A total of 50 female BALB/c mice (age, 6-8 weeks; weight, 18-20 g) were purchased from the Experimental Animal Center of Shandong University (Jinan, China). All experimental procedures were performed in accordance with animal protocols approved by The Second Hospital of Shandong University Animal Care Committee (approval no. KYLL-2020-(KJ)A-0134). All mice were housed in a pathogen-free animal facility, which was maintained at 22-24˚C at 50-60% humidity with 12:12 h light-dark cycle. The mice had easy access to food and water before the experiments. For cardiac diameter and cardiac function assessment, mice were anesthetized with 3% isoflurane, which was subsequently maintained at 1.3%. At the end of study, mice were anesthetized by intraperitoneal injection of pentobarbital sodium (100 mg/kg) and euthanized via exsanguination.

For EAM model construction, murine cardiac α-myosin heavy chain [α614-629 (Ac-SLKLMATLFSTYASAD-OH); GL Biochem (Shanghai), Ltd.] was dissolved in PBS (1 mg/ml) and emulsified 1:1 with complete Freund's adjuvant (CFA; MilliporeSigma). Each mouse was subcutaneously injected with 200 µl of emulsion (containing 200 µg of murine cardiac α-myosin) by inguinal injection or subaxillary injection on day 0 and 7 to induce EAM (12). The control group mice were treated with CFA mixed with PBS following the same procedure as for the EAM group mice. To knockdown Ets-1 expression, a small interfering (si)RNA against mouse Ets-1 was transfected into mouse hearts and a scramble siRNA was employed as a control. The target sequence for Ets-1 was 5'-GCUACCUUCAGUGGUUUCATT-3' and scramble siRNA sequence was 5'-UUCUCCGAACGUGUCACGUTT-3' (Shanghai GenePharma Co., Ltd.). Mice were randomly divided into five groups: i) Control group (n=10); ii) non-treated EAM group (n=10); iii) spironolactone-treated EAM group (n=10); iv) saline-treated EAM group (n=10); and v) siRNA-Ets-1-treated EAM group (n=10). Spironolactone treatment therapy started on day 7 following the emulsion injection. The myocardial injection method was used to transfect cells based on previous study (13). A total of 1x10⁷ UT/30 µl lentivector with siRNA-Ets-1 was injected in multiple sites in the left ventricle of the EAM group mice at day 7. Mice were fed orally via gastric gavage for 4 weeks from day 7 to day 36 after immunization. The calculated daily dosage of 50 mg/kg/day spironolactone per mouse was based on a previous study by Wehr et al (14). Assessment and analysis of the establishment of EAM in the mouse model were performed on day 36. No mice succumbed throughout the duration of the present study.

Serum was collected for ELISA. Blood samples were collected via eyeball removal and were stored at 4˚C overnight to let the serum separate from the blood cells. Aldosterone was quantified using a Aldosterone ELISA kit (cat. no. ZC-38593; Shanghai Zhuo Cai Technology Co., Ltd.) Serum levels of IL-6 and TNF-α were also quantified using IL-6 and TNF-α ELISA kit (cat. no. ZC-37988 and ZC-39024, respectively; Shanghai Zhuo Cai Technology Co., Ltd.), according to the manufacturer's protocol.

Echocardiography was performed on the mice as previously described (15,16). Wall thickness and left ventricular (LV) dimensions, including LV internal dimensions (LVIDs) in diastole, LVID in systole, LV posterior wall (LVPW) of diastole, LVPW of systole and LV posterior diameter in systole, were obtained from the short-axis view. LV ejection fraction (LVEF) was assessed according to the American Society of Echocardiography Guidelines (17). Pulsed-wave Doppler echocardiography was used to measure early (E) and late (A) blood flow velocities via the mitral valve and the E/A ratio was determined.

