I CANALI HCN E IL LORO RUOLO NELL'ECCITABILITÀ CARDIACA E NEURONALE - PARTE I: CARATTERIZZAZIONE DI CELLULE 'SINOATRIAL-LIKE' DA PRECURSORI CD166+ DERIVATI DA MESCS - PARTE II: SCREENING GENETICO DI PAZIENTI AFFETTI DA EPILESSIA DEL LOBO TEMPORALE

TITLE: I CANALI HCN E IL LORO RUOLO NELL’ECCITABILITÀ CARDIACA E NEURONALE PARTE I: CARATTERIZZAZIONE DI CELLULE “SINOATRIAL-LIKE” DA PRECURSORI CD166+ DERIVATI DA mESCs PARTE II: SCREENING GENETICO DI PAZIENTI AFFETTI DA EPILESSIA DEL LOBO TEMPORALE - GENERAL INTRODUCTION HCN channels and the related current (If/h) contribute to many important physiological processes, such as cardiac pacemaker activity and neuronal excitability, in particular setting the membrane resting potential and integrating dendritic signals. - PART I This part of my project was aimed at developing a biological pacemaker, that is a cellular substrate able to induce ectopic spontaneous activity in the host tissue. Our strategy is based on differentiation of embryonic stem cells (ESCs) and selection of cell progenitors based on expression of the marker CD166, at day 8 of mouse ESC differentiation. CD166 is transiently co-expressed with HCN4, a marker of the cardiac pacemaker tissue, during heart development. In culture, CD166-selected cells develop into spontaneously beating cells that express low levels of ventricular genes (Cx43, Kv4.2, HCN2, Nkx2.5) and high levels of genes involved both in SAN development (Tbx18, Tbx3, Isl-1, Shox2) and function (Cx30.2, HCN4, HCN1, CaV1.3). Furthermore CD166+ cells form an autorhythmic syncytium made of cells morphologically similar to and with the electrophysiological properties of murine SAN myocytes. Acetylcholine decreases (−23%) and isoproterenol increases (+57%) their beating rate because of the presence of both the muscarinic and β-adrenergic receptors. In co-cultures, these CD166+ cells are also able to pace neonatal ventricular myocytes at a rate faster than their own. Finally, CD166-selected cells do not express pluripotency genes and don’t induce teratomas in vivo. - PART II Because Ih current is important in the control of neuronal excitability we have carried out a genomic screening of patients affected by temporal lobe epilepsy (TLE) in the search of rare nucleotidic alterations (SNPs) in the genes encoding for the HCN1 and HCN2 isoforms and MiRP1 (KCNE2) which contribute to native Ih. After DNA extraction from blood samples and efforts in PCR condition setting, we’ve been analyzing their sequencings. Up to now, we’ve found 12 synonymous polymorphisms (SNPs) in hHcn2 and only a deletion in hHcn1 (del 2 gly 73-74). All these DNA variations have been already recorded in the specific database (dbSNP) and were not correlated to epilepsy. Furthermore, we’ve not found any alteration in KCNE2 sequence. - GENERAL CONCLUSIONS Due to the importance of HCN channels in cellular excitability, they may represent important targets for therapeutic interventions. A cell-based biological pacemaker development would contribute to ameliorate decisively patient quality of life overcoming the major drawbacks associated with electronic devices. Understanding HCN-mediated epilepsy pathological mechanisms could therefore allow to discover drugs able to limit the channel alterations and thus contribute to improve patient everyday life.