The aging myocardium expresses many gene transcripts that are normally expressed during embryonic and fetal development. Using transcriptome-based and promoter-based techniques, we have worked towards understanding the mechanisms underlying this control of gene expression both in models of development and in aging. Serial analysis of gene expression (SAGE) yields a quantitative, representative and comprehensive differential gene expression profile. We have employed SAGE analysis to generate a quantitative transcript assessment that has proven to be much more rapid and economical than other techniques. We used an RT-PCR based technique to determine the time points where a number of mesodermal and cardiac-restricted gene products are expressed in differentiated EC derived cardiomyocytes, and more recently feeder layers general employed to maintain embryonic stem cells. These latter studies were designed to identify factors that could possibly reprogram cells to become more multipotent.? We have utilized quantitative PCR and in situ hybridizations to analyze the temporal and spatial distribution of a number of the most differentially regulated transcripts identified by SAGE, several of which have shown a cardiac predominance in either fetal or adult heart. The novel gene products identified in this study provide a framework for the analysis of pre- and early cardiac developmental processes in human and mouse embryonic stem cells and are the subject of active investigation. Included among these gene transcripts that have been subjected to follow-up analysis are Cripto and pleiotrophin.? Since the original identification, we have determined that pleiotrophin (PTN), a development-regulated cytokine growth factor that promotes angiogenesis, cell proliferation and differentiation, is prominently expressed in developing myocardium but it is poorly expressed in adult heart. Conversely, in a rat model of myocardial infarction and in human dilated cardiomyopathy, pleiotrophin is markedly up-regulated, suggesting that it may have a functional role in injury repair. To elucidate the effects of PTN on contractile cells, we employed primary cultures of rat neonatal (NN) and adult (A) cardiomyocytes (CM). We found that PTN promotes caspase-mediated genomic DNA fragmentation in a dose- and time-dependent manner. More importantly, it significantly potentiates the apoptotic response of NNCMs to hypoxic stress and to ultraviolet (UV) irradiation, and of ACMs to hypoxia-reoxygenation. We furthermore found that PTN potentiated UV-induced apoptosis is abolished in NNCMs following siRNA-mediated knockdown of endogenous PTN proteins. Mechanistically, PTN antagonizes IGF-1 associated Ser-473 phosphorylation of AKT/PKB, and it concomitantly decreases both BAD and GSK3beta; phosphorylation. Adenoviral expression of constitutively active AKT and lithium chloride mediated inhibition of GSK3beta; also reduced the programmed cell death potentiated by PTN. These latter data indicate that PTN promotes CM cell death, at least partially, through inhibition of AKT signaling. In conclusion, we have uncovered a novel role for PTN in CMs. This cytokine, which in addition to its pro-angiogenic effects in heart, potentiates CM programmed cell death in response to pro-apoptotic stress.
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