PDE Activity Assay in Heart Tissue

PDE activity assay was performed as described previously with modification [12 (link), 13 (link)]. Frozen heart tissue samples were homogenized in PDE assay lysis buffer [40 mM TrisHCl · (pH 7.5), 15 mM β-mercaptoethanol, 20% (vol/vol) glycerol, 1 mM Na₃VO₄, 100 nM Okadaic acid, Abcam protease inhibitor mixture]. cAMP/cGMP–PDE activities were measured using 1 μM cAMP or cGMP substrate in the presence of 4μg/mL calmodulin and 0.8 mM CaCl₂ (for Ca²⁺/CaM-dependent PDE assay). Briefly, PDE assay reactions were assembled on ice until addition of cAMP substrate. Reactions were incubated at 25 °C for 10 min and then were terminated at 100 °C for 1 min. After cooling at RT for 10 min, the remaining cAMP or cGMP level in the reaction lysate was measured by AlphaScreen assay kit (PerkinElmer). PDE1 activities were defined as total PDE activity minus PDE activity in the presence of PDE1 inhibitor IC86340 (15 μM). Activities were normalized to protein concentration.