Troponin I (TnI) is a thin filament contractile protein involved in the regulation of contractility by calcium. In the embryonic myocardium, the slow skeletal TnI gene is expressed, followed by later expression of the cardiac TnI gene. There is a several fold increase in the mRNA for cardiac TnI between fetal and post-natal heart, while the slow skeletal TnI gene is down-regulated in this time period. In order to define mechanisms for this developmental gene switch, the transcriptional regulation of the rat cardiac TnI gene has been studied by in vitro transfection assays. To discern positive or negative regulatory elements which might have differential effects in maturation, transient transfections of stage-specific cardiocytes were undertaken. TnI-luciferase constructs were transfected into fetal (day 19), day 1-2 post-natal and day 4 post-natal rat cardiocytes. Results were normalized by co-transfection of a CMV-β-galactosidase plasmid. Results are expressed as mean and standard error in the table below for the construct indicated. Because the results for 4 day post-natal did not differ from 1-2 day post-natal, only the 1-2 day post-natal are reported. The data indicated that an element within region -524 to -151 acted as a repressor in the fetal but not post-natal cells. To confirm this finding, additional deletion constructs were prepared in which bp -444 to -151 were removed from constructs containing upstream region -896TnI and -2000TnI. Each of these 2 constructs had 2 fold increased activity over the parent constructs when transfected into fetal cells (n=4). We conclude that a developmentally relevant negative regulatory element resides in the region from -444 to -151. Three E-box sites at -435,-380 and -283 are contained within this region. The precise element(s) which mediate this effect is being mapped with a series of additional deletion constructs.

Table 1 Table: Relative luciferase activity in fetal vs. post-natal cardiocytes for TnI luciferase constructs.
Full size table