Heart tissues were fixed using 10% formalin at room temperature for 72 h, dehydrated with an ethanol gradient, embedded in paraffin, sectioned (thickness, 5 µm) and stained with hematoxylin and eosin (H&E). The main steps of H&E staining were the following: Staining with hematoxylin for 10 min at room temperature, washing in tap water, staining with eosin for 3 min at room temperature, washing in distilled water and ethanol (90%), dehydration in ethanol (95%), ethanol (100%). Masson trichome staining was used to stain collagen fibers dark green, which were quantified to assess fibrosis. Masson staining takes place at room temperature and the main steps are the following: Staining with hematoxylin for 5 min, washing with tap water, staining with fuchsin for 5 min, rinsing in distilled water, incubating in phosphotungstic-phophomolybdic acid for 5 min, dyeing aniline blue for 5 min. Paraffin sections underwent immunohistochemistry using a microwave-based antigen retrieval method. Sections were incubated with primary antibodies for rabbit polyclonal collagen I (1:100; cat. no. AF7001; Wuhan Servicebio Technology Co., Ltd.), collagen III (1:500; cat. no. GB111629; Wuhan Servicebio Technology Co., Ltd.), TGF-β1 (1:100 cat. no. AF1027; Wuhan Servicebio Technology Co., Ltd.) and phosphorylated (p)-Ets-1 (1:200; cat. no. CBP1153; Assay Biotechnology Co., Inc.) overnight at 4˚C. Following the primary antibody incubation, samples were incubated with a HRP-conjugated secondary goat anti-rabbit antibody (cat. no. PV9001; Zhongshan Bio-Tech Co., Ltd.) for 30 min at 37˚C. Signals were amplified using an ABC kit (Vector Laboratories, Inc.). Sections were imaged using a confocal FV 1000 SPD laser scanning microscope (Olympus Corporation). Immunohistochemical staining was analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Inc.).

Total protein was extracted from mice myocardium using RIPA lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology). Subsequently, total protein was separated using SDS-PAGE on a 10% gel. Separated protein was then transferred onto a PVDF membrane, which was incubated overnight at 4˚C with the following primary antibodies: total Ets-1 (1:1,000; cat. no. 6258; Cell Signaling Technology, Inc.), p-Ets-1 (1:500; cat. no. CBP1153; Assay Biotechnology Co., Inc.), Smad-2/3 (1:1,000; cat. no. 8685; Cell Signaling Technology, Inc.) and p-Smad-2/3 (1:1,000 dilution; cat. no. 8828; Cell Signaling Technology, Inc.). Following the primary incubation membranes were incubated with an HRP-conjugated secondary antibody for 1 h at room temperature (1:2,500 dilution; cat. no. ZB-2306; OriGene Technologies, Inc.). Protein expression levels were normalized to β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) as an internal control and p-proteins to that of the total protein. Bands were semi-quantified using optical densities, which were analyzed using ImageJ software (v1.8, National Institutes of Health).

Total RNA was extracted from heart tissue using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and converted into complementary DNA using a RevertAid kit (Fermentas; Thermo Fisher Scientific, Inc.), both kits were used according to the manufacturer's protocol. Reactions involved the use of a real-time PCR thermocycler (IQ5 real-time PCR cycler; Bio-Rad Laboratories) with SsoFast EvaGreen Supermix (Bio-Rad Laboratories). The program was 30 sec at 95˚C, then 40 cycles of 96˚C for 5 sec and 56˚C for 10 sec. mRNA expression levels were normalized to the internal reference gene GAPDH. The primer sequences used for qPCR were as follows: MMP-2 forward F, 5'-ACAAGTGGTCCGCGTAAAGT-3' and reverse R, 5'-GTAAACAAGGCTTCATGGGGG-3'; MMP-9 F, 5'-GCCGACTTTTGTGGTCTTCC-3' and R, 5'-GGTACAAGTATGCCTCTGCCA-3'; and GAPDH F, 5'-AGGTCGGTGTGAACGGATTTGGG-3' and R, 5'-TGTAGACCATGTAGTTGAGGTCA-3'. Relative fold change of mRNA expression levels was calculated using the 2^-∆∆CT method (18). Data are representative of three independent experiments.

The enzymatic activity of MMP in myocardial tissues was assayed using gelatin zymography. Samples were electrophoresed via SDS-PAGE on a 10% gel containing 0.1% gelatin. After the gels were washed twice with 2.5% Triton X-100, the gels were incubated in activation buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM CaCl₂, 1 µM ZnCl₂] at 37˚C overnight. Subsequently, gels were stained with Coomassie brilliant blue R-250 solution for 3 h at room temperature. Gels were de-stained with 45% methanol and 10% acetic acid until the bands of lysis become clear. Lytic bands of gelatin digestion were represented by MMP-2 (72 kDa) and MMP-9 (92 kDa) activity.

Statistical analysis was performed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA) and data are presented as the mean ± SD. Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Aldosterone synthase expression levels are high in human myocardium during acute myocarditis (5). Aldosterone serves an important role in the pathophysiology of cardiac remodeling (19). Therefore, aldosterone concentration in EAM mice myocardium was assessed using ELISA. Compared with the controls, aldosterone serum concentrations were significantly increased in EAM mice on day 36 (P<0.05; Fig. 1).

