Human induced pluripotent stem cell-derived atrial cardiomyocytes carrying an 
Atrial fibrillation (AF), the most common cardiac arrhythmia in clinical practice, represents a major public health challenge. Currently over 33 million people worldwide suffer from AF and the burden of the arrhythmia is likely to rise as the population ages . The AF epidemic places tremendous burden on healthcare costs in society with frequent hospitalizations and the associated morbidity and increased mortality . Over the past 15 years, positional cloning and next generation sequencing have identified AF-linked mutations in cardiac ion channels , signaling molecules, and sarcomeric proteins . Although genetic approaches to the mechanisms of AF have provided important insights into the underlying pathophysiology, direct impact of these discoveries to the bedside care of patients has been limited in part because in vitro methods to characterize mutations associated with AF fail to capture the full spectrum of functional changes . Furthermore, cardiac ion channels in transgenic murine models are species specific and may not precisely reproduce the human AF phenotype. There is increasing interest in developing cellular models of disease that are genetically matched to specific patients using human induced pluripotent stem cells (iPSCs) . This approach has been used to generate large numbers of patient-specific iPSC lines and differentiate them into specific cardiac cell types, including atrial, nodal, and ventricular types . Human iPSC-derived atrial (a) CMs have emerged as a new model to not only study AF mechanisms but also test pharmacological responses to antiarrhythmic drugs as they preserve the exact genetic context of the AF-linked mutations . We previously identified SCN5A (encoding cardiac Na v 1.5 sodium channel) mutations associated with AF . Here, we applied iPSC technology as well as a CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated 9) approach performing electrophysiological, pharmacological, and mechanistic analyses to explore the underlying mechanisms of AF-linked SCN5A mutations. We showed that patient-specific AF iPSC-aCMs exhibited a striking in vitro electrophysiological phenotype of AF-linked SCN5A mutations, and transcriptomic analyses suggested that the nitric oxide (NO) signaling pathway modulated late sodium currents (I Na,L ) and triggered AF. 

