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Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201
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 Ca2+ (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.
calcium adenosine 5'-triphosphatase; sarcoplasmic-endoplasmic reticulum calcium; adenovirus vectors
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