Voltage Optical Mapping Research Articles

Abstract 4138278: A Titin Missense Variant Causes Atrial Fibrillation

Background: Rare loss-of-function variants in TTN , the gene encoding the sarcomeric protein titin, have been causally associated with atrial fibrillation (AF). Missense TTN variants ( TTN mv) are generally regarded as benign in relation to dilated cardiomyopathy, but their role in AF is unknown. Hypothesis: TTN mv are associated with clinical outcomes and AF pathogenesis. Aims: We aimed to determine if TTN mv are associated with AF/heart failure (HF) hospitalizations and investigate if a rare missense variant ( TTN -T32756I) caused AF using human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs). Methods: Whole exome sequencing was performed in 131 adults at an urban academic healthcare system who identified as non-Hispanic Black (NHB) or Hispanic/Latinx (HL). Multivariable Cox proportional hazard models were used to assess cumulative risk of AF/HF hospitalization. Isogenic TTN -T32756I iPSC-aCM mutants were generated using the CRISPR-Cas9 technique. We used confocal microscopy to assess sarcomeric integrity, MuscleMotion to assess contractility, and optical voltage mapping and patch-clamping to assess the electrophysiological (EP) properties. Results: We identified 137 TTN mvs in 76/131 (58.0%) of subjects, which were most commonly in the TTN A-band. 64 AF-related and 110 HF-related hospitalizations occurred after a median follow-up time of 4.16 years. Hospitalization risk was higher in TTN mv carriers (HR 1.81, Fig. 1A ). We also identified three patients with early-onset AF who shared a rare missense variant T32756I. TTN-T32756I -iPSC-aCMs displayed aberrant contractility ( Fig. 1B-D ), shortened action potential duration ( Fig. 1G-H ), increased activity of cardiac K + channel ( Fig. 1I ), and dysregulated Ca 2+ homeostasis ( Fig. 1J ) without compromising sarcomeric integrity ( Fig. 1E-F ). The titin-binding protein, Four-and-a-Half Lim domains 2 (FHL2), also has increased binding with KCNQ1 and its modulatory subunit KNCE1 in TTN-T32756I -iPSC-aCMs, which may enhance the slow delayed rectifier K + current ( I ks ). Suppression of FHL2 normalized I ks , supporting FHL2 as a mediator of the increased I ks ( Fig. 1K) . Conclusion: In a single-center multi-ethnic AF cohort, TTN mv were associated with increased AF/HF hospitalization, and the T32756I- TTN mv causes ion channel remodeling and an AF-like EP phenotype. These findings establish a mechanistic link between TTN mv, K + ion channels, and sarcomeric regulatory proteins that may represent a novel therapeutic target.

Acquired control over proliferation and differentiation of ventricular cardiomyocytes to break down the mechanisms of cardiac repair

Abstract Funding Acknowledgements None. Loss of myocardial tissue remains a leading cause of morbidity and mortality by causing heart rhythm disturbances and heart failure. The (pre-)clinical effects of inducing cardiac regeneration by cardiac cell therapy have been disappointing. In order to find new curative options for these disabling cardiac diseases a shift of focus towards steering the heart to regenerate itself is needed. The aim of this study was to generate a robust ventricular cardiomyocyte model in which proliferation and differentiation could be tightly controlled to investigate cardiac regeneration. To accomplish this we have generated a line of immortalized neonatal rat ventricular cardiomyocytes (nrVMs) through expression of the cell cycle regulators cyclin-dependent kinase 4 and cyclin D1 (CDK4-D1). The CDK4-D1 expressing nrVMs were positive for the proliferation marker Ki67 in contrast to control nrVMs. Cardiomyogenic differentiation could still be induced after multiple passages (> passage 10) as evidenced by immunocytochemistry and optical voltage mapping. These contractile and excitable cells were indeed positive for the cardiac-specific sarcomeric proteins alpha actinin and troponin T in a sarcomeric pattern similar to native nrVMs. Passaging of the control nrVM cultures, however, ultimately led to a senescent cell population lacking sarcomeric markers after passage 3. Electrophysiological analysis of immortalized monolayers, after inducing cardiomyogenic differentiation, showed not only uniform propagation of electrical signal throughout the cultures, but also re-entrant activity although at low frequency. In conclusion, these data show that were able to immortalize ventricular cardiomyocytes by preserving their contractile and excitable phenotype upon significant expansion. Further optimisation of this model will lead to a research model of ventricular cardiomyocytes which will give us the opportunity to unravel the process of cardiomyogenesis and uncover mechanisms which could allow us to enhance the ability of the heart to regenerate itself.