SCN5A familial AF kindreds We enrolled a 62-year-old white man who underwent an atrio-ventricular (AV) node ablation and implantation of a single-chamber pacemaker for drug-refractory AF . An electrocardiogram (EKG) from the proband showed ventricular pacing with underlying AF ( A). As the proband reported a family history, ascertainment of family members revealed familial AF in a three-generation kindred ( B). Sequencing SCN5A in the proband and affected members identified a rare non-synonymous coding region mutation (E428K). The heterozygous G→A point mutation conferred substitution of a conserved glutamic acid residue with a lysine residue of SCN5A channel that is highly conserved across vertebrate species ( C–1E). We enrolled a 19-year-old African American man who presented with symptomatic persistent AF at 17 years of age. An EKG from the proband while on AV nodal blocking drugs showed AF with controlled ventricular rates ( A). The patient had a strong family history of AF, with both his mother and maternal grandmother developing AF at a relatively young age ( B). Sequencing SCN5A in the proband identified a rare non-synonymous coding region mutation (N470K) that was highly conserved across vertebrate species ( C and S1D). Genotype-phenotype correlation The SCN5A -E428K proband (I:1, B) who presented with symptomatic AF at 52 years was initially treated with sotalol and then amiodarone. However, he underwent AV node ablation and pacemaker implantation because he developed persistent AF. The proband's daughter (II:2) developed AF when 48 years old but, despite sotalol treatment, she continued to experience symptoms and was eventually treated with rate control therapy. The proband's grandson (III:1) presented with symptomatic AF at 35 years of age and underwent successful pulmonary vein isolation (PVI). He remains asymptomatic on no medications. describes the clinical characteristics of the familial AF kindreds. The SCN5A -N470K proband (I:1) who presented with symptomatic AF at 17 years of age  was initially treated with AV nodal blocking agents but then underwent a successful PVI. The proband's mother (II:2), who developed early-onset AF in the absence of underlying cardiac disease, was initially treated with sotalol but, with frequent recurrences, also underwent a successful PVI. The proband's maternal grandmother presented with minimally symptomatic AF at age 52 years and has successfully been treated with rate control therapy. Generation of AF iPSCs Human iPSCs were generated from the SCN5A -mutation probands and unaffected family members who do not carry the variant (control). Independent clones of each cell line were derived from peripheral blood mononuclear cells by Sendai virus-based reprogramming as previously described . Patient-specific iPSCs were maintained on human recombinant vitronectin-coated plates in mTesR1 Basal medium. The generated iPSCs cell lines expressed pluripotency markers OCT4 and SOX2 ( F). Gene correction of AF iPSCs using CRISPR/Cas9 To validate the pathogenicity of the AF-linked variant SCN5A- E428K, we generated an isogenic-control line by targeted gene correction (GC). Using CRISPR-Cas9 and ribonucleoprotein (RNP) complex containing the GC sequence and homology arms as the repair template, we generated a GC-E428K iPSC line ( A) with positive clones and site-specific GC confirmed by genomic sequencing ( B). The GC-E428K iPSCs expressed the typical pluripotency markers including OCT4 and SOX2 ( F) and maintained a normal karyotype ( C). Generation and characterization of iPSC-aCMs The iPSCs cell lines were first differentiated into CMs using the monolayer-based method and a coated layer of extracellular matrix (ECM), and then a retinoic acid (RA)-based protocol was applied to differentiated cells into iPSC-aCMs as previously described . To analyze the specific types of CMs generated from RA-treated versus control cells, we first examined relative gene expression levels of atrial and ventricular CM markers in iPSC-derived CMs from AF119 family . Real-time qPCR revealed that RA-treated iPSC-CMs exhibited increased expression of atrial markers (NPPA, Kv1.5) and decreased expression of ventricular (MYL2, MYH7) markers . We then examined the percentage of cells expressing ventricular marker MYL2 and atrial markers Kv1.5 and MYL7 . Flow cytometry revealed a significant decrease in the percentage of cells expressing MYL2 in RA-treated cells compared with DMSO-treated cells ( A–S3B). We showed a significant increase in the percentage of cells expressing Kv1.5 in RA-treated cells compared with DMSO-treated cells ( C–S3F). We further examined the second atrial marker MYL7 and ventricular marker MYL2, and confirmed that most RA-treated cells are differentiated into atrial cells ( G–S3J). Meanwhile, we showed a significant increase in the percentage of cells expressing Kv1.5 in RA-treated N470K iPSC-aCMs ( E–F). Abnormal electrophysiological profiles in AF iPSC-aCMs We first determined electrophysiological phenotype of iPSC-aCMs in a monolayer. Multi-electrode array (MEA) recordings were used to characterize cardiac field potential properties of control, E428K, E428K-GC iPSC-aCMs. The E428K atrial iPSC-CMs exhibited faster beating frequency, higher incidence of peak-to-peak interval variability ( A–3C, , and ), and prolonged field potential duration (FPD) compared with controls ( D and 3E). Similar results were found in the second independently derived SCN5A -N470K iPSC-aCMs ( A–S4E, ). We also showed that E428K AF iPSC-aCMs displayed spontaneous arrhythmogenic activity with beat-to-beat irregularity ( and ) suggestive of an AF-like phenotype . Video S1. Normal beating of iPSC-aCMs derived from the unaffected family member (control) who does not harbor the SCN5A-E428K mutation Video S2. Increased beating frequency and arrhythmogenicity in the iPSC-aCMs derived from the AF proband carrying SCN5A-E428K mutation At the single-cell level, we characterized action potential (AP) profiles of the iPSC-aCMs cell lines, AP parameters including action potential duration at 90% repolarization (APD 90 ), maximal upstroke velocity (dV/dt max ), resting membrane potential (RMP), and the time between two adjacent peaks (interval). The E428K mutation iPSC-aCMs displayed shorter interval, faster upstroke, and prolonged APD compared with the control and E428K-GC cells ( A–4E, ). Additionally, triggered-like beats were observed in the E428K mutant AF iPSC-aCMs ( A). In the SCN5A -N470K iPSC-aCMs, we also observed shorter intervals, faster dV/dtmax, and prolonged APD 90 compared with the control cell line, with no significant differences in RMP between the cell lines ( F–S4K, ). We then investigated calcium transients in AF iPSC-aCMs because irregular APs associated with cardiac arrhythmias have been associated with abnormal calcium transients in CMs . The larger calcium transient amplitude exhibited by E428K mutant iPSC-aCMs may be due to differences in the spontaneous rate and sarcoplasmic reticulum calcium loading with the irregular calcium rhythm reflecting irregular AP firing ( A–S5C). AF iPSC-aCMs displayed enhanced I Na,L We characterized sodium currents (I Na ) in iPSC-aCMs by a voltage clamp configuration, and showed that I Na amplitude was not significantly changed in the E428K mutant AF iPSCs ( F and 4G). The AF iPSC-aCMs exhibited a minor hyperpolarization shift but there was no significant change. The V 1/2 was −42.6 ± 3.5 mV, −46.4 ± 3.6 mV, and −44.7 ± 3.0 mV in control, E428K, and E428K-GC iPSC-aCMs ( H). However, there was a marked increase in I Na,L in AF iPSC-aCMs but not in control and E429K-GC cells ( I and 4J). When we determined the biophysical properties of the SCN5A -N470K iPSC-aCMs, it also showed increased I Na,L ( L–S4Q). The enhanced I Na,L may underlie the prolonged FPD, APD, and triggered-like beats ( , A–4E, and ), consistent with a gain-of-function mechanism for the SCN5A mutation. We investigated SCN5A channel protein expression and found that there were no significant differences across the three cell lines ( D). To determine if enhanced I Na,L is caused by E428K or N470K mutation-induced altered gating, we overexpressed the SCN5A -E428K mutation in human embryonic kidney (HEK) 293 cells. However, both the mutation and the wild-type (WT) SCN5A channels failed to exhibit persistent inward sodium currents . Enhanced I Na,L , elicited in SCN5A mutation AF iPSC-aCMs but not in a heterologous expression system, suggested that channel dysfunction only becomes evident in the CM environment. One possible explanation is altered interactions with sodium channel partners, present in aCMs but absent in HEK293 cells. Differential mRNA-level expression of NO signaling pathways in AF iPSC-aCMs To explore potential partners interacting with the cardiac sodium channel, we performed transcriptomic analysis of the E428K control, E428K, and E428K GC iPSC-aCM lines. The major Gene Ontology (GO) categories identified by RNA sequencing and the most significantly modulated in the E428K iPSC-aCMs included cell differentiation, proliferation, and morphogenesis . We selected the NO signaling pathway because studies have shown that I Na,L is enhanced in pathological states and regulated by cellular pathways including calmodulin kinase (CaMK), NO synthase (NOS), phosphoinositide 3-kinase (PI3K)-Akt, and protein kinase C (PKC) . We hypothesized that mRNA-level expression of signaling pathways involved in I Na,L post-translational modification in the AF iPSC-aCMs might differ from control cells. We performed RNA sequencing to explore differential expressed genes (DEGs) of E428K AF iPSC-aCMs, and identified a total of 5,351 genes with 1,688 upregulated and 3,663 downregulated in the AF iPSC-aCMs. GO pathway analysis of DEGs revealed involvement of NO signaling pathways . We showed that genes such as NOS1 , NOS1 adapter protein ( NOS1AP ), NOS interacting protein ( NOSIP ), calmodulin 1 ( CALM1 ), dimethylarginine dimethylaminohydrolase 1 ( DDAH1 ), dihydrofolate reductase ( DHFR ), and lysophospholipase 1 ( LYPLA1 ) involved in NO-mediated signal transduction and positive regulation of NO biosynthetic processes were upregulated in E428K AF iPSC-aCMs but rescued in E428K-GC cells ( A–S7G). Importantly, common genetic variants in genes encoding NOS1 and NOS1AP , important for NO production in CMs, have been associated with arrhythmias . We hypothesized that upregulation of NOS1 and NOS1AP in the NO signaling pathway may underlie enhanced I Na,L in AF iPSC-aCMs. We validated the expression of NOS1 and NOS1AP in E428K and control cells ( A–6C). Increased expression of NOS1 , NOS1AP , and NO regulation of the cardiac sodium channel prompted us to determine if an NO blocker (N-ethylmaleimide) modulated I Na,L . We showed a reduction in I Na,L in the AF iPSC-aCMs after treatment with N-ethylmaleimide ( D). We then determined Nav1.5 nitrosylation across the three cell lines as nitrosylation of Nav1.5 increases I Na,L . We showed that E428K AF iPSC-aCMs increased Nav1.5 nitrosylation which was rescued in E428K-GC cells ( E), indicating increased I Na , L in the mutant AF cell lines was associated with nitrosylation of Nav1.5. We further assessed expression profiles of cardiac ion channels in E428K mutant iPSC-aCMs and showed differential mRNA-level expression ( H–S7N). Notably, upregulation of both ryanodine receptor type 2 ( RyR2 ) and sarcoplasmic endoplasmic reticulum calcium ATPase 2 ( SERCA2 ) may be linked with enhanced calcium handling in AF atrial iPSC-CM lines ( A and J–S7K). Ranolazine rescued abnormal electrophysiological phenotype in AF iPSC-aCMs We showed that 20 μM ranolazine, a I Na,L blocker, significantly reduced I Na,L in AF iPSC-aCMs ( A and 7B) and replicated these findings in the MEA recordings and calcium transient measurement showing that ranolazine reduced the peak-peak interval variability, beating frequency, and calcium transient amplitude of the AF iPSC-aCMs ( C–7H). Our data suggested that targeted inhibition of the I Na,L with ranolazine decreased the aberrant cellular electrophysiological phenotype of the AF iPSC-aCMs. 

