Males, 2 weeks Sprague–Dawley rats, 25–30 g body weight (BW), were used for this study. Animals were fed a standard rat chaw and allowed to drink water ad libitum. They were housed in temperature-controlled (22–25°C) cages with a 12-h dark and 12-h light cycle. This study protocol was approved by the Fuwai Hospital Animal Care and Use Committee. The investigation conforms with the “Guide for the Care and Use of Laboratory Animals” published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and the “Regulation to the Care and Use of Experimental Animals” of the Beijing Council on Animal Care (1996).

After being heparinized (3 IU/g BW) and anesthetized with 50 mg/kg BW of sodium pentobarbital by intraperitoneal injection, rats were subjected with a median sternotomy and the hearts were rapidly excised and placed in ice-cold heparinized K-H solutions. The time taken from opening of the chest to excision of the heart was 1–2 min. Immediately thereafter, the aorta was connected to a standard Langendorff apparatus and perfused with Krebbs–Henseleit (K-H) buffer solution (pH 7.4: in mM: NaCl 118; KCl 4.7; CaCl₂ 2.0; MgSO₄ 1.2; NaHCO₃ 25; KH₂PO₄ 1.2; glucose 11.1), and continuously gassed with 95% O2 and 5% CO2 at pressure of 80 mmHg at 37°C. A pulmonary arteriotomy was performed to allow free drainage of coronary effluent. A water-filled latex balloon connected to a pressure transducer was inserted into the left ventricle to assess the LV function. Parameters, including heart rate (HR), left ventricle systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), were continuously measured. The volume of the latex balloon was adjusted to set LVEDP to 0–10 mmHg during the stabilization phase.

Rats were randomly assigned to one of four experimental groups. There were 6 rats in each group: (1) Sham group; (2) I/R group; (3) I/R+LPA group; (4) I/R+Ki16425+LPA group. The design of the experimental protocol is illustrated in Figure 1. All hearts were equilibrated for 10 min followed by a 20-min treatment period and subjected to 60 min of global ischemia by stopping the K-H buffer perfusion (maintaining 37°C), followed by 30 min of reperfusion. LPA (10 μM) was added into the perfusion fluid before ischemia and throughout reperfusion with or without ki16425 (10 μM).

After 30 min of reperfusion, the isolated hearts were removed and cut into uniform sections of 2–3 mm thickness and incubated for 20 min in 0.1 M sodium phosphate buffer containing 1% 2,3,5-triphenyl tetrazolium chloride (TTC) (Sigma–Aldrich, St. Louis, MO, USA) at 37°C. Viable myocardium showed up as red, and the infarcted area showed up as pale white. The infarct size was calculated using Image-Pro Plus software, and was expressed as a percentage of the total heart size. The level of creatine kinase-MB (CK-MB) was measured from coronary effluent which was collected while reperfusion took place, in order to assess the infarct degree. The method of measurement was by spectrophotometry, using commercial assay kits (Roche, Indianapolis, IN, USA).

The heart was fixed in neutral 10% formalin for 24 h, embedded in paraffin, and cut into 8 μm-thick slices. After de-paraffinating and re-hydratation, sections underwent antigen retrieval by boiling in sodium citrate solution for 2 min. TdT-mediated dUTP nick end labeling (TUNEL) staining was performed using a fluorescence detection kit (Roche, Indianapolis, IN, USA). Thereafter sections were covered with Mounting Medium with DAPI (Invitrogen, California, USA). Images were acquired using an Olympus fluorescence microscope and counted using the Image- Pro Plus software from 10 random fields at 400 × magnification. In order to examine caspase-3 activity, commercial kit reagents (Biovision Research Products, Mountain View, CA, USA), were used in accordance with the procedures outlined by the manufacturer. Samples of whole left ventricular homogenate were prepared and tissues were homogenized in the cell lysis buffer followed by centrifugation. The supernatant was collected and used for the assay. Protein concentration in the lysate was measured and 200 μg lysate protein was incubated in a 96-well plate with 2 × Reaction Buffer (50 μl) at 37°C in the dark. Caspase activity was monitored using a Microplate Reader at 405 nm.

