Apelin-13 attenuates doxorubicin-induced myocardial injury via the PI3K/AKT and ERK/MAPK signaling pathways

Introduction

Doxorubicin (DOX) remains a cornerstone chemotherapeutic agent used in the treatment of various malignancies, including breast cancer, lung cancer, and lymphoma (1-3). However, its clinical utility is significantly limited by its cardiotoxic effects, particularly irreversible myocardial injury. Although DOX-induced cardiotoxicity is commonly dose-dependent, clinical observations have indicated the absence of a definitively safe cumulative dose (4,5). Early signs of myocardial damage, such as electrocardiogram abnormalities and arrhythmias, may be detectable through clinical assessment. Nonetheless, effective preventive and therapeutic strategies for this cardiotoxicity are currently insufficient. Dexrazoxane is the only cardioprotective pharmacological agent approved for clinical use (6); however, its application is constrained by an associated increased risk of secondary malignancies and the inability to administer it concurrently with DOX via intravenous infusion. Consequently, the prevention and treatment of DOX-induced myocardial injury continues to be a primary focus within the field of cardio-oncology.

The pathophysiological mechanisms underlying DOX-induced myocardial injury are multifaceted, involving oxidative stress, apoptosis, autophagy dysregulation, and calcium overload (3,7,8). This form of cardiotoxicity is also characterized by alterations in multiple intracellular signaling cascades, including transforming growth factor-β (TGF-β)/mothers against decapentaplegic homolog (Smad), extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathways, which are central to cellular processes such as proliferation, differentiation, and apoptosis (9-12). Several pharmacological agents that confer cardio protection in the context of DOX-induced injury exert their effects by modulating these pathways (13-15). However, the currently known agents that confer protection against DOX-induced myocardial injury all have their limitations, including dispersed therapeutic targets, incomplete cardioprotective effects, limited applicable patient populations, restricted therapeutic windows for drug administration, substantial heterogeneity of evidence, and difficulty in precisely modulating the intracellular survival/apoptosis signaling pathways in cardiomyocytes. In this study, Apelin-13 was selected. Apelin-13 acts on the apelin receptor (APJ) on the cardiomyocyte membrane, thereby activating the downstream ERK/MAPK and PI3K/AKT signaling pathways, both of which are intimately involved in cell growth, proliferation, survival, and apoptosis. This precise regulatory paradigm of “receptor-signaling pathway-cellular change” provides a more clearly defined mechanism of action than traditional non-specific cardioprotective agents and also offers a theoretical basis for the future development of more precise cardioprotective interventions.

In addition, dysfunction within the renin-angiotensin-aldosterone system (RAAS) has been observed during DOX-induced myocardial injury, and pharmacologic modulation of the RAAS has demonstrated cardioprotective effects in this context (16-19). The APJ, also referred to as angiotensin receptor-like 1, is structurally homologous to the angiotensin II type 1 receptor and demonstrates antagonistic activity in several physiological and pathological contexts (20-22). Apelin is an endogenous ligand of APJ. Depending on the number of amino acids it contains, it is divided into different subtypes, such as Apelin-13, Apelin-17, and Apelin-36, which belong to G protein-coupled receptors. Apelin-13 is the most important subtype in human physiological responses, which plays an important role in the occurrence and development of cardiovascular diseases such as participating in various processes like inflammation, cell apoptosis, and oxidative stress (23). Upon binding to APJ, Apelin-13 activates several downstream signaling pathways, including TGF-β/Smad, ERK/MAPK, and PI3K/AKT, thereby contributing to various cardioprotective effects (24,25). These include attenuation of myocardial ischemia-reperfusion injury, inhibition of myocardial fibrosis, and reduction of oxidative stress and apoptosis (22,26-29). However, the potential protective role and specific mechanisms of Apelin-13 in the setting of DOX-induced myocardial injury remain inadequately characterized.

The present investigation involved the development of a DOX-induced cardiomyopathy model in mice, incorporating Apelin-13 administration to evaluate its effects on cardiac structure and function, cardiomyocyte apoptosis, and myocardial fibrosis. C57 mice were selected for this study based on their genetic stability, well-established modeling techniques in cardiovascular disease research, and extensive literature support. Additionally, a DOX-induced injury model was established using rat H9C2 cardiomyocytes to assess the cellular protective effects of Apelin-13 and to explore its possible underlying molecular mechanisms. H9C2 cardiomyocytes were selected for the in vitro experiments because they are derived from rat embryonic cardiac cells, retain the fundamental characteristics of cardiomyocytes, exhibit high purity, and provide excellent reproducibility in experimental studies. In addition, they are easy to culture, which reduces experimental costs, and have well-established disease models in cardiovascular research. We present this article in accordance with the ARRIVE reporting checklist (available at https://cdt.amegroups.com/article/view/10.21037/cdt-2025-358/rc).