We enrolled a 62-year-old white man who underwent an atrio-ventricular (AV) node ablation and implantation of a single-chamber pacemaker for drug-refractory AF . An electrocardiogram (EKG) from the proband showed ventricular pacing with underlying AF ( A). As the proband reported a family history, ascertainment of family members revealed familial AF in a three-generation kindred ( B). Sequencing SCN5A in the proband and affected members identified a rare non-synonymous coding region mutation (E428K). The heterozygous G→A point mutation conferred substitution of a conserved glutamic acid residue with a lysine residue of SCN5A channel that is highly conserved across vertebrate species ( C–1E). We enrolled a 19-year-old African American man who presented with symptomatic persistent AF at 17 years of age. An EKG from the proband while on AV nodal blocking drugs showed AF with controlled ventricular rates ( A). The patient had a strong family history of AF, with both his mother and maternal grandmother developing AF at a relatively young age ( B). Sequencing SCN5A in the proband identified a rare non-synonymous coding region mutation (N470K) that was highly conserved across vertebrate species ( C and S1D). 

The SCN5A -E428K proband (I:1, B) who presented with symptomatic AF at 52 years was initially treated with sotalol and then amiodarone. However, he underwent AV node ablation and pacemaker implantation because he developed persistent AF. The proband's daughter (II:2) developed AF when 48 years old but, despite sotalol treatment, she continued to experience symptoms and was eventually treated with rate control therapy. The proband's grandson (III:1) presented with symptomatic AF at 35 years of age and underwent successful pulmonary vein isolation (PVI). He remains asymptomatic on no medications. describes the clinical characteristics of the familial AF kindreds. The SCN5A -N470K proband (I:1) who presented with symptomatic AF at 17 years of age  was initially treated with AV nodal blocking agents but then underwent a successful PVI. The proband's mother (II:2), who developed early-onset AF in the absence of underlying cardiac disease, was initially treated with sotalol but, with frequent recurrences, also underwent a successful PVI. The proband's maternal grandmother presented with minimally symptomatic AF at age 52 years and has successfully been treated with rate control therapy. 

Human iPSCs were generated from the SCN5A -mutation probands and unaffected family members who do not carry the variant (control). Independent clones of each cell line were derived from peripheral blood mononuclear cells by Sendai virus-based reprogramming as previously described . Patient-specific iPSCs were maintained on human recombinant vitronectin-coated plates in mTesR1 Basal medium. The generated iPSCs cell lines expressed pluripotency markers OCT4 and SOX2 ( F). 

To validate the pathogenicity of the AF-linked variant SCN5A- E428K, we generated an isogenic-control line by targeted gene correction (GC). Using CRISPR-Cas9 and ribonucleoprotein (RNP) complex containing the GC sequence and homology arms as the repair template, we generated a GC-E428K iPSC line ( A) with positive clones and site-specific GC confirmed by genomic sequencing ( B). The GC-E428K iPSCs expressed the typical pluripotency markers including OCT4 and SOX2 ( F) and maintained a normal karyotype ( C). 

The iPSCs cell lines were first differentiated into CMs using the monolayer-based method and a coated layer of extracellular matrix (ECM), and then a retinoic acid (RA)-based protocol was applied to differentiated cells into iPSC-aCMs as previously described . To analyze the specific types of CMs generated from RA-treated versus control cells, we first examined relative gene expression levels of atrial and ventricular CM markers in iPSC-derived CMs from AF119 family . Real-time qPCR revealed that RA-treated iPSC-CMs exhibited increased expression of atrial markers (NPPA, Kv1.5) and decreased expression of ventricular (MYL2, MYH7) markers . We then examined the percentage of cells expressing ventricular marker MYL2 and atrial markers Kv1.5 and MYL7 . Flow cytometry revealed a significant decrease in the percentage of cells expressing MYL2 in RA-treated cells compared with DMSO-treated cells ( A–S3B). We showed a significant increase in the percentage of cells expressing Kv1.5 in RA-treated cells compared with DMSO-treated cells ( C–S3F). We further examined the second atrial marker MYL7 and ventricular marker MYL2, and confirmed that most RA-treated cells are differentiated into atrial cells ( G–S3J). Meanwhile, we showed a significant increase in the percentage of cells expressing Kv1.5 in RA-treated N470K iPSC-aCMs ( E–F). 

We first determined electrophysiological phenotype of iPSC-aCMs in a monolayer. Multi-electrode array (MEA) recordings were used to characterize cardiac field potential properties of control, E428K, E428K-GC iPSC-aCMs. The E428K atrial iPSC-CMs exhibited faster beating frequency, higher incidence of peak-to-peak interval variability ( A–3C, , and ), and prolonged field potential duration (FPD) compared with controls ( D and 3E). Similar results were found in the second independently derived SCN5A -N470K iPSC-aCMs ( A–S4E, ). We also showed that E428K AF iPSC-aCMs displayed spontaneous arrhythmogenic activity with beat-to-beat irregularity ( and ) suggestive of an AF-like phenotype . Video S1. Normal beating of iPSC-aCMs derived from the unaffected family member (control) who does not harbor the SCN5A-E428K mutation Video S2. Increased beating frequency and arrhythmogenicity in the iPSC-aCMs derived from the AF proband carrying SCN5A-E428K mutation At the single-cell level, we characterized action potential (AP) profiles of the iPSC-aCMs cell lines, AP parameters including action potential duration at 90% repolarization (APD 90 ), maximal upstroke velocity (dV/dt max ), resting membrane potential (RMP), and the time between two adjacent peaks (interval). The E428K mutation iPSC-aCMs displayed shorter interval, faster upstroke, and prolonged APD compared with the control and E428K-GC cells ( A–4E, ). Additionally, triggered-like beats were observed in the E428K mutant AF iPSC-aCMs ( A). In the SCN5A -N470K iPSC-aCMs, we also observed shorter intervals, faster dV/dtmax, and prolonged APD 90 compared with the control cell line, with no significant differences in RMP between the cell lines ( F–S4K, ). We then investigated calcium transients in AF iPSC-aCMs because irregular APs associated with cardiac arrhythmias have been associated with abnormal calcium transients in CMs . The larger calcium transient amplitude exhibited by E428K mutant iPSC-aCMs may be due to differences in the spontaneous rate and sarcoplasmic reticulum calcium loading with the irregular calcium rhythm reflecting irregular AP firing ( A–S5C). 

Na,L We characterized sodium currents (I Na ) in iPSC-aCMs by a voltage clamp configuration, and showed that I Na amplitude was not significantly changed in the E428K mutant AF iPSCs ( F and 4G). The AF iPSC-aCMs exhibited a minor hyperpolarization shift but there was no significant change. The V 1/2 was −42.6 ± 3.5 mV, −46.4 ± 3.6 mV, and −44.7 ± 3.0 mV in control, E428K, and E428K-GC iPSC-aCMs ( H). However, there was a marked increase in I Na,L in AF iPSC-aCMs but not in control and E429K-GC cells ( I and 4J). When we determined the biophysical properties of the SCN5A -N470K iPSC-aCMs, it also showed increased I Na,L ( L–S4Q). The enhanced I Na,L may underlie the prolonged FPD, APD, and triggered-like beats ( , A–4E, and ), consistent with a gain-of-function mechanism for the SCN5A mutation. We investigated SCN5A channel protein expression and found that there were no significant differences across the three cell lines ( D). To determine if enhanced I Na,L is caused by E428K or N470K mutation-induced altered gating, we overexpressed the SCN5A -E428K mutation in human embryonic kidney (HEK) 293 cells. However, both the mutation and the wild-type (WT) SCN5A channels failed to exhibit persistent inward sodium currents . Enhanced I Na,L , elicited in SCN5A mutation AF iPSC-aCMs but not in a heterologous expression system, suggested that channel dysfunction only becomes evident in the CM environment. One possible explanation is altered interactions with sodium channel partners, present in aCMs but absent in HEK293 cells. 

To explore potential partners interacting with the cardiac sodium channel, we performed transcriptomic analysis of the E428K control, E428K, and E428K GC iPSC-aCM lines. The major Gene Ontology (GO) categories identified by RNA sequencing and the most significantly modulated in the E428K iPSC-aCMs included cell differentiation, proliferation, and morphogenesis . We selected the NO signaling pathway because studies have shown that I Na,L is enhanced in pathological states and regulated by cellular pathways including calmodulin kinase (CaMK), NO synthase (NOS), phosphoinositide 3-kinase (PI3K)-Akt, and protein kinase C (PKC) . We hypothesized that mRNA-level expression of signaling pathways involved in I Na,L post-translational modification in the AF iPSC-aCMs might differ from control cells. We performed RNA sequencing to explore differential expressed genes (DEGs) of E428K AF iPSC-aCMs, and identified a total of 5,351 genes with 1,688 upregulated and 3,663 downregulated in the AF iPSC-aCMs. GO pathway analysis of DEGs revealed involvement of NO signaling pathways . We showed that genes such as NOS1 , NOS1 adapter protein ( NOS1AP ), NOS interacting protein ( NOSIP ), calmodulin 1 ( CALM1 ), dimethylarginine dimethylaminohydrolase 1 ( DDAH1 ), dihydrofolate reductase ( DHFR ), and lysophospholipase 1 ( LYPLA1 ) involved in NO-mediated signal transduction and positive regulation of NO biosynthetic processes were upregulated in E428K AF iPSC-aCMs but rescued in E428K-GC cells ( A–S7G). Importantly, common genetic variants in genes encoding NOS1 and NOS1AP , important for NO production in CMs, have been associated with arrhythmias . We hypothesized that upregulation of NOS1 and NOS1AP in the NO signaling pathway may underlie enhanced I Na,L in AF iPSC-aCMs. We validated the expression of NOS1 and NOS1AP in E428K and control cells ( A–6C). Increased expression of NOS1 , NOS1AP , and NO regulation of the cardiac sodium channel prompted us to determine if an NO blocker (N-ethylmaleimide) modulated I Na,L . We showed a reduction in I Na,L in the AF iPSC-aCMs after treatment with N-ethylmaleimide ( D). We then determined Nav1.5 nitrosylation across the three cell lines as nitrosylation of Nav1.5 increases I Na,L . We showed that E428K AF iPSC-aCMs increased Nav1.5 nitrosylation which was rescued in E428K-GC cells ( E), indicating increased I Na , L in the mutant AF cell lines was associated with nitrosylation of Nav1.5. We further assessed expression profiles of cardiac ion channels in E428K mutant iPSC-aCMs and showed differential mRNA-level expression ( H–S7N). Notably, upregulation of both ryanodine receptor type 2 ( RyR2 ) and sarcoplasmic endoplasmic reticulum calcium ATPase 2 ( SERCA2 ) may be linked with enhanced calcium handling in AF atrial iPSC-CM lines ( A and J–S7K). 

We showed that 20 μM ranolazine, a I Na,L blocker, significantly reduced I Na,L in AF iPSC-aCMs ( A and 7B) and replicated these findings in the MEA recordings and calcium transient measurement showing that ranolazine reduced the peak-peak interval variability, beating frequency, and calcium transient amplitude of the AF iPSC-aCMs ( C–7H). Our data suggested that targeted inhibition of the I Na,L with ranolazine decreased the aberrant cellular electrophysiological phenotype of the AF iPSC-aCMs. 

