Tirzepatide attenuates doxorubicin-induced cardiotoxicity via mitochondrial function improvement

Introduction

Doxorubicin (DOX), a cornerstone anthracycline antibiotic, demonstrates potent clinical efficacy and is extensively employed against diverse malignancies, including breast and ovarian cancers (1,2). Despite its broad-spectrum antitumor activity, the cardiotoxicity associated with DOX treatment poses a significant and growing clinical challenge. Reaching specific cumulative doses in the body can lead to myocardial cell damage, culminating in severe, often irreversible cardiomyopathy and potentially fatal heart failure (3). This serious adverse effect critically restricts DOX’s therapeutic utility. Currently, dexrazoxane stands as the sole compound approved by the Food and Drug Administration (FDA) to mitigate DOX-induced cardiotoxicity. However, evidence indicates that dexrazoxane may compromise chemotherapy sensitivity and exacerbate myelosuppression in patients, leading to regulatory restrictions imposed by both the FDA and the European Medicines Agency (EMA) (4). Consequently, the development of safe and effective agents to counteract DOX cardiotoxicity represents an urgent medical need.

Tirzepatide functions as a dual agonist targeting both the glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 receptor (GLP-1R). Its molecular design primarily derives from the GIP sequence, incorporating a C20 fatty diacid modification (5). Beyond its role in type 2 diabetes mellitus (T2DM), glucagon-like peptide-1 (GLP-1) is a pleiotropic hormone with broader therapeutic potential. Its GLP-1R agonists (GLP-1RAs) demonstrate cardioprotective effects (6), which are associated with multiple mechanisms such as improving myocardial energy metabolism, inhibiting apoptosis, and alleviating oxidative stress. Notably, mitochondrial dysfunction has been widely recognized as a central pathological link in DOX-induced cardiotoxicity, characterized by decreased mitochondrial membrane potential (MMP), excessive production of reactive oxygen species (ROS), and impaired adenosine triphosphate (ATP) synthesis (7). Furthermore, studies indicate that activation of GLP-1R signaling can promote myocardial mitochondrial biogenesis, enhance mitochondrial quality, and improve its function (8). Simultaneously, GIP receptor (GIPR) signaling is also involved in regulating cellular metabolism and survival (9). Therefore, as a dual agonist of both GLP-1 and GIPRs, tirzepatide may, through synergistic actions, specifically ameliorate DOX-induced myocardial mitochondrial dysfunction, offering a new direction for exploring the mechanisms underlying its potential cardioprotective effects. To enhance weight loss and diabetes outcomes via complementary pathways, multi-agonists combining GLP-1RAs with peptides like GIP or glucagon (GCG) are under active investigation (10,11). Tirzepatide exemplifies this approach and has demonstrated efficacy in reducing body weight and improving key cardiorenal risk factors in overweight/obese populations (12). Although GLP-1/GIP co-agonists like tirzepatide have shown cardioprotective potential, it remains unclear whether they can alleviate DOX-induced cardiomyopathy by improving mitochondrial function. Based on the potential regulatory effects of GLP-1/GIP dual receptor agonists on mitochondrial function, we hypothesize that tirzepatide may counteract DOX-induced cardiac toxicity by mitigating the associated mitochondrial dysfunction. This study is specifically designed to investigate this underlying mechanism. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2801/rc).

Methods

Animals

C57BL/6 mice were obtained from Jackson Laboratory. The animals used in this study were male, aged 8 to 10 weeks. All animal experiments were performed under a project license (No. 92120251015RB02) granted by the Institutional Animal Care and Use Committee (IACUC) of The 921 Hospital of Joint Logistics Support Force People’s Liberation Army of China, in compliance with National Institutes of Health (NIH) guide for the care and use of animals. Mice were maintained under specific pathogen-free (SPF) conditions within a barrier facility at the Animal Center of the 921 Hospital. Housing consisted of individually ventilated cages with controlled ambient temperature (20–25 ℃) and humidity (50%±5%). Animals had ad libitum access to autoclaved food and water throughout the study period. After a week of acclimatization, the mice were randomly divided into different groups. They received DOX (5 mg/kg, intraperitoneally; MedChemExpress, Monmouth Junction, NJ, USA) or an equivalent volume of the control solution (saline) weekly for 4 weeks (13). During the 4-week DOX treatment period, mice received daily subcutaneous injections of tirzepatide (10 nmol/kg) (14-16). Following a 4-week regimen of DOX-induced cardiotoxicity, weekly observations and weighing of all mice were conducted, and their hearts were collected for subsequent research. Measurements of body weight and the heart weight to body weight ratio (HW/BW) were taken.

