Abstract
BACKGROUND: The ionic mechanism by which catecholamines increase the heart rate is incompletely understood. In this study, we have assessed the roles of sinoatrial node L-type Ca(v)1.3 (α(1D)) Ca(2+) channels, phosphorylation of L-type channel regulatory partner protein Rad (Ras-related RGK GTP-binding protein), and cAMP-dependent regulation of hyperpolarization-activated HCN (hyperpolarization-activated cyclic nucleotide-gated) channels. METHODS: We studied β-adrenergic regulation of heart rate and sinoatrial pacemaker activity in mice lacking Ca(v)1.3 channels and in mice expressing dihydropyridine-insensitive L-type Ca(v)1.2 channels alone or concomitantly expressing cAMP-insensitive HCN4 subunits in a heart-specific and time-controlled manner. We also studied the chronotropic response to sympathomimetics of sinoatrial pacemaker myocytes under conditions of specific inhibition of cAMP-dependent regulation of HCN4 by the cyclic dinucleotide cyclic di-(3',5')-GMP and ablation of PKA (protein kinase A)-dependent phosphorylation of Rad. RESULTS: Mutant mice with knockout of Ca(v)1.3 and cAMP-insensitive HCN4 subunits in the heart lacked diurnal variation in heart rate and failed to increase their heart rate after administration of catecholamines or during physical activity. Selective pharmacological inhibition of Ca(v)1.3 prevented the enhancement of pacemaker activity by sympathomimetics or by direct activation of adenylate cyclase, as well as by phosphodiesterase inhibitors, when cAMP-dependent regulation of HCN was simultaneously silenced. Upregulation of Ca(v)1.3 and HCN-mediated funny current (I(f)) accounted for the total change in diastolic current on activation of β-adrenoceptors, explaining the loss of chronotropic effect of catecholamines. Preventing PKA phosphorylation of Rad abrogated the chronotropic response to sympathomimetics of intact hearts under HCN blockade, or in pacemaker myocytes on preventing cAMP-dependent regulation of HCN4, respectively. CONCLUSIONS: PKA phosphorylation of Rad, which disinhibits Ca(v)1.3 channels and cAMP-dependent activation of HCN channels, are key effectors in β-adrenergic regulation of cardiac pacemaker activity and can sustain positive chronotropic effects independently. These findings on Rad-mediated regulation of Ca(v)1.3 and HCN channels unravel the ionic mechanisms underlying the catecholaminergic acceleration of the heart rate.