Twelve male Sprague-Dawley rats, aged 8-weeks-old and weighing 220–260 g, were supplied by Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The rats were housed at constant temperature (22±2°C) and humidity (60%) under a 12-h light/dark cycle, and provided with ad libitum access to water and food for 1 week prior to initiation of the experiment. All animal care and experimental procedures were performed according to the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health (National Institutes of Health publication no. 85-23) (24). The present study was approved by the Ethics Committee of Third Hospital of Shijiazhuang [Shijiazhuang, China; approval no. 054(2018)].

Following an acclimatization period of one week, the rats were randomly assigned to control and ISO groups (n=6). Rats in the ISO and control groups were intraperitoneally administered ISO (5 mg/kg) or equal volume of saline daily for one week to induce HF as previously described (25).

After 4 weeks following successful establishment of the model of HF, the rats were intraperitoneally anesthetized using urethane (1.2 g/kg). To evaluate left ventricular function, a heparin-filled catheter connected to a pressure transducer (Chengdu Taimeng Technology Co., Ltd., Chengdu, China) was inserted into the left ventricle from the right carotid artery to measure hemodynamic parameters, including left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), heart rate (HR), maximum contraction velocity (+dp/dt_max) and maximum relaxation velocity (-dp/dt_max). Following recording, rats were sacrificed, and their hearts were immediately dissected and frozen at −80°C for subsequent experimentation. Additionally, blood was collected from the right common carotid artery and centrifuged at 1,370 × g at 4°C for 10 min to obtain plasma, which was collected and stored at −80°C for subsequent analysis.

Plasma lactate dehydrogenase (LDH), creatine kinase (CK) and CK-muscle/brain (CK-MB) levels were determined using the corresponding assay kits (cat. nos. A020-2, A032, H197, respectively; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocols.

The plasma levels of BNP (cat. no. JYM00013; Wuhan Colorful Gene Biological Technology, Wuhan, China) and GDF11 (cat. no. E-EL-R0463c; Elabscience Biotechnology Co., Ltd, Wuhan, China) were determined using the corresponding assay kits according to the manufacturer's protocols.

H9C2 rat cardiac myoblasts, a cardiomyoblast cell line derived from the heart tissue of embryonic rats (Chinese Academy of Sciences Cell Bank, Shanghai, China), were cultured in Dulbecco's Modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare, Logan, UT, USA) and 1% penicillin-streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C in 5% CO₂. H9C2 cells were categorized into three groups based on treatment: i) Control group (cells were cultured in DMEM for 24 h and then treated with saline at 37°C for 24 h); ii) ISO group (cells were cultured in DMEM for 24 h and then treated with 10 µM ISO at 37°C for 24 h); and iii) ISO + rGDF11 group [cells were pre-incubated with different concentrations (0.5, 5 or 50 nM) of rGDF11 protein (cat. no. 1958-GD; R&D Systems, Inc., Minneapolis, MN, USA) (22) at 37°C for 24 h and then treated with 10 µM ISO at 37°C for 24 h].

H9C2 cells were inoculated in 6-well culture plates and transfections were performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Specific siRNA against GDF11 (5′-GCCAGUGCGAGUACAUGUUTTdTdT-3′) and scrambled siRNA (5′-UUCUCCGAACGUGUCACGUdTdT-3′; negative control) were obtained from Ambion (cat. no. AM16708; Thermo Fisher Scientific, Inc.). For each reaction, 5 µl of siRNA, 5 µl of Lipofectamine 2000 and 95 µl of Opti-MEM™ reduced-serum medium (Invitrogen; Thermo Fisher Scientific, Inc.) were mixed at room temperature followed by incubation at 37°C for 20 min. Opti-MEM (800 µl) was subsequently added drop-wise to each well. H9C2 cells were then added to the resultant mixture. Following transfection for 6 h, the cell culture medium was replaced and cells were further incubated at 37°C for 24 h prior to treatment with ISO.

The proliferation of H9C2 cells cultured at a density of 1×10⁴ cells/well in 96-well plates was measured using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's protocols. The absorbance of the cells at 450 nm was determined using a microplate reader and normalized to that of the control group.

The extent of cellular injury was monitored by LDH release. H9C2 cells were cultured at a density of 1×10⁶ cells/well in 6-well plates. Following treatment, the culture medium was analyzed to determine LDH activity using a LDH assay kit (Nanjing Jiancheng Bioengineering Institute) with a microplate reader.

