Characterization and Expression of Sphingosine 1-Phosphate Receptors in Human and Rat Heart

Abstract

Aim: Sphingosine 1-phosphate (S1P), sphingolipid derivatives are known anti-inflammatory, anti-apoptotic, and anti-oxidant agent. S1P have been demonstrated to have a role in the cardiovascular system. The purpose of this study was to understand the precise expression and distribution of S1P receptors (S1PRs) in human and rat cardiovascular tissues to know the significance and possible implementation of our experimental studies in rat models. Methods and Results: In this study, we investigated the localization of S1PRs in human heart samples from cardiac surgery department, University of Verona Hospital and rat samples. Immunohistochemical investigation of paraffin-embedded sections illustrated diffused staining of the myocardial samples from human and rat. The signals of the human heart were similar to those of the rat heart in all chambers of the heart. The immunohistochemical expression levels correlated well with the results of RT-PCR-based analysis and western blotting. We confirmed by all techniques that S1PR1 expressed strongly as compared to S1PR3, and are uniformly distributed in all chambers of the heart with no significant difference in human and rat myocardial tissue. S1PR2 expression was significantly weak while S1PR4 and S1PR5 were not detectable in RT-PCR results in both human and rat heart. Conclusion: These results indicate that experimental studies using S1PR agonists on rat models are more likely to have a potential for translation into clinical studies, and second important information revealed by this study is, S1P receptor agonist can be used for cardioprotection in global ischemia-reperfusion injury.

Keywords: cardioprotection; heart; human; ischemia-reperfusion injury; rat; sphingosine 1-phosphate receptors.

FIGURE 1

A comparative study of expression of S1PR between human (n = 10) and rat (n = 10) heart in all chambers by quantitative real-time RT-PCR. Data are represented as the mean ΔCt ± SD (ΔCt = Ct value of target mRNA- Ct value of 18S rRNA); therefore, greater expression is equivalent to smaller ΔCt value of mRNA. S1PR1 and S1PR3 mRNA are more abundantly expressed in all chambers of heart as compared to S1PR2. Inversely, S1PR4 and S1PR5 are not detectable in both species (^≠ p ≤ 0.05, ^{≠ ≠} p ≤ 0.001 represent human, ^∗p ≤ 0.05, ^∗∗p ≤ 0.001 represent rat).

FIGURE 2

Detection of S1PR1 (A) and S1PR3 (B) proteins in human and heart by Western blotting. Analysis of S1PR1 and S1PR3 was performed using 50 μg of total proteins of human and rat heart from all four chambers. For S1PR1 detection, the membrane was developed for only 1 min using an ECL(+) kit (Amersham-Pharmacia Biotech), whereas 45 min was necessary for S1PR3. S1PR1 was significantly high as compared to S1PR3 (p ≤ 0.05, S1PR1 versus S1PR3 in both species and all chambers). S1PR1 and S1PR3 were uniformly distributed in all part of heart in both species.

FIGURE 3

Representative images of S1PR1 and S1PR3 expression in human and rat heart in right atrium, left atrium, right ventricle and left ventricle (A, M = human right atrium, E, Q = rat right atrium), (B, N = human right ventricle, F, R = rat right ventricle), (C, O = human left atrium, G, S = rat left atrium), (D, P = human left ventricle, H, T = rat left ventricle), (I–L are control samples treated same except primary antibody incubation. S1PR1 expressed in all chambers of both species similar, while S1PR3 expressed mildly less in rat samples but also uniform distribution in all chambers). Magnification 20x.