In addition, we observed a similar extent of protein synthesis induction after PGE2 stimulation as with the well\known hypertrophic \adrenoceptor agonist phenylephrine (Fig?EV1B)

In addition, we observed a similar extent of protein synthesis induction after PGE2 stimulation as with the well\known hypertrophic \adrenoceptor agonist phenylephrine (Fig?EV1B). activate MEF2, but a comprehensive analysis of GPCR activators that regulate MEF2 has to our knowledge not been performed. Here, we tested several GPCR agonists regarding their ability to activate a MEF2 reporter in neonatal rat ventricular myocytes. The inflammatory mediator prostaglandin E2 (PGE 2) strongly activated MEF2. Using pharmacological and protein\based inhibitors, we exhibited that PGE 2 regulates MEF2 VP3.15 via the EP 3 receptor, the subunit of Gi/o protein and two concomitantly activated downstream pathways. The first consists of Tiam1, Rac1, and its effector p21\activated kinase 2, the second of protein kinase D. Both pathways converge on and inactivate histone deacetylase 5 (HDAC5) and thereby de\repress MEF2. in inflamed hearts. Results PGE2 activates MEF2 through the EP3 receptor To identify unknown GPCR\dependent signaling pathways that regulate MEF2 activity, we conducted a screening experiment using neonatal rat ventricular myocytes (NRVMs). NRVMs were infected with an adenovirus harboring a MEF2\reporter (3xMEF2\Luc), which responds to endogenous MEF2. Thereafter, the cells were stimulated with different GPCR agonists for 24?h in serum\free medium. Much like previous reports, endothelin\1 (100?nM) activated MEF2 to a high extent (Backs (Herman Myomaxin(Fig?1B). MEF2 activation is commonly associated with the induction of hypertrophic gene programs. Likewise, TNFA PGE2 increased the mRNA levels of the hypertrophy marker (Fig?1B) and induced cellular hypertrophy of NRVMs, which correlated with the induction of MEF2 activity (Fig?1C). In addition, we observed a similar extent of protein synthesis induction after PGE2 activation as with the well\known hypertrophic \adrenoceptor agonist phenylephrine (Fig?EV1B). Next, we aimed to determine the receptor involved in PGE2\mediated MEF2 activation. The four different isoforms of PGE2 receptors (EP1, EP2, EP3, EP4) show a different degree of G protein coupling and expression patterns (Woodward P?P?P?P?and and and and and test, vehicle vs. ET1 or vs. PGE2, and relevance of these results, we used the LPS\induced endotoxemia model to induce myocardial inflammation. The inflammatory effect in this model was confirmed by increased mRNA levels of pro\inflammatory cytokines including interleukin 6 (recognized pathways upstream to MEF2 in inflammation. Phosphorylation of PKD at Ser\744/Ser\748 and of HDAC5 was significantly elevated, and we observed a slight but statistically significant increase of PAK2 phosphorylation at Thr\402 (Fig?7D). Open in a separate window Physique 7 LPS\induced endotoxemia prospects to myocardial inflammation, increased myocardial PGE 2 levels, and MEF2 activationMEF2\lacZ reporter mice (BALB/c background, 6C12?weeks old) were treated with 7?mg/kg lipopolysaccharide from (O111:B4) or saline intraperitoneally and were sacrificed after 24?h. mRNA levels of different inflammatory cytokines, as indicated. The graphs show relative mRNA levels, fold increase compared to saline\treated controls, normalized to 18s content. PGE2 was quantified after mechanical homogenization of deeply frozen hearts using nano\liquid chromatography\tandem mass spectrometry. Histologic and macroscopic stainings of \galactosidase activity in MEF2\lacZ reporter mice show MEF2 activation (blue cells and VP3.15 precipitates, respectively) in the myocardium upon LPS treatment. Saline\treated littermates served as control. Level bar of histological stainings is usually 100?m. Quantification of whole\heart stainings is shown in the right panel (pixel intensity in blue channel normalized to total intensity). Induction of PKD, HDAC5, and PAK2 in myocardial inflammations. Immunoblots were performed on extracts of hearts of saline\ and LPS\treated mice. Representative blots are shown in the left panel, and the quantification is in the right panel. Data information: Values are imply??s.e.m. The exact values are shown in the bottom of the bar graphs. *and data, we provide evidence that cardiac inflammation in a sepsis model induces activation of MEF2. Thus, the unmasked upstream signaling pathway consisting of PGE2, EP3, Rac1, and PKD may provide a number of new drug target candidates for the VP3.15 treatment of heart failure in the specific establishing of inflammatory causes. Cyclooxygenase inhibitors, such as acetylsalicylic acid, are generally used as anti\inflammatory, anti\nociceptive, and anti\thrombotic brokers (Woodward investigations using, e.g., EP3\, PKD\, or MEF2D\deficient mice in the context of inflammatory cardiomyopathies are warranted to determine a critical role of the above mentioned signaling pathways and may facilitate translational studies toward personalized strategies to combat inflammatory cardiomyopathies..