Developmental Changes And The Role Of Lysophosphatidic Acid Signaling In Postnatal Rodent Heart

Congenital heart disease (CHD) is the most common class of human congenital malformation. Cardiac surgery is the main treatment for CHD, which face many problems to be solved in China. Timing of surgical treatment of many kinds of complex CHD is usually the short period after birth, when the function of heart is not mature. Comprehending the regulatory mechanism of postnatal heart development is undoubtedly the primary task of surgical intervention in CHD. Although the heart development in embryonic stage has been widely studied, but little is known about the process of immature myocardium turning into mature myocardium and the key mechanism regulating this shift.Lysophospholipids (LP) are cell membrane-derived small signaling lipids that regulate a broad range of physiological and pathological processes through membrane-bound G protein-coupled receptors (GPCRs). The glycerophospholipid lysophosphatidic acid (LPA) and the sphingolipid sphingosine1-phosphate (SIP) are two representative lysophospholipids (LPs) that act as extracellular signals in vertebrates. Expression of both LPA receptor and SIP receptor was observed in mouse heart from E8.5-E12.5, suggesting that lysophospholipid signaling is critical for proper development of the cardiovascular system. But until now, little is known about the role of LP signaling during cardiac development. We previously reported that LPA3and/or LPA1mediated LPA-induced hypertrophy of neonatal cardiac myocytes. To further explore the functions of LP signaling in heart development and functional maturation, we measured the expression patterns of10confirmed LP receptors during perinatal and postnatal cardiac development in rats and explored the possible role of LPA signaling in postnatal myocardial growth and functional maturation.The study includes three aspects as follows:Part1Developmental changes in lysophospholipid receptor expression in rodent heart——from near-term fetus to adultTo investigate the function of LP signaling in heart organogenesis and maturation, we measured the expression of10confirmed LP receptors (Lparl-5and S1prl-5) in rat heart from embryonic day19.5(E19.5d) to postnatal week12(P12w). The expression of Lpar3mRNA peaked at37-fold higher than adult expression at P1d, while the expression levels of Lpar1and Lpar4increased markedly after Pld and peaked at19-fold and48-fold of adult expression on P7d. The expression levels of all three receptor mRNAs were significantly reduced by P21d and remained low thereafter. Expression of the corresponding receptor proteins also peaked during the early postnatal period but the subsequent decline was less dramatic from P14d to P12w compared to mRNA expression. In contrast, S1prl and Slpr3exhibited more gradual developmental changes. Although early expression was higher than mature expression (3-to6-fold), these receptors were still strongly expressed at P12w. The other isotypes examined, Lpar2, Lpar5, S1pr4, and S1pr5, were very weakly expressed at all developmental stages. Analysis of receptor distribution within the developing heart (P1d) revealed that Lparl, Lpar3, and Lpar4were expressed in the myocardium of all four chambers but not in valves, while Lpar3was also uniquely expressed in the aorta and coronary vessels. Western blots revealed that the developmental changes in Lparl, Lpar3, and Lpar4protein expression mirrored changes in β-actin and β-tubulin expression. The increase in Lpar1and Lpar4receptors from Pld to P7d corresponds to the period of rapid myocardial growth and functional maturation. Moreover, the relatively high expression of Lparl, Lpar3, and Lpar4in the late prenatal rat heart suggests that these LPA receptors may also contribute to organogenesis.Part2Lysophosphatidic acid stimulates proliferation of immature cardiomyocytesAccording to the results of part1, we investigated the role of LPA signaling in proliferation of immature cardiomyocytes during early neonatal life. For the first time, we show that the proliferative potential of neonatal rat cardiomyocytes is powerfully stimulated by the sustained activation of the LPA signaling. Not only Pld cardiomyocytes but also P4d, which was suggested having lost their proliferative potential, could proliferate stimulated by LPA in dose-dependent and time-dependent pattern. LPA-induced proliferaiton was completely abrogated by Ki16425, an Lparl/Lpar3antagonist, while the Lpar3agonist OMPT promoted proliferation in immature cardiomyocyte. In addition, the relative higher expression of Lpar3in cardiomyocytes suggests that Lpar3may play more important role in cardiomyocytes than cardiac fibroblasts. So we speculate that Lpar3mediated LPA-induced proliferation of immature cardiomyocytes. Furthermore, flow cytometric analysis showed that the cell number of S phase was promoted by LPA, which suggest that LPA signaling may affect cell cycle to stimulate the proliferation of immature cardiomyocyte.Part3Effect of Lysophosphatidic acid in contraction and excitation-contraction coupling of postnatal cardiomyocyteThe purpose of this part was to prove that LPA involved in contraction and excitation-contraction coupling by inducing calcium mobilization in immature cardiomyocytes. Unfortunately, we found that LPA had no effect on the contraction and calcium transient in isolated cardiomyocytes at postnatal14days of age, which was expected do. But consistent with the hypothesis, LPA did not change the contraction and calcium transient in isolated cardiomyocytes at postnatal21days of age when the function of contraction matured. Western blot analysis showed that the expressions of Lparl, Lpar3and Lpar4were down-regulated at P14d, which was not paralleled the developmental changes of these three receptors in whole heart. It suggests that LPA signaling have lost the effect of developmental modulation in cardiomyocytes at P14d. Moreover, in vivo cardiac echocardiography were performed in normal SD rats at postnatal day1,7,14,21,28, and postnatal week8and12. The results showed that ejection fraction increased within the first7days, then decreased until day14, and became stable afterward, which indicated that contraction function of myocardium may mature at P14d. Based on these results, we thought part of the original hypothesis, LPA signaling was not involved in contraction and excitation-contraction coupling in mature cardiomyocyte, may was proved.