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Introduction: Cardiovascular sequelae of targeted cancer therapies may provide novel insights into cardiovascular biology and disease pathogenesis, including atrial fibrillation (AF). The Bruton tyrosine kinase (BTK) inhibitor ibrutinib has revolutionized treatment for B-cell malignancies but increases the incidence of AF compared with conventional chemotherapy. Recent data in mouse models have shown that ibrutinib mediated-AF likely results not from inhibition of BTK (“on target” effect), but from off-target inhibition of a different kinase, C-terminal Src kinase (CSK). The signaling pathway by which CSK inhibition results in AF remains unknown and thus may represent a novel mechanism for AF molecular pathogenesis. Methods: To study how CSK inhibition results in AF in a human derived model, we created atrial specific cardiomyocytes from human induced pluripotent stem cells (hiPSC-aCMs) from a healthy control. We studied cellular monolayers of hiPSC-aCMs using extracellular field potential (EFP) recordings obtained by the Nanion CardioExcyte 96 system after 24 hour exposure to vehicle, ibrutinib, or the second generation BTK inhibitor acalabrutinib. Results: Chamber specificity of hiPSC-aCMs was confirmed by Western blot analysis of subtype-specific markers. Treatment with ibrutinib for 24 hours resulted in striking increase in beat-to-beat variability in spontaneous contraction, an in vitro correlate of arrhythmogenic behavior (mean coefficient-of-variance/second following treatment 0.835 for ibrutinib vs. 0.198 for acalabrutinib vs. 0.165 for DMSO vehicle control, p Conclusion: These experiments provide a novel method of screening for arrhythmogenic potential of targeted cancer therapies and validate atrial-specific hiPSC-CMs as a model system for the study of CSK mediated AF.

Background: While the cardiac sodium channel ( SCN5A ) variants S1103Y and R1193Q have been associated with LQT3 and drug-induced arrhythmia, they are too common to be highly arrhythmogenic: S1103Y allele frequency is 8.1% in Africans and R1193Q 6.1% in East Asians. We have previously reported that S1103Y had no effect on QT in a large biobank or on action potential duration (APD) in iPSC-CMs. Here, we identify a mechanism underlying control of normal baseline repolarization with these variants and study whether the mechanism is generalizable. Methods: Late sodium current (I Na-L ), I Kr , and sensitivity to the blockers dofetilide (I Kr ) and GS967 (I Na-L ) were measured in natural or genome-edited iPSC-CMs carrying S1103Y, R1193Q, or R518X, a common KCNQ1 nonsense variant in the Jervell Lange-Nielsen (JLN) syndrome and unaffected family members. Results: Although APD was unaffected by S1103Y, I Na-L was increased 2~16-fold in SY/YY compared to SS cells (pKr was increased in SY/YY ([SY] 1.2 pA/pF, n=17, pKr and I Na-L and increased dofetilide sensitivity. In R518X JLN iPSC-CMs, I Ks was absent as expected but I Kr was increased (1.9 pA/pF, n=5), and editing R518X to wild-type increased I Ks (1.3 pA/pF, n=10) and dramatically decreased I Kr (0.2 pA/pF, n=4). Conclusions: S1103Y and R1193Q increase I Na-L but unexpectedly do not affect baseline repolarization. We hypothesize that increased I Kr through as yet undetermined mechanisms maintains normal repolarization but at the cost of increased sensitivity to I Kr block. The findings with R518X suggest this may be a general mechanism. Our data show that preposition to genetically- or drug-mediated arrhythmias is determined by a complex interaction among multiple repolarizing currents that is not fully captured by heterologous expression systems.

Background— The D1275N SCN5A mutation has been associated with a range of unusual phenotypes, including conduction disease and dilated cardiomyopathy, as well as atrial and ventricular tachyarrhythmias. However, when D1275N is studied in heterologous expression systems, most studies show near-normal sodium channel function. Thus, the relationship of the variant to the clinical phenotypes remains uncertain. Methods and Results— We identified D1275N in a patient with atrial flutter, atrial standstill, conduction disease, and sinus node dysfunction. There was no major difference in biophysical properties between wild-type and D1275N channels expressed in Chinese hamster ovary cells or tsA201 cells in the absence or presence of β1 subunits. To determine D1275N function in vivo, the Scn5a locus was modified to knock out the mouse gene, and the full-length wild-type (H) or D1275N (DN) human SCN5A cDNAs were then inserted at the modified locus by recombinase mediated cassette exchange. Mice carrying the DN allele displayed slow conduction, heart block, atrial fibrillation, ventricular tachycardia, and a dilated cardiomyopathy phenotype, with no significant fibrosis or myocyte disarray on histological examination. The DN allele conferred gene-dose–dependent increases in SCN5A mRNA abundance but reduced sodium channel protein abundance and peak sodium current amplitudes (H/H, 41.0±2.9 pA/pF at −30 mV; DN/H, 19.2±3.1 pA/pF, P P Conclusions— Although D1275N produces near-normal currents in multiple heterologous expression experiments, our data establish this variant as a pathological mutation that generates conduction slowing, arrhythmias, and a dilated cardiomyopathy phenotype by reducing cardiac sodium current.