Abstract Aims Doxorubicin is a powerful chemotherapeutic agent for cancer, whose use is limited due to its potential cardiotoxicity. Semaglutide (SEMA), a novel analog of glucagon-like peptide-1 (GLP-1), has received widespread attention for the treatment of diabetes. However, increasing evidence has highlighted its potential therapeutic benefits on cardiac function. Therefore, the objective of this study was to examine the efficacy of semaglutide in ameliorating doxorubicin-induced cardiotoxicity. Methods and results Doxorubicin-induced cardiotoxicity is an established model to study cardiac function. Cardiac function was studied by transthoracic echocardiography and invasive hemodynamic monitoring. The results showed that semaglutide significantly ameliorated doxorubicin-induced cardiac dysfunction. RNA sequencing suggested that Bnip3 is the candidate gene that impaired the protective effect of semaglutide in doxorubicin-induced cardiotoxicity. To determine the role of BNIP3 on the effect of semaglutide in doxorubicin-induced cardiotoxicity, BNIP3 with adeno-associated virus serotype 9 (AAV9) expressing cardiac troponin T (cTnT) promoter was injected into tail vein of C57/BL6J mice to overexpress BNIP3, specifically in the heart. Overexpression of BNIP3 prevented the improvement in cardiac function caused by semaglutide. In vitro experiments showed that semaglutide, via PI3K/AKT pathway, reduced BNIP3 expression in the mitochondria, improving mitochondrial function. Conclusion Semaglutide ameliorates doxorubicin-induced mitochondrial and cardiac dysfunction via PI3K/AKT pathway, by reducing BNIP3 expression in mitochondria. The improvement in mitochondrial function reduces doxorubicin-mediated cardiac injury and improves cardiac function. Therefore, semaglutide is a potential therapy to reduce doxorubicin-induced acute cardiotoxicity. Keywords: BNIP3, Doxorubicin-induced cardiotoxicity, Mitochondria, PI3K/AKT, Semaglutide Graphical abstract Image 1 [49]Open in a new tab 1. Introduction Doxorubicin (DOX), an anthracycline antibiotic commonly used to treat a range of cancers, including breast cancer, bladder cancer, ovarian cancer, gastric cancer, lung cancer, thyroid cancer, and advanced esophageal squamous cell carcinoma, is one of the most effective anticancer drugs available [[50][1], [51][2], [52][3], [53][4], [54][5]]. However, several side effects limit its therapeutic value, including cardiotoxicity, which may cause irreversible loss in cardiac function and subsequently, heart failure [[55]6,[56]7]. Currently, dexrazoxane is the only FDA-approved cardioprotective agent specifically indicated in patients receiving anthracycline chemotherapy. Nevertheless, it is worth noting that dexrazoxane may potentially trigger adverse effects such as bone marrow suppression, liver toxicity, and the development of secondary malignant tumors [[57]8]. Therefore, more efficient and safer cardioprotective drugs for chemotherapy-related cardiotoxicity are needed. Antihyperglycemic medications, such as glucagon-like peptide-1 (GLP-1) receptor agonists, have been found to improve left ventricular function in patients with acute myocardial infarction and severe systolic dysfunction [[58]9,[59]10]. Additionally, they improved left ventricular ejection fraction and functional status in patients with chronic heart failure [[60]11]. While the cardioprotective effects of GLP-1 receptor agonist are well-established, their effect on doxorubicin-induced cardiotoxicity has not been well investigated. Given the role of semaglutide, one GLP-1 analog, in cardioprotection, it is plausible that semaglutide may offer protection against doxorubicin-induced cardiac dysfunction. Therefore, our present study examined the effect of semaglutide on doxorubicin-induced cardiotoxicity and investigated its underlying mechanisms. 2. Materials and methods 2.1. Animals Six-week-old C57/BL6J mice were obtained from the laboratory animal center of Daping Hospital, Third Military Medical University (Chongqing, China). The mice were housed in and treated at the Animal Centre of Daping Hospital, where they were maintained under specific pathogen-free, environmentally controlled (temperature: 20–25 °C; humidity: 50 ± 5%) barrier conditions in individual ventilated cages and fed with sterile food and water ad libitum. Following a week of acclimatization, the mice were randomly assigned to individual groups. The mice were administered doxorubicin (i.p. 5 mg/kg; MedChemExpress, Monmouth Junction, NJ) or an equivalent volume of the vehicle (saline) once a week for 4 weeks [[61]12]. Semaglutide (12 μg/kg; Novo Nordisk, Copenhagen, Denmark) was subcutaneously injected daily for 6 weeks as shown in [62]Fig. S1A [[63]13]. For the overexpression of BNIP3, the mice received a single intravenous injection of an adeno-associated serotype 9 (HBAAV2/9-cTNT-m-Bnip3-2 × flag-EGFP) viral vector or negative control (HBAAV2/9-cTNT-EGFP) (Hanbio Biotechnology, Shanghai, China), via the tail vein at a concentration of 5 × 10^10 viral genome per mouse and studied after 4 weeks ([64]Fig. S1B). At the end of the experiment, all mice were anesthetized with isoflurane (5%) and euthanized by cervical dislocation. The experimental procedures received ethical approval from the Institutional Animal Care and Use Committee of the Third Military Medical University (ethical approval number: AMUWEC20229024), adhering to the guidelines stated in the NIH Guide for the Care and Use of Laboratory Animals. 2.2. Echocardiography Echocardiography was performed using a high-resolution ultrasound imaging system (Vevo 2100, Visual Sonics, Canada). Briefly, the mice were anesthetized with 1.5% isoflurane and 98.5% O[2] and placed in a supine position on a temperature-controlled heating platform to maintain a body temperature of 37 °C. The parasternal long axis plane was used to calculate the systolic function index during the M-type measurement, using the Vevo analysis software (Vevo LAB 5.5.1), taking the average of at least three consecutive heartbeats. The investigators performing these studies were blinded to the drug interventions. 2.3. Invasive hemodynamics Isoflurane (3% for induction; 1.5% for maintenance mixed with 98.5% oxygen) was used to anesthetize the animals. The mice were placed on regulated heating pads to keep their body temperatures at 37 °C and after having their tracheas intubated were subsequently artificially ventilated, using a volume-controlled ventilator trach. The left external jugular vein was cannulated with a polyethylene catheter to provide fluids. The right carotid artery was used as the entry point for a 1.4 F micro tip pressure-conductance micro-catheter (SPR-839, Millar Instruments, Houston, TX), which was inserted past the aortic valve and into the left ventricle (LV). Following a 5-min stabilization period, the signals were continuously collected using a pressure-volume (P-V) conductance system coupled to a Power Lab 16/30 data collection system (AD Instruments, Colorado Springs, CO), at a sampling rate of 1000 samples/s. The abdominal inferior vena cava (IVC) was briefly occluded, to allow construction of variably loaded pressure-volume loops, from which LV end-systolic (ESPVR) and end-diastolic pressure-volume relations (EDPVR) were calculated [[65]14]. 2.4. Histological analysis The heart samples were initially fixed in 4% paraformaldehyde at 4 °C for 1–2 days, followed by dehydration in ethanol. Afterwards, the samples were cleared with xylene and then embedded in paraffin. Subsequently, the samples were sliced into sections with a thickness of 4 μm. In order to assess cardiac morphology, fibrosis, and cardiac myocyte area (CSA), the tissue sections were subjected to specific staining procedures. Hematoxylin and eosin (H&E) staining (G1120, Solarbio, Beijing, China), Masson's trichrome staining (G1345, Solarbio), and wheat germ agglutinin (WGA) staining ([66]W11261, Thermo Fisher Scientific, Waltham, MA) were utilized, following standard protocols. The sections were visualized using an Olympus SlideView VS200 microscope (Olympus, Hamburg, Germany). Quantitative analysis of the staining was conducted in a blinded manner using Image J software. 2.5. Cardiac marker enzymes measurements BNP (HB533-Mu, Hengyuan Biological Technology, Shanghai, China), CK-MB (HB761-Mu, Hengyuan Biological Technology) and LDH (HB1402-Mu, Hengyuan Biological Technology) serum levels were measured using commercial kits. LDH release was measured using an LDH cytotoxicity assay kit (HY-K1090, MedChemExpress). All commercially available kits were used according to the manufacturer's instructions. 