Methods

Experimental animals

The schematic figure of the study is shown in Figure 1. A total of 40 adult male (30,31) C57BL/6J mice (8 weeks old; weight range: 20–22 g) were obtained from SPF (Beijing) Biotechnology Co., Ltd. (License No. SCXK [Beijing] 2019-0010, Beijing, China). These mice were housed in a specific pathogen-free (SPF) animal facility under controlled environmental conditions. The ambient temperature was maintained at 23±1 °C, with a relative humidity of 55±10%, regulated in real time by a central air conditioning system to ensure stable environmental parameters. The light cycle was set to 12 hours (7:00 a.m. to 7:00 p.m.) with an illumination intensity of 180±20 Lx. Three mice were housed per cage.

Figure 1 Schematic figure of the study. CCK-8, Cell Counting Kit-8; DOX, doxorubicin; HE, hematoxylin and eosin; TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling.

Following one week of acclimatization in an isolated, barrier-controlled environment under standardized housing conditions, the mice were randomly allocated to experimental groups.

Humane endpoints were established in this study to minimize both the number of experimental animals used and their suffering. The experiment was terminated early and the mice were euthanized if any of the following conditions occurred: (I) body weight loss of ≥20% from baseline; (II) persistent lethargy, unresponsiveness, or inability to feed or drink spontaneously; (III) severe respiratory distress or overt signs of heart failure; (IV) markedly reduced locomotor activity or inability to ambulate normally; (V) unrelievable pain or other severe adverse reactions. Upon meeting any of the above criteria, the animals were immediately euthanized by cervical dislocation under anesthesia.

All animal experiments were performed under a project license (No. 2024kjt105) granted by the Ethics Committee of The Affiliated Cardiovascular Hospital of Shanxi Medical University, in compliance with institutional guidelines for the care and use of animals. A protocol was prepared before the study without registration.

Experimental cells

Rat H9C2 cardiomyocyte cells were obtained from the Cell Bank of the Chinese Academy of Sciences (American Type Culture Collection). The cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (high glucose; Gibco, Life Technologies Corporation, Thermo Fisher Scientific, Grand Island, New York, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), under standard culture conditions.

Reagents and equipment

DOX (DOX hydrochloride) and Apelin-13 were purchased from MedChemExpress (MedChemExpress LLC, Monmouth Junction, New Jersey, USA).

Primary antibodies against B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved caspase-3, ERK, phosphorylated ERK (p-ERK), AKT, phosphorylated AKT (p-AKT), TGF-β, Smad2/3, and Smad7, along with appropriate goat anti-rabbit or goat anti-mouse secondary antibodies, were purchased from Proteintech. The Cell Counting Kit-8 (CCK-8) was acquired from Biosharp Life Sciences Corporation (Beijing Labgic Technology Co., Ltd., Beijing, China). The one-step terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) apoptosis detection kit (fluorescein isothiocyanate-labeled, green fluorescence) was purchased from KeyGen Biotech Co., Ltd. (Nanjing, China). Additional equipment included a small animal ultrasound imaging system (Vevo 3100 LT, Toronto, Canada) and a pathological slide scanner (KF-PRO-005; KFBIO, Ningbo, China).

Experimental grouping and drug intervention

The 40 healthy male C57 mice were randomly assigned to four groups with all mice receiving intraperitoneal injections as follows: Control group (n=8, normal saline 4 mL/d), DOX group (n=11, DOX 2.5 mg/kg every other day, cumulative dose of 20 mg/kg), Apelin-13 group (n=11, Apelin-13 0.1 µmol/kg, once daily), Apelin-13 + DOX group (n=10, Apelin-13 0.1 µmol/kg, once daily; DOX 2.5 mg/kg, every other day, cumulative dose of 20 mg/kg). Dosages for DOX and Apelin-13 were determined based on preliminary experimental findings and existing literature (26,29,32). In the Apelin-13 + DOX group, Apelin-13 and DOX were administered on the same day with DOX being administered 1–2 hours after the administration of Apelin-13. The total experimental duration was 35 days. The timeline of drug administration is shown in Figure S1.

In the in vitro model, H9C2 cardiomyocytes from the same batch were seeded into 6-well plates and allowed to reach 90% confluence before random allocation into four groups: Control group (normal DMEM medium), Apelin-13 group (normal DMEM medium + Apelin-13 1 µM), DOX group (normal DMEM medium + DOX 2 µM), Apelin-13 + DOX group (normal DMEM medium, first treated with Apelin-13 1 µM, followed by DOX 2 µM after 1 hour). Drug concentrations for in vitro interventions were selected based on preliminary assays and previous reports (26,28,29).

Echocardiographic assessment

The echocardiographic assessment was conducted after 35 days of drug intervention. Following weight measurement, a high-resolution small animal ultrasound imaging system was employed to assess left ventricular structure [left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD)] and function [left ventricular ejection fraction (LVEF) and fractional shortening (FS)] using two-dimensional, M-mode, and Doppler echocardiography. All imaging measurements were conducted by two independent investigators who were blinded to the experimental group allocations.

