C3H10T1/2 cells, obtained from University of Chicago Molecular Oncology Laboratory (Chicago, IL, USA), were grown in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, EMD Millipore, Billerica, MA, USA). Lentiviruses (Lv) overexpressing GFP or Islet-1/GFP (multiplicity of infection=20) (GeneChem Co., Ltd., Shanghai, China), and 5 µg/ml polybrene (GeneChem Co., Ltd.) were mixed together and added to the culture medium of cells when they reached 30% confluence. The culture medium was replaced following culturing at 37°C in 5% CO₂ for 24 h. Fluorescence microscopy (Eclipse Ti-s; Nikon Corporation, Tokyo, Japan) was used to observe the GFP expression after 3 days. The infection efficiency was assessed with flow cytometry (BD FACSCanto II, BD Biosciences, San Jose, CA, USA), and prepared by BD FACS Diva version 3.0. The experiment was divided into 3 groups: The blank group, the Lv-GFP group (GFP cells) and the Lv-islet-1 group (cells infected with a plasmid overexpressing Islet-1/GFP). The Lv-islet-1 group was further divided into the following subgroups: Islet-1-1 week, Islet-1-2 weeks, Islet-1-3 weeks and Islet-1-4 weeks based on the lentiviral infection time.

C3H10T1/2 cells (1×10⁵ cells/well) were plated in 24-well plates on 1×1 cm² glass coverslips. Then, the cells were fixed in absolute acetone for 15 min at 4°C. Following 3 washes with PBS, the cells on the glass coverslips were blocked with goat serum (dilution, 1:20, ZSGB-BIO, Beijing, China), washed again, and incubated with the primary anti-cardiac troponin T(cTnT) monclonal antibody (ab209813; 1:400; Abcam, Cambridge, MA, USA) overnight at 4°C. Then, the cells were washed with PBS and incubated with a Cy3-conjugated secondary antibody (CW0159S; 1:150; Beijing Cowin Bioscience Co., Ltd., Beijing, China) for 1 h at 37°C. Following washing with PBS, 4′,6-diamidino-2-phenylindole was added for 3 min. Following the final wash, images were acquired under a fluorescence microscope (BX51; Olympus Corporation, Tokyo, Japan) and prepared by Nikon NIS-element AR 4.0 software. A total of six fields of view were assessed, and three replicates were performed.

Cellular RNA was extracted from the blank group, the Lv-GFP group and the Lv-islet-1 groups (Islet-1-1, Islet-1-2, Islet-1-3 and Islet-1-4 weeks) according to the instructions of the RNA extraction reagent kit (RP120; BioTeke Corporation, Beijing, China) and subjected to reverse transcription by PrimeScript™ RT Master Mix kit (RR047A; Takara Biotechnology Co., Ltd., Dalian, China). The cDNA was amplified (RR047A; Takara Biotechnology Co., Ltd.), using the reaction conditions of: 40 cycles of 95°C for 30 sec, 95°C for 5 sec and 60°C for 40 sec. Each reaction contained one blank well, and the samples of each group included three replicate wells. β-actin was used as the internal control. The relative expression levels of the genes were calculated using the 2-ΔΔCq method (21). The changes in the gene expression levels of GATA4, Nkx2.5 and Mef2c were assessed at all time points. The primer sequences of the genes are provided in Table I.

Formaldehyde (1%) was added to the samples to cross-link the protein-DNA complexes. The ChIP trials were conducted using a ChIP assay kit (Merck KGaA Darmstadt, Germany). Following cross-linking, the DNA was fragmented by sonication (Bioruptor UCD-200; Diagenode, Liège, Belgium) consisting of 25 cycles of 30 sec each, with an interval of 30 sec to cool down. Then, the protein-DNA complexes were precipitated with the following antibodies: Histone H3 (acetyl K9; ab10812; 3 µg/µl), general control of amino acid biosynthesis protein 5 (Gcn5; ab18381; 7 µg/µl), P300 (ab14984; 5 µg/µl), DNMT1 (ab87656; 5 µg/µl), DNMT3a (ab2850; 9 µg/µl) or DNMT3b (ab2851; 9 µg/µl), All antibodies purchased from Abcam and incubated overnight on a shaker at 4°C. DNA was extracted using the ChIP assay kit. The experiment included both a positive control (DNA precipitated by the RNA polymerase II antibody) and a negative control (DNA precipitated by normal mouse IgG), these antibodies all part of the assay kit noted above and used according to the manufacturer's protocols. The amount of extracted DNA was determined by qPCR (RR420A; Takara Biotechnology Co., Ltd., Dalian, China), the thermocycler conditions and the method of quantification strictly followed the ChIP assay kit protocols. The primer sequences and annealing temperatures of the ChIP-qPCR reaction are presented in Table II.