2.6. Oxidative stress detection and cell viability Reactive oxygen species (ROS) production was measured in vivo using DHE staining [[67]15] (S0063, Beyotime, Shanghai, China) and in vitro using MitoSOX Red staining [[68]16] (HY-D1055, MedChemExpress). In brief, cryosections of fresh heart samples or on coverslips were stained with DHE (5 μmol/L) or MitoSOX Red (5 μmol/L) in the dark at 37 °C for 30 min. The stained samples were then examined under an Olympus SlideView VS200 microscope in a blinded manner. Cell viability was determined using a calcein/PI cell viability/cytotoxicity assay kit [[69]17] (C2015 M, Beyotime). After incubation with the calcein/PI buffer for 30 min, live cells (green) and dead cells (red) were observed under an Olympus SlideView VS200 microscope. 2.7. Seahorse respirometry Seahorse respirometry was using the Seahorse XFe24 Extracellular Flux Analyzer and Seahorse XF24 FluxPaks (Agilent Technologies, Waldbronn, Germany). To evaluate oxygen consumption rates (OCR), primary cardiomyocytes were seeded in an assay microplate at 4 × 10^4 cells/well in growth medium. After treatment with indicated reagents for indicated period, the culture medium was replaced with Seahorse XF DMEM buffer. The OCR was assessed at the basal level and following metabolic perturbations with 1.5 μmol/L oligomycin, 1 μmol/L FCCP (mitochondrial uncoupler), and 0.5 μmol/L rotenone, and antimycin A. Calculations were performed using the Agilent Seahorse Wave Software for Agilent Seahorse XF analyzers (Seahorse Bioscience). 2.8. TUNEL staining TUNEL staining of heart slices used a one-step TUNEL In Situ Apoptosis Kit (E-CK-A331, Elabscience, Wuhan, China). Images were obtained using Olympus SlideView VS200 microscope. 2.9. Immunofluorescence staining Heart sections were deparaffinized and rehydrated using a gradient elution method with xylene and ethanol. Antigen retrieval was performed by incubation in citrate antigen retrieval solution (P0081, Beyotime) at 95 °C for 20 min. The cells were fixed with 4% paraformaldehyde for 15 min at room temperature. The prepared heart sections and cells were blocked by incubation in QuickBlock™ Immunostaining Blocking Solution (P0260, Beyotime) at 37 °C for 30 min, followed by overnight incubation at 4 °C with primary antibodies against γ-H2AX (1:200, ab81299, Abcam, Cambridge, MA), cardiac troponin T (1:200, ab209813, Abcam), and BNIP3 (1:200, ab109362, Abcam). The slides were then incubated with Alexa Fluor 488™ secondary antibody (1:200, A-11008, Thermo Fisher Scientific) or AlexaFluor555™ secondary antibody (1:200, A-11035, Thermo Fisher Scientific) in the dark at 37 °C for 1 h. The nucleus was counterstained with DAPI (C1006, Beyotime, China). Each step was followed by washing with PBS three times for 5 min. The images were captured using laser confocal microscopy and analyzed with the Olympus Fluoview FV300 version 3C Acquisition Software. Quantitative analysis of the fluorescence intensity was conducted in a blinded manner using Image J software. 2.10. RNA-seq analysis The hearts from C57/BL6J mice, with or without semaglutide treatment, were collected for further analysis. RNA isolation, quality control, library construction, and sequencing were carried out by Shanghai Applied Protein Technology ([70]https://www.aptbiotech.com). The library preparations were then subjected to sequencing on an Illumina Novaseq 6000 platform. Subsequent bioinformatics analyses were performed using RStudio (Version 2022.07.2 Build 576). Differential expression analyses were conducted using DESeq2 (R packages v1.38.1) with a cut-off set at a 1.5-fold change and a significance threshold of P < 0.05 [[71]18,[72]19]. Pathway enrichment analysis was carried out using cluster Profiler (R packages v4.6.0) to examine the KEGG pathways [[73]20]. Enrichment analysis for Gene Ontology (GO) was conducted by utilizing GSEABase (R packages v1.60.0). Plots were generated using enrichplot (v1.18.3), ggplot2 (v3.4.1), cowplot (v1.1.1), and pheatmap (v1.0.12) packages. The RNA sequencing (RNA-seq) data reported in this paper have been deposited into sequence read archive (SRA) database under BioProject accession number PRJNA1053606 ([74]https://trace.ncbi.nlm.nih.gov/Traces/study/?acc=PRJNA1053606&o=ac c_s%3Aa). 