Using patient-specific iPSC-aCMs, we showed that an AF-linked SCN5A mutation recapitulated electrophysiological phenotype with enhanced I Na,L , APD prolongation, and triggered-like activity. RNA sequencing of AF iPSC-aCMs revealed differential expression of NO signaling pathways potentially underlying enhanced I Na,L . Targeted inhibition of the I Na,L with ranolazine reduced arrhythmogenic activity and irregular beating in AF iPSC-aCMs. Our findings showed patient-specific AF iPSC-aCMs exhibited striking in vitro phenotypes of AF-linked SCN5A mutations and transcriptomic analyses suggested that the NO signaling pathway modulated IN a,L and triggered AF. Modeling iPSC-aCMs Patient-specific iPSC-aCMs are particularly suited to modeling AF-causing mutations as they elicit cell autonomous phenotypes. Despite variable penetrance of AF and modulation of clinical expression of ion channel mutations by co-morbidities such as hypertension—the so called two-hit hypothesis —iPSC-based models may uncover distinct EP phenotypes and pharmacological responses at the cellular level. Such an approach not only enables us to examine the AF phenotype in different genetic backgrounds but also provides important insights into genotype-phenotype correlations, paving the way for a more personalized approach to AF therapy. Importantly, atrial embryonic stem cell CMs have been used to model pre-clinical testing of atrial-specific AADs, and the use of iPSC-aCMs can complement this model by deriving iPSCs from individual patients with AF. A recent study modeled familial AF by generating iPSCs from two siblings and showed that the common genetic background of the patients induced ion channel remodeling and created a substrate for AF . However, this report used unrelated controls and failure to GC the AF-causing genes may have provided only limited insights into underlying pathophysiological mechanisms of familial AF. Here, we generated and characterized patient-specific atrial iPSCs from the AF proband, unaffected family member not harboring the SCN5A mutation, and the GC-isogenic control line. Our results not only confirmed the E428 variant as AF causing and uncovered a potential role for the NO signaling pathway in modulating I Na,L and triggered AF but also highlighted the importance of isogenic-control iPSC lines in modeling familial AF. The I Na,L and AF We showed that SCN5A mutant iPSC-aCMs displayed prolonged corrected FPD (FPDc)  and enhanced I Na,L , and ranolazine, a I Na,L blocker, reversed the AF-like phenotype  with reduced peak-peak interval variability, beating frequency, triggered-like beats, and calcium transient amplitude, supporting our hypothesis that increased I Na,L is arrhythmogenic and leads to AF. Support for our hypothesis comes from a study by , who also reported that enhanced I Na,L is arrhythmogenic and facilitates AF by prolonging the APD to increase the excitability of atrial myocytes and triggered activity. An increased I Na,L (often referred to as a gain of function in inherited arrhythmia syndromes) represents persistent depolarization of the atrial myocyte with prolongation of the APD, which corresponds to prolonged FPDc in our monolayer recording . Several diseases, including the long QT syndrome and dilated cardiomyopathy, have been associated with mutations in SCN5A , and a number of reports have linked SCN5A gain-of-function mutations and AF . Although the mechanisms whereby some mutations produce AF and others generate ventricular phenotypes like long QT syndrome remains unclear, a possible explanation for the phenotypic variability in sodium channel-linked disease may be related to chamber- or disease-specific interactions between the channel and its partners . Notably, both mutations failed to increase the I Na,L when expressed in HEK293 cells , suggesting that sodium channel dysfunction only becomes evident in the milieu of atrial CMs and potentially altered expression of cellular signaling pathways such as NO linked with I Na,L in AF. One possible explanation is altered interactions with sodium channel partners, present in atrial CMs but absent in HEK293 cells. While we recorded irregular beats with abnormal AP shape that were suggestive of afterdepolarization-like triggered beats as reported by in iPSCs, the possibility that the beats may represent abnormal automaticity should be considered. We showed that SCN5A -E428K iPSC-aCMs displayed prolonged FPDc  and enhanced I Na,L , and ranolazine, a I Na,L blocker, reversed the AF-like phenotype  with reduced peak-peak interval variability, beating frequency, and calcium transient amplitude, supporting our hypothesis that the irregular beats represented afterdepolarization triggered-like beats. The signaling pathways regulating the I Na,L are complex and include calcium/ CaMK, NOS, PKC, PI3K-Akt, and other signaling kinases . One study showed that CaMKIIδ phosphorylated the cardiac sodium channel and enhanced I Na,L , with another reporting that NO enhanced I Na,L under pathophysiological conditions . It is also important to note that the SCN5A -E428K mutation is localized to the interdomain linker between domains 1 and 2. As activation of CaMKII is dependent on the interaction with calcium-calmodulin (CaM) at the inter domain linker region of Nav1.5, it is possible that the E428K mutation leads to increased CaMKII phosphorylation of the cardiac sodium channel and enhanced I Na,L . Using human iPSC-aCMs, we showed for the first time that enhanced I Na,L associated with an AF-linked SCN5A mutation is in part modulated by the NO signaling pathway. RNA sequencing showed that NOS1 and NOS1AP were both upregulated in AF iPSC-aCMs and I Na,L was reduced by the NO inhibitor in aCMs. Collectively, our data suggest that that NO signaling plays an important role in modulating SCN5A and AF pathogenesis. While the precise mechanism(s) by which an SCN5A mutation modulates the NO signaling pathway remains unclear, one possibility is that the E428K variant alters the transcriptional regulation of NO genes. This is supported by our RNA sequence data  and studies showing that SCN5A is not only a regulator of gene transcriptional networks controlling cancer invasion  but also gene expression in neuronal and skeletal muscle cells . Targeted medicine and clinical implications Despite recent advances in catheter-based therapies, symptomatic AF is often treated with antiarrhythmic drugs . However, ∼50% of patients experienced symptomatic recurrence of AF within 6 months of starting membrane-active drugs . The limited pharmacological success of antiarrhythmic therapy for AF is due in part to the heterogeneity of the underlying substrate and failure to target therapy in individual patients . Therefore, an in vivo modeling system that assesses response to targeted therapy would be a major advancement. One potential implication of our findings is the development of mechanism-based therapies for AF. In our kindred family, members with AF carrying the SCN5A mutation did not respond to non-mechanism-based antiarrhythmic therapy. However, based on our findings using patient-specific iPSC-aCMs, it is plausible that a mechanism-based therapy such as the FDA-approved drug ranolazine may be more efficacious as it specifically blocks the I Na,L . Furthermore, some antiarrhythmic drugs are associated with serious toxicities, including drug-induced long QT syndrome, highlighting the importance of screening platforms for precision medicine. Modeling AF with patient-specific iPSC-aCMs has important implications for drug screening and drug discovery as well as enabling individualized assessments of efficacy and safety that guide clinicians toward personalizing therapy even before administering them to patients . Limitations The predictive power of iPSC-CMs to serve as a platform for patient-specific studies may be limited by their immaturity compared with adult CMs, and it is possible that the electrophysiological phenotype of iPSC-aCMs does not fully recapitulate that of human adult atrial CMs. Recently, paracrine factors, including triiodothyronine, insulin-like growth factor-1, and dexamethasone, have been shown to enhance ventricular iPSC-CM maturity . However, less is known about the maturity of iPSC-aCMs and how paracrine factors or extracellular matrix influence their maturity; further studies are required to address these uncertainties. Another limitation of the current study is that even though we showed that upregulated NO signaling may underlie enhanced I Na,L in the AF E428K iPSC-aCMs, precisely how the mutation alters NO pathway gene expression remains unclear. This is an important question with broad implications and may reveal mechanistic insights into how ion channel activity modulates gene expression. In summary, our findings showed that patient-specific iPSC-aCMs exhibited a striking electrophysiological phenotype of an AF-linked mutation that produced minimal changes in vitro , and suggested a link between enhanced I Na,L , transcriptional regulation of NO signaling, and triggered AF. These findings not only provide insights into underlying cellular mechanisms of AF-linked mutations but also highlight potential mechanism-based therapies for this common arrhythmia. 

Patient-specific iPSC-aCMs are particularly suited to modeling AF-causing mutations as they elicit cell autonomous phenotypes. Despite variable penetrance of AF and modulation of clinical expression of ion channel mutations by co-morbidities such as hypertension—the so called two-hit hypothesis —iPSC-based models may uncover distinct EP phenotypes and pharmacological responses at the cellular level. Such an approach not only enables us to examine the AF phenotype in different genetic backgrounds but also provides important insights into genotype-phenotype correlations, paving the way for a more personalized approach to AF therapy. Importantly, atrial embryonic stem cell CMs have been used to model pre-clinical testing of atrial-specific AADs, and the use of iPSC-aCMs can complement this model by deriving iPSCs from individual patients with AF. A recent study modeled familial AF by generating iPSCs from two siblings and showed that the common genetic background of the patients induced ion channel remodeling and created a substrate for AF . However, this report used unrelated controls and failure to GC the AF-causing genes may have provided only limited insights into underlying pathophysiological mechanisms of familial AF. Here, we generated and characterized patient-specific atrial iPSCs from the AF proband, unaffected family member not harboring the SCN5A mutation, and the GC-isogenic control line. Our results not only confirmed the E428 variant as AF causing and uncovered a potential role for the NO signaling pathway in modulating I Na,L and triggered AF but also highlighted the importance of isogenic-control iPSC lines in modeling familial AF. 

