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 limi ting factors in the expression and Ca2+ transport function of recombinant s arcoplasmic-endoplasmic reticulum Ca2+ (SERCA) isoforms. Titration experime nts showed that all COS-1 cells and myocytes in culture could be infected b y an adenovirus titer of 10 plaque-forming units (pfu) per seeded cell. Rai sing the adenovirus titer further yielded higher protein expression up to a n asymptotic limit for functional, membrane-bound SERCA protein. The asympt otic 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 man ifested tight muscle specificity. However, its efficiency was lower than th at of the nonspecific cytomegalovirus (CMV) promoter. At any rate, identica l maximal levels of SERCA expression were obtained with the CMV and the cTn T promoter, as long as the viral titer was adjusted to compensate for trans cription efficiency. A maximal threefold increase of total SERCA protein ex pression over the level of the endogenous SERCA of control myocytes was rea ched (a sevenfold increase compared with the endogenous SERCA of the same i nfected 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 fo r 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 r ecombinant SERCA1 or SERCA2a, respectively. This information is deemed nece ssary for studies attempting to modify myocardial cell function by manipula tion of SERCA expression.