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.

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Sarco‐endoplasmic reticulum Ca2+‐ATPase from fast skeletal (SERCA1) or cardiac muscle (SERCA2a) was expressed in embryonic chicken and neonatal rat cardiac myocytes by adenovirus vectors, with c‐myc tags on both constructs to compare expression and distinguish exogenous from endogenous SERCA2a in myocytes. Expression of the two isoforms was similar (approximately 3‐fold higher than endogenous SERCA). However, SERCA1 activity was 2‐fold greater than SERCA2a activity, due to intrinsic differences in turnover rates. Activation of both exogenous SERCA isoforms by Ca2+ was displaced to slightly lower [Ca2+], suggesting that the overexpressed isoforms were independent of phospholamban. In fact, phospholamban and calsequestrin expression were unchanged. Decay time constants of cytosolic Ca2+ transients from cells overexpressing SERCA1 were reduced by 30–40 % and half‐widths by 10–15 % compared to controls. SERCA2a overexpression produced much less acceleration of transients in chick than in rat, and less acceleration than SERCA1 overexpression in either species. There was no significant change in resting [Ca2+], peak amplitudes, or in the amount of Ca2+ releasable by caffeine from overexpression of either SERCA isoform. However, the amplitudes of the transients increased with SERCA1 overexpression when pacing frequency limited refilling of the sarcoplasmic reticulum. It is concluded that total SERCA transport velocity has a primary effect on the decay phase of transients. Transport velocity is affected by SERCA isoform turnover rate, temperature, and/or SERCA copy number.

Abstract-Collateral effects of exogenous sarcoendoplasmic reticulum Ca 2ϩ ATPase (SERCA) expression were characterized in neonatal rat and chicken embryo cardiac myocytes, and the conditions required to produce acceleration of Ca 2ϩ transients with minimal toxicity were established. Cultured myocytes were infected with adenovirus vector carrying the cDNA of wild-type SERCA1, an inactive SERCA1 mutant, or enhanced green fluorescence protein under control of the cytomegalovirus promoter. Controls were exposed to empty virus vector. Each group was tested with and without phenylephrine (PHE) treatment. Under conditions of limited calf-serum exposure, the infected rat myocytes manifested a more rapid increase in size, protein content, and rate of protein synthesis relative to noninfected controls. These changes were not accompanied by reversal to fetal transcriptional pattern (as observed in hypertrophy triggered by PHE) and may be attributable to facilitated exchange with serum factors. SERCA virus titers Ͼ5 to 6 plaque-forming units per cell produced overcrowding of ATPase molecules on intracellular membranes, followed by apoptotic death of a significant number of rat but not chicken myocytes. Enhanced green fluorescence protein virus and empty virus also produced cytotoxic effects but at higher titers than SERCA. Expression of exogenous SERCA and enhancement of Ca 2ϩ transient kinetics could be obtained with minimal cell damage in rat myocytes if the SERCA virus titer were maintained within 1 to 4 plaque-forming units per cell. Expression of endogenous SERCA was unchanged, but expression of exogenous SERCA was higher in myocytes rendered hypertrophic by treatment with PHE than in nontreated controls. Key Words: SERCA Ⅲ gene therapy Ⅲ heart Ⅲ adenovirus Ⅲ calcium transients T he sarcoendoplasmic reticulum Ca 2ϩ ATPase (SERCA) pumps Ca 2ϩ from the cytosol back into the sarcoplasmic reticulum (SR) after myocardial contraction, thereby coordinating contractile tension and relaxation kinetics. Ca 2ϩ uptake by the SR has been reported to be inadequate in failing human heart 1,2 as a consequence of reduced SERCA activity, 3 SERCA protein expression, 4,5 and SERCA mRNA levels. 6 On the other hand, it was shown in experimental models that uptake of cytosolic Ca 2ϩ by the SR can be accelerated by expression of exogenous SERCA genes and consequent increase of the ATPase copy number in cardiac myocytes. [7][8][9] In fact, isolated failing human cardiac myocytes have shown improved performance after overexpression of exogenous SERCA. 10 Recombinant adenovirus has proven to be a very effective vector for delivery of exogenous SERCA cDNA into cardiomyocytes, 11 with 100% efficiency of infection compared with 5% to 10% efficiency by other transfection methods. 12 The positive benefits of exogenous SERCA expression on Ca 2ϩ homeostasis continues to be characterized by several laboratories, whereas collateral effects of gene expression have received little attention. We have observed important side effects that are much more eviden...