Echocardiography

Echocardiographic evaluations were carried out in the week following the completion of treatment in compliance with previously established protocols (17). In summary, these assessments were performed in a blinded fashion by independent observers unaware of the animals’ treatment groups under continuous anesthesia with 1.5–2% isoflurane, using a high-resolution micro-ultrasound system (Vevo 2100, VisualSonics, Toronto, Canada) designed for small-animal research. Left ventricular systolic function was assessed by calculating the ejection fraction (EF) and fractional shortening (FS).

Histological analysis

Cardiac tissues were processed for histological examination. Briefly, samples were fixed in 4% paraformaldehyde at 4 ℃ for 24–48 hours. Following fixation, they underwent dehydration through a graded ethanol series, were cleared in xylene, and embedded in paraffin. Finally, serial sections of 4 µm thickness were obtained using a microtome. To evaluate cardiac morphology and fibrosis, tissue sections were subjected to hematoxylin and eosin (H&E; G1120, Solarbio, Beijing, China) and Masson’s trichrome (G1345, Solarbio) staining according to established protocols. H&E staining was applied for qualitative observation of myocardial fibers morphological changes, while Masson’s trichrome staining was used for quantitative analysis of cardiac interstitial fibrosis. Stained sections were then examined under an Olympus SlideView VS200 microscope (Olympus, Hamburg, Germany). For the quantitative analysis of fibrosis, a blinded method was adopted to perform image analysis utilizing Image J software: six non-overlapping visual fields were randomly selected from each section, the perivascular tissue area was strictly excluded during the analysis, and the percentage of myocardial fibrotic area to the total myocardial area was calculated as the quantitative parameter for cardiac fibrosis.

Transmission electron microscopy (TEM)

For TEM, heart samples were immersion-fixed in 3% glutaraldehyde and post-fixed in 1% osmium tetroxide. Following dehydration through an ascending acetone series, tissues were infiltrated and embedded in Epon 812 resin. Semithin sections (stained with methylene blue) and ultrathin sections (contrasted with uranyl acetate and lead citrate) were prepared for observation under a JEM-1400FLASH transmission electron microscope. Three randomly selected myocardial tissue fragments were analyzed per mouse, and all electron microscopy observations and analyses were performed by the same observer in a blinded manner.

Cell culture

Primary neonatal rat ventricular cardiomyocytes (NRVMs) were isolated from 1- to 3-day-old Sprague-Dawley rats using a differential adhesion-centrifugation method, based on established protocols (18). In brief, neonatal rats were anesthetized with isoflurane and euthanized via cervical dislocation. The hearts were quickly removed and transferred into ice-cold ADS buffer with the following composition (in mmol/L): 120 NaCl, 20 HEPES, 8 NaH₂PO₄, 6 glucose, 5 KCl, and 0.8 MgSO₄ (pH adjusted to 7.4). Ventricular tissues were rinsed with fresh buffer, minced into fragments smaller than 1 mm³, and then washed several times until the supernatant became clear. Subsequent enzymatic digestion was carried out in a mixture of 2 mL collagenase II and 2 mL ADS buffer under constant stirring conditions (180 rpm, 37 ℃, 8 min). The resulting cell digest was filtered through a sterile mesh into a pre-cooled 50 mL centrifuge tube, and 1 mL of fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) was added to terminate digestion. To deplete fibroblasts through differential adhesion, the filtered cell suspension was placed into a culture dish and maintained at 37 ℃ under 5% CO₂ for 2 h. This short-term plating allowed fibroblasts to attach rapidly to the dish surface, whereas most cardiomyocytes stayed in suspension. The supernatant, now enriched with cardiomyocytes, was carefully removed and centrifuged at 2,000 g for 5 min. The pelleted cells were gently resuspended in a minimal volume of FBS. Further purification was achieved by density-gradient centrifugation. The cardiomyocyte-enriched suspension was layered onto a Percoll gradient (GE17-0891-01, Sigma-Aldrich, St. Louis, MO, USA) prepared with sterile ADS buffer and centrifuged at 1,800 g for 45 min. The cardiomyocyte band, located in the intermediate layer of the gradient, was carefully collected. Finally, the isolated NRVMs were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% FBS and 1% penicillin-streptomycin, and maintained at 37 ℃ in a humidified 5% CO₂ incubator for 24 h prior to experimental use. For these experiments, the cells were treated with DOX (1 µM) (19), either individually or in conjunction with tirzepatide (1 µM), based on appropriate concentrations and durations before proceeding to further experimental evaluations.