Fast creation of data-driven low-order predictive cardiac tissue excitation models from recorded activation patterns

Excitable systems give rise to important phenomena such as heat waves, epidemics and cardiac arrhythmias. Understanding, forecasting and controlling such systems requires reliable mathematical representations. For cardiac tissue, computational models are commonly generated in a reaction–diffusion framework based on detailed measurements of ionic currents in dedicated single-cell experiments.Here, we show that recorded movies at the tissue-level of stochastic pacing in a single variable are sufficient to generate a mathematical model. Via exponentially weighed moving averages, we create additional state variables, and use simple polynomial regression in the augmented state space to quantify excitation wave dynamics. A spatial gradient-sensing term replaces the classical diffusion as it is more robust to noise. Our pipeline for model creation is demonstrated for an in-silico model and optical voltage mapping recordings of cultured human atrial myocytes and only takes a few minutes.Our findings have the potential for widespread generation, use and on-the-fly refinement of personalised computer models for non-linear phenomena in biology and medicine, such as predictive cardiac digital twins.

Cardiac-specific deletion of BRG1 ameliorates ventricular arrhythmia inmice with myocardial infarction.

Malignant ventricular arrhythmia (VA) after myocardial infarction (MI) is mainly caused by myocardial electrophysiological remodeling. Brahma-related gene 1 (BRG1) is an ATPase catalytic subunit that belongs to a family of chromatin remodeling complexes called Switch/Sucrose Non-Fermentable Chromatin (SWI/SNF). BRG1 has been reported as a molecular chaperone, interacting with various transcription factors or proteins to regulate transcription in cardiac diseases. In this study, we investigated the potential role of BRG1 in ion channel remodeling and VA after ischemic infarction. Myocardial infarction (MI) mice were established by ligating the left anterior descending (LAD) coronary artery, and electrocardiogram (ECG) was monitored. Epicardial conduction of MI mouse heart was characterized in Langendorff-perfused hearts using epicardial optical voltage mapping. Patch-clamping analysis was conducted in single ventricular cardiomyocytes isolated from the mice. We showed that BRG1 expression in the border zone was progressively increased in the first week following MI. Cardiac-specific deletion of BRG1 by tail vein injection of AAV9-BRG1-shRNA significantly ameliorated susceptibility to electrical-induced VA and shortened QTc intervals in MI mice. BRG1 knockdown significantly enhanced conduction velocity (CV) and reversed the prolonged action potential duration in MI mouse heart. Moreover, BRG1 knockdown improved the decreased densities of Na+ current (INa) and transient outward potassium current (Ito), as well as the expression of Nav1.5 and Kv4.3 in the border zone of MI mouse hearts and in hypoxia-treated neonatal mouse ventricular cardiomyocytes. We revealed that MI increased the binding among BRG1, T-cell factor 4 (TCF4) and β-catenin, forming a transcription complex, which suppressed the transcription activity of SCN5A and KCND3, thereby influencing the incidence of VA post-MI.

