Characterization of promoter elements of the rabbit cardiac sarcoplasmic reticulum Ca(2+)-ATPase gene required for expression in cardiac muscle cells.
The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) plays a critical role in the contractile performance of cardiac and slow-twitch skeletal muscle by restoring cytosolic calcium to low resting levels during the contractile cycle. We have previously shown that SERCA2 expression in the heart is altered by a number of pathophysiological stimuli. In an effort to define molecular mechanisms regulating expression of the SERCA2 gene in cardiac muscle cells, deletions of a 1460-bp promoter fragment were generated and inserted into a luciferase reporter plasmid. Promoter constructs were transiently transfected into embryonic cardiocytes and skeletal muscle cell lines Sol 8 and C2C12 in vitro and injected into adult myocardium in vivo. Results demonstrate that sequences from the transcription start site to -284 are both necessary and sufficient for high-level transcription of the reporter gene in differentiating muscle cells and in fetal cardiocytes in culture. We further demonstrate that this promoter fragment is highly active in vivo when injected into rat hearts, suggesting that the same regulatory elements are functional in vivo as well as in vitro. The region of the gene from -284 to -658 exerts a modest positive effect in cardiocytes and Sol 8 myotubes but exerts a negative effect in C2C12 fast skeletal muscle cells. This initial analysis of transcriptional regulation of the SERCA2 gene will serve as a foundation for the study of alterations of expression of the gene in pathological conditions.
Fisher
S A
SA
Department of Medicine, University of Vermont College of Medicine, Burlington.
Buttrick
P M
PM
Sukovich
D
D
Periasamy
M
M
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000235:genetics*; D002478:Cells, Cultured; D002642:Chick Embryo; D015870:Gene Expression; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*; D014162:Transfection
D012333:RNA, Messenger; D000252:Calcium-Transporting ATPases