RICERCA DI MUTAZIONI NEL CANALE PACEMAKER HCN4 IN PAZIENTI AFFETTI DA DISTURBI DEL RITMO CARDIACO

Cardiovascular diseases are widespread and they are a major cause of death in the western part of the world. Therefore, identifying their causes precisely with the aim of establishing a preventive and therapeutic strategy has become subject of research also for their social and economic impact. Many studies have now clearly established that the genetic characteristics of a person can be a background predisposing to the onset of various diseases including cardiac arrhythmias. For example, various mutations in genes that code for ion channels are known and their accessory proteins that lead to the development of heart diseases such as the long QT syndrome and bradycardia. My laboratory of the PhD has shown a correlation between a specific mutation in the hHCN4 (Hyperpolarization-activate Cyclic Nucleotide-gated channel) and sinus bradycardia. The screening is a part of research of monogenic diseases and in particular the focus is on genes directly involved in the genesis of the rhythm (HCN channels) and on interacting proteins (CAV3, MiRP1, FLNA). In my thesis I performed a genetic screening in order to identify polymorphisms and mutations in the gene encoding for the hHCN4 pacemaker channel. The HCN4 protein channel is an isoform of the HCN family responsible of the If current. This channel is more expressed at the level of the sinus node, an anatomical region of the heart in which the source of the heart rhythm is located. SAN (sinus atrial node) cells are able to generate action potentials in an autonomous way that spread through the conduction system allowing the contraction of the heart. The current If plays a key role in the slow diastolic depolarization phase, where it leads the membrane potential to threshold values for a new action potential. The heart rate is determined by the duration of the diastolic depolarization phase: the principal current responsible is the If current. The genetic analysis was performed on the gene coding for the channel hHCN4 in patients with cardiac arrhythmias (sinus bradycardia, sinus tachycardia and atrial fibrillation) in order to identify genetic mutations causally related to the pathological phenotype. Among the various polymorphisms we found, the attention was focused on the R524Q mutation in HCN4, identified in patients of the same family with inappropriate sinus tachycardia (IST): it is a non-conservative missense mutation in heterozygosis (located on exon 4, position 1571G> A mRNA, codon CGG in CAG). R524 is located in the initial part of the C-linker, an important region which connects the last transmembrane domain (S6) to CNBD and which has a function in modulating channel gating. To test whether the R524Q polymorphism affects biophysical properties of the HCN4 pacemaker channel and consequently determines sinus tachycardia in patients carrying the mutation, we conducted electrophysiological experiments in heterologous cells (HEK 293) transfected with plasmids expressing the gene respectively hHCN4 wild type, mutated homozygous and heterozygous (hHCN4 coexpression of plasmids with wild-type and mutated hHCN4 homozygous). In some families that have inheritance of the disease, it was not possible to identify a mutation in HCN4. In collaboration with a specialized center that employs NGS (New Generation Sequencing) technology, the screening has been extended to other genes involved in heart disease. We selected two families: the first with bradycardia and atrial fibrillation, and the second with atrial fibrillation and tachycardia. The first experiments on probands have led to the identification of mutations that will be interesting to investigate in the future. In a vitro system we characterized the I57T mutation found in MiRP1, identified in the second family.