Body weight and organ weight

Following echocardiographic assessment, mice were euthanize by cervical dislocation under anesthesia, and all procedures were performed by experienced personnel in accordance with ethical requirements. and tissues including the heart, bilateral lungs, liver, and kidneys were excised and weighed. One leg was collected to isolate the tibia for length measurement to normalize heart weight. The heart was dissected, and left ventricular annular myocardial tissue was fixed in 4% paraformaldehyde, followed by paraffin embedding. Paraffin blocks were preserved for subsequent analyses, while the remaining left ventricular myocardial tissue and bilateral kidney tissues were stored in cryovials at −80 °C for future experimental procedures. Bilateral lung and liver tissues were weighed for both wet and dry weights after natural air-drying; wet-to-dry weight ratios were calculated for statistical analysis. One mouse in the DOX group died during the echocardiography procedure. During processing, samples with suboptimal fixation were excluded from histological and protein analyses to ensure data quality. The final sample sizes for histological and molecular analyses were: Control group, n=8; DOX group, n=9; Apelin-13 group, n=7; Apelin-13 + DOX group, n=8. All groups had a sample size greater than 6, which is sufficient for statistical analysis.

Hematoxylin and eosin (HE) staining

The number of cross-sections analyzed for each animal was between 8 and 10. The results were analyzed using a blind method, conducted by personnel who were unaware of the grouping information. Collagen was measured as the percentage of collagen volume in the selected 8–10 cross-sections, and the average value was used for statistical analysis. Paraffin-embedded left ventricular myocardial tissue was sectioned into 3–4 µm slices and mounted on poly-L-lysine–treated slides to prevent detachment. Sections underwent standard deparaffinization using xylene and ethanol gradient rehydration, followed by hematoxylin staining for 3 minutes. After phosphate-buffered saline (PBS) washing and differentiation, sections were stained with eosin for 3 minutes. Subsequently, sections were dehydrated, cleared, and mounted. Scanning was performed using a pathological slide scanner, and cross-sectional area (CSA) was quantitatively analyzed with ImageJ software.

Masson staining

Myocardial tissue sections were deparaffinized and rehydrated. After hematoxylin staining for 5 minutes and PBS washing, sections were counterstained with Ponceau S for 5–10 minutes, immersed in glacial acetic acid, stained with phosphomolybdic acid for 4 minutes, followed by aniline blue staining for 5 minutes, and briefly rinsed with glacial acetic acid. After graded alcohol dehydration, clearing, and mounting, sections were scanned using a pathological scanner. Quantitative analysis of collagen volume fraction (CVF) was conducted using ImageJ software.

TUNEL staining

Sections of myocardial tissue were deparaffinized, rehydrated through graded alcohol, and permeabilized with proteinase working solution incubated at 37 °C for 30 minutes. Sections were washed three times with PBS for 5 minutes each. Positive controls and labeling were performed following the manufacturer’s protocol. After mounting with an anti-fluorescence quenching agent, sections were observed under fluorescence microscopy. The cardiomyocyte apoptosis index was quantified using ImageJ software.

Immunohistochemistry

Immunohistochemistry was performed to evaluate the expression of type I collagen (COL 1A1), type III collagen (COL 3A1), and TGF-β in myocardial tissue. Paraffin-embedded myocardial sections underwent deparaffinization, dehydration, and antigen retrieval using a citrate buffer under high pressure. Sections were blocked with immunohistochemical blocking solution for 1 to 2 hours, then incubated overnight at 4 °C with diluted primary antibodies (Mouse anti-COL 1A1 and Mouse anti-COL 3A1, both at 1:1,000, Rabbit anti-TGF-β was diluted at a ratio of 1:200). Following rewarming and triple PBS washing, sections were incubated with secondary antibodies (both Goat-Anti-Mouse IgG H + L and Goat-Anti-Rabbit IgG H + L were diluted at a ratio of 1:100) at 37 °C for 35 minutes and washed with PBS. Staining was developed using 3,3'-diaminobenzidine, counterstained with hematoxylin, and rinsed with double-distilled water. After mounting, sections were scanned, and analysis was conducted using Fiji (ImageJ) software. COL 1A1 and COL 3A1 expression were quantified as the percentage of positive staining area, while TGF-β expression was assessed by staining intensity measured as average optical density (OD) of positive regions.

Western blot

Western blot (WB) was performed to assess the expression levels of Bax, Bcl-2, ERK, p-ERK, AKT, p-AKT, TGF-β, Smad2/3, and Smad7 proteins in mouse myocardial tissue. Approximately 10 mg of frozen left ventricular myocardial tissue was homogenized and lysed in pre-cooled cell lysis buffer supplemented with protease and phosphatase inhibitors using a tissue homogenizer and ultrasonic homogenizer. Protein concentration was determined by bicinchoninic acid assay. Protein samples were loaded onto SDS-PAGE gels for electrophoretic separation. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes, which were blocked with 5% skim milk or bovine serum albumin for 2 hours. Membranes were incubated overnight at 4 °C with diluted primary antibodies (Bax 1:2,000; Bcl-2 1:2,000; ERK 1:2,000; p-ERK 1:1,000; AKT 1:5,000; p-AKT 1:5,000; TGF-β 1:1,000; Smad2/3 1:2,000; Smad7 1:500) on a shaker. Following incubation with secondary antibodies (Goat Anti-Rabbit or Goat Anti-Mouse, both at 1:2,000) for 1 hour at room temperature, enhanced chemiluminescence reagent was applied for detection. Band intensities were quantified using Image-Lab software. WB was also conducted to detect Bax, Bcl-2, cleaved caspase-3, ERK, p-ERK, AKT, and p-AKT proteins in rat H9C2 cardiomyocytes. Protein extraction from cells cultured in 6-well plates was performed using enhanced radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors. Protein quantification, sample preparation, denaturation, electrophoresis, and detection procedures mirrored those described for tissue protein analysis.

