Chymase enzymatic activity in CM

Chymase activity in CMs was determined by HPLC, as previously described [15]. Because cardiac chymase adheres to proteoglycans within the cell membrane, a 1M NaCl buffer solution was substituted for the typical normal salt (125 mM NaCl) buffer for the determination of chymase activity. Radiolabeled (¹²⁵I) (Perkin Elmer Life and Analytical Sciences, Inc., Waltham, MA) rat Ang-(1-12) (GenScript USA, Inc., Piscataway, NJ) was used as a substrate for the determination of chymase activity. Selection of Ang-(1-12) as the substrate for the chymase activity assay is based on our previous demonstration of higher enzymatic activity of rat chymase with Ang-(1-12) compared to Ang I [15].