Caspase-3 activity was determined to investigate the apoptosis of cells. Following treatment, H9C2 cells cultured at a density of 1×10⁶ cells/well in 6-well plates were homogenized using 50 mM of potassium phosphate buffer. Following centrifugation at 10,000 × g at 4°C for 10 min, the supernatant was analyzed using the caspase-3 activity kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's protocols. Briefly, 50 µl supernatant was mixed with 10 µl caspase-3 substrate Ac-DEVD-pNA and 40 µl buffer solution, and then incubated at 37°C for 2 h, and absorbance at 450 nm was measured, reflecting cleavage of the colorimetric substrate. Finally, values were normalized to those of the control group.

Apoptotic cell death was determined by Hoechst 33258 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) staining. In brief, H9C2 cells were cultured at a density of 1×10⁶ cells/well in 6-well plates. Following treatment, the cells were fixed with 1 ml of 4% paraformaldehyde at 37°C for 30 min. Then, the cells were incubated in 1 ml PBS containing 10 µM Hoechst 33258 at 37°C for 30 min and observed using fluorescence microscopy (Olympus Corporation, Tokyo, Japan) from five random fields (×200). Normal nuclei stained blue and apoptotic nuclei were identified as condensed or fragmented nuclei stained bright blue. The rate of apoptosis was calculated as follows: Apoptotic rate=No. of apoptotic cells/Total no. of cells ×100.

Intracellular ROS generation was estimated using dihydroethidium (DHE; Sigma-Aldrich; Merck KGaA) fluorescent staining. Briefly, following treatment as aforementioned, H9C2 cells cultured at a density of 1×10⁴ cells/well in 96-well plates were washed with PBS and incubated with DHE (10 µM) at 37°C for 30 min in the dark. Then, DHE was removed by washing with PBS, and the fluorescence intensity was measured using a fluorescence plate reader (Tecan Infinite M200, Mannedorf, Switzerland) at excitation/emission wavelength of 488/610 nm, respectively. The values were normalized to those of the control group.

Following treatment as aforementioned, H9C2 cells cultured at a density of 1×10⁶ cells/well in 6-well plates were homogenized using 50 mM potassium phosphate buffer. Following centrifugation at 10,000 × g at 4°C for 10 min, the MDA concentration of the supernatant was analyzed using an MDA assay kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocols and the optical density (OD) was measured at 532 nm using a microplate reader. The obtained values were standardized to total protein content, which was determined via a bicinchoninic acid (BCA) protein assay kit.

Protein extracted from heart tissues or H9C2 cells was quantified using a BCA protein assay kit. Proteins (50 µg) were separated via 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and blocked using 5% non-fat milk at room temperature for 1 h. Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: Anti-B-cell lymphoma 2 (Bcl-2; 1:1,000; cat. no. 3498; Cell Signaling Technology, Inc., Danvers, MA, USA); anti-Bcl-2-associated X protein (Bax; 1:1,000; cat. no. 5023; Cell Signaling Technology, Inc.); anti-nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2; 1:1,000; cat. no. 19013-1; ProteinTech Group, Inc., Chicago, IL, USA); anti-Nox4 (1:1,000; cat. no. 14347-1; ProteinTech Group, Inc.); anti-GDF11 (1:1,000; cat. no. AF1958; R&D Systems, Inc.) and anti-GAPDH as an internal control (1:2,000; cat. no. 10494-1; ProteinTech Group, Inc.). Following three washes in TBS-Tween 20 (0.5 ml/l), the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:1,000; cat. no. A0208; Beyotime Institute of Biotechnology) at room temperature for 1 h. Target bands were detected using the SuperSignal West Pico Chemiluminescent Substrate (Pierce; Thermo Fisher Scientific, Inc.), and band intensity was quantified using ImageJ 1.48i software (National Institutes of Health, Bethesda, MD, USA). The experiment was repeated three times.

Total RNA was extracted from heart tissues or H9C2 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and 1 µg RNA was subjected to reverse transcription using first-strand cDNA synthesis kit (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocols. The temperature protocol was as follows: 42°C for 30 min and 85°C for 5 min. Real-time PCR analysis was performed with the ABI 7500 FAST system, using an SYBR^® Green RT-PCR kit (Toyobo Life Science, Osaka, Japan) according to the manufacturer's protocols. The thermocycling conditions consisted of an initial, single cycle of 2 min at 94°C, followed by 40 cycles of 15 sec at 94°C, 20 sec at 60°C and 30 sec at 70°C. As an internal control, β-actin primers were used for RNA template normalization. All PCRs were performed in triplicate. The primers used to determine gene expression were as follows: Rat GDF11, forward 5′-TGGGGAGCAGGCAAGGGGTAG-3′, reverse, 5′-TGCCCGTGGTAAGTGCTCAGAA-3′; and rat β-actin, forward 5′-TACCACATCCAAGGAAGGCAGCA-3′ and reverse, 5′-TGGAATTACCGCGGCTGCTGGCA-3′. Fold changes in GDF11 expression normalized to β-actin expression were calculated using the 2^−ΔΔCq method (26).