CCK-8 cell viability assay

Rat H9C2 cardiomyocytes were seeded in 96-well plates at a density of 5,000 cells per well. When cell confluence reached approximately 90%, cell viability was assessed using the CCK-8 assay kit (Biosharp). Preliminary concentration gradient experiments demonstrated that Apelin-13 promoted cell viability within the 0.5–2 µM range, with maximal effect at 1 µM, whereas DOX inhibited viability in a dose-dependent manner, with 2 µM producing significant cytotoxicity. Specifically, the mean cell viability was 61% at 1 µM DOX and 49.2% at 2 µM DOX. Although 1 µM DOX induced a statistically significant reduction in viability, subsequent experiments (flow cytometry and WB) using this concentration yielded less reproducible results, likely due to variations in cell density and drug distribution. In contrast, treatment with 2 µM DOX consistently induced clear morphological changes under microscopy and produced stable, reproducible results in flow cytometry and western blot analyses. Therefore, 2 µM DOX was selected for all subsequent experiments.

Cells were divided into four groups: Control, Apelin-13 (1 µM), DOX (2 µM), and Apelin-13 + DOX (Apelin-13 administered first, followed by DOX after 1 hour). After 24 hours of treatment, 10 µL of CCK-8 reagent was added to each well, and incubation continued for 2 hours. Absorbance was measured at 450 nm to determine cell survival rates.

Flow cytometry

Cardiomyocyte apoptosis was quantified using Annexin V-propidium iodide (PI) double staining. Following 24 hours of drug treatment, H9C2 cells were washed with PBS, detached using phenol red-free trypsin, and washed twice with PBS. Cells were resuspended in 1× Annexin V binding buffer at approximately 5×10⁵ cells/mL. A volume of 500 µL of cell suspension was transferred to a 15 mL centrifuge tube, followed by the addition of 5 µL Annexin V-FITC and mixing. Subsequently, 5 µL PI was added, gently mixed, and samples were incubated in the dark at room temperature for 15 minutes. Apoptosis was analyzed within one hour using a BD FACS Calibur flow cytometer.

Statistical analysis

Data analysis was performed using SPSS and GraphPad Prism 5 software. Quantitative results are presented as mean ± standard error of the mean (SEM). The normality of data distribution was assessed using the Shapiro-Wilk test. All comparisons were performed using two-sided tests. Inter-group differences were assessed using one-way analysis of variance (ANOVA), followed by Bonferroni post hoc tests for multiple comparisons. Statistical significance was set at P<0.05. Sample size was determined using the resource equation method. For a completely randomized design with four experimental groups (three treatment groups and one control group), the degrees of freedom for error (E) were calculated as E = total number of animals − number of groups. E should be between 10 and 20 to ensure adequate statistical power. Based on this principle, the total sample size was set at 40 animals (Control group, n=8; DOX group, n=11; Apelin-13 group, n=11; Apelin-13 + DOX group, n=10). This allocation accounted for potential mortality and ensured sufficient power for the planned analyses. Post hoc power analysis indicated that for key outcome measures, the statistical power exceeded 0.8 with a two-tailed significance level of α=0.05.

Results

Effects of Apelin-13 on general condition of DOX-induced cardiomyopathy mice

The general condition of mice in each group was monitored throughout the experiment. Mice in the control group maintained good health, exhibiting normal feeding behavior, glossy fur, and steady weight gain. In contrast, mice in the DOX group demonstrated progressively decreased activity, poor responsiveness to stimuli, reduced food intake, fur loss, and minimal to no weight gain, with some experiencing weight loss. One mouse developed pericardial effusion, while two exhibited pleural effusion and ascites. Compared with the DOX group, the Apelin-13 + DOX group demonstrated improved general condition, with no occurrences of pericardial or pleural effusion; however, weight gain did not significantly improve. Body weight, heart weight, and liver and lung wet weight/dry weight ratios for the four groups are presented in Table 1. The weight change curves of the mice in each group during the experiment are shown in Figure S2.

Effects of Apelin-13 on cardiac function in DOX-induced cardiomyopathy mice

The echocardiography results showed that compared with the controls, the left ventricular end diastolic diameter (LVEDD) and left ventricular end systolic diameter (LVESD) of the mice in the DOX group were significantly increased (P<0.05). Compared with the mice in the DOX group, the LVEDD and LVESD of the Apelin-13 + DOX group were significantly decreased (P<0.05). The heart rate of the mice in the DOX group showed an increasing trend compared with the controls, and compared with the mice in the DOX group, the heart rate of the mice in the Apelin-13 group showed a decreasing trend. Mice in the DOX group exhibited significantly reduced LVEF and FS compared to controls (P<0.05). In the Apelin-13 + DOX group, LVEF and FS were significantly increased relative to the DOX group (P<0.05), indicating improved cardiac function (Figure 2, Table 2).