Abstract P3140: Extracellular Vesicle MicroRNA Cargo Drives Ventricular Arrhythmia In Heart Failure Patients By Recapitulating Developmental Genes

Heart Failure (HF) is the leading cause of death worldwide, of which arrhythmias account for 50%. The relationship between HF and arrhythmias is closely intertwined. Since novel biomarkers associated with HF that lead to these arrhythmogenic events are an area of unmet need, we aimed to identify the specific plasma Extracellular vesicles (EV)-driven microRNA cargo that could predispose ventricular cardiomyocytes to arrhythmia during HF as well as delineate targetable pathways to rescue the arrhythmic phenotype. We treated human pluripotent stem cell-derived ventricular cardiomyocytes (hPSC-VCM) with plasma-derived EVs from HF and control samples and observed significantly prolonged action potential duration in HF-treated hPSC-VCM (94.41 ± 16.13 msec) using optical voltage mapping. Small RNA cargo of the plasma EVs were sequenced to reveal 26 downregulated and 70 upregulated differentially expressed miRNAs between HF and control. Upon deconvolution analysis, many of these plasma miRNA transcripts were found to be enriched in the brain, RBCs, monocytes and other tissues. We also sequenced the long-RNA transcriptome of the recipient cardiomyocytes. We prioritized 33 targets for qRT-PCR validation based on predicted protein-coding targets of miRNA, and 10 of these mRNAs were different between HF and control EV-treated hPSC-VCMs. Interestingly, some mRNAs (EIF4E, NEDD4, ID3, ID1, CDKN1A, HSPD1) are known to be expressed during development. Pathway enrichment analysis of the validated targets converged in TGF beta signaling and Hippo pathways, implicated in cardiac developmental arrhythmogenic abnormalities. In conclusion, circulating EV small RNA cargo of HF patients can alter the electrophysiological properties of cardiomyocytes, many of which are derived, predominantly from the brain. More importantly, the arrhythmic phenotype is triggered when there is a recapitulation of developmental genes induced by the EV-derived miRNAs. While these miRNAs could be identified as putative biomarkers for SCD, targeting these developmental genes may be a fruitful therapeutic approach to rescue electrical remodeling in HF patients.

PO-01-108 ELUCIDATING THE MECHANISM OF ATRIAL FIBRILLATION IN LAMIN A/C HEART DISEASE USING HUMAN IPSC-DERIVED ATRIAL CARDIOMYOCYTES

Mutations in the LMNA gene, encoding the nuclear envelope proteins lamin A/C, cause cardiac arrhythmias, conduction disease, and dilated cardiomyopathy. Malignant arrhythmias such as atrial fibrillation (AF) and ventricular tachycardia are common and pose an increased risk of stroke and sudden cardiac death. To investigate the etiological mechanisms by which a heterozygous pathogenic LMNA-S143P mutation causes early-onset AF using mature human iPSC-derived atrial cardiomyocytes (hiPSC-aCM) generated from a large kindred with a strong cardiac phenotype. Detailed phenotyping of a 4-generation family pedigree and generation of hiPSCs from the proband using a comprehensive approach for the differentiation of mature hiPSC-aCMs. Action potential duration was measured by optical voltage mapping. Calcium transients were measured by confocal microscopy. The proband (III-3) is a 42-year-old female of European and Asian descent who developed symptomatic early-onset AF at age 35. She underwent direct current cardioversion (DCCV), pulmonary vein isolation, and implantation of a biventricular pacemaker. Cardiac MRI showed an enlarged left atrium with preserved left ventricular systolic function (EF 56%). A detailed family history revealed early-onset AF and AV block, congestive heart failure (CHF), and sudden death (Fig. 1). Genetic testing of the proband revealed a pathogenic missense mutation in LMNA (c427T>Cp. Ser143Pro). Mature LMNA-S143P-hiPSC-aCMs displayed prolongation of the atrial action potential duration (APD90) with delayed afterdepolarizations, abnormal calcium transients, and AF as compared to wild-type hiPSC-aCMs. Detailed phenotyping of a large LMNA kindred revealed a phenotype of AF, cardiac conduction disease, dilated cardiomyopathy, and sudden death. Our hiPSC-aCM studies uncovered a triggered mechanism for the AF with ion channel remodeling and pave the way for targeting calcium handling proteins as a potential therapeutic strategy in LMNA associated arrhythmias.

Mechano-electrical interactions and heterogeneities in wild-type and drug-induced long QT syndrome rabbits.

