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1 Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224; and 2 Howard Hughes Medical Institute, Stanford University Medical Center, Stanford, California 94305
Rapid development of
transgenic and gene-targeted mice and acute genetic manipulation via
gene transfer vector systems have provided powerful tools for
cardiovascular research. To facilitate the phenotyping of genetically
engineered murine models at the cellular and subcellular levels and to
implement acute gene transfer techniques in single mouse
cardiomyocytes, we have modified and improved current enzymatic methods
to isolate a high yield of high-quality adult mouse myocytes (5.3 ± 0.5 × 105 cells/left ventricle, 83.8 ± 2.5%
rod shaped). We have also developed a technique to culture these
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-contraction coupling and receptor-mediated
signaling, remained intact, but the contraction kinetics were
significantly slowed. Furthermore, gene delivery via recombinant
adenoviral infection was highly efficient and reproducible. In adult
1/2-adrenergic receptor (AR)
double-knockout mouse myocytes, adenovirus-directed expression of
either 1- or 2-AR, which occurred in
100% of cells, rescued the functional response to -AR agonist
stimulation. These techniques will permit novel experimental settings
for cellular genetic physiology.
excitation-contraction coupling; -adrenergic signaling
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