RUOLO DEL CANALE HCN4 NEL SISTEMA DI CONDUZIONE CARDIACO

Cardiac pacemaking is generated and modulated by the coordinated action of several processes. In mammals heart rhythm originates from specialized myocytes of the sinoatrial node (SAN) due to the presence of the spontaneous diastolic depolarization. The If (“funny” or pacemaker) current, whose molecular constituents are the Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels, and mainly HCN4, is considered to play the most important role for the maintenance of cardiac automaticity. Most of the data on the role of this current were collected by in vitro experiments and only recently initial data on the role of If in intact animal have become available in our laboratory thanks to the knockout mouse technology, applied to the Hcn4 gene. We therefore set to further investigate the role of the HCN4 channels in the adult by creating a transgenic mouse line in which the Hcn4 gene can be deleted in a conditional and inducible manner, using the Cre/LoxP system. We recorded a reduced contribution of the If current evaluated in vivo by telemetric ECG recordings, and in vitro by single cell electrophysiology. In our ci-HCN4 knockout mice, deletion of HCN4 consistently leads the progressive development of severe bradycardia (~50% reduction) and AV block which always progressed to heart arrest and death of the animals in about 5 days; in control mice the same experiments did not result any rate modification or premature death. In ciHCN4-KO animals the residual If was also sensitive to β-AR modulation, and the permanence of rate response to β-AR stimulation was observed both in vivo and in vitro, but the β-AR response of ciHCN4-KO animals was not able to reach the maximum value. Similar results were obtained by means of pharmacologic selective block using the If-specific reducing agent Ivabradine. The finding that If removal leads to AV conduction block was novel and the exact role of the If current in this contest still requires careful analysis and understanding. We therefore set to carry out an initial evaluation of the electrical properties of single cells isolated from ciHCN4-KO AVN and compare them to those of wild type AVN cells: cells isolated from ciHCN4-KO AV nodes showed a decrease in the If current density, and normal kinetics. In conclusion our results indicate that the functional presence of the HCN4 protein in the SAN and AVN of adult mice is required to maintain the heart rate to levels compatible with life.