The LPA3-/- mice used in this study were described previously (Yang et al., 2002; Ye et al., 2005; Choi et al., 2008). All mice were bred from a BALBc strain, and backcrossed for more than 10 generations. This study was carried out in accordance with the “Regulation to the Care and Use of Experimental Animals” of the Beijing Council on Animal Care study (1996). The protocol was approved by the Fuwai Hospital Animal Care and Use Committee.

The culture of NRCMs isolated from 1- to 3-day-old Sprague-Dawley rats was carried out as previously described (Chen et al., 2002). DMEM containing 10% fetal bovine serum, penicillin/streptomycin (1,000 U/ml each) with 100 mM 5-Bromo-2-deoxyUridine was added to inhibit the growth of cardiac fibroblasts. After 24 h of culture, the cells were washed and starved overnight in serum-free medium prior to use within experiments.

Small interfering RNA (siRNA) (Invitrogen, Carlsbad, CA, USA) for LPA₃ was transfected into cardiomyocytes using Lipofectamine™ RNAiMAX according to the manufacturer's protocol, as previously described (Yang et al., 2013b). Stealth siRNA duplex target sequences were 5′-UACACCACCACCAUGAUGAAGAAGG-3′ and 5′-CCUUCUUCAUCAUGGUGGUGGUGUA-3′. The sequence for the stealth siRNA low-GC duplex was used as a negative control. The cells were transfected with the stealth siRNA at 20 nM.

NRCMs were placed on coverslips in six-well culture plates and treated with LPA (5 μM) or ISO (1 μM) for 24 h, or pretreated with propranolol (1 μM) for half an hour before LPA/ISO stimulation. In the transfection experiments, cells were transfected with siRNA of LPA₃ or the negative control for 24 h, then exposed to LPA or ISO for 24 h. The proportion of cells staining positively for α-actinin was measured to determine the degree of cardiomyocyte hypertrophy. For α-actinin staining, cells were fixed with 4% paraformaldehyde for 30 min, and subsequently permeabilized with 0.1% Triton X-100 for 10 min and 1% BSA for 30 min at room temperature. After washing, cells were incubated with mouse monoclonal antibody against α-actinin (1:250, Abcam, Cambridge, MA, USA) at 37°C for 2 h and a fluorescein-conjugated goat-anti-mouse IgG (1:300, Zhongshan Jinqiao Biotechnology, Beijing, China) at 37°C for 1 h. DAPI staining marked the nuclei. The cells were visualized under a fluorescence microscope (Zeiss, Oberkochen, Germany) and laser confocal microscopy (Leica, Wetzlar, Germany). At least 100 cardiomyocytes in 20–30 fields were examined in each group.

Micro-osmotic pumps (model 1002, Alzet) releasing ISO (Sigma, St. Louis, MO, USA; 60 mg/kg/day in PBS) or vehicle (PBS) were implanted subcutaneously into age-matched (8–10 weeks old) male LPA3-/- mice and wild-type littermates after being anesthetized by intraperitoneal administration of tribromoethanol (400 mg/kg). Hypertrophy was assessed by echocardiography using a VisualSonics Vevo 2100 ultrasound system (VisualSonics, Inc., Toronto, ON, Canada). Ventricular measurements in M-mode were taken before and 13 days after implantation. The following day mice were sacrificed for histological and molecular analysis.

Myocardial infarction (MI) experiments were performed on 8–10 week-old LPA3-/- and wild-type mice. The MI model was characterized as previously described (Fan et al., 2015). Mice were anesthetized by intraperitoneal administration of tribromoethanol (400 mg/kg) and ventilated with a rodent respirator, then the left anterior descending coronary artery was permanently occluded using a 7-0 polypropylene suture, and the occlusion was confirmed by blanching of the anterior wall of the left ventricle. For non-infarcted controls, mice underwent sham operation where the ligature around the left anterior descending coronary artery was not tied. Animals were recovered from anesthesia under warm conditions with normal ventilation. Four weeks after surgery, cardiac function was assessed by echocardiography using a VisualSonics Vevo 2100 ultrasound system (VisualSonics, Inc.), then animals were sacrificed and hearts were excised for further analysis.

Hearts were fixed in 10% formalin overnight at room temperature and embedded in paraffin. Subsequently, the hearts were cut serially from the apex to the base. Each short-axis section (5 μm), collected with an interval of 200 μm, was stained with Picrosirius red for morphometric analysis. The experiments were executed following standard procedures. The infarct size was calculated as a percentage of the total left ventricle (LV) wall circumference from each of the three LV sections. Scar circumstance was calculated using Image Analysis Software.

WGA staining was carried out to measure the cross-sectional area of cardiac myocytes. Following antigen retrieval using citric acid buffer (10 mM, pH 6.0), the sections were blocked with 1% bovine serum albumin for 30 min at room temperature and then incubated with Alexa Fluor®594-WGA (Invitrogen) for 1 h at room temperature. The slides were washed three times in PBS, mounted using an anti-fade mounting media containing DAPI (Zhongshan Jinqiao Biotechnology), and imaged was performed using a Leica DM6000B microscope.

