Comparison of SERCA1 and SERCA2a expressed in COS-1 cells and cardiac myocytes.
Cultured COS-1 cells, as well as chicken embryonic and neonatal rat cardiac myocytes, were infected with recombinant adenovirus vectors to define limiting factors in the expression and Ca2+ transport function of recombinant sarcoplasmic-endoplasmic reticulum Ca(2+) (SERCA) isoforms. Titration experiments showed that all COS-1 cells and myocytes in culture could be infected by an adenovirus titer of 10 plaque-forming units (pfu) per seeded cell. Raising the adenovirus titer further yielded higher protein expression up to an asymptotic limit for functional, membrane-bound SERCA protein. The asymptotic behavior of SERCA expression was not transcription related but was due to posttranscriptional events. The minimal (-268) cardiac troponin T (cTnT) promoter was a convenient size for adenovirus vector construction and manifested tight muscle specificity. However, its efficiency was lower than that of the nonspecific cytomegalovirus (CMV) promoter. At any rate, identical maximal levels of SERCA expression were obtained with the CMV and the cTnT promoter, as long as the viral titer was adjusted to compensate for transcription efficiency. A maximal threefold increase of total SERCA protein expression over the level of the endogenous SERCA of control myocytes was reached (a sevenfold increase compared with the endogenous SERCA of the same infected myocytes due to reduction of endogenous SERCA after infection). In contrast with previous reports [Ji et al. Am. J. Physiol. 276 (Heart Circ. Physiol. 45): H89-H97, 1999], a higher kinetic turnover was demonstrated for the SERCA1 compared with the SERCA2a isoform as shown by a 5.0- versus 2.6-fold increase in calcium uptake rate accompanying maximal expression of recombinant SERCA1 or SERCA2a, respectively. This information is deemed necessary for studies attempting to modify myocardial cell function by manipulation of SERCA expression.
Sumbilla
C
C
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Cavagna
M
M
Zhong
L
L
Ma
H
H
Lewis
D
D
Farrance
I
I
Inesi
G
G
D000256:Adenoviridae; D000818:Animals; D000831:Animals, Newborn; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D002642:Chick Embryo; D003587:Cytomegalovirus / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D014162:Transfection; D020107:Troponin T / Q000235:genetics
D011994:Recombinant Proteins; D020107:Troponin T; D000252:Calcium-Transporting ATPases; D002118:Calcium