Cardiac myocytes were isolated as reported previously [26 (link), 27 (link)]. Briefly, each rat heart was cannulated through the aorta on an Easycell System for Cardiomyocyte Isolation (Harvard Apparatus, Holliston, MA) and perfused at 37°C with calcium-free buffer for 5 min, and then switched to digestion buffer for 20-25min. The heart was pulled from the cannula and the ventricles were transferred to a 60-mm sterile dish containing 11 mL of transfer buffer and cut into small pieces. The minced tissue was incubated in a 37°C water bath for 10 min. The cell suspension was filtered through a 100-μm mesh cell strainer (BD Biosciences, San Jose, CA) to remove tissue debris and spun at 420 rpm at room temperature for 2 min. The resultant pellet portion underwent further processing for CM chymase activity and for CM mRNA expression of rMCPs.