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 in vitro differentiated EC derived cardiomyocytes. Results show that the earliest contractions occur on Day 5.5 of differentiation (3+2.5 or 4+1.5). Some contractile protein genes (beta MHC) are expressed before the third day of differentiation, but we show that a clear induction of expression of alpha MHC and MLC2V starts relatively later (5+0.5 and 3+1 for alpha MHC and MLC2V respectively). GATA-4, a transcription factor involved in the regulation of cardiac contractile protein gene expression is highly present in the earliest stages of differentiation, and from 3+1.5 days of differentiation, its expression is reduced to the level of adult heart tissue. This indicates that this time period corresponds to an early stage of cardiac differentiation. We have used the protocols of SAGE (and microarrays) to analyze expressed sequence profiles during these time periods. A total of 150,354 tags have been sequenced from three P19 EC cell-based libraries (undifferentiated P19 cells, differentiation Days 3+0.5 and 3+3.0). Ten percent of the 43,432 unique tags matched sequences in the NR GenBank database, while more than 30% of the unique tags did not match any known mouse sequence. Temporally-restricted induction or repression of transcripts at each differentiation stage examined was a common feature of the developmental profiles, suggesting that differentiation-specific processes are necessary for pre- and early cardiac development. Analysis of the normalized tag frequency with differentiation revealed significant changes (p<0.01) in expression of 357 gene products, some of which we now know to also be altered in aging mouse myocardium (microarray-based analyses). We have also 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. Two promoter regions that control the expression of genes important to cardiac function (Na/Ca exchanger and the sarco(endo)plasmic reticulum Ca ATPase, SERC) have also been analysed. We have shown that an upstream portion of the multipartitite Na/Ca exchanger promoter is regulated at least in part by GATA-4 and GATA-6 transcription factors, which may control, at least in part, the expression of the Na/Ca exchanger both during development and with aging. Recently we also demonstrated the critical role of SP1 factors in the regulation of the human SERCA2 gene promoter in neonatal rat cardiomyocytes. The control of this promoter depends on the binding of these factors to specific cis-elements located in the promimal promoter.

National Institute of Health (NIH)
National Institute on Aging (NIA)
Intramural Research (Z01)
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