A variant noncoding region regulates Prrx1 and predisposes to atrial arrhythmias

Rationale Atrial Fibrillation (AF) is the most common cardiac arrhythmia diagnosed in clinical practice. Genome-wide association studies have identified AF-associated common variants across 100+ genomic loci, but the mechanism underlying the impact of these variant loci on AF susceptibility in vivo has remained largely undefined. One such variant region, highly associated with AF, is found at 1q24, close to PRRX1, encoding the Paired Related Homeobox 1 transcription factor. Objective To identify the mechanistic link between the variant region at 1q24 and AF predisposition. Methods and results The mouse orthologue of the noncoding variant genomic region (R1A) at 1q24 was deleted using CRISPR genome editing. Among the genes sharing the topologically associated domain with the deleted R1A region (Kifap3, Prrx1, Fmo2, Prrc2c), only the broadly expressed gene Prrx1 was downregulated in mutants, and only in cardiomyocytes. Expression and epigenetic profiling revealed that a cardiomyocyte lineage-specific gene program (Mhrt, Myh6, Rbm20, Tnnt2, Ttn, Ckm) was upregulated in R1A−/− atrial cardiomyocytes, and that Mef2 binding motifs were significantly enriched at differentially accessible chromatin sites. Consistently, Prrx1 suppressed Mef2-activated enhancer activity in HL-1 cells. Mice heterozygous or homozygous for the R1A deletion were susceptible to atrial arrhythmia induction, had atrial conduction slowing and more irregular RR intervals. Isolated R1A−/− mouse left atrial cardiomyocytes showed lower action potential upstroke velocities and sodium current, as well as increased systolic and diastolic calcium concentrations compared to controls. Conclusion The noncoding AF variant region at 1q24 modulates Prrx1 expression in cardiomyocytes. Cardiomyocyte-specific reduction of Prrx1 expression upon deletion of the noncoding region leads to a profound induction of a cardiac lineage-specific gene program and to propensity for AF. These data indicate that AF-associated variants in humans may exert AF predisposition through reduced PRRX1 expression in cardiomyocytes. FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Fondation Leducq

Integration of Scandinavian genetic data with UK biobank data implicates the RBM20 gene with atrial fibrillation pathogenesis

Purpose Atrial fibrillation (AF) is the most common sustained arrhythmia. It carries a large healthcare burden and is associated with serious complications. The arrhythmia has a substantial genetic component and is associated with several structural genes, including the gene TTN. A recent large genome-wide association study on AF found an association to RBM20. The RBM20 gene is a splicing factor targeting TTN, RYR2 and CAMK2D among other cardiac genes. Using Next-Generation Sequencing and data derived from the UK Biobank, we aimed to reveal the role of RBM20 in AF. Methods and results We examined the burden of rare (Minor allele frequency (MAF)<0.01%) RBM20 loss-of-function (LOF) variants in whole-exome sequencing data from the UK Biobank (n=175,280). AF was defined by ICD9/10, while individuals without AF were used as controls. Association tests aggregating rare variants in RBM20 using the Efficient Variant-Set Mixed Model Association Test (SMMAT) were performed to assess the effect of LOF RBM20 variants, adjusted for age, sex and principal components. We identified 33 LOF variants in RBM20, which were significantly enriched in AF (P=0.0087). To examine the effect of rare missense RBM20 variants in the splicing of TTN, we screened an in-house cohort of 531 Scandinavian early-onset AF patients using targeted sequencing. We filtered for rare (MAF<0.1%) and deleterious (defined as combined annotation dependent depletion score >20) variants and identified nine missense variants and three novel LOF variants in RBM20. To evaluate the effect of these RBM20 variants, we constructed a series of human RBM20 single nucleotide base exchange mutants. The splicing activity of the variants was measured with RT-qPCR on HEK293 cells transfected with a TTN241–3 splicing reporter. Four of these variants resulted in a significantly altered splicing activity in TTN, with the largest effect observed for LOF variants. In order to examine the biological effect of RBM20 variants on structural changes in atrial tissue, we used a Norwegian Brown rat animal model with loss of RBM20. In this model, Transmission Electron Microscopy revealed altered sarcomere and mitochondrial structure in its atrial cardiomyocytes. Furthermore, nanopore RNA sequencing of atrial tissue from the aforementioned animal model indicated altered expression in several key cardiac genes, including TTN and PITX2. Conclusion Rare RBM20 LOF variants are significantly enriched in AF cases, seen in a large population of 175,000 individuals. We demonstrated that the effect of LOF RBM20 on alternative TTN splicing can be detected on an individual level in patients with AF. Studies using an animal model indicates that LOF in RBM20 may affect atrial function through altered expression of several genes in the atria, and may cause structural changes in the atrial cardiomyocytes. This suggests that RBM20 may be involved in AF pathogenesis mediated through an atrial cardiomyopathy. FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Novo Nordisk Foundation Pre-Graduate Scholarships (NNF18OC0053094)The Hallas Møller Emerging Investigator grant (Novo Nordisk Foundation (NNF17OC0031204))