Figure 2 Apelin-13 attenuates cardiac dysfunction in DOX-induced cardiomyopathy. Representative echocardiographic images from the four groups: Control group, DOX group, Apelin-13 group, and Apelin-13 + DOX group. DOX, doxorubicin.

Effects of Apelin-13 on cardiomyocyte arrangement and CSA

DOX administration caused cardiomyocyte disorganization and a loosened myocardial structure, accompanied by a lower CSA. These pathological changes were notably mitigated following Apelin-13 treatment (P<0.05) (Figure 3A-3C).

Figure 3 Effects of Apelin-13 on cardiomyocyte morphology and myocardial fibrosis in DOX-induced cardiomyopathy. (A) Representative HE-stained myocardial sections showing cardiomyocyte arrangement across the Control, DOX, Apelin-13, and Apelin-13 + DOX groups (scale bar =100 µm). (B) Representative images of cardiomyocyte CSA from HE-stained myocardial sections (scale bar =50 µm). (C) Quantitative analysis of CSA. CSA was significantly decreased in the DOX group compared to the Control group (**, P<0.01) and significantly increased in the Apelin-13 + DOX group compared to the DOX group (^##, P<0.01). (D) Representative images of myocardial fibrosis assessed by Masson’s trichrome staining (scale bar =100 µm). (E) Quantitative evaluation of CVF using ImageJ. CVF was significantly increased in the DOX group compared to the Control group (*, P<0.05) and significantly decreased in the Apelin-13 + DOX group compared to the DOX group (^#, P<0.05). CSA, cross-sectional area; CVF, collagen volume fraction; DOX, doxorubicin; HE, hematoxylin and eosin.

Effects of Apelin-13 on CVF in DOX cardiomyopathy mice

Myocardial fibrosis is a hallmark of DOX-induced cardiac injury. Masson staining demonstrated prominent collagen fiber accumulation in the cardiomyocyte interstitium of DOX-treated mice, with significantly elevated CVF compared to controls (*, P<0.05). Treatment with Apelin-13 reduced myocardial fibrosis and significantly decreased CVF (#, P<0.05) (Figure 3D,3E).

Effects of Apelin-13 on expression levels of myocardial fibrosis-related proteins COL 1A1, COL 3A1, and TGF-β in DOX cardiomyopathy mice

Immunohistochemical analysis indicated that, relative to controls, mice in the DOX group exhibited increased expression of COL 1A1 and COL 3A1 within cardiomyocyte interstitial spaces, as evidenced by significantly higher percentages of positive staining area (*, P<0.05). Intracellular expression of TGF-β, identified by brown granular staining, was also significantly elevated, with an increased average OD value (*, P<0.05). Apelin-13 treatment significantly reduced COL 1A1 and COL 3A1 expression in the interstitium (#, P<0.05) and decreased intracellular TGF-β expression (#, P<0.05) compared to the DOX group (Figure 4A-4D).

Figure 4 Effects of Apelin-13 on myocardial collagen expression and TGF-β/Smads signaling pathway in DOX-induced cardiomyopathy. (A) Representative immunohistochemical staining for COL 1A1, COL 3A1, and TGF-β in myocardial tissue. COL 1A1 and COL 3A1 appear as brown clusters or granules in the interstitial space, and TGF-β as brown intracytoplasmic granules (scale bar =100 µm). (B-D) Quantitative analyses of immunohistochemical staining. (B) Percentage of positively stained area for COL 1A1. (C) Percentage of positively stained area for COL 3A1. (D) Average OD for TGF-β. Expression levels of all three markers were significantly increased in the DOX group compared to the Control group (P<0.05) and significantly reduced in the Apelin-13 + DOX group compared to the DOX group (^#, P<0.05). (E) Representative western blot images for TGF-β, Smad7, and Smad2; GAPDH was used as a loading control (70 µg protein per lane). (F-H) Densitometric analysis of band intensity for TGF-β (F), Smad7 (G), Smad2 (H). Data are expressed as mean ± SEM (n=3 per group). Compared to the control group: *, P<0.05; ***, P<0.001; compared to the DOX group: ^#, P<0.05; ^##, P<0.01; ^###, P<0.001. DOX, doxorubicin; OD, optical density; SEM, standard error of the mean; Smad, mothers against decapentaplegic homolog; TGF-β, transforming growth factor-β.

Effects of Apelin-13 on TGF-β/smads signaling pathway-related protein expression levels in DOX cardiomyopathy mice

The TGF-β/Smad signaling pathway plays a critical role in tissue and organ fibrosis. DOX-induced myocardial injury in mice is associated with alterations in this pathway. Western blot analysis demonstrated that, compared to the control group, the expression levels of TGF-β and Smad2 proteins were significantly increased in the DOX group (*, P<0.05; ***, P<0.001), whereas Smad7 protein expression was decreased (*, P<0.05). Apelin-13 treatment inhibited these DOX-induced changes (#, P<0.05) (Figure 4E-4H).