2.11. RT-qPCR After extracting total RNA from the hearts using RNAiso plus reagent (9108, TaKaRa, Dalian, China) according to the manufacturer's instructions [[75]21], cDNA was obtained using the PrimeScriptTM RT reagent Kit with gDNA Eraser (RR047A, Takara, China). Subsequently, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was conducted using the SYBR Premix Ex Taq II (RR820A, TaKaRa, China) to detect the levels of targeted mRNAs with the 7900HT Fast RealTime PCR system (Applied Biosystems). GAPDH was utilized as the internal control. Each reaction was performed in triplicate. The values were normalized to GAPDH to calculate the relative RNA expression levels. The primer sequences used for mRNA expression detection are listed in [76]Supplementary Table S1. 2.12. Transmission electron microscopy The hearts were prefixed with 3% glutaraldehyde and then postfixed with 1% osmium tetroxide. They were dehydrated in a series of acetone solutions, infiltrated with Epon 812 for an extended period, and finally embedded. The semithin sections were stained with methylene blue, while ultrathin sections were cut using a diamond knife and stained with uranyl acetate and lead citrate. The sections were examined using a JEM-1400FLASH Transmission Electron Microscope. 2.13. Mitochondrial assays The JC-1 mitochondrial membrane potential assay kit (C2003S, Beyotime) was utilized to measure the mitochondrial membrane potential (MMP). Following a single wash with PBS, the cells were incubated with 500 μL of staining working buffer per well for 20 min. After two washes with staining buffer solution, the covering liquid was replaced with Dulbecco's Modified Eagle Medium (DMEM) ([77]11965092, Thermo Fisher Scientific). Finally, the cell fluorescence was observed using a fluorescence microscope. The mitochondrial permeability transition pore (PT-pore) assay kit (C2009S, Beyotime) was used to detect the opening of PT-pore. Each well was incubated with 500 μL of staining working solution for 30 min, followed by replacing the incubation liquid with pre-warmed DMEM and incubating further for 30 min under light-avoiding conditions. The nuclei were counterstained with Hoechst33342 (C1017, Beyotime). Subsequently, the cell fluorescence was observed under a fluorescence microscope after two washes with PBS. Mitotracker Red (M7512, ThermoFisher Scientific) was preloaded into live cells before conducting immunofluorescence experiments. Subsequently, immunocytochemistry was carried out. Using Image J software, a blinded quantitative study of the fluorescence intensity was performed. 2.14. Cell culture Neonatal (1-3 day-old) Sprague-Dawley rats were used to isolate primary ventricular cardiomyocytes (NRVMs) [[78]22]. Briefly, neonatal rats were anesthetized by isoflurane and then euthanized by cervical dislocation. The hearts were removed and immediately placed in ADS buffer (pre-cooled, composed of 120 mmol/L NaCl, 20 mmol/L HEPES, 8 mmol/L NaH[2]PO[4], 6 mmol/L glucose, 5 mmol/L KCl, 0.8 mmol/L MgSO[4], pH 7.4). Subsequently, the hearts were transferred into fresh ADS buffer and diced into pieces smaller than 1 mm^3. Then, the heart tissues were repetitively rinsed with sterile ADS until the rinse fluid became clear. The tissues were gathered and placed into a sterile 25 ml bottle along with 2 ml of collagenase II solution and 2 ml of ADS. The digestion process was conducted by continuously agitating the mixture at a speed of 180 rpm, maintained at a temperature of 37 °C, for an 8-min-duration. The suspensions were filtered into 50 ml centrifuge tubes that were kept chilled in ice. Into each tube, 1 ml of fetal bovine serum (FBS, Gibco) was added. After a 5-min centrifugation at 2000 g, the supernatant was discarded. The pellets were suspended in FBS and subsequently seeded into a petri-dish. The dish was incubated at 37 °C, with a CO[2] concentration of 5%, for a period of 2 h. This process allowed for the separation of cardiomyocytes and fibroblasts. Then, the supernatant was carefully transferred into a 50 ml centrifuge tube. High purity cardiomyocytes were isolated from fibroblasts by employing a Percoll gradient (GE17-0891-01, Sigma-Aldrich) in sterile ADS buffer. Following centrifugation at 1800 g for 45 min, the