Cells viability assay

Using the cell counting kit-8 (CCK-8) kit (Dojindo Molecular Technologies, Kumamoto, Japan), cell viability was determined as per the manufacturer’s guidelines. The optical density (OD) at 450 nm was detected with a microplate reader. The results of the cell viability assay verified the reliability of the experimental system (Figure S1), and the optimal effective concentration of tirzepatide for all subsequent experiments was identified as 1 µM through concentration-effect curve analysis.

Mitochondrial assays

MMP was assessed using the enhanced MMP assay kit with JC-1 (C2003S, Beyotime, Shanghai, China). This assay utilizes the potential-dependent fluorescent probe JC-1, which accumulates in mitochondria and shifts from emitting red fluorescence (as J-aggregates) to green fluorescence (as monomers) upon mitochondrial depolarization. To assess MMP, cells were subjected to JC-1 staining. Briefly, after a PBS wash, 500 µL of JC-1 working solution was added per well and incubated for 20 min at 37 ℃ in the dark. Cells were then rinsed twice with kit-specific buffer, and the medium was replaced with fresh DMEM (11965092, Thermo Fisher Scientific, Waltham, MA, USA) prior to fluorescence microscopy imaging. Fluorescence images were captured, and the ratio of red to green fluorescence intensity was quantified using ImageJ as a measure of MMP. Six randomly selected microscope fields were used for MMP analysis.

Statistical analysis

Data are presented as the mean ± standard error of the mean (SEM). All statistical analyses were performed using GraphPad Prism software (version 9.0, GraphPad Software), unless otherwise stated. All experiments included at least three independent biological replicates. The exact sample size (N) for each group and the number of technical replicates are detailed in the corresponding figure legends. The Brown-Forsythe test was utilized to evaluate variance among groups, revealing no significant differences. For comparisons involving more than two groups, one-way analysis of variance (ANOVA) was performed, with post-hoc analysis conducted using Tukey’s or Dunnett’s test as appropriate. Survival analysis employed the Kaplan-Meier method, and results were compared using the log-rank test. P<0.05 indicated statistically significant differences.

Results

Tirzepatide alleviates DOX cardiotoxicity

A chronic model of DOX-induced myocardial injury was employed to assess the role of tirzepatide in this context. In agreement with prior studies (20), administration of DOX [5 mg/kg/week, intraperitoneal injection (i.p.)] successfully recapitulated the expected decline in cardiac function. The impairment in cardiac function was reflected by significant decreases in both left ventricular EF and FS, decreased body weight, and a lower heart weight to tibia length ratio (HW/TL) (Figure 1A-1E). The DOX-induced cardiac dysfunction may have contributed to the reduced survival rate observed in this group compared to the control group (Figure 1F). Notably, these abnormal parameters were almost completely reversed by treatment with tirzepatide (administered subcutaneously at 10 nmol/kg/day) (Figure 1A-1F).

Figure 1 Tirzepatide alleviates DOX-induced cardiac dysfunction. To establish a DOX-induced cardiac injury model, male C57BL/6J mice received weekly intraperitoneal administrations of DOX (5 mg/kg) over 4 weeks. Concurrently, tirzepatide was administered daily through subcutaneous injections at 10 mg/kg for 5 weeks, after which experimental measurements were performed. (A-C) Cardiac function was evaluated by echocardiography, including M-mode imaging (A), left ventricular EF% (B), and left ventricular FS% (C) (n=6 for each group). (D) Body weight. (E) HW/TL (n=6 per group). (F) The survival rates of mice (n=15 per group). Data are mean ± SEM. *, P<0.05; **, P<0.01. Data were analyzed using one-way ANOVA followed by Tukey’s post-hoc test (B-E) or Kaplan-Meier survival analysis (F); the results were compared using the log-rank test. ANOVA, analysis of variance; CON, control; DOX, doxorubicin; EF, ejection fraction; FS, fractional shortening; HW/TL, heart weight to tibia length ratio; SEM, standard error of the mean; TIRZ, tirzepatide.