Data were presented as the mean ± standard error of the mean. Statistical analysis was performed using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA). Comparisons between two rat groups were conducted using Student's t-tests. Comparisons between >2 groups were performed using one-way analyses of variance followed by a Tukey's test. P<0.05 was considered to indicate a statistically significant difference.

The results of hemodynamic analysis revealed that ISO treatment induced a significant increase in LVEDP, and a decrease in LVSP and ± dp/dt_max in the ISO group compared with the control group (Fig. 1A-D). A significant difference in HR was not observed; however, the mean value was markedly lower in the ISO group than in the control group (Fig. 1E). The results indicated that ISO treatment induced cardiac dysfunction in rats.

ISO-induced HF was associated with increased plasma levels of myocardial injury markers, including LDH, CK and CK-MB. Compared with the control group, the plasma levels of LDH, CK and CK-MB increased significantly following ISO treatment (Fig. 1F-H). Additionally, plasma BNP levels, an important biomarker of HF, were significantly elevated following ISO treatment compared with the control group (Fig. 1I). These findings suggested that ISO treatment induced HF in rats.

As presented in Fig. 2A-C, the levels of GDF11 protein and mRNA expression were significantly increased in the heart tissues of ISO-treated rats compared with in the control group; however, the mean plasma GDF11 levels were markedly unchanged. Additionally, to demonstrate the effects of ISO on GDF11 production in cardiomyocytes, H9C2 cells were treated with ISO for 24 h. As presented in Fig. 2D and E, the levels of GDF11 protein and mRNA expression in H9C2 cells were significantly increased following ISO treatment compared with the control.

To investigate the roles of GDF11 in ISO-treated H9C2 cells, rGDF11 was used in the following experiments. rGDF11 significantly reduced the proliferation (Fig. 3A) and increased LDH levels (Fig. 3B) in ISO-treated H9C2 cells in a dose-dependent manner compared with ISO treatment only; 50 nM rGDF11 was selected for use in subsequent experiments. It was revealed that, compared with ISO treatment, 50 nM rGDF11 significantly reduced the proliferation (Fig. 4A), and increased the LDH release (Fig. 4B) and apoptosis of H9C2 cells, represented by the increase in Bax/Bcl-2 protein expression, caspase-3 activity and apoptotic rate in Hoechst 33258 staining (Fig. 4C-G).

ISO treatment often induces oxidative stress injury that is reflected by increased ROS and MDA concentrations (27). As presented in Fig. 5A and B, the concentrations of ROS and MDA were significantly increased in the ISO group compared with in the control group. rGDF11 in addition to ISO treatment further increased ROS and MDA concentrations compared with ISO treatment only.

To further investigate the potential molecular mechanisms underlying ISO-induced HF, the expression levels of the Nox subunits, Nox2 and Nox4, in H9C2 cells were determined via western blot analysis (Fig. 5C). The results revealed that Nox4 protein levels were significantly increased in the ISO group compared with in the control group and were further upregulated following rGDF11 treatment; however, no significant alterations in Nox2 expression were observed (Fig. 5D and E).

To further investigate the effects of GDF11 on ISO-treated H9C2 cells, GDF11 was downregulated by siRNA-mediated knockdown (Fig. 6A). Compared with the scrambled siRNA group, silencing of GDF11 significantly increased the proliferation (Fig. 6B), and significantly reduced the LDH release (Fig. 6C) and apoptosis of ISO-treated cells, represented by the decrease in Bax/Bcl-2 protein expression, caspase-3 activity and apoptotic rate in Hoechst 33258 staining (Fig. 6D-G).

GDF11 knockdown also significantly decreased the concentrations of ROS (Fig. 6H) and MDA (Fig. 6I) in ISO-treated H9C2 cells compared with the control. Additionally, western blot analysis demonstrated that the levels of Nox4 protein expression were downregulated in the GDF11 knockdown group compared with in the scrambled siRNA group; however, the levels of Nox2 expression were not significantly altered (Fig. 6J-L).