Abstract P498: Polycystin-1 Regulates Excitation-contraction Coupling In Atrial Cardiomyocytes

Patients with Autosomal Dominant Polycystic Kidney disease (ADPKD) have multiple cardiovascular manifestations, including increased susceptibility to arrhythmias. Mutations in polycystin-1 (PC1) encoding gene accounts for 85% cases of ADPKD, whereas mutations in polycystin-2 (PC2) only accounts for 15%. In kidney cells, PC1 interacts with PC2 to form a protein complex at the primary cilia to regulate calcium influx via PC2. However, cardiomyocytes are non-ciliated cells and the role of both PC1 and PC2 in atrial cardiomyocytes remains unknown. We have previously demonstrated that PC1 regulates action potentials and calcium handling to fine-tune ventricular cardiomyocyte contraction. Here, we hypothesize that PC1 regulates action potentials and calcium handling in atrial cardiomyocytes independent of PC2 actions. To test this hypothesis, we differentiated human induced pluripotent stem cells (iPSC) into atrial cardiomyocytes (iPSC-aCM) using previously published protocols. To determine the contribution of PC1/PC2 in atrial excitation-contraction coupling, protein expression was knocked down utilizing specific siRNA constructs, for each protein, or a universal control siRNA transfected using lipofectamine RNAiMAX. We measured action potentials using the potentiometric dye FluoVolt and intracellular calcium with Fura-2 AM or Fluo-4. Changes in fluorescence were monitored using a multiwavelength IonOptix system. iPSC-aCM were paced at 2 Hz to synchronize the beating pattern using field electrical stimulation. Our data shows that PC1 ablation significantly decreased action potential duration at 50% and 80% of repolarization, by 24% and 23%, respectively. Moreover, we observed that PC1 knockdown significantly reduced calcium transient amplitude elicited by field electrical stimulation without changes in calcium transient decay. Interestingly, PC2 knockdown did not modify calcium transients in atrial cardiomyocytes (iPSC-aCM). Our data suggest that PC1 regulates atrial excitation-contraction coupling independent of PC2 actions. This study warrants further investigation into atrial dysfunction in ADPKD patients with PC1 mutations.

Abstract P445: A Combinatorial Approach Comprehensively Improves The Maturation Of Human Induced Pluripotent Stem Cell Derived Atrial Cardiomyocytes

Introduction: The limited success of pharmacological approaches to atrial fibrillation ( AF ) is due to limitations of in vitro and in vivo models and inaccessibility of human atrial tissue. Patient-specific induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) are a robust platform to model the heterogeneous myocardial substrate of AF, but their immaturity limits their fidelity. Objective: We hypothesized that a combinatorial approach of biochemical (triiodothyronine [ T3 ], insulin-like growth factor-1 [ IGF-1 ], and dexamethasone; collectively TID ), bioenergetic (fatty acids [ FA ]), and electrical stimulation ( ES ) will enhance electrophysiological ( EP ), structural, and metabolic maturity of iPSC- a CMs. Methods: We assessed maturation with whole cell patch clamping, calcium transients, immunofluorescence (IF), Seahorse Analyzer, contractility assay, RT-PCR, Western Blotting, and RNA sequencing (RNAseq). Using a time series with RNAseq we identified signaling pathways and transcriptional regulation that drive EP, structural, and metabolic atrial development and compared iPSC-aCM maturity with human aCMs (haCMs) obtained from the same patient. Results: TID+FA+ES significantly improved structural organization and cell morphology ( Fig. 1a ), enhanced membrane potential stability and improved depolarization ( Fig. 1b ), improved Ca 2+ kinetics with faster and increased Ca 2+ release from sarcoplasmic reticulum ( Fig. 1c ), and increased expression of Na + , Ca 2+ , and K + channels, markers of structural maturity, FA metabolism, and oxidative phosphorylation ( Fig. 1d ). There was no difference in each parameter between TID+FA+ES iPSC-aCMs and haCMs from the same patient. Conclusion: Our optimized, combinatorial TID+FA+ES approach markedly enhanced EP, structural, and metabolic maturity of human iPSC-aCMs, which will be useful for elucidating the genetic basis of AF developing precision drug therapies.