Apelin-13 reduces DOX-induced mouse cardiomyocyte apoptosis

Apoptosis contributes significantly to DOX-induced myocardial injury. Western blot results indicated that DOX administration markedly increased the expression of the pro-apoptotic protein Bax (**, P<0.01) and decreased the anti-apoptotic protein Bcl-2 (**, P<0.01), resulting in a significantly reduced Bcl-2/Bax ratio (**, P<0.01). Apelin-13 administration reversed these alterations (Figure 5A-5D).

Figure 5 Effects of Apelin-13 on apoptosis-related protein expression and cardiomyocyte apoptosis in DOX-induced cardiomyopathy. (A) Representative western blot images of Bax and Bcl-2 protein expression in myocardial tissue, with GAPDH used as the internal control (loading amount: 35 µg). (B-D) Quantitative densitometric analyses: Bax (B), Bcl-2 (C), Bcl-2/Bax ratio (D). (E) TUNEL staining of myocardial sections. Apoptotic cells are indicated by green fluorescence, and nuclei of normal cardiomyocytes by blue fluorescence. In merged images, normal cells appear blue and apoptotic cells appear blue-green (magnification ×200). (F) Quantification of TUNEL-positive cells, presented as the percentage of total cells. Data are expressed as mean ± SEM (n=3). Compared to the control group: **, P<0.01; ***, P<0.001; compared to the DOX group: ^#, P<0.05; ^##, P<0.01; ^###, P<0.001. Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; DAPI, 4',6-diamidino-2-phenylindole; DOX, doxorubicin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SEM, standard error of the mean; TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling.

TUNEL staining revealed an elevated apoptosis rate in myocardial tissue from the DOX group (***, P<0.001), which was reduced following Apelin-13 treatment (^###, P<0.001) (Figure 5E,5F).

Effects of Apelin-13 on PI3K/AKT and ERK/MAPK signaling pathways in DOX-induced cardiomyopathy

No significant differences were observed in total AKT and ERK protein expression among groups (P>0.05). However, p-AKT and p-ERK levels were significantly decreased in the DOX group compared to controls, with lower p-AKT/AKT and p-ERK/ERK ratios (P<0.05). Apelin-13 treatment significantly increased p-AKT and p-ERK levels, restoring the ratios compared to the DOX group (P<0.05). Compared with the Control group, the Apelin-13 group exhibited a decreasing trend in p-AKT and p-ERK protein levels; however, no statistically significant differences were observed in the p-AKT/AKT and p-ERK/ERK ratios (P=0.055) (Figure 6).

Figure 6 Effects of Apelin-13 on AKT and ERK signaling in DOX-induced cardiomyopathy. (A) Representative western blots of total AKT and p-AKT in myocardial tissue, with GAPDH as the internal control (loading amount: 60 µg). (B-D) Quantitative analyses of: AKT (B), p-AKT (C), p-AKT/AKT ratio (D). (E) Representative western blots of total ERK and p-ERK, with GAPDH as the internal control (loading amount: 35 µg). (F-H) Quantitative analyses of: ERK (F), p-ERK (G), p-ERK/ERK ratio (H). Data are expressed as mean ± SEM [n=3; (G,H) n=6]. Compared to the control group: **, P<0.01; ***, P<0.001; compared to the DOX group: ^#, P<0.05; ^##, P<0.01. AKT, protein kinase B; DOX, doxorubicin; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p-AKT, phosphorylated AKT; p-ERK, phosphorylated ERK; SEM, standard error of the mean.

Apelin-13 improves survival rate of DOX-treated H9C2 cardiomyocytes

Preliminary concentration gradient experiments using the CCK-8 assay evaluated the effects of Apelin-13 and DOX on H9C2 cardiomyocytes. Apelin-13 had no significant effect on cell proliferation at concentrations of 0.01–0.1 µM but promoted cell viability at 0.5–2 µM, with the highest activity observed at 1 µM. At concentrations exceeding 5 µM, the proliferative effect diminished and demonstrated a trend toward inhibiting cell viability. Consequently, 1 µM was selected for subsequent experiments (Figure 7A).

Figure 7 Effects of Apelin-13 and DOX on H9C2 cardiomyocyte viability assessed by CCK-8 assay. (A) Cell viability following treatment with increasing concentrations of Apelin-13. (B) Dose-dependent cytotoxicity of DOX on H9C2 cardiomyocytes. (C) Comparative analysis of cell viability among the Control, Apelin-13, DOX, and Apelin-13 + DOX groups. Data are expressed as mean ± SEM. Compared to the control group: **, P<0.01; ***, P<0.001; compared to the DOX group: ^###, P<0.001. CCK-8, Cell Counting Kit-8; DOX, doxorubicin; SEM, standard error of the mean.

H9C2 cells treated with DOX at concentrations ranging from 0 to 5 µM exhibited a dose-dependent decrease in viability. A concentration of 2 µM DOX was chosen for further experimentation (Figure 7B).

Using the CCK-8 assay, Apelin-13 pretreatment significantly improved the survival rate of DOX-treated H9C2 cardiomyocytes compared to the DOX-only group (P<0.001), indicating a protective effect against DOX-induced cytotoxicity (Figure 7C).

