iPSC-derived human cardiomyocytes from atrial fibrillation patients show increased frequency rate and alteration of If current

Atrial Fibrillation (AF) is the most common type of cardiac arrhythmias. Since in aged population AF is frequently observed as a complication of other cardiovascular disorders, it is difficult to understand the pathophysiologic causes of the disease. Genetic mutations and variants have been associated with AF but their characterization is often problematic because of the lack of a proper cellular system. Patient-derived induced pluripotent stem cells (iPSC) may represent a suitable cellular model. We characterized a family with three siblings affected by persistent AF whose early onset suggest a genetic basis. We isolated dermal fibroblasts from two AF and one healthy subjects and generated iPSC transducing them with the Yamanaka’s factors (OCT4, KLF4, SOX2, c-MYC). AF- and control-derived iPSC were spontaneously differentiated into CMs (AF-CM) through embryoid bodies formation. Around day 12 spontaneous beating areas appeared; the beating clusters were dissected and cultured up to 30-60 days. CM differentiation was confirmed by RT-PCR and immunocytochemical analyses. Around day 30, we performed electrophysiological and molecular analyses. Spontaneous action potentials (AP) were recorded from beating clusters. Beating rate was significantly higher in AF-CMs of both patients than in controls. We then analyzed the pacemaker current If in dissociated cells. We found that If density was larger (-5.2±1.1 pA/pF n=5, -8.3± 1.0 pA/pF n=9) and activated at more positive potentials in AF-CMs of both patients (V1/2 -72.5±4.5 mV n=6, -72.1±2 mV n=9) than in control CM (-3.1±0.4 pA/pF; V1/2, -82.7±2.2 mV, n=11) . Nevertheless, cells responded normally to isoproterenol stimulation. qPCR analysis of cardiac proteins and channels identified a higher expression of the HCN4 isoform, in agreement with functional data. In order to identify the genetic causes of these alterations, we performed whole-exome analysis. Results showed that the three siblings carry two single nucleotide heterozygous missense mutations in cellular filaments, that are absent in 7,000 control genomes. We are presently trying to link these genetic mutations with electrophysiological alterations in order to endorse cellular filaments as novel therapeutic targets for AF. In conclusion our data demonstrate that iPSC are a valid cellular model to study complex pathologies with genetic alterations not directly linked to ion channels.