SummaryHuman induced pluripotent stem cell (hiPSC)-derived atrial cardiomyocytes (CMs) hold great promise for elucidating underlying cellular mechanisms that cause atrial fibrillation (AF). In order to use atrial-like hiPSC-CMs for arrhythmia modeling, it is essential to better understand the molecular and electrophysiological phenotype of these cells. We performed comprehensive molecular, transcriptomic, and electrophysiologic analyses of retinoic acid (RA)-guided hiPSC atrial-like CMs and demonstrate that RA results in differential expression of genes involved in calcium ion homeostasis that directly interact with an RA receptor, chicken ovalbumin upstream promoter-transcription factor 2 (COUP-TFII). We report a mechanism by which RA generates an atrial-like electrophysiologic signature through the downstream regulation of calcium channel gene expression by COUP-TFII and modulation of calcium handling. Collectively, our results provide important insights into the underlying molecular mechanisms that regulate atrial-like hiPSC-CM electrophysiology and support the use of atrial-like CMs derived from hiPSCs to model AF.

Background: Epidemiological studies have established obesity as an independent risk factor for atrial fibrillation (AF), but the underlying pathophysiological mechanisms remain unclear. Reduced cardiac sodium channel expression is a known causal mechanism in AF. We hypothesized that obesity decreases Nav1.5 expression via enhanced oxidative stress, thus reducing I Na , and enhancing susceptibility to AF. Methods: To elucidate the underlying electrophysiological mechanisms a diet-induced obese mouse model was used. Weight, blood pressure, glucose, F 2 -isoprostanes, NOX2 (NADPH oxidase 2), and PKC (protein kinase C) were measured in obese mice and compared with lean controls. Invasive electrophysiological, immunohistochemistry, Western blotting, and patch clamping of membrane potentials was performed to evaluate the molecular and electrophysiological phenotype of atrial myocytes. Results: Pacing-induced AF in 100% of diet-induced obese mice versus 25% in controls ( P <0.01) with increased AF burden. Cardiac sodium channel expression, I Na and atrial action potential duration were reduced and potassium channel expression (Kv1.5) and current ( I Kur ) and F 2 -isoprostanes, NOX2, and PKC-α/δ expression and atrial fibrosis were significantly increased in diet-induced obese mice as compared with controls. A mitochondrial antioxidant reduced AF burden, restored I Na , I Ca,L , I Kur , action potential duration, and reversed atrial fibrosis in diet-induced obese mice as compared with controls. Conclusions: Inducible AF in obese mice is mediated, in part, by a combined effect of sodium, potassium, and calcium channel remodeling and atrial fibrosis. Mitochondrial antioxidant therapy abrogated the ion channel and structural remodeling and reversed the obesity-induced AF burden. Our findings have important implications for the management of obesity-mediated AF in patients. Graphic Abstract: A graphic abstract is available for this article.

Lymphoid-specific tyrosine phosphatase (LYP), a member of the protein tyrosine phosphatase (PTP) family of signaling enzymes, is associated with a broad spectrum of autoimmune diseases. Herein we describe our structure-based lead optimization efforts within a 6-hydroxy-benzofuran-5-carboxylic acid series culminating in the identification of compound 8b, a potent and selective inhibitor of LYP with a Ki value of 110 nM and more than 9-fold selectivity over a large panel of PTPs. The structure of LYP in complex with 8b was obtained by X-ray crystallography, providing detailed information about the molecular recognition of small-molecule ligands binding LYP. Importantly, compound 8b possesses highly efficacious cellular activity in both T- and mast cells and is capable of blocking anaphylaxis in mice. Discovery of 8b establishes a starting point for the development of clinically useful LYP inhibitors for treating a wide range of autoimmune disorders.

PEST-domain-enriched tyrosine phosphatase is an important positive regulator of anaphylaxis. Pharmacological inhibition of its activity together with glucocorticoid administration provide an effective rescue for PSA in mice.

A frameshift (fs) mutation in the natriuretic peptide precursor A (NPPA) gene, encoding a mutant atrial natriuretic peptide (Mut-ANP), has been linked with familial atrial fibrillation (AF) but the underlying mechanisms by which the mutation causes AF remain unclear. We engineered 2 transgenic (TG) mouse lines expressing the wild-type (WT)-NPPA gene (H-WT-NPPA) and the human fs-Mut-NPPA gene (H-fsMut-NPPA) to test the hypothesis that mice overexpressing the human NPPA mutation are more susceptible to AF and elucidate the underlying electrophysiologic and molecular mechanisms. Transthoracic echocardiography and surface electrocardiography (ECG) were performed in H-fsMut-NPPA, H-WT-NPPA, and Non-TG mice. Invasive electrophysiology, immunohistochemistry, Western blotting and patch clamping of membrane potentials were performed. To examine the role of the Mut-ANP in ion channel remodeling, we measured plasma cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) activity in the 3 groups of mice. In H-fsMut-NPPA mice mean arterial pressure (MAP) was reduced when compared to H-WT-NPPA and Non-TG mice. Furthermore, injection of synthetic fs-Mut-ANP lowered the MAP in H-WT-NPPA and Non-TG mice while synthetic WT-ANP had no effect on MAP in the 3 groups of mice. ECG characterization revealed significantly prolonged QRS duration in H-fsMut-NPPA mice when compared to the other two groups. Trans-esophageal (TE) atrial pacing of H-fsMut-NPPA mice