A Variant Noncoding Region Regulates Prrx1 and Predisposes to Atrial Arrhythmias

Rationale: Atrial Fibrillation (AF) is the most common cardiac arrhythmia diagnosed in clinical practice. Genome-wide association studies have identified AF-associated common variants across 100+ genomic loci, but the mechanism underlying the impact of these variant loci on AF susceptibility in vivo has remained largely undefined. One such variant region, highly associated with AF, is found at 1q24, close to PRRX1, encoding the Paired Related Homeobox 1 transcription factor. Objective: To identify the mechanistic link between the variant region at 1q24 and AF predisposition. Methods and Results: The mouse orthologue of the noncoding variant genomic region (R1A) at 1q24 was deleted using CRISPR genome editing. Among the genes sharing the topologically associated domain with the deleted R1A region (Kifap3, Prrx1, Fmo2, Prrc2c), only the broadly expressed gene Prrx1 was downregulated in mutants, and only in cardiomyocytes. Expression and epigenetic profiling revealed that a cardiomyocyte lineage-specific gene program (Mhrt, Myh6, Rbm20, Tnnt2, Ttn, Ckm) was upregulated in R1A-/- atrial cardiomyocytes, and that Mef2 binding motifs were significantly enriched at differentially accessible chromatin sites. Consistently, Prrx1 suppressed Mef2-activated enhancer activity in HL-1 cells. Mice heterozygous or homozygous for the R1A deletion were susceptible to atrial arrhythmia induction, had atrial conduction slowing and more irregular RR intervals. Isolated R1A-/- mouse left atrial cardiomyocytes showed lower action potential upstroke velocities and sodium current, as well as increased systolic and diastolic calcium concentrations compared to controls. Conclusions: The noncoding AF variant region at 1q24 modulates Prrx1 expression in cardiomyocytes. Cardiomyocyte-specific reduction of Prrx1 expression upon deletion of the noncoding region leads to a profound induction of a cardiac lineage-specific gene program and to propensity for AF. These data indicate that AF-associated variants in humans may exert AF predisposition through reduced PRRX1 expression in cardiomyocytes.

Modelling of atrial fibrillation at physiologically relevant scales enabled by massive expansion of native human atrial cardiomyocytes

