Jérôme Leroy

Spatiotemporal Dynamics of β-Adrenergic cAMP Signals and L-Type Ca2+ Channel Regulation in Adult Rat Ventricular Myocytes Role of Phosphodiesterases

Steady-state activation of cardiac β-adrenergic receptors leads to an intracellular compartmentation of cAMP resulting from localized cyclic nucleotide phosphodiesterase (PDE) activity. To evaluate the time course of the cAMP changes in the different compartments, brief (15 seconds) pulses of isoprenaline (100 nmol/L) were applied to adult rat ventricular myocytes (ARVMs) while monitoring cAMP changes beneath the membrane using engineered cyclic nucleotide-gated channels and within the cytosol with the fluorescence resonance energy transfer–based sensor, Epac2-camps. cAMP kinetics in the two compartments were compared to the time course of the L-type Ca2+ channel current (ICa,L) amplitude. The onset and recovery of cAMP transients were, respectively, 30% and 50% faster at the plasma membrane than in the cytosol, in agreement with a rapid production and degradation of the second messenger at the plasma membrane and a restricted diffusion of cAMP to the cytosol. ICa,L amplitude increased twice slower than cAMP at the membrane, and the current remained elevated for ≈5 minutes after cAMP had already returned to basal level, indicating that cAMP changes are not rate-limiting in channel phosphorylation/dephosphorylation. Inhibition of PDE4 (with 10 &mgr;mol/L Ro 20-1724) increased the amplitude and dramatically slowed down the onset and recovery of cAMP signals, whereas PDE3 blockade (with 1 &mgr;mol/L cilostamide) had a minor effect only on subsarcolemmal cAMP. However, when both PDE3 and PDE4 were inhibited, or when all PDEs were blocked using 3-isobutyl-l-methylxanthine (300 &mgr;mol/L), cAMP signals and ICa,L declined with a time constant >10 minutes. cAMP-dependent protein kinase inhibition with protein kinase inhibitor produced a similar effect as a partial inhibition of PDE4 on the cytosolic cAMP transient. Consistently, cAMP-PDE assay on ARVMs briefly (15 seconds) exposed to isoprenaline showed a pronounced (up to ≈50%) dose-dependent increase in total PDE activity, which was mainly attributable to activation of PDE4. These results reveal temporally distinct β-adrenergic receptor cAMP compartments in ARVMs and shed new light on the intricate roles of PDE3 and PDE4.

Phosphodiesterase 4B in the cardiac L-type Ca2+ channel complex regulates Ca2+ current and protects against ventricular arrhythmias in mice

β-Adrenergic receptors (β-ARs) enhance cardiac contractility by increasing cAMP levels and activating PKA. PKA increases Ca²⁺-induced Ca²⁺ release via phosphorylation of L-type Ca²⁺ channels (LTCCs) and ryanodine receptor 2. Multiple cyclic nucleotide phosphodiesterases (PDEs) regulate local cAMP concentration in cardiomyocytes, with PDE4 being predominant for the control of β-AR-dependent cAMP signals. Three genes encoding PDE4 are expressed in mouse heart: Pde4a, Pde4b, and Pde4d. Here we show that both PDE4B and PDE4D are tethered to the LTCC in the mouse heart but that β-AR stimulation of the L-type Ca²⁺ current (ICa,L) is increased only in Pde4b-/- mice. A fraction of PDE4B colocalized with the LTCC along T-tubules in the mouse heart. Under β-AR stimulation, Ca²⁺ transients, cell contraction, and spontaneous Ca²⁺ release events were increased in Pde4b-/- and Pde4d-/- myocytes compared with those in WT myocytes. In vivo, after intraperitoneal injection of isoprenaline, catheter-mediated burst pacing triggered ventricular tachycardia in Pde4b-/- mice but not in WT mice. These results 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 Ca²⁺-induced Ca²⁺ release in cardiomyocytes.

