Abstract 16750: Phosphodiesterase 4B in the L-Type Calcium Channel Complex Regulates Cardiac Calcium Current and Protects Against Ventricular Arrhythmias
Beta-adrenergic receptors (β-AR) enhance cardiac contractility by increasing cAMP levels and activating PKA. PKA increases Ca2+-induced Ca2+ release (CICR) by phosphorylating L-type Ca2+ channels (CaV1.2) and ryanodine receptors (RyR). Multiple cyclic nucleotide phosphodiesterases (PDEs) regulate β-AR-dependent cAMP signals, among which the PDE4 family is dominant. Three genes encoding PDE4 are expressed in heart: PDE4A, PDE4B and PDE4D. Ablation of the PDE4D gene in mice causes misregulation of RyR2 and results in late-onset cardiomyopathy. Here, using the patch clamp technique we show that isoprenaline (Iso) stimulation (100 nM, 15s) of the L-type Ca2+ current (ICa,L) (+58.8±3.4%, n=62) is potentiated by the selective PDE4 inhibitor Ro20–1724 (10 µM) (+103.3± 7.7%, n=37, p<0.001) in wild type adult mouse ventricular myocytes (WT). Using specific antibodies, we show that both PDE4B and PDE4D are tethered to CaV1.2 in macromolecular signaling complexes in the mouse heart. Iso stimulation (100 nM, 15 s) of ICa,L is increased in ventricular myocytes from PDE4B deficient mice (PDE4B-/-, +78.3±3.6%, n=46, p<0.01) whereas the effect of Ro20–1724 on Iso is blunted. In contrast, ICa,L regulation is normal in PDE4A-/- and PDE4D-/- mice. Immunostaining indicates that CaV1.2 and a fraction of PDE4B are colocalized along T-tubules in mouse. Iso stimulation (100 nM, 15 s) of Ca2+ transients (DR) and cell contraction (DL) is enhanced in PDE4B-/- (DR=30.0±1.4%, DL=9.3±0.7%, n=47) and PDE4D-/- myocytes (DR=31.0±1.6%, DL=9.2±0.7%, n=47) compared to WT (DR=24.1±1.3%, DL=5.9±0.5%, n=51, p<0.01). PDE4B-/- and PDE4D-/- myocytes also display increased spontaneous Ca2+ release events upon Iso (4.5±1.1 per 20s, n=47 and 4.5±0.9 per 20s, n=47, respectively) compared to WT (1.4±0.5 per 20s, n=51, p<0.01). In vivo, after IP injection of Iso (0.2 mg/kg), catheter-mediated burst pacing triggers ventricular tachycardia in PDE4B-/- mice but not in WT (4/10 versus 0/9, respectively, p<0.05). These results constitute the first evidence for a role of PDE4B in the heart. They identify PDE4B in the CaV1.2 complex as a critical regulator of ICa,L during β-AR stimulation, and suggest that distinct PDE4 subtypes are important for normal regulation of CICR in cardiac myocytes.
- © 2010 by American Heart Association, Inc.