Funding Acknowledgements Type of funding sources: Public hospital(s). Main funding source(s): LUMC Background Current in vitro models of atrial fibrillation have limited translational potential due to a lack of relevant human physiology or the inability to reach the high activation frequencies present in human atrial fibrillation. Absence of relevant models is the result of a general deficit of readily available and standardized sources of well-differentiated human atrial cardiomyocytes. Therefore, we aimed to immortalize native human atrial cardiomyocytes to produce natural and standardized lines of these cells. Methods Human fetal atrial cardiomyocytes were transduced with a lentiviral vector directing myocyte-specific and doxycycline-inducible expression of simian virus 40 large T antigen. Addition of doxycycline to the culture medium pushed cardiomyocytes towards a highly proliferative phenotype (proliferation up to 10^12 cells). These cells were labelled hiAMs (human immortalised Atrial Myocytes). After differentiation upon doxycycline removal, hiAM cells were characterized using various molecular, biological and electrophysiological assays. Results Following cardiomyogenic differentiation, hiAMs no longer expressed the proliferation marker Ki67, revealed striated α-actinin and troponin T staining patterns and displayed synchronous contractions. Optical voltage mapping of hiAM monolayers revealed excitable cells showing homogeneous spreading of action potentials at 22.5 ± 3.1 cm/s with a mean APD80 of 139 ± 22 ms. Addition of flecainide (10 µM) to hiAM monolayers decreased the conduction velocity by 35% and increased the APD80 by 107%. Dofetilide (10 nM) addition had no effect on the conduction velocity, but did increase the APD80 by 81%. Due to their scalability, monolayers of hiAMs as big as 10 cm2 showing homogenous action potential propagation could easily be created. Following high-frequency electrical pacing, rotors could be induced with an average activation frequency of 7.5 ± 0.9 Hz. Infusion of flecainide during arrhythmic activity resulted in termination of the rotor in 18 of 24 attempts (75%), whereas addition of 0.1% DMSO (vehicle control) did not result in termination in any of the attempts. Dofetilide infusion did not result in termination. However, it did lower the average activation frequency to 2.1 ± 0.7 Hz. Conclusion We have generated first-of-a-kind lines of human atrial cardiomyocytes, allowing massive cell expansion under proliferation conditions and robust formation of cross-striated, contractile and excitable cardiomyocytes after differentiation. These characteristics allow, for the first time, the modelling, at a large-scale, of human atrial arrhythmias with frequencies similar to human atrial fibrillation. With the generation of hiAMs, a user-friendly, clinically-relevant and much-anticipated human atrial research model has been produced. Abstract Figure. hiAM AF Model

The SCN5A point mutation M1875T, associated with familial atrial fibrillation, causes a gain-of-function effect of the cardiac Nav1.5 channel in atrial cardiomyocytes

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): British Heart Foundation Leducq Foundation Background The point mutation M1875T in the SCN5A gene, which encodes the pore-forming α-subunit of the cardiac voltage-gated Na+ channel Nav1.5, has been associated with familial atrial fibrillation (AF), but its effects on atrial cardiomyocyte electrophysiology is unclear. Aim To investigate the effect of the point mutation M1875T on atrial electrophysiological parameters. Methods In a novel heterozygous knock-in murine model (Scn5a-M1875T+/-), whole-cell patch clamp electrophysiology was used to investigate Na+ currents in left atrial (LA) cardiomyocytes isolated from hearts of young adult mice (10-16 weeks). LA microelectrode and optical mapping recordings were used to study action potential (AP) characteristics. Cardiac size and function were measured by transthoracic echocardiography. Atrial Scn5a gene and Nav1.5 protein expression were assessed by Rt-PCR and Western blot. Results The Na+ current was increased in cardiomyocytes isolated from Scn5a-M1875T+/- LA (wildtype (WT) -22.7 ± 0.9 pA/pF (N = 14, n = 115); Scn5a-M1875T+/- -28.3 ± 1.1 pA/pF (N = 15, n = 117)). Scn5a-M1875T+/- intact isolated superfused LA had an elevated AP amplitude (100 ms pacing cycle length (PCL): WT 86.4 ± 0.9 mV (N = 8, n = 24); Scn5a-M1875T+/- 91.2 ± 0.7 mV (N = 8, n = 25)) and a faster peak upstroke velocity (100 ms PCL: WT 127.98 ± 3.28 mV/ms; Scn5a-M1875T+/- 142.80 ± 3.98 mV/ms). AP duration (APD) was not different apart from a small APD shortening at slow rates. Echocardiography revealed no difference in size and function at the age of investigation. Atrial Scn5a gene and Nav1.5 protein expression were comparable. When challenged with flecainide (1 µM), Scn5a-M1875T+/- LA showed less conduction slowing than WT (100 ms PCL: WT -10.43 ± 1.27 cm/s (N = 12); Scn5a-M1875T+/- -6.10 ± 1.34 cm/s (N = 12)). 5 µM flecainide caused significant increase in WT refractoriness (7/12 atria lost 1:1 capture at PCL ≤ 120 ms) compared to Scn5a-M1875T+/- (1/12). Conclusion(s): SCN5A point mutation M1875T increases the Na+ current in atrial cardiomyocytes and intact atria, leading to a faster AP upstroke and an attenuated response to flecainide. Abstract Figure 1: Current-Voltage relationship