cardiomyocytes were separated and placed in the intermediate layer. The isolated cells were then cultured in a 5% CO[2] incubator at 37 °C for 24 h using DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. The primary cardiomyocytes were incubated in a microplate, and then infected with the AAV9 (Hanbio Biotechnology, Shanghai, China). The medium was replaced 6–8 h after infection, and subsequent treatments were performed after 24 h. 2.15. Mitochondrial fractionation, western blot analyses, and immunoprecipitation Mitochondrial and cytoplasmic fractions were extracted from cardiomyocytes using the Cell Mitochondria Isolation kit (C3601, Beyotime Biotechnology). Protein expression levels in both cardiac tissues and NRVMs were measured by Western blot analysis. Total protein samples were collected, and their concentrations were measured using a BCA protein assay kit (PC0020, Solarbio). Afterwards, 50 mg of the protein samples were subjected to SDS-PAGE for separation and then transferred onto nitro-cellulose membranes (GE Healthcare Life Sciences, Logan, UT). To block non-specific binding, the membranes were treated with 5% non-fat milk for 1 h and then incubated overnight at 4 °C with the corresponding primary antibodies. The blotted membranes were probed with the following antibodies, that included rabbit anti-BNIP3 antibody (1: 1,000, ab109362, Abcam), rabbit anti-PI3K p85 antibody (1: 1,000, ab191606, Abcam), rabbit anti-p-PI3K p85 antibody (1: 1,000, ab182651, Abcam), rabbit anti-AKT antibody (1:1,000, 4691T, Cell Signaling Technology, Danvers, MA), rabbit anti-p-AKT antibody (1:1,000, 4060T, Cell Signaling Technology), rabbit anti-VDAC1 antibody (1:5000, ab154856, Abcam), and rabbit anti-GAPDH antibody (1:10,000, 10494-1-AP, Proteintech, Wuhan, China). Following incubation, the membranes were washed with a wash buffer to remove any excess primary antibodies. Fluorescent-labeled goat anti-rabbit IgG (5151, Cell Signalling Technology) was then used to detect the primary antibodies, and the resulting protein bands were visualized using the Odyssey Western Blot Detection System (LI-COR Biotechnology, Lincoln, Nebraska, NE). The relative protein expression levels were determined based on the gray intensity of the bands. Phosphorylated BNIP3 was detected by immunoprecipitation. The cell lysates were immunoprecipitated with phospho-Ser/Thr antibody (Abcam, ab17464) and then immunoblotted with anti-BNIP3 antibody. For immunoprecipitation, IgG was used as negative control (NC) and anti-BNIP3 antibody was used as positive control (PC) 2.16. Statistical analysis The data are presented as mean ± SEM. GraphPad Prism 9.0 (GraphPad Software) was used for statistical analyses. Experiments were independently conducted at least 3 times, with specific N numbers and technical replicates provided in each figure. Variance between groups was assessed using a Brown-Forsythe test, which showed no significant differences. One-way analysis of variance (ANOVA) was used to compare more than two groups, followed by Tukey's post-hoc test when different groups were compared among each other. Differences between the two groups were assessed using a 2-tailed unpaired Student's t-test. Survival analysis was performed utilizing the Kaplan-Meier method, and the outcomes were subsequently compared using the log-rank test. 3. Results 3.1. Semaglutide ameliorates doxorubicin-induced cardiac dysfunction To investigate the potential role of semaglutide in doxorubicin-induced cardiotoxicity, we established a mouse model of chronic doxorubicin-induced myocardial injury ([79]Fig. S1A). Consistent with other reports [[80]23], the doxorubicin (i.p. 5 mg/kg/week) mouse model has decreased body weight, heart size, and heart weight/tibia length ratio ([81]Fig. 1A-C), and increased serum levels of lactate dehydrogenase (LDH) and creatine kinase MB (CK-MB) ([82]Fig. 1D-E). The cardiac function was impaired by doxorubicin, shown by increased serum brain natriuretic peptide (BNP) concentration ([83]Fig. 1F), and decreased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) and increased left ventricular internal dimension-systole (LVIDs) and left ventricular internal dimension-diastole (LVIDd) ([84]Fig. 1G). Furthermore, doxorubicin induced a rightward shift in the steady-state pressure-volume (P-V) loop and led to a reduction in dp/dt[min], dp/dt[max], and stroke volume (SV) ([85]Fig. 1H). In addition, there was a decrease in the slope of the LV end-systolic pressure-volume relationship (ESPVR) ([86]Fig. 1I) acquired during variable loading, subsequent to inferior vena cava occlusion in doxorubicin-treated mice. The doxorubicin-mediated impairment in cardiac function may have contributed to the decreased survival rate relative to the control group ([87]Fig. 1J). The above-mentioned aberrant parameters were almost completely abrogated by semaglutide (s.c. 12 μg/kg/day) ([88]Fig.1A-J). Fig. 1. [89]Fig. 1 [90]Open in a new tab SEMA ameliorates doxorubicin-induced cardiac dysfunction. C57/BL6J mice were used to establish a mouse model of doxorubicin-induced myocardial injury by the intraperitoneal (i.p.) injection of doxorubicin (5 mg/kg/week) for 4 weeks. Before the induction of cardiac dysfunction by doxorubicin treatment, semaglutide (SEMA,12 μg/kg/day) was subcutaneously injected daily for one week and continued for another 5 weeks, including one week after the last doxorubicin injection, after which time the parameters were collected. A-C: Body weight (A), representative whole heart pictures (B), and the ratio of heart weight to tibia length (HW/TL) (C) (n = 6 per group). D-F: Serum levels of lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), and brain natriuretic peptide (BNP) (n = 6 per group). G: Cardiac function determined by echocardiography, including typical M-mode echocardiogram pictures, left ventricular ejection fraction (EF%), left ventricular fractional shortening (FS%), left ventricular internal dimension-systole (LVIDs), and left ventricular internal dimension-diastole (LVIDd) (n = 6 per group). H and I: Cardiac function determined by invasive hemodynamics, including representative left ventricular pressure-volume (P-V) loops at steady state, dp/dt[min] (maximal relaxation rate), dp/dt[max] (maximum rate of pressure development), stroke volume (SV), representative left ventricular end-systolic pressure volume relationship (ESPVR) slope and load independent parameters of LV function: ESPVR (n = 6 per group). J: Mouse survival rates during the six week-experimental period (n = 15 per group). Statistical analysis was performed using one-way ANOVA, followed by Tukey's post-hoc test (A, C-I). Kaplan-Meier survival analysis was used to evaluate the survival rate of mice, and the results were compared using the log-rank test (J). *p < 0.05, **p < 0.01. The functional data were corroborated by histopathological studies. Doxorubicin caused severe myocardial fiber breakdown and disorder, shown by H&E staining ([91]Fig. 2A), accompanied with increased interstitial fibrosis and decreased cardiomyocyte cross-sectional area, shown by Masson trichrome and WGA staining ([92]Fig. 2B-C). These histopathological aberrations caused by doxorubicin were partially or completely reversed by semaglutide treatment. TUNEL staining also showed that semaglutide decreased the increased apoptosis of cardiomyocytes in doxorubicin-treated mice ([93]Fig. 2D), which may, at least in part, account for the protective effects of semaglutide on doxorubicin-induced cardiotoxicity. Fig. 2. [94]Fig. 2 [95]Open in a new tab SEMA attenuates doxorubicin-induced myocardial damage. Doxorubicin (DOX)-induced cardiac dysfunction was established as shown in [96]Fig. 1, and then the hearts were harvested for histological analyses. A: H&E staining for the assessment of myocardial injury. Scale bar = 50 μm. B: Masson staining for the assessment of myocardial fibrosis. Scale bar = 50 μm. Representative image (B1) and quantification (B2) of the myocardial fibrotic area (n = 6 per group). C: Wheat germ agglutinin (WGA) staining for the assessment of myocyte size. Scale bar = 20 μm. Representative image (C1) and quantification (C2) of cross-sectional area (CSA) (n = 6 per group). D: TUNEL staining for the assessment of apoptosis. Scale bar = 20 μm. Representative images (D1) and quantification (D2) of TUNEL-positive cardiomyocytes (n = 6 per group). Statistical analysis was performed using one-way ANOVA, followed by Tukey's post-hoc test. *p < 0.05, **p < 0.01. 