed as a single value, with the mean ± SD. *p \u0026lt; 0.05 indicates significant differences between each intervention group and the control group. #p \u0026lt; 0.05 indicates significant differences between each exercise group and the DOX group.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8426076/v1/6b9b83c67437d909bc752801.jpg"},{"id":102175158,"identity":"21729943-ee0c-42ab-ad4e-74bb70c1658c","added_by":"auto","created_at":"2026-02-09 05:55:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1442010,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8426076/v1/d9b99f49-22aa-444b-98ec-a16238701b7c.pdf"},{"id":100371402,"identity":"63580999-400e-4eaa-a64f-df91f166a0af","added_by":"auto","created_at":"2026-01-16 08:09:59","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":27747333,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfigure.zip","url":"https://assets-eu.researchsquare.com/files/rs-8426076/v1/22c88104b00ba9a4433e6b92.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exercise improves doxorubicin-induced cardiotoxicity by regulating mitochondrial quality control through activation of the SIRT1-FOXO1 pathway","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eDoxorubicin (DOX) has become a vital treatment for specific cancers, such as certain childhood leukemias and breast cancer, due to its broad-spectrum and highly effective anticancer properties \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. However, its inherent dose-dependent toxicity can affect multiple organs, including the heart, liver, kidneys, and gonads, thereby severely limiting the drug's clinical application potential \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. DOX-induced cardiotoxicity is due to persistent cardiomyocyte damage, which may ultimately lead to congestive heart failure. Currently, the mechanism by which DOX affects cardiac function remains incompletely elucidated; however, it is known to be driven by multiple factors, including mitochondrial dysfunction, oxidative stress, apoptosis, and impaired autophagy flux \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Dexrazoxane is currently the only drug approved by the FDA for mitigating DOX-induced cardiotoxicity. However, its clinical application is subject to strict limitations due to the potential for reduced tumor sensitivity and an increased risk of bone marrow suppression \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In this context, exploring safe and effective non-pharmacological intervention strategies holds significant clinical significance for preventing or mitigating DOX-related cardiac injury and enhancing treatment safety for patients.\u003c/p\u003e \u003cp\u003eSilent Information Regulator 1 (SIRT1) is an NAD+-dependent deacetylase that participates in critical physiological processes, such as cellular senescence, oxidative stress, and energy metabolism, by regulating multiple key targets \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. SIRT1 is primarily localized within the cell nucleus, but under specific conditions, it can translocate to the cytoplasm, thereby regulating its downstream target, forkhead box protein O1 (FOXO1). Mitochondrial quality control (MQC) is a crucial mechanism for maintaining the integrity and function of the intracellular mitochondrial network, primarily involving processes such as mitochondrial biogenesis, dynamics (fusion and fission), and mitochondrial autophagy \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The SIRT1-FOXO1 pathway plays a central regulatory role in maintaining MQC by coordinating the aforementioned processes. Specifically, SIRT1-mediated deacetylation of FOXO1 enhances its transcriptional activity on downstream targets such as peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), superoxide dismutase (SOD), and microtubule-associated protein light chain 3 (LC3), thereby promoting mitochondrial biogenesis, reactive oxygen species (ROS) neutralisation, and the clearance of damaged organelles \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Given the pivotal role of the SIRT1-FOXO1 pathway in sustaining MQC, we hypothesize that specific activation of this pathway could effectively ameliorate DOX-induced mitochondrial dysfunction.\u003c/p\u003e \u003cp\u003eAs a non-pharmacological intervention, exercise plays a significant role in the prevention and management of cardiovascular disease. Its cardioprotective effects have been demonstrated to be closely associated with improvements in MQC, a process involving multifaceted regulatory mechanisms \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Exercise promotes mitochondrial biogenesis by activating the PGC-1α signaling pathway, thereby increasing the number and membrane surface area of mitochondria \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Concurrently, exercise enhances endogenous antioxidant defenses, reducing excessive ROS accumulation and mitigating the oxidative damage it induces \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. These mechanisms act synergistically to maintain mitochondrial homeostasis and safeguard normal cardiac function. Notably, the cardioprotective effects of exercise are influenced by multiple factors, with exercise intensity being a significant factor. Low-intensity swimming effectively enhances skeletal muscle PGC-1α mRNA expression in SD rats \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In comparison, MIE produces a more sustained elevation in PGC-1α mRNA expression, which significantly exceeds the transient and limited increase induced by HIE \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Therefore investigating the differential regulatory mechanisms of exercise intensity on MQC and its downstream pathways holds significant importance for elucidating precise protective strategies against cardiovascular disease.\u003c/p\u003e \u003cp\u003eThis study established a DOX-induced cardiac injury mouse model to systematically examine the cardioprotective effects of exercise and compare the effects of different exercise intensities. The research further focused on the SIRT1-FOXO1 signalling pathway and the MQC mechanism, aiming to elucidate their potential roles and provide a scientific foundation for the precise clinical application of exercise therapy.\u003c/p\u003e"},{"header":"2 Result","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Effects of different exercise intensities on DOX-induced cardiac function and structure in mice\u003c/h2\u003e \u003cp\u003eThis study first established a DOX-induced mouse cardiac injury model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Analysis of physiological parameters revealed that DOX treatment significantly reduced mouse heart weight (HW), body weight (BW), and the heart-to-body weight ratio (HW/BW) compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-E). All exercise interventions effectively prevented DOX-induced decrease in heart weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Echocardiographic assessment revealed that both LVEF and LVFS were significantly lower in the DOX-treated group compared to the control group. In the exercise intervention group, LIE showed no significant improvement in cardiac function, whereas MIE markedly improved LVEF and LVFS in DOX-treated mice. Conversely, HIE further reduced both LVEF and LVFS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Histopathological analysis further revealed marked ventricular dilatation accompanied by vacuolar degeneration of cardiomyocytes in the DOX-only group. No significant dilatation or vacuolar degeneration was observed in the right ventricles of any exercise intervention group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Meanwhile, H\u0026amp;E staining revealed that single DOX treatment disrupted myocardial tissue architecture, whereas myocardial cells in the exercise groups exhibited a tighter and more orderly arrangement. Masson staining results revealed that DOX treatment significantly increased the area of myocardial fibrosis, while MIE effectively reduced this pathological change. No significant improvement was observed in the LIE and HIE groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH-I).\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Effects of different exercise intensities on DOX-induced myocardial oxidative stress and apoptosis\u003c/h2\u003e \u003cp\u003eFirst, we measured ROS levels in myocardial tissue. The results showed that DOX treatment significantly increased myocardial ROS production. In contrast, MIE effectively reduced this effect, suggesting that MIE may exert its action by enhancing the endogenous antioxidant defense system. Although LIE and HIE partially alleviated ROS accumulation, their effects were not significant (Fig.\u0026nbsp;2A-B), reflecting differences in redox regulation among varying exercise intensities. In terms of energy metabolism, DOX significantly reduced myocardial ATP content, suggesting mitochondrial dysfunction. MIE intervention significantly restored ATP levels, thereby improving mitochondrial oxidative phosphorylation efficiency and energy metabolism balance, whereas the LIE and HIE groups showed no significant improvement (Fig.\u0026nbsp;2C). To further evaluate apoptosis induced by oxidative damage, we employed TUNEL staining for detection. Results showed that DOX significantly increased the apoptosis rate of cardiomyocytes, while MIE effectively inhibited this process. In contrast, the number of apoptotic cells in the LIE and HIE groups remained significantly higher than that in the control group (Fig.\u0026nbsp;2D-E), indicating their weaker anti-apoptotic effects. In summary, MIE effectively alleviates DOX-induced myocardial oxidative stress, improves mitochondrial function, and inhibits apoptosis, whereas LIE and HIE did not demonstrate significant protective effects in these aspects.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Effects of different exercise intensities on DOX-induced MQC imbalance\u003c/h2\u003e \u003cp\u003eThe results in Fig.\u0026nbsp;3A show that DOX treatment led to an abnormal increase in myocardial mitochondrial number, a fragmented morphology, and the accumulation of autophagic vacuoles. MIE effectively reversed these morphological abnormalities. Although autophagic vacuoles decreased in the LIE and HIE groups, they remained higher than in the control group, suggesting that MIE may promote expected degradation during autophagy. Further Western blot analysis was used to measure the expression of proteins related to mitochondrial dynamics. In the DOX group, mitochondrial fusion proteins MFN1 and MFN2 showed decreased expression, indicating suppression of the fusion process. Both MIE and HIE restored their protein expression to normal levels, whereas LIE did not exhibit a significant reversal effect (Fig.\u0026nbsp;3B-C). Concurrently, DOX induces increased expression of the fission regulatory protein DRP1, indicating that DOX promotes the fission process, ultimately disrupting the fusion-fission equilibrium and shifting it toward the fission state. Additionally, DOX suppressed the expression of PGC-1α, a key factor in mitochondrial biogenesis, and upregulated the levels of PARK2, an autophagy-related protein. MIE effectively reversed these abnormal protein expressions (Fig.\u0026nbsp;3D-E). In summary, the results of this study indicate that MIE can effectively improve the MQC imbalance caused by DOX, while LIE and HIE did not demonstrate the same degree of protective effect.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 MIE activates the SIRT1-FOXO1 pathway in DOX-damaged cardiomyocytes\u003c/h2\u003e \u003cp\u003eThis study compared the protein expressions of SIRT1 and FOXO1 in the myocardial tissues of five groups of experimental mice. Western blot results showed that DOX treatment significantly reduced the protein expression levels of SIRT1 and FOXO1 (Fig.\u0026nbsp;4A-B), suggesting that it may inhibit the activity of the SIRT1-FOXO1 pathway, thereby affecting downstream oxidative stress and metabolic regulatory functions. In the exercise intervention group, MIE effectively reversed DOX-induced suppression of SIRT1 and FOXO1 expression, while HIE also showed some upregulating effects. Conversely, LIE further reduced the expression of both proteins. Further assessment of FOXO1 acetylation levels revealed that DOX significantly increased FOXO1 acetylation, while MIE intervention markedly suppressed this excessive acetylation phenomenon. LIE and HIE showed no significant ameliorative effects. The results indicate that MIE effectively activates the SIRT1-FOXO1 signaling pathway in DOX-damaged cardiomyocytes, whereas LIE and HIE exert limited regulatory effects on this pathway.\u003c/p\u003e "},{"header":"3 Discussion","content":"\u003cdiv class=\"Heading\"\u003e\u003cb\u003e\u003c/b\u003e\u003c/div\u003e \u003cp\u003eAs a widely used anthracycline antibiotic, DOX exhibits significant antitumor effects, but its severe cardiotoxicity limits its clinical application \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Therefore, identifying strategies that can effectively mitigate DOX-induced cardiac damage has become a significant focus of research in this field. The main findings of this study include: (1) MIE significantly ameliorates DOX-induced cardiac dysfunction and myocardial structural abnormalities; (2) MIE exerts its cardioprotective effects by modulating MQC; (3) The SIRT1-FOXO1 axis is a key pathway through which MIE mitigates DOX-induced cardiotoxicity.\u003c/p\u003e \u003cp\u003eOur study confirms that exercise intervention can alleviate DOX-induced cardiotoxicity, with exercise intensity being the key determinant of its efficacy. MIE significantly ameliorates DOX-induced cardiac dysfunction and myocardial structural abnormalities, while LIE yields limited benefits. HIE did not demonstrate any improvement in cardiac function. This finding aligns with existing research on the critical role of exercise intensity. Notably, while long-term regular exercise has been proven to exert cardiovascular protective effects through multiple mechanisms \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and even improve the prognosis of heart failure patients \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, exercise intensity remains the core factor determining both its protective efficacy and safety. In particular, HIE may induce myocardial ischemia by substantially increasing myocardial oxygen consumption. Simultaneously, it enhances oxygen metabolism, leading to excessive ROS production and lactic acid accumulation, which in turn triggers oxidative damage and apoptosis \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, ultimately resulting in myocardial structural injury \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The findings of this study further reinforce the understanding that in the DOX-induced cardiotoxicity model, MIE demonstrated the most beneficial effects in alleviating oxidative stress and reducing apoptosis, confirming the critical role of exercise intensity. In summary, the alleviation of DOX-induced cardiotoxicity through exercise intervention is related to exercise intensity, and MIE is considered an intervention strategy with greater cardioprotective potential.\u003c/p\u003e \u003cp\u003eAnother finding of this study is that MIE improves DOX-induced cardiotoxicity by regulating MQC. Mitochondrial dysfunction is one of the core mechanisms underlying DOX-induced cardiotoxicity. Studies have demonstrated that DOX exerts its antitumor effects through sustained activation of the Hippo pathway, while simultaneously inducing significant myocardial mitochondrial damage. This mechanism constitutes a key basis for its adverse cardiac effects \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. This study observed a decrease in myocardial ATP synthesis capacity, disruption of mitochondrial ultrastructure, and dysregulation of key MQC proteins such as PGC-1α, MFN1, MFN2, and DRP1 following DOX treatment, consistent with previous research reports \u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Notably, we observed downregulation of mitochondrial fusion proteins MFN1/2 and upregulation of fission protein DRP1 following DOX treatment, indicating impaired mitochondrial fusion and excessive activation of fission. This imbalance leads to the fragmentation of the mitochondrial network and induces respiratory chain uncoupling, ultimately impairing the efficiency of energy metabolism \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. These findings suggest that DOX disrupts mitochondrial dynamics by promoting fission and inhibiting fusion. MIE effectively reverses these abnormal protein expressions, restoring the structural and functional integrity of mitochondria, suggesting that its cardioprotective effects are closely associated with reestablishing mitochondrial dynamic equilibrium. In summary, MIE ultimately disrupts the vicious cycle between DOX-induced mitochondrial dysfunction and myocardial injury by regulating the MQC balance.\u003c/p\u003e \u003cp\u003eThis study further clarifies that the activation of the SIRT1-FOXO1 pathway plays a core regulatory role in the process of MIE improving the cardiotoxicity of DOX. Experimental results indicate that DOX treatment significantly suppresses SIRT1 expression and increases FOXO1 acetylation levels, whereas MIE effectively reverses these changes, restoring pathway activity. Further mechanistic studies indicate that MIE promotes mitochondrial biogenesis mediated by PGC-1α, mitochondrial fusion involving MFN1/2, and PARK2-associated autophagic clearance by activating the SIRT1-FOXO1 pathway, thereby synergistically restoring mitochondrial functional integrity. The findings are highly consistent with previous studies, indicating that SIRT1, as a key regulator of energy metabolism and oxidative stress, is crucial for maintaining MQC \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. For instance, Yao et al. found that SIRT1 regulates mitophagy to promote the clearance of damaged mitochondria \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Mitochondria are highly dynamic organelles, and the dynamic equilibrium between mitochondrial fusion and fission is a crucial mechanism for maintaining mitochondrial homeostasis \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Ding M et al. demonstrated that SIRT1 activates PGC-1α and inhibits DRP1-mediated excessive fission \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Furthermore, SIRT1 also modulates the PGC-1α/PDK2/PARL axis to promote mitochondrial fusion \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. These studies collectively demonstrate that SIRT1 plays multiple roles in coordinating mitochondrial homeostasis. Our research further confirms that MIE reverses DOX-induced myocardial MQC imbalance by activating the upstream SIRT1-FOXO1 pathway, which regulates mitochondrial biogenesis, fusion-fission, and autophagy processes.\u003c/p\u003e \u003cp\u003eThe innovation of this study lies in systematically elucidating the role of different exercise intensities in regulating MQC through the SIRT1-FOXO1 pathway in DOX-induced cardiotoxicity, confirming MIE as the optimal intervention intensity. This discovery not only deepens our understanding of the protective mechanisms of exercise on the heart but also provides a theoretical basis and potential intervention targets for clinically preventing and improving chemotherapy-related cardiac dysfunction. However, it is worth noting that our study still has some limitations. First, the SIRT1-FOXO1 signaling pathway may not be the sole mechanism through which exercise affects mitochondria; exercise may also exert effects via other signaling pathways, which warrants further investigation. Second, we subjected mice to endurance training at varying intensities, but the effects of different intensities of resistance training remain unknown. Future studies could incorporate resistance training at different intensities and combine it with more comprehensive exercise physiology metrics to provide more refined exercise protocols. Despite these limitations, our findings still suggest potential therapeutic strategies for DOX-induced cardiotoxicity.\u003c/p\u003e"},{"header":"4 Conclusion","content":" \u003cp\u003eIn summary, our findings indicate that MIE significantly ameliorates DOX-induced cardiotoxicity, with a protective effect superior to that of LIE and HIE. Mechanistically, MIE effectively alleviates DOX-induced MQC imbalance by activating the SIRT1-FOXO1 signaling pathway. These findings support the beneficial role of exercise in mitigating DOX-related cardiac toxicity and provide a basis for optimizing exercise protocols for patients undergoing anthracycline-based chemotherapy.\u003c/p\u003e"},{"header":"5 Materials and methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e5.1 Animals and treatments\u003c/h2\u003e \u003cp\u003eEight-week-old C57BL/6 male mice were purchased from the Animal Experiment Center of Xi'an Jiaotong University. They were housed in an SPF environment (temperature: 20\u0026ndash;25\u0026deg;C, humidity: 50%) with free access to water and food. One week of adaptive feeding preceded the experiment, followed by random assignment to five groups: control group (Ctrl), doxorubicin model group (DOX), doxorubicin\u0026thinsp;+\u0026thinsp;low-intensity exercise group (DOX\u0026thinsp;+\u0026thinsp;LIE), doxorubicin\u0026thinsp;+\u0026thinsp;moderate-intensity exercise group (DOX\u0026thinsp;+\u0026thinsp;MIE), and doxorubicin\u0026thinsp;+\u0026thinsp;high-intensity exercise group (DOX\u0026thinsp;+\u0026thinsp;HIE). The intervention period for all mice was 4 weeks. Control group mice received weekly intraperitoneal injections of saline (5 mg/kg). In comparison, mice in the other groups received intraperitoneal injections of DOX at the same dose (5 mg/kg)\u003csup\u003e34\u003c/sup\u003e. Echocardiography was used to assess the success of modeling. All experimental procedures were approved by the Experimental Center of Shaanxi Provincial People's Hospital.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e5.2 Exercise protocols\u003c/h2\u003e \u003cp\u003eThe FT-201 small animal treadmill was used with electrical stimulation parameters set at 25 V, 0.34 mA, and 2 Hz \u003csup\u003e35\u003c/sup\u003e. The exercise capacity testing protocol was as follows: initial speed, 8.5 m/min; incline, 0\u0026deg;; duration, 9 min. Then, increase the speed to 10 m/min, incline to 5\u0026deg;, and maintain for 3 minutes. Subsequently, increase speed by 2.5 m/min every 3 min (maximum 40 m/min) and incline by 5\u0026deg; every 9 min (maximum 15\u0026deg;). Fatigue Criteria (meet any one of the criteria): Remaining on the grid for \u0026ge;\u0026thinsp;10 seconds; Time spent on grid\u0026thinsp;\u0026gt;\u0026thinsp;50%; Transitioning from normal posture to prone position with hind limbs trailing for \u0026gt;\u0026thinsp;30 seconds. Record speed, slope, and total running distance during fatigue episodes. Based on test results, mice were assigned to different exercise intensity groups (LIE: 55\u0026ndash;60% Vmax; MIE: 75% Vmax; HIE: 90% Vmax). They exercised 5 days a week: 40 minutes/day during weeks 1\u0026ndash;2, 50 minutes/day during weeks 3\u0026ndash;4, and 60 minutes/day during weeks 5\u0026ndash;8. Control mice received routine care without exercise intervention. All mice were deeply anesthetized with 5% isoflurane(26675-46-7, MCE) 24 hours after the conclusion of the experiment until they lost the righting reflex and responded to toe pinch. Euthanasia was then promptly performed by cervical dislocation. Cardiac tissue