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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Cell-specific expression of SERCA, the exogenous Ca²⁺ transport ATPase, in cardiac myocytes

Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201-1503

Submitted 3 July 2003 ; accepted in final form 23 October 2003

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 -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.

cardiac myocytes; cell-specific expression; adenovirus vectors; calcium transport

This article has been cited by other articles:

				A. M. Prasad, H. Ma, C. Sumbilla, D. I. Lee, M. G. Klein, and G. Inesi Phenylephrine hypertrophy, Ca2+-ATPase (SERCA2), and Ca2+ signaling in neonatal rat cardiac myocytes Am J Physiol Cell Physiol, June 1, 2007; 292(6): C2269 - C2275. [Abstract] [Full Text] [PDF]