Phosphodiesterase 8A (PDE8A) regulates excitation-contraction coupling in ventricular myocytes

Abstract

In ventricular myocytes, activation of protein kinase A (PKA) by 3'-5' cyclic adenosine monophosphate (cAMP) increases the force of contraction by increasing L-type Ca(2+) channel currents (I(Ca)) and sarcoplasmic reticulum (SR) Ca(2+) release during excitation-contraction coupling. Cyclic-nucleotide phosphodiesterases (PDEs) comprise a large family of enzymes whose role in the cell is to regulate the spatial and temporal profile of cAMP signals by controlling the degradation of this second messenger. At present, however, the molecular identity and functional roles of the PDEs expressed in ventricular myocytes are incompletely understood. Here, we tested the hypothesis that PDE8A plays a critical role in the modulation of at least one compartment of cAMP and hence PKA activity during beta-adrenergic receptor (betaAR) activation in ventricular myocytes. Consistent with this hypothesis, we found that PDE8A transcript and protein are expressed in ventricular myocytes. Our data indicate that evoked [Ca(2+)](i) transients and I(Ca) increased to a much larger extent in PDE8A null (PDE8A(-/-)) than in wild-type (WT) myocytes during beta-adrenergic signaling activation. In addition, Ca(2+) spark activity was higher in PDE8A(-/-) than in WT myocytes. Our data indicate that PDE8A is a novel cardiac PDE that controls one or more pools of cAMP implicated in regulation of Ca(2+) movement through cardiomyocyte.

Figure 1

PDE8A expression in cardiac tissue and effect its absence on Ca²⁺ transient. (A) PDE8A immunohistochemistry detection in mouse ventricles. (Ai) PDE8A^−/− ventricle section stained for β-Gal activity. Lower left inset shows a section from a WT animal used as negative control. (Aii) staining on an isolated WT and PDE8A^−/− myocyte (see also supplementary Fig.2). Due to the presence of a nuclear localization sequence (NLS) fused at the 3′ of the LacZ gene, β-Gal activity is confined to the nucleus of PDE8A expressing cells. (B) Western Blot gels of PDE8A immunoprecipitates (IP) and their supernatants (SN) from ventricle (V) or cardiomyocytes (CM) from WT and PDE8A^−/− animals. Recombinant PDE8A is used as positive control (n=3). (C) PDE activity assay on IP in presence of 10nmol/L cAMP as substrate and absence or presence of 100μmol/L IBMX (n=3). (D) Typical fluo-4 traces of Ca²⁺ transients at 2 mmol/L Ca²⁺ and 1.0-Hz field stimulation, in basal state and after 2 min of 100 nmol/L Isoproterenol (ISO) stimulation. (E) Statistics for Ca²⁺ transient peak amplitude fold increase over basal at increasing concentration of ISO (n=31 to 51 cells from 5 to 6 hearts for each group; * P<0.05 WT vs. 8A^−/−).

Figure 2

Effects of lack of PDE8A on ISO-stimulated I_Ca and SR leak. (A) Average normalized I_Ca in WT and 8A^−/− myocytes before and after 100 nmol/L ISO stimulation (n=6 cells from 2 hearts for each group; ***P<0.005). (B) Confocal line-scan images showing spontaneous Ca²⁺ sparks in WT and 8A^−/− cells and statistic on calculated spark frequency (n=42 to 56 cells from 5 hearts for each group; ***P<0.005 WT vs. 8A^−/−). (C) Average spark frequency before and after 2 minute exposure to 100 nmol/L Isoproterenol (ISO) (n=20 to 24 cells from 5 hearts for each group; **P<0.01 WT basal vs. 8A^−/− basal, *P<0.05 WT basal vs. WT ISO). (D) SR loading measured by caffeine induced Ca²⁺ peak in resting cells and after 5 minutes of 100 μmol/L Tetracaine exposure (n=12 to 16 cells from 3 hearts for each group; *P<0.05 WT+Tetracaine vs. 8A^−/− +Tetracaine).