samples were collected for subsequent analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e5.3 Echocardiography\u003c/h2\u003e \u003cp\u003eCardiac structure and function were assessed using echocardiography (Vevo 3100) prior to exercise intervention and 8 weeks after exercise. Parameters measured: Left ventricular end-diastolic diameter (LVIDd), Left ventricular end-systolic diameter (LVIDs), Left ventricular systolic posterior wall thickness (LVPWs), Left ventricular diastolic posterior wall thickness (LVPWd), Ejection fraction (EF%), Fractional shortening (FS%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e5.4 Histological analysis\u003c/h2\u003e \u003cp\u003eThe heart specimens were fixed in 4% paraformaldehyde, followed by dehydration with graded ethanol, clearing with xylene, and embedding in paraffin. Sections were dewaxed with xylene and hydrated with graded ethanol solutions, then stained using haematoxylin and eosin (H\u0026amp;E), Masson's trichrome, and TUNEL staining kits. Stained sections were embedded in neutral resin, examined under an optical microscope, and imaged using a digital pathology slide scanner (LG-S80). Scan and view using Saiviewer-1.0.9 software. Quantify Masson-stained sections with Aipathwell software. Count TUNEL-positive nuclei under an upright fluorescence microscope (Nikon, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e5.5 ATP content detection\u003c/h2\u003e \u003cp\u003ePlace fresh tissue in pre-chilled lysis buffer and homogenize. Centrifuge for 15 minutes, collect the supernatant, and determine the total protein concentration in the supernatant using the Bradford method. Dilute the ATP assay reagent. Add equal volumes of supernatant and diluted reagent to a 96-well plate. Record the emission intensity at 560 nm using a chemiluminescence detector. Normalize the relative ATP levels to the protein content of each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e5.6 Reactive oxygen species detection\u003c/h2\u003e \u003cp\u003eThaw frozen sections at room temperature and blot dry. Add autofluorescence quencher for 5 minutes, then rinse under running water. Add ROS staining solution (D7008, Sigma, Shanghai) dropwise and incubate at 37\u0026deg;C in a dark incubator for 30 minutes. Cells were counterstained with DAPI for nuclei and sealed with an anti-fluorescence quenching mounting medium. Images were acquired using an upright fluorescence microscope (Nikon, Japan). Aipathwell software was used for quantitative analysis of fluorescence intensity per unit area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e5.7 Transmission electron microscope\u003c/h2\u003e \u003cp\u003eIsolate fresh mouse left ventricular heart tissue samples (1 mm\u0026sup3;), fix with 2% osmium tetroxide and 1% uranyl acid, rinse with ultrapure water, dehydrate with ethanol, permeate and embed in Eponate 12 resin, prepare sections, and perform double staining with uranin and lead citrate. Analyze under a transmission electron microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e5.8 Western blot analysis\u003c/h2\u003e \u003cp\u003eCardiac tissue was lysed in pre-chilled RIPA lysis buffer. After centrifugation, the supernatant was collected. Protein concentration was determined using the BCA assay kit (Servicebio, Wuhan, China). An equal volume of protein was mixed with loading buffer, denatured, and transferred to a membrane. Incubate at room temperature for 1 hour, then incubate overnight at 4\u0026deg;C with primary antibodies: anti-SIRT1 (1:1000), anti-FOXO1 (1:1000), anti-Ac-FOXO1 (1:1000), anti-MFN1 (1:1000), anti-MFN2 (1:1000), anti-PGC-1α (1:1000), anti-PARK2 (1:1000), anti-DRP1 (1:1000), and anti-GAPDH. HRP-labeled secondary antibody (1:3000, Servicebio, Wuhan) was incubated at room temperature for 1 hour. ECL chemiluminescence was developed, and images were captured using an SCG-W3000 chemiluminescence reader. Image Quant TL software was used to calculate the gray values of the bands.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e5.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The normality of data distribution was assessed using the Shapiro-Wilk test (α\u0026thinsp;=\u0026thinsp;0.05), and homogeneity of variance among groups was confirmed by Levene's test. Comparisons of protein expression levels among different exercise intensity groups were performed using one-way analysis of variance (ANOVA). When statistically significant differences existed between groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), post hoc multiple comparisons were conducted using the Least Significant Difference (LSD) test to identify specific groups exhibiting differences. Statistical analysis was performed using Graphpad Prism 10.0, with a significance level set at α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003eEthics statement\u003c/p\u003e\n\u003cp\u003eThe animal study was approved by the Ethics Committee of Shaanxi Provincial People\u0026apos;s Hospital, with ethics approval number SPPH-LLBG-06-3.1. The study was conducted in accordance with the local legislation and institutional requirements. This study is reported in accordance with the ARRIVE guidelines.