Previous results have demonstrated that inflammatory cytokines serve an important role in the progression of post-myocarditis cardiac remodeling (20). On day 36, H&E staining demonstrated that inflammatory cell infiltration was present in the EAM group compared with the control groups. However, spironolactone treatment significantly reduced cell infiltration in the EAM group (Fig. 2A). To evaluate the levels of inflammatory cytokines in the EAM myocardium, TNF-α and IL-6 serum concentrations were quantified using ELISA. Compared with the control groups, the serum concentrations of TNF-α and IL-6 significantly increased in the EAM group (P<0.05) but significantly decreased in the EAM model with spironolactone treatment (P<0.05; Fig. 2B and C).

Echocardiography was used to assess cardiac function on day 36 of the experiment. It was determined that the LVEF, E/A ratio, LV chamber size and wall thickness did not differ between animals in each group. At day 36, compared with the control groups, a significantly lower E/A ratio was observed in EAM mice (P<0.05), which was accompanied by a significant increase in the LVPW (P<0.05). Spironolactone treatment significantly ameliorated the reduced E/A ratio and LVPW in EAM mice (P<0.05). However, there was no statistically significant difference in LVEF or LVID between the four groups investigated (P>0.05). These results indicated that EAM mice may exhibit LV hypertrophy and diastolic dysfunction at day 36, but there was no evidence of LV dilatation and systolic dysfunction (Table I).

Subsequently, whether Ets-1 participated in EAM-induced cardiac fibrosis was investigated. Ets-1 protein expression levels were determined using immunohistochemistry (Fig. 3A) and western blotting (Fig. 3B). The results demonstrated that total Ets-1 and p-Ets-1 protein expression levels were significantly increased (P<0.05; Fig. 3C and D) in EAM mice myocardium. Subsequently, the effect of spironolactone on myocarditis-induced Ets-1 activation was investigated. Compared with the myocarditis mice groups, Ets-1 protein expression levels and phosphorylation were significantly reduced by spironolactone treatment (P<0.05).

TGF-β1 performs a crucial role in cardiac fibrosis, whereby Ets-1 participates in matrix remodeling in response to TGF-β1(21). In the present study, the effect of spironolactone on the TGF-β1/Smad-2/3 signaling pathway was explored. The protein expression level of TGF-β1 in the myocardium was determined by immunohistochemistry. The results demonstrated that TGF-β1 protein expression levels were significantly increased in EAM mice compared with the control groups (P<0.05). However, these increased protein expression levels were significantly reduced with spironolactone treatment (P<0.05; Fig. 4A and B). Smad proteins are the downstream signaling molecules of the TGF family and are also involved in myocardial remodeling (22). The effect of spironolactone on p-Smad-2/3 protein expression levels in EAM mice was assessed. Western blotting demonstrated that spironolactone significantly inhibited the myocarditis-induced increase of p-Smad-2/3 in myocardial tissue (P<0.05; Fig. 4C and D). These results indicated that spironolactone may inhibit Ets-1 activation via the TGF-β1/Smad-2/3 signaling pathway in EAM mice.

Aldosterone antagonists are established therapeutics for patients with heart failure (23). The Masson's trichrome staining method was used to assess the degree of myocardial fibrosis. The results demonstrated that in the EAM group fibrosis was significantly increased (P<0.05; Fig. 5A and B). As reported in previous studies, the present study demonstrated that spironolactone significantly inhibited myocardium fibrosis in EAM mice (P<0.05). Furthermore, in EAM mice, significantly increased protein expression levels of collagens I and III were observed in the myocardium (P<0.05; Fig. 5C and D). Spironolactone treatment significantly decreased the protein expression levels of collagen I and III in the EAM group (P<0.05). Ets factors are important mediators of ECM remodeling (24). To further examine the effects of Ets-1 on cardiac fibrosis induced by myocarditis, Ets-1 expression was silenced using siRNA. Compared with the control, Ets-1 protein expression levels were significantly reduced by siRNA transfection (P<0.05; Fig. S1). Immunohistochemistry analysis demonstrated that the quantities of collagen I and collagen III significantly decreased in the siEts-1 group compared with EAM group (P<0.05, Fig. 5). These results indicated that spironolactone may limit myocarditis-induced fibrosis that is mediated by the inhibition of Ets-1.

MMPs may also serve a central role in ECM remodeling (25). RT-qPCR and gelatin zymography demonstrated that the mRNA expression levels and the activity of MMP-2 and MMP-9 increased significantly in the EAM group compared with the controls, whereas spironolactone treatment significantly attenuated this effect (P<0.05; Fig. 6). Furthermore, the role of Ets-1 in MMP mRNA expression and activity in EAM mice was explored. The results demonstrated that compared with EAM mice, the increased mRNA expression levels and activity of MMP-2 and MMP-9 were significantly downregulated by Ets-1-siRNA (P<0.05). These results therefore indicated that spironolactone may inhibit the expression and activation of MMP-2 and MMP-9 via Ets-1 activation suppression.