Apelin-13 reduces DOX-induced H9C2 cardiomyocyte apoptosis

Apoptosis in DOX-treated H9C2 cells was assessed by Annexin V and PI double staining. Compared to the control group, DOX treatment significantly increased both early and late apoptotic cell populations (**, P<0.01; ***, P<0.001). Apelin-13 pretreatment reduced DOX-induced early and late apoptosis (##, P<0.01; ###, P<0.001) (Figure 8A-8D).

Figure 8 Effects of Apelin-13 on DOX-induced apoptosis in H9C2 cardiomyocytes. (A) Flow cytometry results showing apoptosis profiles across the Control, Apelin-13, DOX, and Apelin-13 + DOX groups. (B) Early apoptosis rate. (C) Late apoptosis rate. (D) Cell survival rate in each group. (E-I) Expression levels of apoptosis-related proteins. (E) Representative western blots of Bax, Bcl-2, and cleaved caspase-3; GAPDH was used as the internal loading control (35 µg protein/lane). (F) Gray value analysis of Bcl-2. (G) Gray value analysis of Bax. (H) Bcl-2/Bax ratio. (I) Gray value analysis of cleaved caspase-3 expression. Data are expressed as mean ± SEM [(B-D) n=2; (F-H) n=3; (I) n=5]. Compared to the control group: *, P<0.05; **, P<0.01; ***, P<0.001; compared to the DOX group: ^#, P<0.05; ^##, P<0.01; ^###, P<0.001. Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; DOX, doxorubicin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SEM, standard error of the mean.

Western blot analysis of cardiomyocyte proteins demonstrated that DOX significantly increased the expression of the pro-apoptotic protein Bax (*, P<0.05), decreased the anti-apoptotic protein Bcl-2 (*, P<0.05), and reduced the Bcl-2/Bax ratio (*, P<0.05), which was consistent with in vivo results in C57 mice. Additionally, DOX elevated cleaved caspase-3 protein levels (*, P<0.05). These DOX-induced changes were reversed by Apelin-13 treatment (#, P<0.05) (Figure 8E-8I).

Effects of Apelin-13 on PI3K/AKT and ERK/MAPK signaling pathways in DOX-induced H9C2 cardiomyocytes

No significant differences were observed in total AKT and ERK protein expression among groups. However, compared to the control group, the DOX group demonstrated significant lower p-AKT and p-ERK protein levels, as well as decreased p-AKT/AKT and p-ERK/ERK ratios (*, P<0.01). Apelin-13 pretreatment significantly increased p-AKT and p-ERK expression and restored these ratios relative to the DOX group (#, P<0.01). These findings indicate that DOX downregulates p-AKT and p-ERK in H9C2 cardiomyocytes, and Apelin-13 pretreatment mitigates this effect (Figure 9).

Figure 9 Expression of AKT, p-AKT, ERK, and p-ERK in H9C2 cardiomyocytes. (A-D) AKT and p-AKT expression in cardiomyocyte lysates. (A) Representative western blots of total AKT and p-AKT, with GAPDH as the internal control (loading amount: 60 µg). (B) Gray value analysis of total AKT. (C) Gray value analysis of p-AKT. (D) p-AKT/AKT ratio. (E-H) ERK and p-ERK expression in cardiomyocyte lysates. (E) Representative western blots of total ERK and p-ERK, with GAPDH as the internal control (loading amount: 35 µg). (F) Gray value analysis of total ERK. (G) Gray value analysis of p-ERK. (H) p-ERK/ERK ratio. Data are expressed as mean ± SEM (n=3). Compared to the Control group: *, P<0.05; **, P<0.01. Compared to the DOX group: ^#, P<0.05; ^##, P<0.01; ^###, P<0.001. AKT, protein kinase B; DOX, doxorubicin; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p-AKT, phosphorylated AKT; p-ERK, phosphorylated ERK; SEM, standard error of the mean.

Discussion

The present findings indicate that Apelin-13 improves the overall condition of mice with DOX-induced cardiomyopathy, mitigates heart failure symptoms, reduces the incidence of complications such as pericardial and pleural effusion, and enhances cardiac function. Histologically, Apelin-13 reduced DOX-induced cardiomyocyte atrophy and myocardial fibrosis. This antifibrotic effect may be associated with modulation of the TGF-β/Smads signaling pathway. Furthermore, Apelin-13 decreased DOX-induced cardiomyocyte apoptosis. Additional analyses revealed that DOX downregulated p-AKT and p-ERK expression, whereas Apelin-13 reversed these changes. In vitro studies using H9C2 cardiomyocytes demonstrated that DOX suppressed cell viability in a concentration-dependent manner. Apelin-13 promoted cell growth at concentrations of 0.5–2 µM, and attenuated DOX-induced cellular damage. Apelin-13 also reduced DOX-induced apoptosis, likely through the reversal of DOX-mediated inhibition of p-AKT and p-ERK expression. These observations indicate that the cardioprotective effect of Apelin-13 is mediated, at least in part, through activation of the PI3K/AKT and ERK/MAPK signaling pathways.

