Cell-specific promoter in adenovirus vector for transgenic expression of SERCA1 ATPase in cardiac myocytes.
Adenovirus-mediated transfer of cDNA encoding the chicken skeletal muscle sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) yielded selective expression in cultured chick embryo cardiac myocytes under control of 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 with a pattern identical to that of the endogenous SERCA2a ATPase. Relative 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 was more active than the cTnT promoter, an upper limit for transgenic expression of functional enzyme was reached under control of either promoter by adjustment of the adenovirus plaque-forming unit titer of infection media. Cytosolic Ca2+ concentration transients and tension development of whole myocytes were also influenced to a similar limit by transgenic expression of SERCA1 under control of either promoter. Our experiments demonstrate that a cell-specific protein promoter in recombinant adenovirus vectors yields highly efficient and selective transgene expression of a membrane-bound and functional enzyme in cardiac myocytes.
Inesi
G
G
Department of Anatomy, University of California School of Medicine, San Francisco 94143, USA.
Lewis
D
D
Sumbilla
C
C
Nandi
A
A
Strock
C
C
Huff
K W
KW
Rogers
T B
TB
Johns
D C
DC
Kessler
P D
PD
Ordahl
C P
CP
D000255:Adenosine Triphosphate / Q000378:metabolism; D000260:Adenoviruses, Human; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002642:Chick Embryo; D018076:DNA, Complementary / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007763:Lac Operon / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009200:Myocardial Contraction; D009206:Myocardium / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D014162:Transfection; D014336:Troponin / Q000235:genetics; D020107:Troponin T
D018076:DNA, Complementary; D007527:Isoenzymes; D008164:Luminescent Proteins; D014336:Troponin; D020107:Troponin T; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium