Abstract 18050: Phosphodiesterase 4B Controls Cyclic AMP Accumulation in a Defined Submembrane Compartment of Neonatal Cardiac Myocytes
Type 4 cyclic nucleotide phosphodiesterases (PDE4s) contribute to the shaping of the cAMP signals in cardiac myocytes. Using a genetic approach, it has been shown that inactivation of PDE4B (PDE4BKO) or PDE4D isoform increases the propensity to arrhythmias and induces hypertrophy and heart failure. Distinct steps in excitation-contraction coupling are affected, as PDE4B ablation augments the function of the L-type calcium channel, whereas PDE4D removal affects the activity of the ryanodine receptor. Here we have further investigated how PDE4B affects cAMP signaling in different compartments of cultured neonatal cardiomyocytes (NCMs). To this aim, mouse NCMs from wild type (WT) and PDE4BKO animals were infected with FRET-based cAMP sensors that are distributed throughout the cytosol or localized to the plasma membrane to perform real time measurements of cAMP in response to maintained β-AR stimulation by isoprenaline (Iso, 10 nM). Local concentration of cAMP after Iso stimulation was derived from the FRET data. The kinetic properties of cAMP accumulation in the cytosol of WT and PDE4BKO NCMs were identical. Conversely, when the response to Iso was assessed at the plasma membrane a clear difference in cAMP dynamics was observed in PDE4BKO. Contrary to WT, cAMP level in PDE4BKO NCMs reached a steady state. The difference between the two genotypes was abolished by PDE4 inhibition with Rolipram (10 µM). Western blot analysis indicated that a PDE4B long form is the major form expressed in NCMs. This long form is activated by PKA in vitro. Consistent with this data, treatment with the PKA inhibitor H89 (20 µM) prior to Iso, promoted cAMP accumulation at the plasma membrane of WT cells but not of PDE4BKO cells. Using selective β1-AR and β2-AR antagonists (CGP20712A and ICI118551, 1 µM), we showed that PDE4B ablation affects β1-AR but not β2-AR responses at the plasma membrane. Our findings demonstrate a restricted function for PDE4B in the submembrane compartment of cardiomyocytes. Indeed, the PDE4B long form is the major PDE4 isoform controlling β1-AR cAMP signals at the plasma membrane, which could explain the predominant role of PDE4B in the regulation of L-type calcium channel activity and the increased propensity to arrhythmias observed in PDE4BKO animals.
- © 2012 by American Heart Association, Inc.