Na,L and AF We showed that SCN5A mutant iPSC-aCMs displayed prolonged corrected FPD (FPDc)  and enhanced I Na,L , and ranolazine, a I Na,L blocker, reversed the AF-like phenotype  with reduced peak-peak interval variability, beating frequency, triggered-like beats, and calcium transient amplitude, supporting our hypothesis that increased I Na,L is arrhythmogenic and leads to AF. Support for our hypothesis comes from a study by , who also reported that enhanced I Na,L is arrhythmogenic and facilitates AF by prolonging the APD to increase the excitability of atrial myocytes and triggered activity. An increased I Na,L (often referred to as a gain of function in inherited arrhythmia syndromes) represents persistent depolarization of the atrial myocyte with prolongation of the APD, which corresponds to prolonged FPDc in our monolayer recording . Several diseases, including the long QT syndrome and dilated cardiomyopathy, have been associated with mutations in SCN5A , and a number of reports have linked SCN5A gain-of-function mutations and AF . Although the mechanisms whereby some mutations produce AF and others generate ventricular phenotypes like long QT syndrome remains unclear, a possible explanation for the phenotypic variability in sodium channel-linked disease may be related to chamber- or disease-specific interactions between the channel and its partners . Notably, both mutations failed to increase the I Na,L when expressed in HEK293 cells , suggesting that sodium channel dysfunction only becomes evident in the milieu of atrial CMs and potentially altered expression of cellular signaling pathways such as NO linked with I Na,L in AF. One possible explanation is altered interactions with sodium channel partners, present in atrial CMs but absent in HEK293 cells. While we recorded irregular beats with abnormal AP shape that were suggestive of afterdepolarization-like triggered beats as reported by in iPSCs, the possibility that the beats may represent abnormal automaticity should be considered. We showed that SCN5A -E428K iPSC-aCMs displayed prolonged FPDc  and enhanced I Na,L , and ranolazine, a I Na,L blocker, reversed the AF-like phenotype  with reduced peak-peak interval variability, beating frequency, and calcium transient amplitude, supporting our hypothesis that the irregular beats represented afterdepolarization triggered-like beats. The signaling pathways regulating the I Na,L are complex and include calcium/ CaMK, NOS, PKC, PI3K-Akt, and other signaling kinases . One study showed that CaMKIIδ phosphorylated the cardiac sodium channel and enhanced I Na,L , with another reporting that NO enhanced I Na,L under pathophysiological conditions . It is also important to note that the SCN5A -E428K mutation is localized to the interdomain linker between domains 1 and 2. As activation of CaMKII is dependent on the interaction with calcium-calmodulin (CaM) at the inter domain linker region of Nav1.5, it is possible that the E428K mutation leads to increased CaMKII phosphorylation of the cardiac sodium channel and enhanced I Na,L . Using human iPSC-aCMs, we showed for the first time that enhanced I Na,L associated with an AF-linked SCN5A mutation is in part modulated by the NO signaling pathway. RNA sequencing showed that NOS1 and NOS1AP were both upregulated in AF iPSC-aCMs and I Na,L was reduced by the NO inhibitor in aCMs. Collectively, our data suggest that that NO signaling plays an important role in modulating SCN5A and AF pathogenesis. While the precise mechanism(s) by which an SCN5A mutation modulates the NO signaling pathway remains unclear, one possibility is that the E428K variant alters the transcriptional regulation of NO genes. This is supported by our RNA sequence data  and studies showing that SCN5A is not only a regulator of gene transcriptional networks controlling cancer invasion  but also gene expression in neuronal and skeletal muscle cells . 

Despite recent advances in catheter-based therapies, symptomatic AF is often treated with antiarrhythmic drugs . However, ∼50% of patients experienced symptomatic recurrence of AF within 6 months of starting membrane-active drugs . The limited pharmacological success of antiarrhythmic therapy for AF is due in part to the heterogeneity of the underlying substrate and failure to target therapy in individual patients . Therefore, an in vivo modeling system that assesses response to targeted therapy would be a major advancement. One potential implication of our findings is the development of mechanism-based therapies for AF. In our kindred family, members with AF carrying the SCN5A mutation did not respond to non-mechanism-based antiarrhythmic therapy. However, based on our findings using patient-specific iPSC-aCMs, it is plausible that a mechanism-based therapy such as the FDA-approved drug ranolazine may be more efficacious as it specifically blocks the I Na,L . Furthermore, some antiarrhythmic drugs are associated with serious toxicities, including drug-induced long QT syndrome, highlighting the importance of screening platforms for precision medicine. Modeling AF with patient-specific iPSC-aCMs has important implications for drug screening and drug discovery as well as enabling individualized assessments of efficacy and safety that guide clinicians toward personalizing therapy even before administering them to patients . 

The predictive power of iPSC-CMs to serve as a platform for patient-specific studies may be limited by their immaturity compared with adult CMs, and it is possible that the electrophysiological phenotype of iPSC-aCMs does not fully recapitulate that of human adult atrial CMs. Recently, paracrine factors, including triiodothyronine, insulin-like growth factor-1, and dexamethasone, have been shown to enhance ventricular iPSC-CM maturity . However, less is known about the maturity of iPSC-aCMs and how paracrine factors or extracellular matrix influence their maturity; further studies are required to address these uncertainties. Another limitation of the current study is that even though we showed that upregulated NO signaling may underlie enhanced I Na,L in the AF E428K iPSC-aCMs, precisely how the mutation alters NO pathway gene expression remains unclear. This is an important question with broad implications and may reveal mechanistic insights into how ion channel activity modulates gene expression. In summary, our findings showed that patient-specific iPSC-aCMs exhibited a striking electrophysiological phenotype of an AF-linked mutation that produced minimal changes in vitro , and suggested a link between enhanced I Na,L , transcriptional regulation of NO signaling, and triggered AF. These findings not only provide insights into underlying cellular mechanisms of AF-linked mutations but also highlight potential mechanism-based therapies for this common arrhythmia. 