Various endogenous bioactive peptides contribute to cardiovascular development and function. Apelin, an endogenous peptide initially isolated from bovine stomach in 1998, is cleaved by angiotensin-converting enzyme into several active fragments, including Apelin-13, Apelin-16, Apelin-17, and Apelin-19. Among these, Apelin-13 exhibits high biological activity. Apelin and its receptor, APJ, comprise the apelin/APJ system, a regulatory axis considered a novel component of the RAAS (20,33-36). Upon ligand binding, the apelin/APJ system activates various downstream signaling cascades that mediate cardioprotective effects. Previous investigations by Masri et al. demonstrated that Apelin, via APJ binding, activates the PI3K/AKT pathway, which subsequently promotes nitric oxide production and exerts anti-apoptotic effects (32,34,37). Studies by Liu et al. and Gunes et al. further revealed that the apelin/APJ axis confers anti-apoptotic effects through modulation of PI3K/AKT, ERK1/2, and caspase signaling pathways (22,27). Additionally, Lv et al. reported that Apelin suppresses myocardial fibrosis by regulating the TGF-β/Smads pathway, whereas Zhang et al. indicated that the anti-fibrotic action of Apelin post-myocardial infarction may involve NF-κB pathway inhibition (28,38). Apelin also appears to confer cardio protection in the context of ischemia-reperfusion injury by suppressing oxidative stress responses (24-26). For instance, research by Zhong et al. demonstrated that Apelin decreased NADPH oxidase activity and superoxide anion (O₂⁻) levels in myocardial infarction models, thereby reducing oxidative stress and ischemia-reperfusion injury (39).

Apelin exerts protective effects on the cardiovascular system through multiple signaling pathways. The cardioprotective potential of the apelin/APJ system against DOX-induced myocardial injury has attracted increasing attention in recent years. Hamada et al. observed that mice lacking the APJ gene exhibited exacerbated DOX-induced myocardial injury compared to wild-type C57 mice, whereas APJ overexpression attenuated damage in H9C2 cardiomyocytes (40). Given the mechanistic similarities between DOX and cisplatin-based chemotherapeutics, findings by Zhang et al. demonstrating that Apelin alleviates cisplatin-induced myocardial injury via the PI3K/AKT and MAPK pathways support the broader relevance of Apelin in chemotherapy-associated cardiotoxicity (29). It is possible to develop analogues or modified versions of Apelin-13 for the treatment and prevention of DOX-induced myocardial damage. Currently, dexrazoxane is the only drug approved by the Food and Drug Administration (FDA) for reducing the cardiotoxicity of anthracycline drugs. However, the clinical application of dexrazoxane also has its limitations (41), such as potentially increasing the incidence of severe leukopenia (42). Dexrazoxane is mostly used in the advanced stages of cancer and can only be used when the cumulative dose of DOX reaches a certain level (43). Due to concerns about interfering with the anti-tumor activity of chemotherapy drugs, it cannot be used at the beginning of chemotherapy. The safety and efficacy of dexrazoxane still need further research to confirm. Compared to dexrazoxane, Apelin is one of the hormones that can be secreted and synthesized by the body to regulate normal physiological functions. Previous studies have shown that Apelin has protective effects on the heart, lung tissue, kidneys, and brain tissue. However, the application timing and effective dose for preventing and treating DOX-induced myocardial damage still require further research.

However, this study has several limitations. The pathophysiology of DOX-induced myocardial injury involves multiple complex mechanisms, and the apoptotic pathways are multifactorial. It remains to be determined whether additional signaling cascades contribute to the cardioprotective effects of Apelin-13. Furthermore, the potential impact of Apelin-13 on the anti-tumor efficacy of DOX remains unclear and warrants further investigation in future studies.

Conclusions

In this study, conducted in mice and H9C2 cardiomyocytes, it was confirmed that Apelin-13 can alleviate the inhibitory effect of DOX on P-AKT and P-ERK, thereby reducing the myocardial damage caused by DOX. Early administration of Apelin-13 plays an important role in the prevention and treatment of DOX-induced myocardial damage. However, the mechanism of Apelin-13’s reversal effect when administered after that DOX has clearly caused myocardial damage still requires further investigation. Future research will further explore the effects of Apelin-13 administered at different time points on DOX-induced myocardial damage, providing more theoretical basis for the prevention and treatment of DOX-induced myocardial damage and the clinical application of Apelin-13. In addition, it is necessary to develop a novel formulation of Apelin-13 with a prolonged half-life to extend the duration of drug action and better meet clinical needs.

Acknowledgments

We would like to acknowledge the hard and dedicated work of all the staff that implemented the intervention and evaluation components of the study.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://cdt.amegroups.com/article/view/10.21037/cdt-2025-358/rc

Data Sharing Statement: Available at https://cdt.amegroups.com/article/view/10.21037/cdt-2025-358/dss

Peer Review File: Available at https://cdt.amegroups.com/article/view/10.21037/cdt-2025-358/prf

Funding: This research was funded by the Scientific Research Incentive Program Project of The Affiliated Cardiovascular Hospital of Shanxi Medical University (No. XYS20230104).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://cdt.amegroups.com/article/view/10.21037/cdt-2025-358/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. 2024kjt105) granted by the Ethics Committee of The Affiliated Cardiovascular Hospital of Shanxi Medical University, in compliance with institutional guidelines 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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