Phosphoinositide 3-Kinase γ Protects Against Catecholamine-Induced Ventricular Arrhythmia Through Protein Kinase A–Mediated Regulation of Distinct Phosphodiesterases

Background— Phosphoinositide 3-kinase &ggr; (PI3K&ggr;) signaling engaged by &bgr;-adrenergic receptors is pivotal in the regulation of myocardial contractility and remodeling. However, the role of PI3K&ggr; in catecholamine-induced arrhythmia is currently unknown. Methods and Results— Mice lacking PI3K&ggr; (PI3K&ggr;−/−) showed runs of premature ventricular contractions on adrenergic stimulation that could be rescued by a selective &bgr;2-adrenergic receptor blocker and developed sustained ventricular tachycardia after transverse aortic constriction. Consistently, fluorescence resonance energy transfer probes revealed abnormal cAMP accumulation after &bgr;2-adrenergic receptor activation in PI3K&ggr;−/− cardiomyocytes that depended on the loss of the scaffold but not of the catalytic activity of PI3K&ggr;. Downstream from &bgr;-adrenergic receptors, PI3K&ggr; was found to participate in multiprotein complexes linking protein kinase A to the activation of phosphodiesterase (PDE) 3A, PDE4A, and PDE4B but not of PDE4D. These PI3K&ggr;-regulated PDEs lowered cAMP and limited protein kinase A–mediated phosphorylation of L-type calcium channel (Cav1.2) and phospholamban. In PI3K&ggr;−/− cardiomyocytes, Cav1.2 and phospholamban were hyperphosphorylated, leading to increased Ca2+ spark occurrence and amplitude on adrenergic stimulation. Furthermore, PI3K&ggr;−/− cardiomyocytes showed spontaneous Ca2+ release events and developed arrhythmic calcium transients. Conclusions— PI3K&ggr; coordinates the coincident signaling of the major cardiac PDE3 and PDE4 isoforms, thus orchestrating a feedback loop that prevents calcium-dependent ventricular arrhythmia.

PI3Kγ Protects against Catecholamine-Induced Ventricular Arrhythmia through PKA-mediated Regulation of Distinct Phosphodiesterases

Background— Phosphoinositide 3-kinase &ggr; (PI3K&ggr;) signaling engaged by &bgr;-adrenergic receptors is pivotal in the regulation of myocardial contractility and remodeling. However, the role of PI3K&ggr; in catecholamine-induced arrhythmia is currently unknown. Methods and Results— Mice lacking PI3K&ggr; (PI3K&ggr;−/−) showed runs of premature ventricular contractions on adrenergic stimulation that could be rescued by a selective &bgr;2-adrenergic receptor blocker and developed sustained ventricular tachycardia after transverse aortic constriction. Consistently, fluorescence resonance energy transfer probes revealed abnormal cAMP accumulation after &bgr;2-adrenergic receptor activation in PI3K&ggr;−/− cardiomyocytes that depended on the loss of the scaffold but not of the catalytic activity of PI3K&ggr;. Downstream from &bgr;-adrenergic receptors, PI3K&ggr; was found to participate in multiprotein complexes linking protein kinase A to the activation of phosphodiesterase (PDE) 3A, PDE4A, and PDE4B but not of PDE4D. These PI3K&ggr;-regulated PDEs lowered cAMP and limited protein kinase A–mediated phosphorylation of L-type calcium channel (Cav1.2) and phospholamban. In PI3K&ggr;−/− cardiomyocytes, Cav1.2 and phospholamban were hyperphosphorylated, leading to increased Ca2+ spark occurrence and amplitude on adrenergic stimulation. Furthermore, PI3K&ggr;−/− cardiomyocytes showed spontaneous Ca2+ release events and developed arrhythmic calcium transients. Conclusions— PI3K&ggr; coordinates the coincident signaling of the major cardiac PDE3 and PDE4 isoforms, thus orchestrating a feedback loop that prevents calcium-dependent ventricular arrhythmia.

0164: Validation of two-dimensional speckle tracking strain for assessment of early right ventricular dysfunction: in vivo and ex vivo study

Background Right ventricular (RV) dysfunction is a major determinant of long-term survival in congenital heart diseases. Early echo detection of RV failure is mandatory, but recent indices need to be validated. Aims Objectives were to: (1) validate standard and strain echo indices for evaluation of RV systolic function, compared to hemodynamic parameters; (2) assess the accuracy of these indices for early detection of RV failure. Methods Combined RV overload as observed in repaired tetralogy of Fallot was surgically reproduced in 2-month-old piglets (n=6). Age-matched piglets were used as controls (n=4). RV function was evaluated at baseline and 4 months of follow-up by standard and strain echo indices, compared to conductance catheter. Sarcomere shortening and calcium transients were recorded in RV isolated myocytes (IonOptix). Contractile reserve was assessed by in-vivo (dobutamine 5µg/kg) and ex-vivo (isoprenaline 100nM) β adrenergic stimulation. The integrity of T-tubules was controlled after Di-4-Anepps labeling. Results 4 months after surgery, hemodynamic RV ejection fraction (FEVD) was significantly decreased (29.7% [26.2-34] vs 42.9% [40.7-48.6], p Conclusion In this model, both standard and strain echo indices allowed the detection of early impairments of RV function and cardiac reserve, which are associated with cardiac excitation-contraction coupling alterations.