Familial AF kindred We enrolled two three-generation families with familial AF in a clinical-DNA biorepository. At enrollment, we asked all patients to complete a detailed medical and drug history and a symptom questionnaire and perform detailed phenotyping of probands and family members as previously described. We used the Vanderbilt University Medical Center and University of Illinois at Chicago (UIC) Institutional Review Board-approved protocol to enroll participants following informed written consent. Whole blood was collected for genomic DNA extraction and analysis from the proband and family members that consented to participate in the registry. Mutation screening of the PCR-amplified exons was performed by direct sequencing using the National Heart, Lung, and Blood Institute-supported Resequencing and Genotyping Service as previously described. All variants were validated by re-sequencing an independent PCR-generated amplicon from the subject. Generation of iPSC lines The iPSC lines were generated as previously described . Three independent clones of each cell line were derived from peripheral blood by Sendai virus-based reprogramming. Human iPSCs were maintained on human recombinant vitronectin-coated plates in mTesR1 Basal medium (Gibco). The iPSCs were differentiated into iPSC-aCMs as we previously described . Briefly, we treated cells with 1 μM RA for 5 days with media changes every other day, and a low-glucose metabolic selection step was then used to enrich CM culture. Human iPSC-aCMs showed typical expression of cardiac-specific marker cardiac troponin-T (cTnT), and atrial-specific marker Kv1.5. GC of AF atrial iPSCs using CRISPR-Cas9 We edited iPSCs heterozygous for SCN5A p.E428K using CRISPR-Cas9. Briefly, we designed allele-specific guide (g) RNAs (5′-CATCGCTGAGACCAAGGAGA-3′) using the CRISPR program to target exon 10 containing the mutant allele. The iPSCs were treated with 10uM Y-27632 for an hour pre-electroporation, and then dissociated into single cells and electroporated with an RNP complex of Cas9 and sequence-guided (sg)RNA along with single-stranded oligodeoxynucleotides (ssODN) containing the WT template using the Neon Transfection System (Invitrogen). Immediately after electroporation, cells were transferred into one well of a Matrigel-coated 24-well plate containing 500 μL of mTesR with 10 μM Y-27632. After expansion for several days, we used half the cells to analyze editing efficiency with next-generation sequencing. Upon confirming editing efficiency, we then manually sorted 96 individual cells to establish single-cell colonies. A total of 21 edited clones were selected for expansion and verification of GC by sequencing. In addition to edited clones, we selected two unedited clones that were exposed to the genome-editing pipeline but remained unmodified. Karyotype analysis Karyotype analysis was performed by the Cytogenetics laboratory at WiCell Research Institute Inc. Cells were harvested and chromosomes were analyzed using the giemsa trypsin wright (GTW) banding method. Twenty metaphase cells were analyzed, all of which were concluded to have a normal karyotype (46, XY). RNA sequencing We extracted RNA with Maxwell RSC simplyRNA Cells Kit (Promega AS1390) based on manufacturer's instructions. RNA samples were normalized to 250 ng, and library prep was carried out using the Universal Plus mRNA-Seq kit (NuGen 0520-A01) as written in the product manual (NuGen M01485 v5). In brief, RNA underwent poly-A selection, enzymatic fragmentation, and generation of double-stranded cDNA using a mixture of Oligo (dT) and random priming. The cDNA underwent end repair, ligation of dual-index adaptors, strand selection, and 15 cycles of PCR amplification. We measured purified library concentrations with the Qubit 1X dsDNA HS Assay Kit (Invitrogen Q33231), and libraries were pooled in equimolar amounts based on the Qubit concentration and TapeStation average size and run on MiniSeq for index balancing. The final, purified pool was quantified by qPCR. Raw reads were aligned to the reference genome hg38 using STAR . ENSEMBL gene expression was quantified using FeatureCounts . Normalized and differential expression statistics were computed using edgeR , and p values were adjusted for multiple testing using the false discovery rate (FDR) correction of Benjamini and Hochberg. We identified DEGs with q value <0.05 and a log 2 fold change ≥2 between control and AF iPSC-aCMs. We performed unsupervised hierarchical clustering of all DEG using the Euclidean distance and complete linkage method and generated volcano plots using R. Up- and downregulated genes were analyzed separately using the DAVID functional annotation tool against the GO Biological Process (GO) database. MEA recordings of atrial iPSC-CMs We used an MEA recording system (MEA2100-Lite-System) to record iPSC-aCMs with the spontaneously beating iPSC-aCMs plated on fibronectin-coated MEA plates. The FPD in both SCN5A -E428K and control iPSC-CMs was determined by pacing at 2 Hz. Fridericia's formula was used to standardize the beat rate-associated dispersed FPD:FPDc = FPD/(Beat period) 0.3 . We recorded the extracellular electrograms in DMEM/HEPES at 37°C with the data analyzed using MC-Rack Program (Multichannel Systems). We added ranolazine to the extracellular solution at the desired final concentration. Patch-clamp recordings of iPSC-aCMs Patch-clamp measurements were performed in whole-cell configurations using an Axopatch 200B amplifier controlled by pClamp10 software through an Axon Digidata 1440A . For measurement of sodium currents, external solution contains 50 mM NaCl, 2 mM CaCl 2 , 100 mM CsCl, 1 mM MgCl 2 , 10 mM glucose, 10 mM HEPES, and 0.001 mM nifedipine (pH 7.4 with CsOH). Pipette solution contained 135 mM CsCl, 10 mM NaCl, 2 mM CaCl 2 , 5 mM EGTA, 10 mM HEPES, and 5 mM Mg-ATP (pH 7.2 with CsOH). The current data were analyzed with pCLAMP Software . Sodium current densities were calculated according to whole-cell current amplitude and capacitance values obtained from the amplifier following electronic subtraction of the capacitive transients . The AP was obtained using the current-clamp mode by injecting stimulus current (2–4 ms, 20–200 pA) at a frequency of 1 Hz . The current-clamp recordings were recorded in a solution containing 140 mM NaCl, 5.4 mM KCl, 1 mM MgCl 2 , 10 mM glucose, 2.0 mM CaCl 2 , and 10 mM HEPES (pH 7.4 with NaOH). The pipette solution contained 120 mM KCl, 1 mM MgCl 2 , 10 mM HEPES, 2 mM Mg-ATP, and 10 mM EGTA (pH 7.3 with KOH). The AP data were analyzed with pCLAMP Software (Molecular Devices). Statistics Data are presented as mean ± standard deviation (SD). For datasets with normal distributions, statistical significance was determined by Student's t test (two-tailed) for two groups or one-way ANOVA for multiple groups with post-hoc Bonferroni corrections. RNA sequencing data availability The RNA sequencing data in this article were deposited in the GEO database and the accession number is GSE154084 . 