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors declare that financial support was received for the research, authorship, and/or publication of this article. This work was thankfully supported by the Shaanxi Provincial People's Hospital Talent Support Program (2021BJ-04) and Shaanxi Provincial Key Industry Innovation Chain Project (2023-ZDLSF-21).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJF: Conceptualization, methodology, Investigation and Research, writing-first draft. QR: Software, formal analysis, visualization. YL: Software, validation. CD: Data Management, Validation. WQ: Investigation and Research, Data Management. BW: Data Management, Visualization. HZ and LS: Resource Provision, Funding Acquisition, Writing-Review and Editing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlradwan, I. et al. Empowering chemotherapy-induced antitumor immunity by multi-targeted synergistic combination nanomedicine for triple-negative breast cancer. \u003cem\u003eMater. Today Bio\u003c/em\u003e. \u003cb\u003e35\u003c/b\u003e, 102445. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.mtbio.2025.102445\u003c/span\u003e\u003cspan address=\"10.1016/j.mtbio.2025.102445\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArrigoni, R., Jirillo, E. \u0026amp; Caiati, C. 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Physiol.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 636. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphys.2016.00636\u003c/span\u003e\u003cspan address=\"10.3389/fphys.2016.00636\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cardiotoxicity, Doxorubicin, Mitochondrial quality control, Exercise intensity, SIRT1-FOXO1 pathway","lastPublishedDoi":"10.21203/rs.3.rs-8426076/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8426076/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDoxorubicin (DOX) is a highly effective broad-spectrum chemotherapeutic agent, yet its clinical application is severely limited by dose-dependent cardiotoxicity, for which preventive strategies are lacking. Exercise has emerged as a promising non-pharmacological intervention; its cardioprotective effects may involve silent information regulator 1 (SIRT1), though the precise mechanisms remain unclear. This study aimed to investigate the effect of exercise intensity on DOX-induced cardiotoxicity through the SIRT1-FOXO1 pathway and mitochondrial homeostasis. Using a murine model of DOX-induced cardiac injury, we implemented exercise regimens of varying intensities. Results demonstrated that moderate-intensity exercise (MIE) significantly attenuated DOX-induced cardiac dysfunction, apoptosis, and oxidative stress, while favorably regulating proteins essential for mitochondrial biogenesis and dynamics.Mechanistically, MIE activated myocardial SIRT1, leading to deacetylation of FOXO1, which enhanced antioxidant defenses and conferred cardioprotection. Notably, low-intensity exercise (LIE) offered only modest benefits, whereas high-intensity exercise (HIE) exacerbated myocardial oxidative stress and injury.Collectively, our findings establish that MIE alleviates DOX-induced cardiotoxicity by activating the SIRT1-FOXO1 pathway and restoring mitochondrial homeostasis, thereby providing a critical experimental foundation for developing tailored exercise regimens to prevent chemotherapy-related cardiac dysfunction..\u003c/p\u003e","manuscriptTitle":"Exercise improves doxorubicin-induced cardiotoxicity by regulating mitochondrial quality control through activation of the SIRT1-FOXO1 pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-14 12:39:55","doi":"10.21203/rs.3.rs-8426076/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"75617b18-6f4a-4401-9efe-ae8a644794e8","owner":[],"postedDate":"January 14th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61080787,"name":"Health sciences/Cardiology"},{"id":61080788,"name":"Biological sciences/Cell biology"},{"id":61080789,"name":"Biological sciences/Drug discovery"},{"id":61080790,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-02-09T05:54:50+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-14 12:39:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8426076","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8426076","identity":"rs-8426076","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}