The Role Of Prostaglandin E₂ Receptor 3 Subtype?EP3? In Eccentric Cardiac Hypertrophy Of Mice

Objective Prostaglandin E2 receptor subtype 3(EP3), a Giprotein-coupled receptor activated by prostaglandin E2, plays a particular role in cardioprotection. To suggest a new prevention strategy against cardiac remodeling, the present experiments used a mouse with targeted disruption of EP3 gene to determine whether a cardiac phenotype developed and preliminary investigate the possible mechanism.Methods Cardiac morphology and function were mainly analyzed by Vevo2100 Imaging System in 16- to 18-week-old male EP3 receptor knockout(EP3KO) mice and littermate wild-type(WT) mice as the control group. Ventricular myocytes were isolated and measured for length and width. Masson's trichrome staining was taken to assess the collagen deposition and fibrosis. Real-time PCR was used to analyze some genes including members of the cardiac fetal gene program, extracellular matrix compounds, matrix metalloproteinases(MMPs), tissue inhibitors of metalloproteinases and enzymes for synthesis of prostaglandin E2(PGE2), as well as the EP receptors. Western blot was used to detect the expression and activity of proteins, including MMP-2 and key components of some signaling pathways, such as MAPK/ERK, PI3K-AKT/PKB-GSK3? and Ca2+-calcineurin(CaN) pathway.Results1. The expression of enzymes for synthesis of PGE2 and the EPreceptors:The mRNA level of EP3 and its subtype(EP3?, ? and ?) were significantly lower in EP3 knockout mice than in WT controls, but no differences were found in other EP receptors between two animal groups.2. The changes of cardiac structure and ventricular function:Accompany with the significantly increasing in left ventricular(LV) end-diastolic and end-systolic volume, the relative LV mass and the ratio of heart weight and body weight were significantly increased. However, there was no difference in the thickness of ventricular wall. These suggested eccentric cardiac hypertrophy might occur in the EP3 KO mice. The severely decreasing of LV ejection fraction and fractional shortening, along with the reduced mitral valve E/A radio, indicated that the EP3 KO mice was suffering from worse cardiac function. Besides, the up-regulated markers of cardiac disease, such as natriuretic peptide type B and ?-skeletal actin, could be detected in the hearts of EP3 KO mice.3. Morphology of isolated ventricular myocyte:The myocytes from hearts of EP3 KO mice showed a greater increase in cell length and the radio of length to width, while no change in cell width.4. Myocardial extracellular matrix remodeling in EP3 KO mice:Masson's trichrome staining demonstrated an increased level of interstitial fibrosis in EP3 KO mice as compared with WT litter mates. Elevated mRNA levels of the matrix genes collagen type I and III as well as the collagen binding proteoglycan biglycan could be detected by real-time PCR, which prompted the increase of collagen synthesis in EP3 KO mice. Meanwhile, the mRNA and protein expression levels of MMP-2 were both markedly reduced in EP3-deficient heart, which means the suppressed degradation of matrix collagen.5. Signaling pathway analysisWestern blot showed a declined phosphorylation level of ERK1/2 in the heart of EP3 KO mice, while there was no significant change of total ERK1/2, GSK3?, phospho-GSK3?, CaN protein content, or CaN activity.Conclusions Inactivation of the EP3 in vivo is sufficient to induce eccentric cardiac hypertrophy and fibrosis, as well as the impaired heart function. These changes happened basis for lack of EP3 may cause eccentric cardiomyocyte growth, and disorder the balance of synthesis and decomposition of collagen. MAPK/ERK pathway may be involved in this process, because EP3 ablation results in a obvious decline in phosphorylation of ERK1/2. Overall, PGE2-EP3 is necessary to maintain the normal growth and development of the heart.