We enrolled two three-generation families with familial AF in a clinical-DNA biorepository. At enrollment, we asked all patients to complete a detailed medical and drug history and a symptom questionnaire and perform detailed phenotyping of probands and family members as previously described. We used the Vanderbilt University Medical Center and University of Illinois at Chicago (UIC) Institutional Review Board-approved protocol to enroll participants following informed written consent. Whole blood was collected for genomic DNA extraction and analysis from the proband and family members that consented to participate in the registry. Mutation screening of the PCR-amplified exons was performed by direct sequencing using the National Heart, Lung, and Blood Institute-supported Resequencing and Genotyping Service as previously described. All variants were validated by re-sequencing an independent PCR-generated amplicon from the subject. 

The iPSC lines were generated as previously described . Three independent clones of each cell line were derived from peripheral blood by Sendai virus-based reprogramming. Human iPSCs were maintained on human recombinant vitronectin-coated plates in mTesR1 Basal medium (Gibco). The iPSCs were differentiated into iPSC-aCMs as we previously described . Briefly, we treated cells with 1 μM RA for 5 days with media changes every other day, and a low-glucose metabolic selection step was then used to enrich CM culture. Human iPSC-aCMs showed typical expression of cardiac-specific marker cardiac troponin-T (cTnT), and atrial-specific marker Kv1.5. 

We edited iPSCs heterozygous for SCN5A p.E428K using CRISPR-Cas9. Briefly, we designed allele-specific guide (g) RNAs (5′-CATCGCTGAGACCAAGGAGA-3′) using the CRISPR program to target exon 10 containing the mutant allele. The iPSCs were treated with 10uM Y-27632 for an hour pre-electroporation, and then dissociated into single cells and electroporated with an RNP complex of Cas9 and sequence-guided (sg)RNA along with single-stranded oligodeoxynucleotides (ssODN) containing the WT template using the Neon Transfection System (Invitrogen). Immediately after electroporation, cells were transferred into one well of a Matrigel-coated 24-well plate containing 500 μL of mTesR with 10 μM Y-27632. After expansion for several days, we used half the cells to analyze editing efficiency with next-generation sequencing. Upon confirming editing efficiency, we then manually sorted 96 individual cells to establish single-cell colonies. A total of 21 edited clones were selected for expansion and verification of GC by sequencing. In addition to edited clones, we selected two unedited clones that were exposed to the genome-editing pipeline but remained unmodified. 

Karyotype analysis was performed by the Cytogenetics laboratory at WiCell Research Institute Inc. Cells were harvested and chromosomes were analyzed using the giemsa trypsin wright (GTW) banding method. Twenty metaphase cells were analyzed, all of which were concluded to have a normal karyotype (46, XY). 

We extracted RNA with Maxwell RSC simplyRNA Cells Kit (Promega AS1390) based on manufacturer's instructions. RNA samples were normalized to 250 ng, and library prep was carried out using the Universal Plus mRNA-Seq kit (NuGen 0520-A01) as written in the product manual (NuGen M01485 v5). In brief, RNA underwent poly-A selection, enzymatic fragmentation, and generation of double-stranded cDNA using a mixture of Oligo (dT) and random priming. The cDNA underwent end repair, ligation of dual-index adaptors, strand selection, and 15 cycles of PCR amplification. We measured purified library concentrations with the Qubit 1X dsDNA HS Assay Kit (Invitrogen Q33231), and libraries were pooled in equimolar amounts based on the Qubit concentration and TapeStation average size and run on MiniSeq for index balancing. The final, purified pool was quantified by qPCR. Raw reads were aligned to the reference genome hg38 using STAR . ENSEMBL gene expression was quantified using FeatureCounts . Normalized and differential expression statistics were computed using edgeR , and p values were adjusted for multiple testing using the false discovery rate (FDR) correction of Benjamini and Hochberg. We identified DEGs with q value <0.05 and a log 2 fold change ≥2 between control and AF iPSC-aCMs. We performed unsupervised hierarchical clustering of all DEG using the Euclidean distance and complete linkage method and generated volcano plots using R. Up- and downregulated genes were analyzed separately using the DAVID functional annotation tool against the GO Biological Process (GO) database. 

We used an MEA recording system (MEA2100-Lite-System) to record iPSC-aCMs with the spontaneously beating iPSC-aCMs plated on fibronectin-coated MEA plates. The FPD in both SCN5A -E428K and control iPSC-CMs was determined by pacing at 2 Hz. Fridericia's formula was used to standardize the beat rate-associated dispersed FPD:FPDc = FPD/(Beat period) 0.3 . We recorded the extracellular electrograms in DMEM/HEPES at 37°C with the data analyzed using MC-Rack Program (Multichannel Systems). We added ranolazine to the extracellular solution at the desired final concentration. 

Patch-clamp measurements were performed in whole-cell configurations using an Axopatch 200B amplifier controlled by pClamp10 software through an Axon Digidata 1440A . For measurement of sodium currents, external solution contains 50 mM NaCl, 2 mM CaCl 2 , 100 mM CsCl, 1 mM MgCl 2 , 10 mM glucose, 10 mM HEPES, and 0.001 mM nifedipine (pH 7.4 with CsOH). Pipette solution contained 135 mM CsCl, 10 mM NaCl, 2 mM CaCl 2 , 5 mM EGTA, 10 mM HEPES, and 5 mM Mg-ATP (pH 7.2 with CsOH). The current data were analyzed with pCLAMP Software . Sodium current densities were calculated according to whole-cell current amplitude and capacitance values obtained from the amplifier following electronic subtraction of the capacitive transients . The AP was obtained using the current-clamp mode by injecting stimulus current (2–4 ms, 20–200 pA) at a frequency of 1 Hz . The current-clamp recordings were recorded in a solution containing 140 mM NaCl, 5.4 mM KCl, 1 mM MgCl 2 , 10 mM glucose, 2.0 mM CaCl 2 , and 10 mM HEPES (pH 7.4 with NaOH). The pipette solution contained 120 mM KCl, 1 mM MgCl 2 , 10 mM HEPES, 2 mM Mg-ATP, and 10 mM EGTA (pH 7.3 with KOH). The AP data were analyzed with pCLAMP Software (Molecular Devices). 

Data are presented as mean ± standard deviation (SD). For datasets with normal distributions, statistical significance was determined by Student's t test (two-tailed) for two groups or one-way ANOVA for multiple groups with post-hoc Bonferroni corrections. 

The RNA sequencing data in this article were deposited in the GEO database and the accession number is GSE154084 . 

D.D. and L.H. conceived the project, designed the experiments, analyzed the data, and wrote the manuscript. L.H. performed electrophysiological experiments. M.Z. carried out cell culture, and generated atrial and GC of iPSC lines with the help of H.C. H.C., O.T.L., M.Z., A.S., E.L., and S.-W.Y. performed flow cytometry, calcium handling, qPCR, western blotting, and other molecular analyses. B.C. assisted with obtaining the clinical data. M.M.C., L.F., and J.R. analyzed RNA sequencing data. S.-G.O. and J.C.W. assisted with designing the iPSC differentiation approaches. All authors revised the manuscript. 

The authors declare no competing interests. 