Electromechanical reciprocity - comprising electro-mechanical (EMC) and mechano-electric coupling (MEC) - provides cardiac adaptation to changing physiological demands. Understanding electromechanical reciprocity and its impact on function and heterogeneity in pathological conditions - such as (drug-induced) acquired long QT syndrome (aLQTS) - might lead to novel insights in arrhythmogenesis. Our aim is to investigate how electrical changes impact on mechanical function (EMC) and vice versa (MEC) under physiological conditions and in aLQTS. To measure regional differences in EMC and MEC in vivo, we used tissue phase mapping cardiac MRI and a 24-lead ECG vest in healthy (control) and IKr-blocker E-4031-induced aLQTS rabbit hearts. MEC was studied in vivo by acutely increasing cardiac preload, and ex vivo by using voltage optical mapping (OM) in beating hearts at different preloads. In aLQTS, electrical repolarization (heart rate corrected RT-interval, RTn370) was prolonged compared to control (P<0.0001) with increased spatial and temporal RT heterogeneity (P<0.01). Changing electrical function (in aLQTS) resulted in significantly reduced diastolic mechanical function and prolonged contraction duration (EMC), causing increased apico-basal mechanical heterogeneity. Increased preload acutely prolonged RTn370 in both control and aLQTS hearts (MEC). This effect was more pronounced in aLQTS (P<0.0001). Additionally, regional RT-dispersion increased in aLQTS. Motion-correction allowed us to determine APD-prolongation in beating aLQTS hearts, but limited motion correction accuracy upon preload-changes prevented a clear analysis of MEC ex vivo. Mechano-induced RT-prolongation and increased heterogeneity were more pronounced in aLQTS than in healthy hearts. Acute MEC effects may play an additional role in LQT-related arrhythmogenesis, warranting further mechanistic investigations. KEY POINTS: Electromechanical reciprocity comprising excitation-contraction coupling (EMC) and mechano-electric feedback loops (MEC) is essential for physiological cardiac function. Alterations in electrical and/or mechanical heterogeneity are known to have potentially pro-arrhythmic effects. In this study, we aimed to investigate how electrical changes impact on the mechanical function (EMC) and vice versa (MEC) both under physiological conditions (control) and in acquired long QT syndrome (aLQTS). We show that changing the electrical function (in aLQTS) results in significantly altered mechanical heterogeneity via EMC and, vice versa, that increasing the preload acutely prolongs repolarization duration and increases electrical heterogeneity, particularly in aLQTS as compared to control. Our results substantiate the hypothesis that LQTS is an ‛electro-mechanical', rather than a 'purely electrical', disease and suggest that acute MEC effects may play an additional role in LQT-related arrhythmogenesis.

PO-01-241 ROLE OF THE TWO-PORE K+CHANNEL TREK1 IN REGULATING HEART FAILURE-INDUCED VENTRICULAR ARRHYTHMIA

Despite therapeutic advances, heart failure (HF) remains a leading cause of death in the United States and is growing in prevalence. Although disease-induced defects in ion channel excitability and expression put patients at higher risk for life-threatening ventricular arrhythmias, the exact mechanisms by which HF leads to proarrhythmic ion channel dysfunction are not completely understood. Notably, several recent studies have identified important roles for the two-pore background K+ channel TREK1 in regulating sinus rhythm, atrial arrhythmia, and ventricular excitability. In the present study, we examined a putative role for TREK1 dysfunction in promoting heart failure-induced ventricular arrhythmia. We subjected wild type (WT) mice and mice with cardiac-specific TREK1 deficiency (TREK1cKO) to transaortic constriction (TAC) surgery as a model of heart failure or to a sham procedure. Over the course of 6-12 weeks following surgery, we examined structural and electrical remodeling via echocardiography and subsurface ECGs. At the end of the time course, ventricular cells were isolated and used for patch clamp, Ca2+/contractility imaging, and immunostaining. A subset of hearts were isolated for optical voltage mapping. We observed that TREK1cKO mice experienced greater reductions in cardiac function earlier in the development of pressure overload compared to WT mice, which was maintained throughout the time course. Additionally, TREK1cKO mice exhibited proarrhythmic changes to ECG parameters over 6-12 weeks of pressure overload including action potential duration (APD) prolongation, QTc prolongation, and QRS widening. However, the TREK1cKOs also experienced faster conduction, lower APD heterogeneity, and lower ventricular arrhythmia inducibility at 6 weeks post-surgery. Overall, these results demonstrate a potentially complex role of TREK1 in regulating ventricular excitability, ion dynamics, and arrhythmia susceptibility in the context of pressure overload-induced heart failure.