Tirzepatide mitigates DOX-induced myocardial damage

In the control and tirzepatide groups, myocardial fibers were arranged in a regular and compact manner with intact myofiber continuity and normal nuclear morphology, and no structural damage was observed. In the DOX group, significant DOX-induced myocardial injury was noted, characterized by the disarrangement and structural disruption of myocardial fibers. In the DOX + tirzepatide group, following treatment with tirzepatide, the disarrangement of myocardial fibers was significantly ameliorated, and the structural integrity of myofibers was markedly restored (Figure 2A). Histopathological analysis further corroborated the functional findings, as H&E staining revealed severe destruction and disarray of myocardial fibers, accompanied by interstitial fibrosis demonstrated by Masson’s trichrome staining (Figure 2B,2C). These DOX-induced histopathological abnormalities were partially or completely ameliorated following tirzepatide treatment, suggesting its protective efficacy may be attributable, in part, to this ameliorative effect.

Figure 2 Tirzepatide mitigates DOX-induced myocardial damage. After confirming DOX-induced cardiac dysfunction (Figure 1), heart tissues were collected for histology. (A) H&E staining for the assessment of myocardial injury. Scale bar =10 μm. In the DOX group, arrows indicate the disarrangement and structural disruption of myocardial fibers; in the DOX + TIRZ group, arrows indicate that the disarrangement of myocardial fibers was significantly ameliorated, and the structural integrity of myofibers was markedly restored. (B,C) Masson’s trichrome staining reveals myocardial fibrosis, including representative images (B) and fibrotic area quantification (C). Scale bar =10 μm (n=6 per group). Data are mean ± SEM. **, P<0.01. Data were analyzed using one-way ANOVA followed by Tukey’s post-hoc test. ANOVA, analysis of variance; CON, control; DOX, doxorubicin; H&E, hematoxylin and eosin; SEM, standard error of the mean; TIRZ, tirzepatide.

Tirzepatide confers resistance to DOX-elicited morphological disruption of myocardial mitochondria in vivo

The heart, as a highly energy-demanding organ, relies critically on mitochondrial function for its operation. Although mitochondria serve as the primary energy source for cardiomyocytes, they simultaneously generate substantial amounts of ROS, leading to mitochondrial damage (21). During the development of DOX-induced cardiomyopathy, mitochondrial dysfunction has been identified as the pivotal factor driving the pathogenesis (22,23). Consistent with prior reports, DOX caused mitochondrial cristae rarefaction and disorganization, whereas tirzepatide alleviated these morphological defects in mitochondria (Figure 3).

Figure 3 Tirzepatide protects against DOX-induced myocardial mitochondrial morphological injury in vivo. Core mitochondrial parameters evaluated comprised: ultrastructural integrity, cristae morphology, degree of swelling, extent of vacuolation, and matrix density. Representative TEM images depicting the mitochondrial structure of cardiac muscle. Scale bar =200 nm. CON, control; DOX, doxorubicin; TEM, transmission electron microscopy; TIRZ, tirzepatide.

Tirzepatide counteracts DOX-induced myocardial mitochondrial dysfunction in vitro

Consistent with in vivo findings, our in vitro data demonstrated that tirzepatide effectively reversed DOX-induced mitochondrial dysfunction in primary cultured cardiomyocytes in a concentration-dependent manner, as measured by cell viability assay (Figure S1). Based on concentration-response profiling, 1 µM tirzepatide was determined to be the optimal and effective dose for all subsequent experiments. DOX significantly reduced MMP in cardiomyocytes, evidenced by increased green fluorescence and decreased red fluorescence in JC-1 staining. These pathological alterations were substantially attenuated by tirzepatide co-treatment (Figure 4A,4B and Figure S2).

Figure 4 Tirzepatide counteracts DOX-induced myocardial mitochondrial dysfunction in vitro. Primary cardiomyocytes were pretreated with tirzepatide (1 μM) for 2 h, followed by 24 h exposure to DOX (1 μM) or saline (vehicle control). (A,B) JC-1 staining for assessment of MMP. Representative image (A) and quantification (B) of JC-1 aggregates/monomers. Scale bar =20 μm (n=6 per group). Data are mean ± SEM. **, P<0.01. Data were analyzed using one-way ANOVA followed by Tukey’s post-hoc test. ANOVA, analysis of variance; CON, control; DOX, doxorubicin; MMP, mitochondrial membrane potential; SEM, standard error of the mean; TIRZ, tirzepatide.