Total RNA was extracted from mice ventricular tissue and cultured cardiomyocytes using Trizol, and was then quantified using a Nanodrop 2000 spectrophotometer. cDNA was generated from total RNA (1 μg) using M-MLV reverse transcriptase and oligo(dT)15 primer. qRT-PCR was performed using a SYBR Green PCR Master Mix and an Applied Biosystems 7500 (ABI, Foster City, CA, USA). The primer pairs used in this study are listed in Table 1.

All data are presented as mean ± SEM. Differences among groups were assessed by a one-way analysis of variance (ANOVA) followed by a post-hoc Tukey's test. Comparisons between two groups were performed using a Student's t-test. All statistical analyses were performed with GraphPad Prism 6.0. A value of p < 0.05 was considered to indicate a statistically significant difference.

To test whether LPA₃ is involved in an agonist of β-AR, ISO-induced hypertrophy, we used RNAi technology to knock down LPA₃ expression in neonatal rat cardiomyocytes (Figure 1A). Cardiomyocyte size and the expression of atrial natriuretic factor (ANP) and brain natriuretic factor (BNP) mRNA were significantly increased after LPA or ISO treatment (Figures 1B–D). LPA₃ interference resulted in a significant reduction in LPA-induced cardiomyocyte hypertrophy, but had no effect on ISO-induced ANP and BNP mRNA expression (Figure 1B) or cardiomyocyte size (Figures 1C,D). These results suggest that LPA₃ is not involved in ISO-induced cardiomyocyte hypertrophy in vitro.

To further explore whether the β-AR participates in LPA-induced cardiomyocyte hypertrophy, a β-AR antagonist, propranolol, was used. After propranolol (1 μM) pretreatment for 30 min, ISO failed to increase cardiomyocyte size and ANP and BNP mRNA levels. However, LPA-induced cardiomyocyte hypertrophy was not significantly inhibited (Figures 2A–C). Taken together, our results indicate that ISO and LPA promote cardiomyocyte hypertrophy in vitro by independent mechanisms.

We further assessed whether the absence of LPA₃ attenuates ISO-induced cardiac hypertrophy in vivo. LPA3-/- and wild-type mice were subcutaneously implanted with an osmotic pump delivering ISO (60 mg/kg/day) for 2 weeks. Echocardiology data showed that left ventricular anterior and posterior wall thickness, and the left ventricular weight/body weight ratio were significantly increased in wild-type mice after ISO infusion (Figures 3A,C–E). LPA3-/- mice showed similar responses to ISO infusion. Furthermore, heart rate was significantly increased in both wild-type and LPA3-/- mice after ISO infusion (Table 2, Figure 3B).

Morphological data show that ISO infusion resulted in a significant increase in heart size (Figure 4A), heart to body ratio (Figure 4B), and cardiomyocyte cross-sectional area in wild-type mice (Figures 4C,D). However, there was no attenuation of these hypertrophic responses in LPA3-/- mice (Figures 4A–D). Furthermore, we assessed the mRNA levels of genes associated with hypertrophy in the four treatment groups. We observed a similar alteration to ANP expression in both LPA3+/+ and LPA3-/- hearts after ISO treatment (Figure 4E). These findings support the hypothesis that LPA₃ deficiency does not affect ISO-induced cardiac hypertrophy in vivo.

Since it is known that LPA-induced hypertrophy is different from the pathological hypertrophy induced by the β-AR, we decided to investigate further the role of LPA₃-induced hypertrophy in pathological ventricular remodeling. At 4 weeks after MI, LPA3+/+ animals showed marked increases in the heart weight/body weight ratio, levels of ANP mRNA expression, and cardiomyocyte cross-sectional area (Figures 5A–D). Compared with LPA3+/+ infarcted mice, there was a tendency for a reduced heart weight/body weight ratio (Figure 5A), a notable decrease in ANP mRNA levels (Figure 5B), and a significant decrease in cardiomyocyte cross-sectional area (Figures 5C,D) in LPA3-/- mice post-MI. These results indicate that LPA₃ is involved in cardiac hypertrophy after MI.

Meanwhile, we further investigated the effect of LPA₃ deficiency on cardiac function post-MI. LPA3-/- mice manifested no pathological abnormalities with respect to cardiac function at baseline. After MI challenge, the ejection fraction percentage (EF%) was lower in both LPA3+/+ and LPA3-/- mice, but LPA3-/- mice exhibited more pronounced LV dilatation and more severe contractile dysfunction (Table 3, Figures 5E,F). Sirius red staining at 4 weeks after MI further displayed a significantly enlarged infarct size in LPA3-/- mice compared with wild-type control animals (P < 0.05; Figures 5G,H). These findings suggest that LPA₃ might play a protective role against MI.

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.