Male Lewis rats (n=24; aged 8 weeks; weight 180–200 g) were purchased from Vital River Laboratories Co., Ltd. (Beijing, China) and were maintained in our animal facilities with air-conditioning at Shantou University Medical College, under constant temperature and humidity conditions with a 12:12-h light-dark cycle. All rats had free access to food and water. Throughout the studies, all animals were treated in accordance with the institutional guidelines for animal experiments. Ethical approval was obtained from the Institutional Review Board of Shantou University Medical College (Shantou, China).

Ethical approval was obtained from the Institutional Review Board of Shantou University Medical College (Shantou, China). HuMSCs were prepared as previously described (10). A total of 5 patients were involved in the study, and written informed consent was obtained from all patients. Human umbilical cords from consenting patients who underwent full-term Caesarian sections were collected immediately into a sterilized 50 ml tubes, washed with phosphate-buffered saline (PBS), and cut into 2- to 3-cm thick sections. Following dissection of the arteries and veins, the remaining tissue, the Wharton's jelly, was sectioned into smaller fragments and transferred to 75 cm² flasks containing Dulbecco's Modified Eagle's medium/F12 media (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 µg/ml penicillin/streptomycin (Beyotime Institute of Biotechnology, Haimen, China), 1 g/ml amphotericin B (Gilead Sciences, Inc., Foster City, CA, USA), 5 ng/ml epidermal growth factor (EGF; Invitrogen; Thermo Fisher Scientific, Inc.), and 5 ng/ml basic fibroblast growth factor (bFGF; Sigma-Aldrich; Merck KGaA). Cultures remained undisturbed for 5–7 days at 37°C in 5% CO₂ to allow migration of cells from the explants. Subsequently, the media was replaced.

Purified porcine cardiac myosin (Sigma-Aldrich; Merck KGaA) was dissolved in 0.01 M PBS and emulsified with an equal volume of complete Freund's adjuvant supplemented with10 mg/ml Mycobacterium tuberculosis (Sigma-Aldrich; Merck KGaA). On day 0, rats received a single immunization at two subcutaneous sites (both footpads) with a total of 0.2 ml emulsion per rat. Rats not subjected to immunization were included as a controlgroup (n=8).

A total of 16immunized rats were randomly divided into two groups. Rats were randomly assigned into the following two groups: Control group, 0.2 ml PBS only (n=8);HuMSCs group, 0.2 ml HuMSCs (1×10⁶ cells/animal; n=8). A total of 10 days following myosin injection, HuMSCs or vehicle (PBS) were administered intravenously via the tail vein.

Echocardiography was performed 21 days following the myosin injection. Rats were anesthetized with 1.5–2.0% volume of isoflurane in air (Sigma-Aldrich; Merck KGaA). A 13-MHz probe was placed at the left fourth intercostal space for imaging using two-dimensional echocardiography (AcusonAntares, Siemens AG, Munich, Germany). Left ventricular systolic dimension (LVDs), left ventricular diastolic dimension (LVDd), interventricular septal thickness (IVS), left ventricular posterior wall thickness (LVPW) and fractional shortening (FS%) were measured and recorded as the mean for three beats. Fractional shortening (%) was calculated as [(LVDd-LVDs)/LVDd]x100%. Investigators blinded to the treatment group performed the echocardiography studies and all analyses were performed offline.

Following echocardiographic analyses, rats were sacrificed by cervical dislocation. Hearts were excised above the origin of the great vessels 21 days after the myosin injection. Hearts were fixed in 4% paraformaldehyde for 6 h at 4°C, embedded in paraffin, sectioned to 4-µm thickness, and stained with hematoxylin and eosin (H&E). A cardiovascular pathologist with no knowledge of the experimental groups evaluated H&E-stained sections. Myocardial injury and inflammation were characterized by assigning histoscores to every fifth cross section, according to a previously published 6-tier scoring system (grade 0, no inflammation; grade 1, cardiac infiltration in <5% of the cardiac sections; grade 2, 6–10% infiltration; grade 3,11–30%infiltration; grade 4, 31–50% infiltration; and grade 5, infiltration in >50% of cardiac sections) (12,13).

Heart tissues were homogenized in ice-cold radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Following centrifugation (12,000 × g for 10 min at 4°C), supernatants were collected and the total protein concentration in samples was measured by use of a BCA Protein Assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. For western blotting assays, 30 µg of total protein was separated by 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). Filters were blocked with 5% non-fat dry milk in TBST (20 mM Tris, pH 6.8, 137 mM NaCl, 0.1% Tween-20) overnight at 4°C, washed, and incubated overnight at 4°C with a 1:1,000 dilution of primary antibodies in blocking solution. Washing was conducted four times with TBST, for 10 min each, with constant shaking. The following primary antibodies were used: GAPDH (catalog no. D4C6R), extracellular signal-regulated kinase (ERK)-1/2 (catalog no. 9258), phosphorylated (p)-ERK-1/2 (catalog no. 4668), p38 MAPK (catalog no. 8690) and p-p38 MAPK (catalog no. 4511), which were all purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Glucose-regulated protein 78 (GRP78) (catalog no. SC-13968) and caspase 12 (catalog no. SC-5627) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Membranes were subsequently washed and incubated for 1 h at room temperature with a 1:2,000 dilution of horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody (catalog no. 4050-05; Southern Biotechnology Associates, Inc. USA). Protein bands were visualized using the ECL Plus chemiluminescence kit (Amersham Biosciences, Uppsala, Sweden).

Paraffin-embedded heart tissues were cut into 4-µm thick sections at room temperature. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed using an in situ apoptosis detection kit according to the manufacturer's protocol (Beyotime Institute of Biotechnology). Sections were mounted and examined using light microscopy. For each animal, five sections were scored for apoptotic nuclei. Only nuclei that were clearly located in cardiac myocytes were considered.

Data are expressed as the mean ± standard deviation. Analyses of differences between groups were performed using one-way analyses of variance, followed by Tukey's multiple comparison tests using SPSS software version 13.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

A significant suppression of cardiac function in EAM rats 21 days following cardiac myosin injection was observed. There was significant impairment in the systolic and diastolic components of cardiac contraction as presented in echocardiographic analyses compared with normal rats (P<0.01; Fig. 1A). HuMSC treatment significantly reversed cardiac remodeling with reduced LVDs and LVDd, and increased FS compared with vehicle-treated EAM rats (Table I). There was marked inflammatory cellular infiltration in the control group as identified by H&E staining, whereas HuMSCs-treated rats exhibited much less inflammatory cellular infiltration (Fig. 1A and B). Cardiomyocyte apoptosis was confirmed by TUNEL staining of myocardial tissue slices. Control animals demonstrated numerous TUNEL-positive apoptotic nuclei whereas HuMSCs-treated animals exhibited fewer apoptotic cells (Fig. 1A and C). Additionally, control rats demonstrated LV remodeling with increased LVDd and LVDs, and reduced FS in vehicle-treated EAM rats, when compared with untreated rats (normal group), indicating impaired myocardial function (Fig. 1D).

As assessed by western blot analysis (Fig. 2A), p-ERK1/2 expression was significantly increased in control rats compared with normal rats, suggesting that oxidative stress mediates stimulation of ERK1/2 signaling. HuMSC treatment significantly ameliorated protein expression levels of ERK 1/2 (^##P<0.05 vs. Normal, *P<0.05 vs. Control) and p-ERK1/2 in EAM rats (^##P<0.05 vs. Normal, *P<0.05 vs. Control) (Fig. 2B). p38-MAPK and p-p38MAPK levels did not differ between the three groups (data not shown).

GRP78 is a marker of ER stress. ER stress leads to activation of caspase 12, resulting in cardiomyocyte apoptosis. Significant increases were observed in myocardial GRP78 and caspase 12 expression in EAM animals, indicating the involvement of ER stress. HuMSC-treated animals exhibited significant attenuation of these ER stress markers compared with the control group (Fig. 2B).