Method-related effects of adenovirus-mediated LacZ and SERCA1 gene transfer on contractile behavior of cultured failing human cardiomyocytes.
BACKGROUND
Adenovirus-mediated gene transfer into cardiomyocytes has emerged as an interesting tool to study functional effects of single proteins. However, the functional consequences of cell isolation, cell culture per se and adenovirus-mediated transfer of the LacZ or SERCA1 gene in failing human cardiomyocytes warrant further investigation.


METHODS
Primary cell culture was performed without or after adenovirus-mediated gene transfer of LacZ or SERCA1. Functional behavior of myocytes was assessed under basal conditions (field stimulation, 0.5 Hz, 37 degrees C), and during inotropic stimulation with isoproterenol (ISO; 10(-9)-10(-5) M), [Ca(2+)](o) (1.5-15 mM) or increasing stimulation rates (0.25-2.5 Hz). Results were compared to trabeculae from the same hearts.


RESULTS
Freshly isolated myocytes showed full inotropic competence as compared to multicellular preparations. The response to stimulation with ISO and [Ca(2+)](o), as well as changes in stimulation rate resulted in a maximal increase in fractional cell shortening (FS) to 215+/-24% and 291+/-34%, and a frequency-dependent decline in FS to 46+/-5% of the basal value, respectively. After 48 h of cell culture, basal FS did not change significantly compared to fresh cells but both time to peak shortening and time to 50% relengthening were prolonged. After culture, the concentration-response curve for ISO was significantly shifted to the left (EC(50) 5.16 x 10(-8) vs. 1.12 x 10(-8) M, p<0.05). LacZ gene transfer caused efficient beta-Gal expression without affecting the inotropic responses to ISO or stimulation rate but impaired the contractile amplitude. SERCA1 gene transfer increased FS by 68% vs. LacZ and accelerated relengthening kinetics (+dL/dt 93+/-13 vs. 61+/-8 mum/s, p<0.05 vs. LacZ).


CONCLUSIONS
Contractile responses of isolated human myocytes are comparable to multicellular preparations. The use of primary cell culture and adenovirus infection with CMV-promoter-mediated LacZ expression per se modulates contractile behavior in failing human myocytes. SERCA1 expression markedly improves contractile function. The method-related changes in contractile behavior observed here need to be taken into account in further studies.
Weisser-Thomas
J
J
Georg-August-Universität Göttingen, Abteilung Kardiologie und Pneumologie, Zentrum Innere Medizin, Robert-Koch-Str. 40, 37075 Göttingen, Germany. juttaweisser@hotmail.com
Dieterich
E
E
Janssen
P M L
PM
Schmidt-Schweda
S
S
Maier
L S
LS
Sumbilla
C
C
Pieske
B
B
D000256:Adenoviridae / Q000235:genetics; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics*; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006333:Heart Failure / Q000473:pathology / Q000503:physiopathology*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D007763:Lac Operon; D008297:Male; D008875:Middle Aged; D009200:Myocardial Contraction / Q000187:drug effects / Q000502:physiology*; D009206:Myocardium / Q000473:pathology; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000502:physiology*; D011401:Promoter Regions, Genetic; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D001616:beta-Galactosidase / Q000378:metabolism
D000318:Adrenergic beta-Agonists; D001616:beta-Galactosidase; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506943:ATP2A1 protein, human; D000252:Calcium-Transporting ATPases; D007545:Isoproterenol; D002118:Calcium