Embryonic rat heart derived H9C2 cells, from the cell bank at the Shanghai Institute for Biological Sciences, were cultured in L-DMEM (GIBCO, Grand Island, NY) supplemented with 10% FBS (fetal bovine serum, GIBCO) and antibiotics (50 U/mL penicillin and 50 μg/mL streptomycin, GIBCO) at 37°C in a 95% room air/5% CO2 incubator and sub-cultured when 80% confluence was reached. Before different treatments to H9C2, the medium would be replaced by serum-free DMEM when the H9C2 was grown to 80% confluence. To establish an I/R model in vitro, a Hypoxia-reoxygenation cell model was used in our study. Briefly, after serum-starvation for 18 h and then treating with different concentration of agents, H9C2 was subjected to hypoxia in a controlled hypoxic plastic chamber for 12 h. Reoxygenation was conducted in a normoxic incubator at 37°C after hypoxia for 4 h. For each evaluation, all experiments were conducted in triplicate.

Cell viability was measured using a quantitative colorimetric assay with thiazolyl blue tetrazolium bromide (MTT, AMResco), showing the mitochondrial activity of living cells. H9C2 cells (~3 × 10⁴) were seeded in 24-well plates. After drug treatment as indicated, cells were incubated with 300 μL MTT (final concentration 0.5 mg/mL) for 4 h at 37°C. The reaction was terminated through the addition of 200 μL DMSO. The MTT reaction products were determined by measuring the absorbance at 630 nm in a CHAMELEON micro-plate reader (HIDEX).

Hoechst 33258 (Invitrogen, California, USA) was used to detect the apoptosis of H9C2 in different groups. Briefly, H9C2 was stained with Hoechst 33258 dye solution (5 μg/mL) for 20 min at room temperature, in darkness, and then examined and photographed using a fluorescence microscope. Apoptotic cells were identified as previously described. The percentage of apoptotic cells was calculated as the ratio of the positive cell number to the total cell number by Image J.

The amounts of apoptotic cells induced by hypoxia-reoxygenation were determined using an Annexin V-FITC apoptosis detection kit (SIGMA, USA), according to the manufacturer's instructions, as previously described. Cells in the initial stage of apoptosis were defined as annexin V (+)/propidium iodide (PI) (−), while late apoptotic cells were defined as annexin V (+)/PI (+). Briefly, H9C2 seeded in 6-wells plates were washed twice with cold PBS, then harvested and stained with 5 μl annexin V and 10 μl propidium iodide (PI) per 500 μl cells for 15 min at RT in the dark. Flow cytometric analyses were performed on a FACS Calibur Analyzer (BD). The apoptotic cells, including annexin V + /PI, were counted.

H9C2 was incubated with 2-NBDG, which is a fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2-deoxyglucose, to investigate the glucose uptake ability. H9C2 was seeded in six-well plates. After hypoxia-reoxygenation—as in a previous description—cell culture was replaced by Kreb's buffer without glucose (NaCl [145 mM], KCl [5 mM], CaCl₂ [6 mM], MgCl₂ [1 mM], HEPES Na [25 mM], and NaHCO₃ [10 mM], pH 7.4) and incubated with different concentrations of LPA and insulin for 20 min at 37°C. Thereafter, 2-NBDG (100 μM) (Invitrogen, California, USA) was supplemented at the same culture temperature for an additional 10 min in the dark. Fluorescence was measured in a FACSCalibur Analyzer (BD) by 488 nm (excitation wavelength) channel. Data was collected from 20,000 events for each measured sample.

After immunoblotting, the intensity of the immunoblot bands was detected utilizing a ChemiDoc XRS+ (Bio-Rad) instrument. Antibodies against the following proteins were purchased from Cell Signaling Technology: anti-Bax (1:1,000), anti-Bcl-2 (1:1,000), anti-GSK3beta (1:1,000), anti-phosphorylated GSK3beta (1:1,000), anti-AKT (1:1,000), anti-p-AKT (Ser473; 1:1,000), anti-pThr172-AMPKα (1:1,000), anti-GLUT4 (1:500) and anti-GAPDH (1:10,000; loading control) antibodies. In order to measure the amount of GLUT4 in the plasma membrane, the plasma membrane protein was separated from the cell by using a compartmental protein extraction kit (Millipore, USA).

SPSS16.0 for Windows (SPSS Inc, Chicago, III) was used for data analysis. All values were expressed as mean ± standard error of the mean (SEM). For comparisons between groups, the one-way ANOVA or unpaired Student T-test, was used where appropriate. Post hoc testing used a Bonferroni Correction for multiple comparisons. If the test for normality failed or if the sample was <5, a Fisher Exact Test was used. A P < 0.05 was considered statistically significant.

There were no significant differences among the groups under basal conditions in cardiac LV function before the start of ischemia (Table 1). After 60 min of global ischemia, the I/R group exhibited a significant reduction in heart rate (HR) (Figure 2A) and left ventricle systolic pressure (LVSP) (Figure 2B) and a remarkable increase in LVEDP (Figure 2C) (P < 0.01 vs. sham group, respectively). Compared with the IR group, administration of LPA significantly enhanced the recovery of HR, LVSP and LVEDP within 30 min of reperfusion (Figures 2A–C). Furthermore, the administration of Ki16425 partially blocked the LPA-induced improvement in ventricular systolic and diastolic function after I/R (Figures 2A–C).

The infarct size determined by TTC staining was significantly lower in the IR+LPA group than in the IR group (Figures 3A,B). However, the infarct-limiting effect of LPA was abolished through pretreatment with Ki16425 (Figures 3A,B). The released CK-MB was also measured in each experimental group to determine the degree of myocardial injury. Global ischemia followed by reperfusion significantly increased CK-MB levels, and LPA pretreatment significantly decreased the release of CK-MB compared with IR group (Figure 3C). Similarly, these effects were also partially abolished by Ki16425 (Figure 3C).

As displayed in Figures 4A,B, IR significantly increased the percentage of TUNEL-positive cells. This increase was attenuated through pretreatment with LPA, and Ki16425 attenuated the anti-apoptotic effects of LPA. Consistent with the TUNEL data, IR significantly increased the activity of infantile myocardial caspase-3, which was rescued by pretreatment with LPA (Figure 4C). Compared with the LPA group, co-administration of LPA and Ki16425 significantly increased the apoptosis index and the activity of caspase-3 (Figures 4B,C), which indicated that LPA exerted its anti-apoptotic effects by coupling to its receptor 1/3.

Furthermore, the expression of classic anti-apoptotic (Bcl-2) and pro-apoptotic proteins (Bax) were examined. IR significantly reduced Bcl-2/Bax relative expression, and pretreatment with LPA increased Bcl-2/Bax relative expression (Figures 5A,B). In addition, western blot analyses of heart tissue indicated that IR induced a statistically significant reduction in the phosphorylation of AKT. LPA pretreatment markedly elevated the phosphorylation of AKT and its downstream molecule GSK-3β. However, those effects were abolished by Ki16425 treatment (Figures 5C,D). These results indicate that the cardioprotective effect of LPA is attributable to the activation of the AKT signaling pathway through the LPA receptor 1/3.

Cell viability was decreased when H9C2 suffered from hypoxia-reoxygenation (H/R) which was a cell model of ischemia reperfusion (IR), as shown by the MTT assay. This change was rescued by LPA pretreatment which was administered in a dose-dependent manner (Figure 6C). Moreover, H/R increased the number of apoptotic H9C2, as shown by the Hoechst staining (Figure 6A). LPA preconditioning significantly reduced the extent of H9C2 apoptosis compared with the H/R group (Figure 6B). Apoptosis/necrosis was also evaluated using the Annexin V-FITC Apoptosis Detection Kit. The percentage of apoptosis and necrosis cells in the IR group was significantly higher than the control group (Figures 7A,B). LPA preconditioning significantly protected H9C2 from H/R induced apoptosis. However, when LPA administration was combined with PI3K/AKT inhibitor LY294002, the anti-apoptosis effect of LPA was negated. Consistently, the results of western blot indicated that IR significantly reduced Bcl-2/Bax relative expression, and pretreatment with LPA increased Bcl-2/Bax relative expression (Figures 7C,D).

To investigate the effects of LPA preconditioning on cardiomyocyte glucose uptake, the uptake of fluorescent glucose (2-NBDG) was studied. As shown in Figures 8A,B, after hypoxia 12 h, LPA (5 μM) significantly increased 2-NBDG uptake in H9C2. In the study, the Insulin (0.1 μM) treatment group was used as a positive control. As expected, insulin increased glucose uptake faster than LPA.

As shown in Figures 8C,D, there was no significant difference between the control group and the LPA preconditioning group on GLUT4 translocation from the cytoplasm to the membrane. However, the insulin treatment group—which is a positive control—showed a significant increase of GLUT4 translocation. In addition, the role of AMP-Activated Protein Kinase (AMPK) was investigated in this procedure. As shown in Figures 8C,E preconditioning with 5 μM LPA significantly enhanced the phosphorylation of AMPK (P < 0.05), while insulin did not. These results suggested that LPA was involved in the activation of AMPK pathway but not the translocation of GLUT4.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.