All treatments were administered at a concentration of 10 mM for 48 h of treatment at 37°C. S1P, W146, SB-431542 (SB4), SIS3 and TGF-β were all purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The primary anti-S1PR1, anti-TGF-β and anti-Smad3 antibodies (Cat. no. ab72806, ab31013, ab40854, respectively) were acquired from Abcam (Cambridge, UK). Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were from Hyclone (GE Healthcare, Little Chalfont, UK). The secondary antibodies conjugated to horseradish peroxidase (Cat. no. ab6789) were purchased from Zhongshan Goldenbridge Bio (Beijing, China). The enhanced chemiluminescent (ECL) kit was obtained from Thermo Fisher Scientific (Shanghai, China). For Reverse-transcription quantitative polymerase chain reaction (RT-qPCR), the MagExtractor-RNA kit and ReverTra Ace qPCR RT Master Mix with gDNA Remover kit were all from Toyobo (Cat. no. FSQ-301, Tokyo, Japan). The lactate dehydrogenase (LDH) detection activity assay kit and the commercialized caspase-3 assay kit were purchased from Sigma-Aldrich (cat. no. CASP3F-1KT, Merck KGaA) and Biovision, Inc. (cat. no. 1533-100, Milpitas, CA, USA), respectively.

Neonatal mice were purchased from SLRC Laboratory Animal (Changsha, China). Animals were provided with standard rodent chow and water ad libitum. All animal procedures were approved by the Institutional Animal Care and Use Committee of Jining No.1 People's Hospital (Jining, China). All protocols conformed to the National Research Council's Guide for the Care and Use of Laboratory Animals.

Animals were anesthetized with ether to remove the hearts, which were put in pre-cooled D-hanks medium (Procell, Wuhan, China). The heart tissues were immersed in 0.1% trypsin (Procell) and oscillated overnight at 4°C. Complete medium was added to terminate digestion by incubation at 37°C for 10 min. After discarding the supernatant, the remnant was incubated with 0.08% collagenase II (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) at 37°C for 10 min and the supernatants were pooled. The combined supernatants were centrifuged at 250 × g for 5 min, and DMEM containing 20% FBS was used to re-suspend the precipitates. The cells were then inoculated in a culture dish and incubated for 50 min at 37°C in a humidified atmosphere containing 5% CO₂. The non-adherent cells were aspirated to be re-suspended for a second adherence culture. The final concentration of the cells was adjusted to 5×10⁵ cells/ml and 10 ml cell suspension was mixed with 100 µl bromodeoxyuridine to inhibit the proliferation and differentiation of non-fibroblasts but without any obvious effect on the proliferation of the fibroblasts (25). The cells were cultured under the abovementioned conditions and the medium was changed once every 2–3 days. The growth and morphological changes of cardiomyocytes were observed and recorded every day.

The IR model was established and evaluated as described previously with minor modifications (26). In brief, the culture medium with 20% FBS was centrifuged at 250 × g for 5 min and resuspended in D-hanks medium with a gas mixture of 95% O₂-5% CO₂ for 30 min of incubation prior to hypoxia. To simulate ischemia, the culture plate was transferred into an anoxic incubator for 2 h of incubation with a gas mixture of 95% N₂-5% CO₂. For the reperfusion process, the D-hanks medium was replaced with DMEM containing 20% FBS for 24 h of incubation at 37°C with a gas mixture of 95% O₂-5% CO₂. The established IR model was evaluated by inspecting apoptosis, LDH release and caspase activity. For the apoptosis assay, cardiomyocytes were collected and centrifuged at 250 × g for 5 min. The cells were inoculated in 6-well flat-bottom plates and digested with 0.3 ml 1X trypsin-EDTA in PBS (37°C). The cells were immediately stained in the dark for 30 min according to the instructions of the Annexin V-FITC/PI apoptosis detection kit (cat. no. 88-8005-72, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Finally, cellular apoptosis was determined by flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA) within 1 h. The measurements of LDH release and caspase-3 activity were performed using respective commercial kits based on the manufacturer's instructions.

