2.1 Animals and treatments
All experiments were performed per the Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Experimental Research in Shaoxing People's Hospital. Six-week-old specific pathogen-free male Wistar rats, weighing approximately 200 g, were allowed free access to food and water at the Animal Experimental Center of Shaoxing People's Hospital and maintained at 25 °C under a 12 h light /12 h dark cycle. After 1 week of adaptive feeding, the rats were randomly divided into five groups (six rats in each group), and the control (CON) group was injected with an equal volume of normal saline. In the DOX group, DOX (Cat: HY-15142A, MedChemExpress) was injected into the tail vein once weekly for 4 weeks at a cumulative dose of 16 mg/kg19. The treatment groups were divided into three groups: Urantide (CAS:669089-53-6, Top Science) was injected at 10, 20, and 30μg/kg/day through the tail vein for 7 days consecutively20,21(Figure 1A). Twenty-four rats were randomly divided into four groups: CON, DOX, DOX + Urantide (30μg/kg/day for iv, 7 days), and DOX +Urantide+p38 MAPK-IN-1 (1 mg/kg for iv, the p38 MAPK inhibitor, HY-12839, MCE) to confirm whether p38 MAPK inhibition contributes to urantide-induced protection22(Figure 1B). Finally, the rats were anesthetized by sevoflurane inhalation (RWD Life Science Co., LTD, Guangdong, China).
After considering the importance and necessity of exercise rehabilitation in heart failure23,24, the rats were randomly divided into five groups after adaptive feeding for 1 week (6 rats in each group): CON, DOX, DOX + Exercise (swim once daily, 5 days a week for 6 weeks), DOX+Urantide (administered by tail vein injection at 30μg/kg every time for 7 consecutive days), and DOX+Urantide+Exercise groups. In the first week, rats received adaptive training, which was gradually increased from 10 min to 40 min, and the training time of 40 min was maintained every time from the second week)25 (Figure 1C).
2.2 Transthoracic echocardiography
LVEF and left ventricular fractional shortening (LVFS) were measured using the Philips iE33 system (Philips Medical, Best, the Netherlands) and an s5-1 probe (12–14 MHz), and transthoracic echocardiography images were obtained at the same time. Data analysis was performed using the Philips QLab 9 post-processing software.
2.3 Histological assessment of myocardial injury
Following echocardiography, all rats were anesthetized, and their hearts were removed, rapidly weighed, photographed, and immediately frozen in liquid nitrogen until analysis. Part of the heart was fixed with 4% formalin, embedded in paraffin, and serially sectioned at 5um thickness. Hematoxylin and eosin (HE) staining was used to evaluate the morphology of the myocardium, and Masson staining was used for collagen analysis. All the sections were imaged using a Leica DM3000 biological microscope (Leica, Wetzlar, Germany) at 200× magnification. Quantization was performed using ImageJ software (National Institutes of Health).
2.4 Serological evaluation of myocardial injury
While removing the heart, a blood sample was taken from the tail vein to the procoagulant and centrifuged at 3500rpm for 10 min after stewing. The supernatant was stored at -80℃. LDH (Nanjing Jiancheng Biological Engineering Research Institute) and CK isoenzyme (CK-MB, Nanjing Jiancheng Biological Engineering Research Institute) were detected using enzyme-linked immunosorbent assay kits.
2.5 Transmission electron microscopy
The left ventricular tissues of the rats were fixed overnight with 2.5% glutaraldehyde, washed with buffer solution, and then fixed in 1% osmic acid. After thorough washing, a series of dehydration steps were performed. Finally, the tissues were embedded in Araldite for coronal sectioning and stained with toluidine blue. Changes in the mitochondria and muscle fibers were observed under a Cs-corrected transmission electron microscope (Titan G2 60-300, FEI, Hillsboro).
2.6 Wheat germ agglutinin (WGA) staining
After dewaxing the 5μm thick paraffin sections of the rat heart, the samples were incubated with 5ug/mL WGA (L4895; Sigma-Aldrich) in the dark at 37 °C for 20 min. After washing thrice with phosphate-buffered saline, 4′,6-diamidino-2-phenylindole (DAPI, P36941; Invitrogen) was stained in the dark at 25℃ for 5 min. Images were acquired using a Nikon Ti-U fluorescence microscope (Minato-ku, Tokyo, Japan) and were observed at 100-fold magnification. The size of cardiac myocytes was determined by dividing the total area by the number of cardiac myocytes.
2.7 Apoptosis assay
Paraffin sections of heart tissue were dewaxed with xylene and rehydrated in a fractionated ethanol series. The sections were incubated with protease K for 20 min at 37 °C, and the tissue was incubated with a TdT-mediated dUTP nick end labeling (TUNEL, Roche) reaction mixture for 1 h in the dark at 37 °C. DAPI (P36941; Invitrogen) was stained in the dark at 25℃ for 5 min, and the TUNEL-positive cells were observed under a Nikon Ti-U fluorescence microscope at a magnification of 400.
2.8 Cell culture and treatment
H9C2 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). Cells were maintained at 37°C in a humidified incubator with 5% CO2. The cells were treated with 5 μmol/L DOX for 24 h to establish a toxic cell model26. The cells were treat with 10 nmol/L, 100 nmol/L, 1,000 nmol/L Urantide or 1,000 nmol/L Urantide + 20 nmol/L p38 MAPK-IN-1 (HY-12839, MCE) for 24 h27,28.
2.9 Assessment of mitochondrial membrane potential
H9C2 cells were incubated with 2.5 mmol/L JC‐1 dye (Solarbio, Beijing, China) in the dark at 37 °C for 30 min. Red JC‐1 aggregates represent normal hyperpolarized membrane potential, whereas green JC‐1 monomers represent mitochondrial membrane potential loss. Images were obtained using a fluorescence microscope at 400 × magnification.
2.10 Western blot analysis
Total protein was extracted from frozen tissues or H9C2 cells using radioimmunoprecipitation assay lysis buffer (Beyotime, China) containing phosphatase inhibitor cocktail II (MedChem Express, China). Proteins were separated by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis and electroblotted to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% skim milk for 1 h at 25℃ and hybridized with primary antibodies against B-cell lymphoma 2 (Bcl-2, ab32124, 1:1000), Bcl-2 Associated X-protein (Bax, ab32503, 1:1000), p38 (ab170099, 1:1000), and glyceraldehyde 3-phosphate dehydrogenase (ab8245, 1:1000) at 4 °C overnight. The next day, the membrane was treated with Horseradish peroxidase-labeled goat anti-rabbit or anti-mouse secondary antibodies (A0208, A0216, 1:1000, Beyotime, China), incubated at 25℃ for l h, and visualized using an excellent chemiluminescent substrate detection kit (Cat:32106, Pierce™ ECL Western Blotting Substrate, Thermo Scientific). Finally, the ImageJ software (National Institutes of Health) was used for quantification. The original images are shown in the supplementary material (Fig S1-Fig S23).
2.11 Statistical analysis
For any multiple-group comparisons, a one-way analysis of variance was performed, and the results were presented as mean ± standard deviation. Data were analyzed using SPSS version 20.0 software (SPSS Inc, Chicago, IL, USA). Statistical significance was set at p<0.05.