Discussion

DOX is widely used clinically to treat malignant tumors and demonstrates significant efficacy. However, its serious adverse effects, particularly cardiotoxicity, pose a major concern, as prolonged administration increases heart failure risk (24). While experimental agents like antioxidants and antiapoptotic substances offer potential cardioprotection, they remain far from clinical application (25). Collectively, our data demonstrate that tirzepatide protects against both the functional decline and structural damage induced by DOX in the heart. This protective effect is mediated, at least in part, through the alleviation of mitochondrial dysfunction.

Tirzepatide was developed by engineering GLP-1 activity into the GIP sequence (5). This dual agonist exhibits imbalanced activity, favoring GIPR over GLP-1R: it binds GIPR with affinity comparable to native GIP, but shows approximately five-fold lower affinity for GLP-1R than native GLP-1. Tirzepatide acts as a biased agonist at the GLP-1R, preferentially stimulating cyclic adenosine monophosphate (cAMP) production over β-arrestin recruitment. While it enhances insulin secretion from mouse islets via this receptor, this effect does not translate to human islets, highlighting a significant species-specific divergence (26,27). This study reveals that tirzepatide potentially affords enhanced cardioprotection through synergistic co-activation of both incretin receptors. Substantial clinical evidence demonstrates tirzepatide’s superiority over semaglutide (GLP-1RA) in glycemic control, weight reduction, and cardiovascular outcome improvement—attributes of particular relevance to chemotherapy patients exhibiting frequent metabolic dysregulation (28). Notably, 30–40% of patients manifest suboptimal therapeutic responses to GLP-1 mono-agonist therapy (29), whereas GIPR agonism independently ameliorates myocardial energetic metabolism (30). These findings collectively suggest that dual-receptor co-agonism may extend therapeutic benefits to a broader patient cohort. Future investigations should prioritize direct comparative assessment of cardiotoxicity mitigation efficacy between these agents and delineate the underlying mechanisms governing GIP-mediated cardioprotection.

DOX induces progressive cardiotoxicity: initial subclinical myocardial damage progresses to asymptomatic declines in left ventricular EF and ultimately irreversible heart failure (31). Consequently, echocardiography quantified cardiac function. Remarkably, tirzepatide nearly restored DOX-reduced EF and FS to baseline in mice, demonstrating potent cardioprotective effects. These findings prompted histological assessment (H&E/Masson staining), which notably revealed that tirzepatide significantly attenuated cardiac fibrosis and tissue damage. The precise pathogenesis of DOX-induced cardiotoxicity continues to be defined; however, current evidence implicates several interlinked pathological processes. These primarily encompass mitochondrial impairment, oxidative stress, dysregulated autophagy, inflammatory activation, and the induction of distinct cell death modalities, including ferroptosis, pyroptosis, and apoptosis. ROS generation is regarded as the primary contributor. Anthracyclines induce substantial ROS accumulation within cardiomyocytes, causing severe damage (32). This stems directly from DOX’s quinone moiety: metabolic reduction forms semi-quinone radicals, and subsequent oxygen consumption drives massive ROS production (33). Notably, mitochondria, as the major organelles involved, mediate phospholipid peroxidation in this process (34). Given mitochondria’s pivotal role in DOX cardiotoxicity, we assessed their myocardial structure and function. TEM demonstrated that tirzepatide significantly ameliorated mitochondrial disorganization, restoring membrane integrity, reducing vacuolation, and normalizing cristae architecture. Crucially, this intervention reversed DOX-induced dissipation of MMP.

While revealing tirzepatide’s novel cardioprotective role against DOX-induced cardiotoxicity via mitochondrial enhancement, this study acknowledges critical limitations. Unresolved mechanisms underlying tirzepatide’s mitochondrial regulation require elucidation to identify precise therapeutic targets. Additionally, potential interference with DOX’s antitumor efficacy during co-administration warrants investigation. We therefore commit to further research elucidating these mechanistic underpinnings.

Conclusions

Our findings demonstrate tirzepatide’s cardioprotective efficacy through mitochondrial preservation, attenuating DOX-induced cardiomyocyte damage and cardiac injury. These results establish tirzepatide as a promising therapeutic candidate against chemotherapy-related cardiotoxicity.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2801/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2801/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2801/prf

Funding: This study was supported in part by grants from the Hunan Provincial Health Science Research Project (No. 20256345) and Hunan Provincial Natural Science Foundation (No. 2024JJ9488).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1-2801/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were performed under a project license (No. 92120251015RB02) granted by the Institutional Animal Care and Use Committee (IACUC) of The 921 Hospital of Joint Logistics Support Force People’s Liberation Army of China, in compliance with National Institutes of Health (NIH) guide for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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