Total RNA was extracted from cardiomyocytes treated with 1 µM S1P alone or in combination with 0.4 µM W146 using a MagExtractor-RNA kit. The extracted total RNA was reverse-transcribed into complementary DNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover kit. The primers, which were synthesized by Sangon Biotech (Shanghai, China), had the following sequences: S1PR1 forward, 5′-AACTACACAACGGCAGCAAC-3′ and reverse, 5′-GCAGGCAATGAAGACACTCA-3′; S1PR2 forward, 5′-GGCTCTGTTCCCTGTATTG-3′ and reverse, 5′-GGGCTCACTTTGCTCCTC-3′; S1PR3 forward, 5′-AAATGGCTGCCTTGGAC-3′ and reverse, 5′-CCCATCGGTTTGGTGCT-3′; S1PR4 forward, 5′-ACGATAGGTGCTGTTAGT-3′ and reverse, 5′-CAGATATGCTGCTTCTTT-3′; S1PR5 forward, 5′-TGGTGGTCCTCATCGTCG-3′ and reverse, 5′-GGAGAAGGTGGCAGTGGTAA-3′; TGF-β forward, 5′-GACTACTACGCCAAGGAGGTC-3′ and reverse, 5′-GAGAGCAACACGGGTTCAG-3′; Smad3 forward, 5′-TGTTGGTGGAGGGTGTAG-3′ and reverse, 5′-AGCAGCAGTGAAGGTGAG-3′; β-actin forward, 5′-ACTCTTCCAGCCTTCCTTC-3′ and reverse, 5′-ATCTCCTTCTGCATCCTGTC-3′. RT-qPCR was performed on an ABI7000 fluorescent quantitative PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the following thermocycling procedure: 95°C for 60 sec, followed by 40 cycles of 95°C for 15 sec, 55°C for 15 sec and 72°C for 45 sec. All data were normalized to the housekeeping gene β-actin used as a reference. The relative expression of the target genes was calculated using the 2^−ΔΔCq method (27).

Cardiomyocytes were incubated with 200 ml lysis buffer (25 mM MgCl₂, 5 mM KCl, 20 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, 0.5% (v/v) complete protease inhibitor and Triton X-100). Protein concentrations were determined using a BCA Protein Quantification kit (Vazyma, Nanjing, China) according to the manufacturer's instructions. Cellular protein (50 mg) was separated using 12% SDS-PAGE prior to transfer onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The protein bands were blocked for 1 h in blocking buffer at room temperature. Antibodies (monoclonal rabbit anti-S1PR1, TGF-β and Smad3; 1:500 dilution) were incubated with the membranes overnight at 4°C. Secondary antibodies conjugated to horseradish peroxidase (1:2,000 dilution) were incubated with the membranes for 1 h at room temperature, followed by an ECL assay. The bands were imaged with the ChemiDoc™ XRS Gel image system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and their intensities were measured using Quantity one v4.62 software (Bio-Rad Laboratories, Inc.).

The S1P content was measured by LC-MS/MS according to previously described procedures (28).

Each experiment was performed in triplicate on three independent occasions. Values are expressed as the mean ± standard deviation and analyzed using one-way analysis of variance with the Least Significant Difference post hoc test. The statistical analyses were performed using SPSS 11.5 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The isolated myocardial cells were used for the establishment of an in vitro IR injury model. In the established IR model, the apoptotic rate was ~34% (P<0.01 vs. control). Addition of S1P resulted in a further increase of cell apoptosis up to ~39% (P<0.01). However, exogenous W146 inhibited the increase of cell apoptosis caused by S1P, resulting in an apoptotic rate that was significantly different from that of the control (Fig. 1A and B). The LDH levels and caspase-3 activity were also measured due to the stability of LDH in dead cells and the critical role of caspase-3 in apoptosis. The results demonstrated that in IR-treated cells, LDH levels and caspase3 activity increased by 2.2- and 2.8-fold, respectively (P<0.05), and were further enhanced in IR+S1P-treated cells, resulting in 2.4- and 3.2-fold increases, respectively, compared with the control (P<0.05). However, introduction of W146 inhibited the increases of LDH levels and caspase3 activity caused by S1P with the resulting values being not significantly different from those in the control group (Fig. 1C and D).

After IR injury, the mRNA and protein levels of S1PR1, TGF-β and Smad3 were significantly increased (P<0.01). Exogenous S1P further increased the mRNA and protein expression of S1PR1, TGF-β and Smad3 (P<0.001). In comparison, W146 abolished the stimulatory effects of S1P on S1PR1, TGF-β and Smad3 mRNA and protein expression, resulting in levels that were comparable to those of the control group (Fig. 2).

Induction of IR resulted in upregulation of S1PR1-3 in myocardial cells (P<0.05 or P<0.01). Pretreatment with TGF-β caused a significant increase of S1PR1 mRNA at 4 h (P<0.05). After 24 h of treatment with TGF-β, the levels of S1PR1-3 mRNA were all significantly stimulated (P<0.05 or P<0.01). The expression of S1PR5 mRNA was not detectable, while S1PR4 mRNA appeared to not be significantly affected by TGF-β (Fig. 3A). The protein levels of S1PR1 were also increased by pretreatment with TGF-β for 0, 4 and 24 h (P<0.05 or P<0.01; Fig. 3B). These results suggested that S1PR1 mRNA was more affected by TGF-β than S1PR2 and S1PR3 mRNA. By using TGFβR1 inhibitor SB4 and Smad3 inhibitor SIS3, the stimulatory effects of TGF-β on S1PR1 and S1P were abolished (Fig. 4A and B).