Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology

Citation
Yy. Zhou et al., Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology, AM J P-HEAR, 279(1), 2000, pp. H429-H436
Citations number
24
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Cardiovascular & Hematology Research
Journal title
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
ISSN journal
0363-6135 → ACNP
Volume
279
Issue
1
Year of publication
2000
Pages
H429 - H436
Database
ISI
SICI code
0363-6135(200007)279:1<H429:CAAIOA>2.0.ZU;2-4
Abstract
Rapid development of transgenic and gene-targeted mice and acute genetic ma nipulation via gene transfer vector systems have provided powerful tools fo r cardiovascular research. To facilitate the phenotyping of genetically eng ineered murine models at the cellular and subcellular levels and to impleme nt acute gene transfer techniques in single mouse cardiomyocytes, we have m odified and improved current enzymatic methods to isolate a high yield of h igh-quality adult mouse myocytes (5.3 +/- 0.5 x 10(5) cells/left ventricle, 83.8 +/- 2.5% rod shaped). We have also developed a technique to culture t hese isolated myocytes while maintaining their morphological integrity for 2-3 days. The high percentage of viable myocytes after 1 day in culture (72 .5 +/- 2.3%) permitted both physiological and biochemical characterization. The major functional aspects of these cells, including excitation-contract ion coupling and receptor-mediated signaling, remained intact, but the cont raction kinetics were significantly slowed. Furthermore, gene delivery via recombinant adenoviral infection was highly efficient and reproducible. In adult beta(1)/beta(2)-adrenergic receptor (AR) double-knockout mouse myocyt es, adenovirus-directed expression of either beta(1) -or beta(2)-AR, which occurred in 100% of cells, rescued the functional response to beta-AR agoni st stimulation. These techniques will permit novel experimental settings fo r cellular genetic physiology.