CELL-SPECIFIC PROMOTER IN ADENOVIRUS VECTOR FOR TRANSGENIC EXPRESSIONOF SERCA1 ATPASE IN CARDIAC MYOCYTES

Adenovirus-mediated transfer of cDNA encoding the chicken skeletal mus cle sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) yielded selectiv e expression in cultured chick embryo cardiac myocytes under control o f a segment (-268 base pair) of the cell-specific cardiac troponin T ( cTnT) promoter or nonselective expression in myocytes and fibroblasts under control of a constitutive viral [cytomegalovirus (CMV)] promoter . Under optimal conditions nearly all cardiac myocytes in culture were shown to express transgenic SERCA1 ATPase. Expression was targeted to intracellular membranes and was recovered in subcellular fractions wi th a pattern identical to that of the endogenous SERCA2a ATPase. Relat ive to control myocytes, transgenic SERCA1 expression increased up to four times the rates of ATP-dependent (and thapsigargin-sensitive) Ca2 + transport activity of cell homogenates. Although the CMV promoter wa s more active than the cTnT promoter, an upper limit for transgenic ex pression of functional enzyme was reached under control of either prom oter by adjustment of the adenovirus plaque-forming unit titer of infe ction media. Cytosolic Ca2+ concentration transients and tension devel opment of whole myocytes were also influenced to a similar limit by tr ansgenic expression of SERCA1 under control of either promoter. Our ex periments demonstrate that a cell-specific protein promoter in recombi nant adenovirus vectors yields highly efficient and selective transgen e expression of a membrane-bound and functional enzyme in cardiac myoc ytes.