Increased frequency rate and alteration of If current in iPSC-derived atrial fibrillation human cardiomyocytes.

Atrial fibrillation (AF) is the most common sustained arrhythmia worldwide. The mechanisms underlying AF are not fully understood, but multiple pathophysiological pathways have been suggested. On the contrary, the well-established AF risk factors include male sex, advancing age, diabetes, hypertension, heart failure, myocardial infarction, and valvular heart disease. Nevertheless, much of the AF risk variability remains unexplained, leading investigators to look for novel and genetic risk factors. Since 1940, a number of reports have described rare inherited AF disorders mainly associated with cardiac channel mutations. Recently several GWAS studies have shown the association of AF with peculiar chromosomal loci, and, nowadays, parental AF is considered as one of the main risk factor in offspring. We started characterizing a family where three siblings suffered a common persistent AF. The consanguinity, the variability in risk factors, and the young age of onset of AF in these patients (from 44 to 52 years old) strongly suggested a genetic basis for this form of arrhythmia. Since an extensive single candidate gene analysis, comprising several known AF-associated genes, did not reveal any mutation, we approached the entire exome analysis to identify other sequence abnormalities. Preliminary results show that the three siblings are carrying two different single nucleotide mutations in cellular filaments. These mutations, further confirmed by Sanger sequence analysis, are absent in the rest of the family and have not been described in human exome sequence databases. In parallel, we built the cellular-based human in vitro AF model, starting from patients-derived primary cultures of dermal fibroblasts. These cells were transduced with Yamanaka’s factors OCT4, KLF4, SOX2 and c-MYC, using two different systems: either a mix of retroviruses each of them carrying a single gene, or a single lentiviral particle carrying an omni-comprehensive polycistronic RNA. Colonies of pluripotent stem cells (iPSC) were generated using both systems, but the lentiviral infection showed a higher reprogramming efficiency. Nevertheless, karyotype analysis of five independent clones showed a common chromosomal translocation (t17;19) that was absent in fibroblast primary culture, as well as in retroviral infected iPS clones. Following the assessment of pluripotency, we have differentiated AF-derived iPSC into cardiomyocytes (CMs) by a standard differentiation protocol through embryoid bodies formation. The presence of beating cells allowed us to identify and to isolate clusters of CMs whose electrophysiological properties have been analyzed. Cellular spontaneous action potentials were recorded revealing different traces and frequencies in AF-derived CMs versus the normal counterpart. Moreover, after single CM isolation, we measured funny current (If), strongly related to pacemaker activity. We found that the activation of HCN channels in AF-derived CMs is reached before the normal counterpart, thus suggesting a higher cellular excitability. In conclusion we obtained iPSC-derived human CMs that, as shown, represent a valuable and reliable model of AF. We are presently using our model to understand the link between the identified mutations and the recorded functional alterations. Using this tool we will ultimately be able to identify novel therapeutic targets for this form of cardiac arrhythmia.