CELLULAR MODELS FOR STUDYING AND TREATING ABNORMAL CARDIAC AND NEURONAL EXCITABILITY

Abnormalities in the electrical activity of cardiac and neuronal cells can cause serious pathologies that affect an increasing number of individuals. In the heart, dysfunctions in the autorhythmic activity of its natural pacemaker, the sinoatrial node (SAN), are becoming progressively more common due to population ageing and are currently treated with implantation of an electronic pacemaker. In the brain, a typical disorder due to neuron hyperexcitability is epilepsy, which is the third most common brain disorder. The first part of this thesis studied a cell-based method to generate a biological pacemaker, that is a cellular substrate able to drive the beating rate of the host tissue. Potentially, a biological pacemaker presents several advantages compared to electronic devices: it can respond to nervous system regulation and does not need maintenance or substitution. We adopted the strategy of stem cell differentiation to select early pacemaker precursors. As a selection marker, we used CD166, since it is transiently but specifically expressed in the SAN during embryonic development. Accordingly, here we show by immunofluorescence that CD166 is co-expressed with the SAN marker HCN4 in mouse forming heart at day 10.5. CD166-selected cells were previously shown to express SAN genes. In this study, we functionally characterized CD166-selected cells showing that they present morphological and electrophysiological characteristics of SAN cells, expressing functional HCN and calcium channels. They can respond to autonomic nervous system regulation: isoproterenol increases (+57%) and acetylcholine decreases (-23%) their firing rate. Importantly, they are also able to drive the rate of rat neonatal cardiomyocytes in co-culture, thus acting as a real pacemaker in vitro. We also set the foundations for translating this selection protocol to human cells, showing that CD166 is expressed during human induced pluripotent stem cell (hiPSC) cardiac differentiation. This study demonstrated that at early stages CD166 selects mouse pacemaker precursors that function as SAN-like cells in vitro. Moreover, it opens promising perspective for translating the method to hiPSCs, with the aim of generating patient-specific biological pacemakers. The second part of this thesis presents a study aimed at identifying genetic mutations in epileptic patient and assessing whether they may contribute to the phenotype, in order to provide new targets for pharmacological treatments. We found M54T MiRP1 mutation in an epileptic patient and in her daughter. MiRP1 was shown to act as β-subunit of HCN channels, whose alterations in the brain have been previously linked to the epileptogenic process. Moreover, the same M54T mutation has been recently found in a patient with long-QT and sinus bradycardia, causing a decrease of HCN4, but not of HCN2 current when expressed in neonatal rat cardiomyocytes. Therefore, we decided to evaluate whether M54T mutation was involved in excitability alterations proper of the epileptic disorder, acting on HCN channels. We firstly verify the effect of M54T mutation in CHO cells by co-expressing wild-type (WT) MiRP1, M54T mutant or both (WT/M54T) with either HCN2 or HCN4. Electrophysiological analysis shows that M54T mutation does not affect HCN2 or HCN4 voltage dependence and current densities. We observed a slower activation of HCN2 at -95 and -85 mV and a slower deactivation of HCN4 in the range of -25/-45 mV, however no difference were found at other voltages. Notably, no effect of WT MiRP1 on HCN2 or HCN4 properties was observed. We then replicated the same co-transfection experiments in rat neonatal cortical neurons. Again, we found no difference in HCN2 or HCN4 voltage dependence and current densities, but only a slower HCN2 activation at -85 mV in M54T MiRP1 transfected neurons compared to control and WT. Finally, we assessed whether M54T mutation may alter neuronal excitability acting on other ion channels, by transfecting neurons with only WT or mutated MiRP1. We found that WT MiRP1 induced a two-fold decrease in the threshold of action potential firing and increased their rate, causing an overall increased cell excitability compared to control. This effect was reverted by M54T mutation. These data oppose a possible contribution of M54T mutation to the epileptogenesis and also rule against the previously acknowledged role of MiRP1 in modulating HCN channels.