Cell-specific expression of SERCA, the exogenous Ca2+ transport ATPase, in cardiac myocytes.
We evaluated various constructs to obtain cell-specific expression of the sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA) gene in cardiac myocytes after cDNA transfer by means of transfections or infections with adenovirus vectors. Expression of exogenous enhanced green fluorescent protein (EGFP) and SERCA genes was studied in cultured chicken embryo and neonatal rat cardiac myocytes, skeletal and smooth muscle cells, fibroblasts, and hepatocytes. Whereas the cytomegalovirus (CMV) promoter yielded high levels of protein expression in all cells studied, cardiac troponin T (cTnT) promoter segments demonstrated high specificity for cardiac myocytes. Their efficiency for protein expression was lower than that of the CMV promoter, but higher than that of cardiac myosin light chain or beta-myosin heavy chain promoter segments. A double virus system for Cre-dependent expression under control of the CMV promoter and Cre expression under control of a cardiac-specific promoter yielded high protein levels in cardiac myocytes, but only partial cell specificity due to significant Cre expression in hepatocytes. Specific intracellular targeting of gene products was demonstrated in situ by specific immunostaining of exogenous SERCA1 and endogenous SERCA2 and comparative fluorescence microscopy. The -374 cTnT promoter segment was the most advantageous of the promoters studied, producing cell-specific SERCA expression and a definite increase over endogenous Ca2+ -ATPase activity as well as faster removal of cytosolic calcium after membrane excitation. We conclude that analysis of promoter efficiency and cell specificity is of definite advantage when cell-specific expression of exogenous SERCA is wanted in cardiac myocytes after cDNA delivery to mixed cell populations.
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201-1503, USA.
Hailun
H
Ma
Carlota M
CM
Sumbilla
Iain K G
IK
Farrance
Michael G
MG
Klein
Giuseppe
G
Inesi
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D018076:DNA, Complementary; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008967:Molecular Biology / Q000379:methods; D032383:Myocytes, Cardiac / Q000166:cytology / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D051381:Rats; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases
C506945:Atp2a1 protein, rat; C506948:Atp2a2 protein, rat; D018076:DNA, Complementary; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium