Atrial fibrillation modeling with iPSC derived cardiomyocyte

Atrial fibrillation (AF) is the most frequently diagnosed cardiac arrhythmia and is associated with an increased risk of stroke because of the potential for thrombus formation in atrial blood. AF has traditionally been described as a multifactorial sporadic disease; however recently, genome-wide association studies have led to the identification of multiple loci, encoding ion channels, transcription factors, and signaling molecules, that confer increased susceptibility to this type of arrhythmia. Hence in vitro AF models to identify the genetic basis, to examine cell differentiation, to characterize interactions of cells belonging to cardiovascular lineage, are urgently needed in order to improve the comprehension of the pathophysiological basis of AF. We began to characterize three siblings with a history of persistent AF, who were scheduled to undergo surgical ablation following failed pharmacological treatment. The young age of onset of loneAF in the three siblings indicates a strong genetic basis for this form of arrhythmia. Initially using a candidate gene approach, the genomic DNA of one of the three patients has been analyzed but no relevant sequence variations have been identified in genes previously involved with monogenic AF (KCNQ1,KCNH2, KCNE1, KCNE2, and SCN5A). Therefore we are now in process of genetically characterize the family by screening several genes through gene-chip and exons sequencing analysis. In order to provide a valuable experimental platform to model AF disease, primary cultures of dermal fibroblasts were established from all patients. From these skin fibroblasts we started to generate induced pluripotent stem cells (iPSC) by retroviral infection of OCT4, KLF4, SOX2 and c-MYC factors. We have characterized the pluripotency state of our colonies evaluating colony morphology, alkaline phosphatase staining, expression of pluripotency associated transcription factors (OCT4, SOX2, NANOG, REX1), surface markers (SSEA3, SSEA4, TRA1-60, TRA1-81), and silencing of retroviral transgenes. 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 isolate CMs. We were able to record action potentials (APs) with different waveforms from AF-derived CMs versus a normal counterpart. These results, although very preliminary, are extremely encouraging because they suggest that the differences we observed in electrophysiology, borne by the iPSC derived CMs, maybe really due to genetic alterations, thus making the study of both genomic and transcriptomic signatures of these patients of primary importance in the comprehension of the biological basis of AF.