| Mammalian cardiomyocytes undergo cell division only during embryonic development. This process can be disturbed by many gene mutations, resulting in myocardial hypoplasia Retinoic acid (RA), the biologically active derivative of vitamin A, is an important morphogen during development. Deficiency in vitamin A by dietary deprivation and mutation in Rxra, a nuclear receptor for retinoic acid, will lead to embryonic myocardial hypoplasia. It has been demonstrated that Rxra acts in the epicardium, a thin layer of cells enveloping the myocardium, to influence myocardium proliferation. Previous studies suggested that RXRA induces the secretion of mitogenic factors from the epicardium. In this study, we found that AGN193109, an RA antagonist, and retinoic acid have no effect on the production of mitogenic activity from a rat epicardial cell line, EMC, a mouse epicardial cell line, MEC1, or primary epicardial cells from chick embryos. Rxra knockdown via adenovirus-mediated RNAi does not reduce the mitogen production either. Our data thus do not support the previously proposed model. Nevertheless, the identities of mitogens are of great interest PDGFA was first identified as a candidate mitogen by a systematic receptor tyrosine kinase phosphorylation screening. Pdgfa is expressed in vivo during stages of myocardium proliferation, as determined by in situ hybridization studies. The concentration of PDGFA in the serum-free conditioned medium was estimated to be about 5-10ng/ml. However, immunodepletion of PDGFA from conditioned media only leads to partial reduction in mitogenic activity. Conditional knockout of the Pdgfra gene in the myocardium does not lead to any visible anatomic defects. All these lead to the search for additional factors. An expression microarray study of MEC1 cells suggested Igf2 as another candidate trophic factor. In situ hybridization showed that Igf2 is expressed in E10.5 to E12.5 mouse epicardium. Western blotting showed that IGF2 is present in MEC1-conditioned media at a substantial concentration around 100ng/ml. Neither knocking down endogenous Igf2 by adenovirus-mediated Igf2-RNAi nor an IGF1R inhibitor, AG1024, completely blocked the mitogenic activity, suggesting the presence of additional factors. We propose PDGFA and IGF2 are two mitogens among multiple mitogenic factors that are secreted by epicardium and stimulate myocardium proliferation. |