0270 : CaMKII inhibition prevents cardiac arrhythmias elicited by phosphodiesterases 3 and 4 inhibitors

β-adrenoceptors (β-AR) stimulation increases cardiac function by rising cAMP that activates protein kinase A (PKA) and enhances Ca2+-induced Ca2+- release by phosphorylating proteins such as ryanodine receptors and phospholamban, which are also targets of the Ca2+/Calmodulin Kinase II (CaMKII). Any dysregulation of the β-AR pathway promotes arrhythmias. Local cAMP concentration in heart is mainly regulated by phosphodiesterases (PDE) type 3 and 4. Here, we investigated the proarrhythmic effects of PDE3 and PDE4 inhibition and evaluated the relative contribution of PKA and CaMKII to these mechanisms. An IonOptix system was used to record intracellular Ca2+ transients in isolated adult rat and pig ventricular myocytes (ARVMs and APVMs) loaded with 1μM Fura-2AM and paced at 1Hz. In ARVMs, PDE4 inhibition with Ro20-1724 (Ro, 10μM) potentiated the inotropic effect of the β-AR agonist isoproterenol (Iso, 1nM) but induced spontaneous Ca2+ waves (SCWs) recorded during 10s pacing pauses (2.3±0.2 SCWs/10s, n=15; p These results show that PDE inhibitors exert inotropic effects via PKA but lead to SCWs via both PKA and CaMKII activation. They also suggest the potential use of CaMKII inhibitors as adjuncts to PDE inhibitors to limit their proarrhythmic effects.

0089 : Echocardiography and right ventricular function: validation of functional criteria compared to in-vivo and ex-vivo contractility parameters

Background Right ventricular (RV) dysfunction is a major determinant of long-term survival in congenital heart diseases. Early echocardiographic detection of RV failure is mandatory, but recent parameters need to be validated. Aims Objectives were to: (1) validate standard and strain echocardiographic parameters for evaluation of RV systolic function, compared to hemodynamic parameters; (2) assess the accuracy of these parameters for early detection of RV failure. Methods Combined RV overload as observed in repaired tetralogy of Fallot was surgically reproduced in 2-month-old piglets (n=6). Age-matched piglets were used as controls (n=4). RV function was evaluated at baseline and 4 months of follow-up by standard and strain echocardiographic parameters, compared to hemodynamic (conductance catheter). Sarcomere shortening and calcium transients were recorded in RV isolated myocytes (IonOptix). Contractile reserve was assessed by in-vivo (dobutamine 5奯kg) and ex-vivo (isoprenaline 100nM) ?-adrenergic stimulation. Results 4 months after surgery, hemodynamic RV ejection fraction (FEVD) was significantly decreased (29.7% [26.2-34] vs 42.9% [40.7-48.6], p Conclusion In this model, both standard and strain echocardiographic parameters allowed the detection of early impairments of RV function and cardiac reserve, which are associated with cardiac excitation-contraction coupling alterations.

0039: Phosphodiesterase type 4 inhibition enhances pro-arrhythmic spontaneous Ca2+ waves induced by β-adrenergic stimulation: respective role of protein kinase A and Ca2+/calmodulin kinase II

β-adrenoceptors (β-AR) increase cardiac function by rising cAMP and activating protein kinase A (PKA) which phosphorylates L-type Ca2+ channels, ryanodine receptors (RyR) and phospholamban (PLB). These proteins are also targets of Ca2+/calmodulin kinase II (CaMKII), which is also activated upon β-AR stimulation. Multiple cyclic nucleotide phosphodiesterases (PDEs) regulate cAMP, among which the PDE4 family is overriding in rodent heart. We and others have shown that PDE4 genetic ablation in mice leads to enhanced arrhythmias. Here, we investigated the mechanisms underlying the proarrhythmic effects of PDE4 inhibition. Adult rat ventricular myocytes loaded with 1μM Fura-2AM were stimulated at 1Hz and Ca2+ transients recorded using an Ionoptix® system. Spontaneous calcium waves (SCWs) occurred upon non selective β-AR stimulation with isoproterenol (Iso 1nM) during a 10s pause of electrical stimulation and increased from 0.4±0.3 (n=10) to 2.3±0.2 SCWs/10s (n=15; p