Phosphodiesterase 4D regulates baseline sarcoplasmic reticulum Ca2+ release and cardiac contractility, independently of L-type Ca2+ current

Abstract

Rationale: Baseline contractility of mouse hearts is modulated in a phosphatidylinositol 3-kinase-γ-dependent manner by type 4 phosphodiesterases (PDE4), which regulate cAMP levels within microdomains containing the sarcoplasmic reticulum (SR) calcium ATPase type 2a (SERCA2a).

Objective: The goal of this study was to determine whether PDE4D regulates basal cardiac contractility.

Methods and results: At 10 to 12 weeks of age, baseline cardiac contractility in PDE4D-deficient (PDE4D(-/-)) mice was elevated mice in vivo and in Langendorff perfused hearts, whereas isolated PDE4D(-/-) cardiomyocytes showed increased whole-cell Ca2+ transient amplitudes and SR Ca2+content but unchanged L-type calcium current, compared with littermate controls (WT). The protein kinase A inhibitor R(p)-adenosine-3',5' cyclic monophosphorothioate (R(p)-cAMP) lowered whole-cell Ca2+ transient amplitudes and SR Ca2+ content in PDE4D(-/-) cardiomyocytes to WT levels. The PDE4 inhibitor rolipram had no effect on cardiac contractility, whole-cell Ca2+ transients, or SR Ca2+ content in PDE4D(-/-) preparations but increased these parameters in WT myocardium to levels indistinguishable from those in PDE4D(-/-). The functional changes in PDE4D(-/-) myocardium were associated with increased PLN phosphorylation but not cardiac ryanodine receptor phosphorylation. Rolipram increased PLN phosphorylation in WT cardiomyocytes to levels indistinguishable from those in PDE4D(-/-) cardiomyocytes. In murine and failing human hearts, PDE4D coimmunoprecipitated with SERCA2a but not with cardiac ryanodine receptor.

Conclusions: PDE4D regulates basal cAMP levels in SR microdomains containing SERCA2a-PLN, but not L-type Ca2+ channels or ryanodine receptor. Because whole-cell Ca2+ transient amplitudes are reduced in failing human myocardium, these observations may have therapeutic implications for patients with heart failure.

Figure 1. Assessment of Langendorff heart function

(A) Records of left ventricular pressure (top), dP/dt_max and dP/dt_min (bottom) measured in paced (9Hz) Langendorff-perfused hearts before and after ROL infusion. (B) Mean data for LVP (left), dP/dt_max and dP/dt_min (middle) and % change from baseline following ROL treatment (right), with baseline recorded after a 20 min equilibration period. *P<0.05 vs.WT, **P<0.01 vs. WT

Figure 2. Ca²⁺ transients and I_CaL Measurements

(A) Ca²⁺ transients (upper) and I_CaL (lower) recorded for WT and PDE4D^−/− cardiomyocytes in response to voltage steps (indicated) from –85mV holding potential and a 500 msec ramp to -45mV. (B) Typical Ca²⁺ transients and I_Ca,L at +10mV before and after ROL application. Mean Ca²⁺ transient and I_Ca,L peaks as a function of voltage in the presence and absence of RpcAMPS (C) and ROL (D). *P<0.01 versus control within same group; †P<0.01 versus WT control

Figure 3. Measurements of SR Ca²⁺ content (A) as well as PLN and RyR2 phosphorylation (B&C)

(A) I_NCX evoked by a 10s application of 20mM caffeine in the presence or absence of RpcAMPS or ROL (left). Mean (time) integrated I_NCX to assess SR Ca²⁺ content bottom (right). *P< 0.05 versus WT. (B) Representative Western blot of protein extracts from left ventricular cardiomyocytes to measure phosphorylated PLN (left). Average intensity ratios of pPLN/PLN_total(right). *P< 0.05 versus WT; †P<0.05 vs. PDE4D^−/− (n=5 hearts). (C) Representative WB of protein extracts from left ventricular cardiomyocytes illustrating the effect of PDE4D ablation on phosphorylated RyR2 (pRyR2) levels (left). Mean data showing changes in pRyR2/ RyR2_total ratios in PDE4D^−/− hearts (right). *P<0.05 versus PDE4D WT (n= 4 hearts).

Figure 4. PDE4D interactions with SERCA2a in murine and human myocardium

A shows representative Western Blots (repeated in 3 separate hearts) probing with PDE4D in heart lysates from a PDE4D^−/− mouse as well as for WT mouse heart homogenates that had been immunoprecipitated using control IgG, or using RyR2- or SERCA2a-specific antibodies. Results show that PDE4D antibodies recognized strong bands at MWs of 97kDaltons (corresponding to PDED-3,-7 and -9 splice variants of PDE4D) in immunoprecipitation reactions with anti-SERCA2a antibodies, but not with anti-RYR2 antibodies or with IgG controls. A very weak nonspecific band having MW ~ 110kDaltons was detected in all immunoprecipitation groups as well as in homogenates from the PDE4D-null mice, confirming this is a nonspecific band of unknown origin. B shows representative inputs for immunoprecipitation reactions shown in A. C shows results of SERCA2a immunoprecipitation in human hearts. (D) Diagram summarizing implications of our results in WT (left) and PDE4D^−/− (right) myocardium.