Pro-Arrhythmic Calcium Waves Induced by Phosphodiesterase Type 4 Inhibition upon Beta-Adrenergic Stimulation Involve Both PKA and CamkII

Abstract

β-adrenergic receptor (β-AR) stimulation increases cardiac function by increasing cAMP levels and activating protein kinase A (PKA). PKA enhances Ca2+-induced Ca2+-release by phosphorylating L-type Ca2+ channels, ryanodine receptors and phospholamban (PLB) which are also targets of the Ca2+/Calmodulin Kinase II (CaMKII). Any dysregulation in the β-adrenergic pathway leads to cardiac arrhythmias. Multiple cyclic nucleotide phosphodiesterases (PDEs) regulate local concentrations of cAMP, among which the PDE4 family is overriding in rodent heart. We investigated the proarrhythmic effects of PDE4 inhibition and evaluated the relative contribution of PKA and CaMKII to this mechanism. Action potentials were recorded at a frequency of 1Hz in isolated adult rat ventricular myocytes using the patch-clamp technique. Delayed afterdepolarizations (DADs) observed upon application of the non selective β-AR agonist Isoproterenol (Iso 1nM) after cessation of electrical stimulation during 10s were potentiated by the PDE4 inhibitor Ro20-1724 (Ro 10µM). These DADs correlated with spontaneous calcium waves (SCWs), recorded in myocytes loaded with Fura-2AM (1µM) using an Ionoptix system. Ro potentiated the inotropic and lusitropic effects of Iso and furthered sarcoplasmic reticulum (SR) Ca2+ load leading to SR Ca2+ leak measured in a 0Na+,0Ca2+ solution ±1mM tetracaine. Upon PDE4 inhibition, PLB was phosphorylated not only by PKA but also by CaMKII demonstrating that both kinases were activated. PKA inhibition with H-89 (10µM) suppressed the SCWs and the inotropic and lusitropic effects of Iso±Ro. CaMKII inhibition with KN-93 (10µM) diminished SCWs incidence by 72% without affecting the inotropic effects of Ro. Thus, upon β-AR stimulation, PDE4 inhibition exerts positive inotropic effects via PKA but induces SCWs and DADs via both PKA and CaMKII activation, suggesting the potential use of CaMKII inhibitors as an adjunct to PDE inhibition in cardiac diseases to limit arrhythmias.