Cellular DNA was extracted from the blank group, the negative control group and the Lv-islet-1 groups (Islet-1-1, Islet-1-2, Islet-1-3, and Islet-1-4 weeks) according to the instructions of the DNA extraction kit (Tiangen Biotech Co., Ltd., Beijing, China). The DNA concentrations were determined to ensure that the amount of DNA treated with bisulfite was 350 ng. The volume of the DNA required for each group was calculated based on the concentration. The DNA bisulfite treatment reagent kit (Zymo Research, Irvine, CA, USA) was used. The primers for MS-PCR were designed using the MethPrimer software according to the previously described method (22). The primer sequences for GATA4 and Nkx2.5 are provided in Table III.

A 2% agarose gel was prepared, and the loading volume of the DNA ladder marker and the MS-PCR products was 5 µl. The electrophoresis was run for 45 min at 120 V in 0.25X TAE agarose gel electrophoresis buffer. The agarose electrophoresis results were observed using the chemiluminescence gel imaging system (G:box; Syngene, Cambridge, UK). The gray value of the electrophoresis band was determined using the Quantity One software (version 4.6.2; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Proteins were extracted from the cells using the radioimmunoprecipitation assay reagent (P0013B; Beyotime Institute of Biotechnology, Shanghai, China) containing 1% phenylmethanesulfonyl fluoride (cat. no. ST506; Beyotime Institute of Biotechnology) to prevent protein degradation. The protein concentrations were measured with the bicinchoninic acid method. The protein samples (40 µg protein each well) were mixed with 5X SDS-PAGE buffer (Beyotime Institute of Biotechnology). The sample loading buffer was boiled for 5 min prior to loading onto a 10% SDS-PAGE gel. Following electrophoresis, the proteins were transferred to polyvinylidene fluoride membranes (EMD Millipore). The membranes were cut according to the marker and incubated in 5% non-fat milk with PBS and Tween-20 (PBST, 0.05% Tween-20) for 1 h on a shaker at room temperature to block non-specific protein binding. The primary antibodies used in the present study were as follows: Anti-Islet-1 (EP4182; 1:2,000; Epitomics, Burlingame, CA, USA), anti-Gcn5 (ab18381; 1:1,000; Abcam), anti-P300 (ab14984; 1:1,000; Abcam), anti-DNMT1 (ab87656; 1:1,000; Abcam), anti-DNMT3a (ab2850; 1:1,000; Abcam) and anti-DNMT3b (ab2851; 1:1,000; Abcam). The β-actin antibody (A5441; 1:2,000; Sigma-Aldrich; Merck KGaA) was used as a control. The membranes were incubated with the primary antibodies overnight at 4°C and then washed in PBST 3 times for 10 min and incubated with the corresponding secondary antibody (ZB-2301 and ZB-2305, 1:2,000; ZSGB-BIO) on a shaker at room temperature. Positive bands were detected using a chemiluminescent reaction (EMD Millipore). The image collection and densitometry analyses were performed with the Quantity One analysis software (version 4.6.2; Bio-Rad Laboratories, Inc.).

Each experiment was repeated at least three times. All data were expressed as the mean ± standard deviation. The statistical evaluations were performed using independent samples by using Student's paired t-tests, continuity correction chi-square test and one-way analysis of variance and Dunnett's as a post hoc test. SPSS software (version 17.0; SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. P<0.05 was considered to indicate a statistically significant difference.

The GFP fluorescence results demonstrated that the GFP was stably expressed, indicating that the lentiviral infection was successful (Fig. 1A). The flow cytometry results demonstrated that the infection efficiency reached 91.7% (Fig. 1B). These results ensured the reliability of subsequent experiments. The western blotting results indicated that the C3H10 T1/2 cells had a high level of Islet-1 expression following lentiviral infection compared with the blank group and the control group (Fig. 1C). No visible difference in morphology was observed in untransfected MSCs and the Lv-GFP group (Fig. 2A). However, following Islet-1 transfection, the MSCs became fibroblast-like cells arranged in the same direction, exhibiting a short rod-shaped morphology and had a homogenous direction, a tight arrangement and a strong refraction (Fig. 2A). cTnT immunofluorescence was visibly higher in the Lv-islet-1 group compared with the blank and Lv-GFP groups, indicating that the MSCs expressed the cardiomyocyte-specific protein in the cytoplasm at 4 weeks following Islet-1 infection (Fig. 2B). The detection of cardiomyocyte-specific early-stage transcription factors indicated that the expression of Nkx2.5, GATA4 and myocyte enhancer factor 2C (Mef2c) gradually increased with time, and was highest in the Islet-1-3W group (Fig. 2C). These results suggested that Islet-1 promoted the differentiation of MSCs into cardiomyocyte-like cells.