3.2. Semaglutide protects cardiomyocytes against doxorubicin-induced injury by improvement of mitochondrial function The results of the in vivo experiments were corroborated by the in vitro experiments. In cardiomyocytes in primary culture semaglutide reduced the doxorubicin-mediated decrease in cell viability, determined by calcein in a concentration-dependent manner ([97]Figs. S2A and S2B). Based on the concentration response studies, 1 nM was chosen as the working concentration for semaglutide in subsequent experiments. Doxorubicin decreased cell viability of cardiomyocytes ([98]Figs. S2A and S2B), and increased LDH concentration in the culture medium, which were reversed by semaglutide ([99]Figs. S2A–S2C). Due to the role of reactive oxygen species (ROS) and DNA damage in the doxorubicin-induced cardiotoxicity [[100]24,[101]25], we measured the levels of ROS and γ-H2AX, a DNA double-strand break marker, in the cardiomyocytes. We found that doxorubicin increased ROS levels and γ-H2AX expressions in vivo ([102]Fig. 3A and C) and in vitro ([103]Fig. 3B and D), which were reversed by semaglutide ([104]Fig. 3A-D). It is known that doxorubicin-induced increase in ROS is via a detrimental effect on the mitochondria [[105]26]. To investigate whether mitochondrial ROS contributes to doxorubicin-induced cardiac damage, we used mitoTEMPO, a mitochondrially targeted antioxidant, in doxorubicin-treated cardiomyocytes. Our results showed that mitoTempo reduced the doxorubicin-induced cell mortality and LDH release ([106]Figs. S2D–S2E), confirming the significance of mitochondrial ROS in doxorubicin-induced cardiomyocyte dysfunction. Our present study also showed that doxorubicin increased the permeability transition (PT)-pore opening ([107]Fig. 3E), decreased mitochondrial membrane potential (MMP), assessed by JC-1 aggregate levels ([108]Fig. 3F) and caused morphological defects of the mitochondria in the cardiomyocytes, as evidenced by distorted and reduced cristae density ([109]Fig. 3G). Furthermore, we used extracellular flux analysis to monitor in real-time mitochondrial respiratory capacity in vitro. Doxorubicin markedly reduced mitochondrial oxygen consumption rate (OCR), basal and maximal respiration, and spare respiratory capacity, as well ([110]Fig. 3H). In the presence of semaglutide, the above-mentioned changes were ameliorated ([111]Fig. 3E-H), suggesting that semaglutide ameliorates doxorubicin-induced cardiac injury via improvement of mitochondrial function. Fig. 3. [112]Fig. 3 [113]Open in a new tab SEMA suppresses cardiomyocyte oxidative stress and attenuates mitochondrial damage. Doxorubicin (1 μM) or saline (vehicle) was used to treat primary cardiomyocytes for 24 h with/without combined incubation of semaglutide (SEMA, 1 nM). The cardiomyocytes were pre-incubated in the incubation medium for 2 h before exposure to doxorubicin. A and B: Total reactive oxygen species (ROS) levels in vivo, measured by DHE (A) and mitochondrial ROS levels in vitro measured by MitoSOX Red (B). Representative images (A1 and B1) and quantifications (A2 and B2) of ROS levels (n = 6 per group). DHE staining: scale bar = 50 μm. MitoSOX staining: scale bar = 10 μm. C and D: Immunofluorescence images of γ-H2AX in vivo(C) and in vitro(D). Representative images (C1 and D1) and quantifications (C2 and D2) of fluorescence intensity for γ-H2AX (n = 6 per group), Scale bar = 50 μm. E: Fluorescent images of mitochondrial permeability transition pore (PT-pore) (fluorescence intensity is negatively correlated with PT-pore opening). Representative image (E1) and quantification (E2) of PT-pore fluorescence intensity (n = 6 per group), Scale bar = 50 μm. F: JC-1 staining for assessment of mitochondrial membrane potential (MMP). Representative image (F1) and quantification (F2) of JC-1 aggregates/monomers (n = 6 per group), Scale bar = 50 μm. G: Representative transmission electron microscopy images of mitochondrial morphology of cardiac muscle. Scale bar = 1 μm. H: Mitochondrial oxygen consumption rate (OCR) of primary cardiomyocytes. Representative image (H1) and quantification (H2 - H4) of basal respiration, maximal respiration, and spare respiratory capacity (n = 5 per group). Statistical analysis was performed using one-way ANOVA, followed by Tukey's post-hoc test. *p < 0.05, **p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred