BRADICARDIA E ARRESTO CARDIACO DOVUTI A KNOCKOUT INDUCIBILE E CONDIZIONALE DEL GENE PACEMAKER HCN4

In mammals the heart rhythm originates from specialized myocytes of the sinoatrial node (SAN) due to the presence of a spontaneous diastolic depolarization sustained mainly by the pacemaker If current. Most of the data on the role of this current were collected by in vitro experiment and only recently initial data on the role of If in intact animal have become available. The knockout mouse technology, applied to the Hcn4 gene, the main molecular component of the native pacemaker channel, has demonstrated that HCN4 proteins are necessary to support life during embryonic development, but less clear results were found in the adult animal. 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 report here the functional consequences of a reduced contribution of the If current evaluated in vivo by telemetric ECG recordings, and in vitro by single cell electrophysiology. In our inducible and cardiac specific HCN4 knockout (ci-HCN4-KO) mouse model, ablation of HCN4 leads to progressive development of severe bradycardia (~50% reduction of original rate) and AV block, eventually leading to heart arrest and death of the animals in about 5 days. Similar experiments carried out in control animals did not result in any rate modifications or premature death Electrical properties of single cells isolated from ciHCN4-KO SAN cells revealed a decrease both in spontaneous firing (by ~60%).) and in the If current density (by ~70%). We then evaluated the extent of the gene KO process by western blot and immunolabelling experiments that confirmed the effective reduction of the HCN4 protein within the node. In ciHCN4-KO animals the residual If was normally sensitive to β-AR modulation, and the permanence of rate response to β-AR stimulation was observed both in vivo and in vitro. In conclusion our results indicate that the functional presence of the HCN4 protein in the SAN of adult mice is required to maintain the heart rate to levels compatible with life.