IMPORTANCEThe association between obesity, an established risk factor for atrial fibrillation (AF), and response to antiarrhythmic drugs (AADs) remains unclear.OBJECTIVE To test the hypothesis that obesity differentially mediates response to AADs in patients with symptomatic AF and in mice with diet-induced obesity (DIO) and pacing induced AF. DESIGN, SETTING, AND PARTICIPANTSAn observational cohort study was conducted including 311 patients enrolled in a clinical-genetic registry. Mice fed a high-fat diet for 10 weeks were also evaluated. The study was conducted from January 1, 2018, to June 2, 2019.MAIN OUTCOMES AND MEASURES Symptomatic response was defined as continuation of the same AAD for at least 3 months. Nonresponse was defined as discontinuation of the AAD within 3 months of initiation because of poor symptomatic control of AF necessitating alternative rhythm control therapy. Outcome measures in DIO mice were pacing-induced AF and suppression of AF after 2 weeks of treatment with flecainide acetate or sotalol hydrochloride.RESULTS A total of 311 patients (mean [SD] age, 65 [12] years; 120 women [38.6%]) met the entry criteria and were treated with a class I or III AAD for symptomatic AF. Nonresponse to class I AADs in patients with obesity was less than in those without obesity (30% [obese] vs 6% [nonobese]; difference, 0.24; 95% CI, 0.11-0.37; P = .001). Both groups had similar symptomatic response to a potassium channel blocker AAD. On multivariate analysis, obesity, AAD class (class I vs III AAD [obese] odds ratio [OR], 4.54; 95% Wald CI, 1.84-11.20; P = .001), female vs male sex (OR, 2.31; 95% Wald CI, 1.07-4.99; P = .03), and hyperthyroidism (OR, 4.95; 95% Wald CI, 1.23-20.00; P = .02) were significant indicators of the probability of failure to respond to AADs. Pacing induced AF in 100% of DIO mice vs 30% (P < .001) in controls. Furthermore, DIO mice showed a greater reduction in AF burden when treated with sotalol compared with flecainide (85% vs 25%; P < .01). CONCLUSIONS AND RELEVANCEResults suggest that obesity differentially mediates response to AADs in patients and in mice with AF, possibly reducing the therapeutic effectiveness of sodium channel blockers. These findings may have implications for the management of AF in patients with obesity.

Mutations in multiple genes have been linked with familial atrial fibrillation (AF) but the underlying pathophysiologic mechanisms and implications for therapy remain poorly understood. To characterize the electrophysiological phenotype of an AF-linked SCN5A mutation and assess novel mechanism-based therapies, we generated patient-specific atrial induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from a carrier with an SCN5A-E428K mutation and an unaffected family member using a differentiation protocol optimized for atrial differentiation generated atrial iPSC-CMs. The atrial iPSC-CMs carrying SCN5A-E428K displayed increased window and late Na+ current (INa,L), increased beating frequency and irregularity with triggered beats and prolongation of the action potential duration (APD) versus control atrial iPSC-CMs. The multi-electrode array (MEA) recordings of mutant atrial iPSC-CMs showed spontaneous arrhythmogenic activity with beat-to-beat irregularity. We further showed that targeted inhibition of the INa,L with ranolazine normalized the aberrant electrophysiologic phenotype in the SCN5A-E428K atrial iPSC-CMs and mexiletine reversed the sodium channel gating defects, and reduced beating frequency and irregularity. Our study illustrates the potential use of atrial iPSC-CMs for modeling AF in a dish, elucidating the underlying cellular mechanisms, and identifying novel mechanism-based therapies custom-tailored for individual patients.

(LQT3). A pediatric patient with severe ventricular arrhythmias was recently determined to carry a missense mutation in SCN5A that putatively leads to a replacement of glutamine by proline at residue 1475 (Q1475P). We sought to uncover, through population-based mathematical modeling of ion channel gating: (1) what defects in gating at the molecular level could account for cellular electrophysiological recordings; and (2) whether these alterations to channel gating could reproduce the patient's LQT3 phenotype. Electrophysiology experiments, performed in HEK293 cells, demonstrated that, compared with Wild Type (WT) Na v 1.5 channels, Q1475P channels exhibited: (1) a positive shift in the activation curve; (2) a positive shift in the inactivation curve; (3) faster inactivation; and (4) faster recovery from inactivation. Simulations attempted to recapitulate these results by randomizing parameters in a published Markov model of Na v 1.5 gating, generating a large (n=10,000) population of channel variants, filtering to match WT and Q1475P channel behaviors. This produced subpopulations of channel variants describing the gating of either genotype. The analysis suggested that the channel gating steps most likely to be affected by the mutation include transitions from: (1) open to closed, (2) inactivated to closed; and (3) closed to inactivated. In addition, when simulated Na v 1.5 channels were incorporated into a model of the human ventricular action potential, Q1475P channels were more likely than WT channels to produce arrhythmogenic early afterdepolarizations (EADs). The results provide important insight into this patient's LQT3 phenotype and demonstrate how population-based modeling can generate mechanistic hypotheses about alterations in ion channel gating that result from mutations.