Modified enzymatic methods yielded high-quality adult mouse myocytes (83.8% rod shaped) that maintained 72.5% viability after 1 day in culture and permitted highly efficient adenoviral gene delivery.
This improved method for isolating, culturing, and infecting adult mouse cardiomyocytes enables highly efficient gene transfer and functional characterization for cardiovascular research.
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 x 10(5) 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 beta(1)/beta(2)-adrenergic receptor (AR) double-knockout mouse myocytes, adenovirus-directed expression of either beta(1)- or beta(2)-AR, which occurred in 100% of cells, rescued the functional response to beta-AR agonist stimulation. These techniques will permit novel experimental settings for cellular genetic physiology.
Zhou et al. (Sat,) conducted a other in Cardiovascular research (murine models). Enzymatic isolation, culture, and recombinant adenoviral infection was evaluated on Yield and viability of isolated adult mouse myocytes. Modified enzymatic methods yielded high-quality adult mouse myocytes (83.8% rod shaped) that maintained 72.5% viability after 1 day in culture and permitted highly efficient adenoviral gene delivery.