Retinoic Acid Signalling Is Activated in the Postischemic Heart and May Influence Remodelling

<div><h3>Background</h3><p>All-trans retinoic acid (atRA), an active derivative of vitamin A, regulates cell differentiation, proliferation and cardiac morphogenesis via transcriptional activation of retinoic acid receptors (RARs) acting on retinoic acid response elements (RARE).We hypothesized that the retinoic acid (RA) signalling pathway is activated in myocardial ischemia and postischemic remodelling.</p> <h3>Methods and Findings</h3><p>Myocardial infarction was induced through ligating the left coronary artery in mice. <em>In vivo</em> cardiac activation of the RARs was measured by imaging RARE-luciferase reporter mice, and analysing expression of RAR target genes and proteins by real time RT-PCR and western blot. Endogenous retinoids in postinfarcted hearts were analysed by triple-stage liquid chromatography/tandem mass spectrometry. Cardiomyocytes (CM) and cardiofibroblasts (CF) were isolated from infarcted and sham operated RARE luciferase reporter hearts and monitored for RAR activity and expression of target genes. The effect of atRA on CF proliferation was evaluated by EdU incorporation. Myocardial infarction increased thoracic RAR activity <em>in vivo</em> (p<0.001), which was ascribed to the heart through <em>ex vivo</em> imaging (p = 0.002) with the largest signal 1 week postinfarct. This was accompanied by increased cardiac gene and protein expression of the RAR target genes retinol binding protein 1 (p = 0.01 for RNA, p = 0,006 for protein) and aldehyde dehydrogenase 1A2 (p = 0.04 for RNA, p = 0,014 for protein), while gene expression of cytochrome P450 26B1 was downregulated (p = 0.007). Concomitantly, retinol accumulated in the infarcted zone (p = 0.02). CM and CF isolated from infarcted hearts had higher luminescence than those from sham operated hearts (p = 0.02 and p = 0.008). AtRA inhibited CF proliferation <em>in vitro</em> (p = 0.02).</p> <h3>Conclusion</h3><p>The RA signalling pathway is activated in postischemic hearts and may play a role in regulation of damage and repair during remodelling.</p> </div

Expression of retinoic acid target genes in the infarct zone and effects on cardiofibroblast proliferation.

<p><b>A)</b> Gene expression of retinoic acid target genes in cardiofibroblast (CF) and cardiomyocytes (CM) isolated from left ventricles of infarcted RAR-luciferase reporter hearts 1 week after infarction. RNA was extracted from cells from the infarcted zone and amplified with real time PCR using primers specific for retinol binding protein 1 (RBP1), cytochrome P450 26B1 (CYP26B1), aldehyde dehydrogenase 1A2 (ALDH1A2), and retinoic acid receptors alpha, beta and gamma (RAR α,β,γ). The figure shows test gene expression relative to expression of rpl32, which was similar in both cell types. Data are mean±SD of n = 5 experiments in each group. <b>B)</b> Cardiofibroblasts were isolated from C57BL6 hearts and cultured in medium supplemented with 1 µM all-trans retinoic acid (atRA) and 10 µM EdU, as indicator of cell division. After 96 hours, EdU incorporation was evaluated by flow cytometry. Data are presented as mean±SD of n = 4 in each group.</p

Expression of retinoic acid target genes and endogenous metabolites after infarction.

<p>Wild type C57Bl6 mice were subjected to <i>in vivo</i> ligation of the left coronary artery or sham surgery, and hearts were sampled serially for RNA extraction and amplification of retinoic acid target genes with real time PCR, or proteins were extracted for western blotting. Hearts were also sampled from mice without any surgery (preoperative). Gene expression of retinol binding protein 1 (RBP1) (<b>A</b>), aldehyde dehydrogenase 1A2 (ALDH1A2) (<b>B</b>), and cytochrome p45026B1 (CYP26B1) are shown (<b>C</b>). (n = 6–8 in each group at each time point). <b>D)</b> Representative western blot analysis of retinoic acid transporting and metabolizing proteins one week after induction of infarction or sham operation. Ponceau solution was used as protein loading (control). Histograms show the relative density of RBP1 at 16 kDa and ALDH1A2 at 53–57 kDa in infarcted and sham operated hearts. <b>E)</b> Endogenous retinoic acid metabolite concentrations were evaluated by triple-stage liquid chromatography/tandem mass spectrometry one week after induction of myocardial infarction. The infarcted zone of the left ventricle (INF), the periinfarcted zone (PERIINF) and left ventricles from sham operated hearts (SHAM) were investigated (n = 6). Data are shown as mean±SD.</p

<i>In vitro</i> imaging of retinoic acid signaling after myocardial infarction.

<p>Cardiomyocytes and cardiofibroblasts were isolated from left ventricular tissue from RARE-luciferase reporter hearts one week after myocardial infarction or sham operation (SHAM). Cells were isolated from the infarct (INF) and periinfarct zone (PERIINF). After plating for three hours, non-viable cells were removed and luciferin was added for imaging. The upper panel shows a representative image of one experiment (<b>A</b>). The lower panel shows mean±SD of n = 5 experiments in each group (<b>B</b>). Note that the Y-axis labelling is different.</p

<i>Ex vivo</i> imaging of cardiac retinoic acid signalling after myocardial infarction.

<p>To verify that the signal in the thoracic region detected <i>in vivo</i> originated from hearts, organs were harvested one week after infarction or sham operation following in vivo luciferin injection in RARE-luciferase reporter mice. Organs were placed in a petridish and the luminescence signal quantified. A representative image of one single experiment is shown on the left (<b>A</b>), where hearts, thymus, gastrointestinal tract (GI-tract), liver, lungs, pancreas, spleen and epididymal white adipose tissue from one infarcted and one sham operated animal are shown. When the signal was quantified from 6 independent experiments, the luminescence was exclusively increased in infarcted hearts, and not in sham operated hearts or any other organs (<b>B</b>). Data are shown as mean±SD.</p