Figure 3. Age-Dependent Alterations in RyR2-Channel Complex and Function in PDE4D^−/− Murine Heart.

(A) Immunoblots showing progressive increase in PKA phosphorylation of RyR2 at Ser2808 (second panel) but no change in CaMKII phosphorylation at Ser2814 (third panel) and progressive decrease in calstabin2 binding to the RyR2 complex (fourth panel). PKA catalytic (-C) and regulatory (-RII) subunits are not changed during heart failure (HF) development (fifth and six panels).

(B) Quantification of age-dependent increase in RyR2 PKA phosphorylation (open bars, wt; filled bars, PDE4D^−/−; *p < 0.05 versus wt, #p < 0.05 versus PDE4D^−/−). Data in (B)–(E) and (G) are mean ± SD.

(C) Quantification of age-dependent decrease in calstabin2 in the RyR2 complex (open bars, wt; filled bars, PDE4D^−/−; *p < 0.05 versus wt, #p < 0.05 versus PDE4D^−/−).

(D) Quantification of CaMKII-specific RyR2 phosphorylation showing no significant changes with aging.

(E) Immunoprecipitation of RyR2 showing complete absence of PDE activity in the RyR2 complex in PDE4D^−/− mouse hearts. *p < 0.05 versus wt; #p < 0.05 versus untreated wt. Rol, rolipram; Mil, milrinone.

(F) Single-channel recordings of wt (left) and PDE4D^−/− RyR2 (right) at 3 months of age (top) and 15 months of age (bottom). Current trace from 3-month-old PDE4D^−/− heart shows slightly increased channel open probability (Po) with short openings consistent with increased PKA phosphorylation. At 15 months of age, RyR2 channels from PDE4D^−/− hearts showed significantly increased Po and subconductance states (indicated by dotted lines), as evidenced by current-amplitude histograms. Upper traces represent 5 s; lower traces represent 500 ms. Channel openings are upward; full openings are 4 pA; closed state is indicated by “c.”

(G) Bar graphs summarizing Po, open frequency (Fo), average open time (To), and average closed time (Tc) at 15 months of age in wt and PDE4D^−/− groups. *p < 0.001; n = 10 each.