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  • br Introduction Prostaglandin E PGE signals through


    Introduction Prostaglandin E2 (PGE2) signals through 4 separate G-protein coupled receptor sub-types (EP1, EP2, EP3 and EP4) to elicit a variety of physiological and pathophysiologic effects. EP2 and EP4 increase cAMP levels in the cell via adenylate cyclase activation, whereas EP3 inhibits cAMP production, and EP1 increases Ca2+ levels in the cell, although the cardiac effects of the receptors have not been studied in depth. Our laboratory has studied the role of PGE2 via its EP4 receptor over the last decade and reported that the EP4 receptor causes hypertrophy of cardiac myocytes in vitro; that gssg of the EP4 receptor only in the cardiac myocyte worsens cardiac function after myocardial infarction (MI) and that male mice with cardiomyocyte deletion of the EP4 receptor develop a dilated cardiomyopathy with age that is characterized by reduced ejection fraction, left ventricle dilation, thinning of the posterior wall, and an increased interstitial cellular infiltrate [[1], [2], [3]]. Moreover, we recently reported that PGE2 via its EP3 receptor could reduce contractility of preparations ranging from isolated myocytes to the whole heart by mechanisms that appeared to involve decreased phosphorylation of phospholamban (PLN) [4]. Presumably, this would lead to an inhibitory effect on SERCA, thereby decreasing contraction. Additionally, we reported that the EP3 receptor is upregulated after MI and although the EP4 receptor is also upregulated, it does not appear to be to a similar extent. We thus hypothesized that an imbalance in the EP3/EP4 ratio favoring expression of EP3 would explain some of the reduced contractility observed after MI and that over-expression of EP4 in the same model would improve cardiac function. This was tested in the present study using a mouse model of MI with the use of AAV9-EP4 driven by the myosin heavy chain promoter to overexpress EP4 in the left ventricle.
    Discussion The results of this study show, for the first time, that administration of AAV9-EP4 into the left ventricle of mice with MI is beneficial in improving cardiac function. We have previously reported that male mice lacking the EP4 receptor in cardiomyocytes (EP4-KO) have worsened cardiac function after MI and develop dilated cardiomyopathy with age [2,3]. More recently we also reported that PGE2 via its EP3 receptor reduced contractility whereas stimulation of EP4 had the opposite effect. These effects appeared to involve changes in the phosphorylation of phospholamban. Moreover, we also reported that after MI, expression of the EP3 receptor subtype in the left ventricle was increased to a greater extent than EP4 [4]. We therefore hypothesized that overexpression of EP4 in the heart would improve cardiac function after MI. Thus, in our experiments described here, we used an adeno-associated viral vector driven by the alpha myosin heavy chain promoter to increase the expression of the EP4 receptor in the cardiomyocytes rather than increase the activation of receptors already present. The results support our hypothesis, with significant improvements in ejection fraction and shortening fraction noted, coupled with reductions in left ventricular dimension after MI without effecting infarct size. Overexpression of EP4 also significantly reduced hypertrophy and fibrosis after MI as evidenced by a smaller MCSA and reduced picrosirius red staining in the peri-infarct and remote zone, and the former was confirmed by reductions in β-MHC and BNP mRNA expression. Previously, other studies have shown a protective role for the EP4 receptor in ischemia-reperfusion injury. Xiao et al. [15] reported that global EP4 KO mice had larger infarcts after reperfusion injury while administration of an EP4 agonist had the opposite effect. A study by Hishikari et al. [16], using a rat model described similar findings but also indicated that treatment with an EP4 agonist could reduce cytokine secretion stimulated by the ischemia/reperfusion. Our data using the permanent coronary ligation model with EP4 overexpression agrees with the aforementioned findings except that we did not observe a change in infarct size at the end of the study. Whether this relates to the different models or different time points remains unknown. Since we also recently reported that PGE2 via its EP3 receptor reduced contractility of the heart via mechanism(s) that appeared to involve decreased phospholamban phosphorylation; we examined phosphorylation of phospholamban in these studies. Sham-operated mice who received AAV9-EP4 exhibited a significant increase in phosphorylated phospholamban, consistent with our in vitro data using the EP4 agonist in isolated cardiomyocytes [4]. A similar trend was observed in animals with MI, although the data did not achieve statistical significance. We therefore speculate that the increased EP3 receptor abundance after MI could account, in part, for the decreased contractility observed. The role of the EP3 receptor in cardiac function is controversial. Martin et al. [17] reported that cardiac overexpression of the EP3 receptor attenuated ischemia-induced myocardial injury. However, that paper used an isolated heart perfusion method and curiously, EP3 transgenic animals exhibited a reduced ejection fraction as measured in vivo using MRI. In subsequent studies, we think it will be necessary to identify the impact of EP3 receptor inhibition with simultaneous EP4 overexpression on cardiac function.