The MS-PCR results indicated that the methylation level of the CpG sites on the GATA4 promoter gradually decreased following Islet-1 transfection; the decrease was most significant at week 3 (P<0.05; Fig. 3A). The methylation levels of the CpG sites on the Nkx2.5 gene promoter were higher but did not exhibit significant differences compared with the blank group and Lv-GFP group (Fig. 3B).

The ChIP-qPCR results demonstrated that the levels of histone acetylation on the promoter regions of GATA4 and Nkx2.5 in the Lv-islet-1 group were gradually increased with time; the expression of GATA4 and Nkx2.5 combined with H3K9ac in the C3H10T1/2 cells infected with Lv-Islet-1 gradually increased, with the peak time of binding at week 3 (P<0.05; Fig. 3C). These results indicated that histone acetylation participated in the regulation of GATA4 and Nkx2.5; by contrast, Nkx2.5 may not be affected by DNA methylation.

To elucidate the mechanism underlying the involvement of histones in the regulation of early-stage transcription factors in cardiomyocytes, protein expression of the major HATs, Gcn5 and P300, was assessed. The western blotting results indicated that the expression level of Gcn5 gradually increased following Islet-1 infection: The expression levels at all time points in the Lv-islet-1 group were higher than those in the blank group and Lv-GFP group, and the Islet-1-3 W was highest (Fig. 4A). The expression levels of P300 gradually decreased and the expression levels at all time points in the Lv-islet-1 group were significantly lower than those in the blank group and the negative control group (P<0.05; Fig. 4A).

The ChIP-qPCR results demonstrated that the levels of GATA4 and Nkx2.5 bound with Gcn5 gradually increased following Islet-1 infection, which was consistent with the increased expression of Gcn5 (Fig. 4B). The binding levels at all time points in the Lv-islet-1 group were higher than those in the blank group and the Lv-GFP group (P<0.05; Fig. 4B). The expression of the GATA4 and Nkx2.5 binding with P300 did not significantly change following Islet-1 infection compared with those in the blank group and the Lv-GFP group (P>0.05; Fig. 4C). These results indicated that Islet-1 enhanced the binding level of Gcn5 to the GATA4 and Nkx2.5 promoter regions through the increase in Gcn5 expression.

Previous studies indicated that DNA methylation participated in the Islet-1-induced MSCs differentiation into cardiomyocyte-like cells (23). Therefore, the present study further investigated the underlying mechanism. The western blotting results indicated that the DNMT-1 expression level gradually decreased following Islet-1 infection and that the expression levels at all time points in the Lv-islet-1 group were lower than those in the blank group and the Lv-GFP group (Fig. 5A). The expression level of DNMT-3a in the Lv-islet-1 group gradually increased; the expression levels at all time points in the Lv-islet-1 group were significantly higher than those in the blank group and the Lv-GFP group (P<0.05; Fig. 5A). By contrast, DNMT-3b expression was almost undetectable (data not shown).

ChIP-qPCR analysis identified that the levels of GATA4 bound with DNMT-1 gradually decreased following Islet-1 infection and the binding levels at all time points in the experimental group were significantly lower than those in the blank group and the Lv-GFP group (P<0.05; Fig. 5B, GATA4); the same trend was also demonstrated for the GATA4 bound with DNMT-3a (P<0.05; Fig. 5C, GATA4). Almost no DNMT-3b binding was detected on the GATA4 and Nkx2.5 promoter region (Fig. 5D). In addition, DNMT-1 and DNMT-3a were demonstrated to bind to the Nkx2.5 promoter region, and the level of binding following Islet-1 infection was not significantly different compared with the blank group (P>0.05; Fig. 5B and C, Nkx2.5, respectively). These results indicated that Islet-1 could reduce the DNMT-1 expression level and thus reduce its binding to the GATA4 promoter region. Eventually, the DNA methylation levels in the GATA4 promoter region decreased and GATA4 expression was promoted. However, DNMT-1 did not affect Nkx2.5 expression.