(A) PDE4D3 was detected in human cardiac lysate and in the immunoprecipitated RyR2 complex (IP:RyR2); IP:IgG, negative control.
(B) RyR2 was immunoprecipitated from cardiac homogenates of normal human (N) and heart failure (HF) samples. PDE4D3 binding to RyR2 was significantly decreased in human HF. Increased PKA phosphorylation was detected by a phosphoepitope-specific RyR2-Ser2808 antibody in HF samples.
(C) RyR2 bound PDE4D3 activity was significantly decreased in HF, as evidenced by close-proximity substrate cAMP catalysis (#p < 0.001). Data in (C) and (D) are mean ± SD.
(D) Rolipram (R), a PDE4-specific inhibitor, but not milrinone (M), a PDE3-specific inhibitor, decreased RyR2-associated PDE activity submaximally; C, control (#p < 0.001 versus untreated sample).