PO-01-158 MODELING AN ATRIAL FIBRILLATION-CAUSING TITIN MUTATION USING HUMAN IPSC-DERIVED ATRIAL CARDIOMYOCYTES

Atrial fibrillation (AF) affects more than 33 million people worldwide and, if left untreated, can lead to increased morbidity and mortality. Although AF has been classified as an ‘ion channelopathy’, next-generation sequencing has identified mutations in sarcomeric proteins like TTN, the gene encoding titin, that are associated with AF. Despite progress in understanding the genetic basis of AF, the translation of these discoveries to the bedside care of patients has been limited due to challenges in understanding the myocardial substrate for AF, especially that develops from the mutation in the structural protein. As human atrial tissue is rarely available and animal models are mechanistically limited, our goal is to model AF using mature human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs) by a CRISPR-Cas9-mediated single amino acid substitution of a rare missense ethnic-specific mutation (TTN_T32756I) in an immunoglobulin (Ig)-like domain of the A-band of titin that is enriched in AF-causing pathogenic variants. Isogenic TTN_T32756I hiPSC-aCM mutants were generated using the CRISPR-Cas9 technique. Cells were matured either with metabolic conditioning or co-culturing with atrial fibroblasts in a micropatterned platform. We used confocal microscopy to assess sarcomeric integrity, MuscleMotion to assess contractility, and optical voltage mapping to assess the electrophysiological properties of TTN_T32756I hiPSC-aCMs. T32756I results in a non-conservative amino acid change, which in-silico tools have previously predicted results in an impairment in titin function. Here, we show that TTN_T32756I hiPSC-aCM mutants display aberrant contractility (Fig. 1A-C) and Ca2+ homeostasis (Fig. 1D) leading to abnormal electrophysiology (Fig. 1E-F) at the cellular level without compromising the sarcomeric integrity of the atrial cardiomyocytes (Fig. 1G-H). Our results suggest that a single amino acid change could have a profound effect on titin function, further establishing titin as a major signaling hub for contractility. Ongoing studies will not only identify the key pathways and transcription factors involved in the pathogenesis of TTN_ T32756I mutant but also uncover the impact of the mutation on atrial action potential and cardiac ion channel activity. Our study will shed new light on the mechanisms by which TTN mutation cause AF and identify novel therapeutic targets for patients carrying AF-associated rare variants in the structural genes.

Abstract 10026: Augmentation of Slow Repolarizing Potassium Channel (I Ks ) Links Truncated Titin Mutations to Atrial Fibrillation

Introduction: Rare titin-truncating variants ( TTN tv) are associated with early-onset atrial fibrillation (AF) but the pathogenic mechanisms and therapeutic implications remain unclear. To determine the function of a highly conserved immunoglobulin-like domain of the A-band of TTN , a region enriched in AF-associated variants, we employed CRISPR-Cas9-mediated deletion of 9 amino acids (Δ9) in human induced pluripotent stem cells and then derived atrial cardiomyocytes (hiPSC-aCMs) for electrophysiological (EP) analysis. Hypothesis: We hypothesized that the TTN -Δ9 mutation results in augmentation of the slow delayed rectifier potassium current (I Ks ) resulting in shortened action potential duration (APD). Methods: TTN -WT hiPSC-aCMs (healthy control) and TTN -Δ9 hiPSC-aCMs (isogenic mutant) were matured with metabolic conditioning, electrical stimulation, co-culture with atrial fibroblasts, and micropatterning. We used transmission electron microscopy (TEM) to assess sarcomeric defects, RNA-sequencing and RT-qPCR to assess molecular ion channel remodeling, and high throughput automated patch clamp and optical voltage mapping to assess the EP properties of TTN -Δ9 hiPSC-aCMs. We also performed targeted pharmacological inhibition of I Ks . Results: We showed that TTN -Δ9 hiPSC-aCMs displayed disrupted organization and myofibril assembly ( Fig. 1A-B ), upregulated voltage-gated potassium channel complexes and increased KCNQ1 expression ( Fig 1C-D ), and shortened APD ( Fig. 1E-F ). I Ks was significantly augmented in TTN -Δ9 hiPSC-aCMs ( Fig. 1G-H ). Infusion of the I Ks -specific blocker HMR-1556, and not I Kr blocker dofetilide, fully restored the shortened APD 90 and partially recovered defects in contractility in TTN -Δ9 hiPSC-aCMs compared to TTN -WT hiPSC-aCMs ( Fig. 1I-J ). Conclusion: Our findings suggest that augmentation of I Ks contributes to the pathogenic mechanism of TTN -mediated AF, pointing to I Ks as a potential therapeutic target.

TLR4 pathway induces proarrhythmogenic slow conduction by impaired depolarisation

Abstract Background Cardiac inflammation driven by the Toll-Like-Receptor 4 (TLR4) is correlated to increased risk of arrhythmia. Cardiac arrhythmogenesis and the formation of re-entry tachycardia is highly dependent on conduction velocity (CV) and action potential (AP) duration (APD). As TLR4 induced APD shortening has been shown, we analyze in this study for the first time the TLR4 effect on conductance disturbances in a LPS induced septic mouse model. Methods Systemic activation of TLR4 in mice was achieved by intraperitoneal LPS injection 3.5 hours prior to experiments. In vivo electrophysiological investigation (EPI) was performed using an octapolar transvenous catheter placed to the right heart. Ex vivo experiments were performed on Langendorff-perfused hearts. AP propagation was measured by optical voltage mapping (OVM) with voltage sensitive dye Di-4-ANEPPS. For AP analysis including RMP, intracellular electrical recordings were performed using sharp microelectrodes. Wildtype mice after LPS injection were compared to wildtype mice after vehicle (NaCl) injection or ubiquitous TLR4 knockout (TLR4−/−) mice with LPS or vehicle application. Results In vivo EPI showed a tendency to more atrial fibrillation after LPS injection (+LPS 5/6, +NaCl 2/6, p=0.2). Ventricular stimulation evoked ventricular tachycardia in every LPS treated WT mouse but less in controls (+LPS 6/6, +NaCl 1/6, p=0.01). OVM measured decreased CV in both atria and ventricle after LPS treatment (atria: +LPS: 43.1±3.1cm/s, n=5; +NaCl: 72.6±9.8cm/s, n=10, p=0.04; ventricle: +LPS: 50.2±2.2cm/s, n=6; +NaCl: 67.7±5.0cm/s, n=10, p=0.02). In analysis of AP in atria upstroke velocity was slightly decreased (max.dV/dt: +LPS: 123.1±4.8V/s, n=22; +NaCl: 158.5±5.3V/s, n=39, p=0.04) but highly reduced in ventricle (max.dV/dt: +LPS: 91.8±3.6V/s, n=27; +NaCl: 140.7±6.3V/s, n=35, p&amp;lt;0.0001). RMP in atria was depolarised after LPS injection (+LPS: −70.1±1.9mV, n=22; +NaCl: −81.1±1.2mV, n=39, p=0.004) explaining decreased upstroke velocity and CV slowing in atria by voltage-dependent Na+ channel inactivation. Ventricular RMP was unaffected by LPS injection (+LPS: −75.1±1.1mV, n=44; +NaCl: −76.3±0.9mV, n=55, p=0.83). Therefore the Na+ currents were measured in isolated ventricular cardiomyocytes using whole cell patch clamp revealing the maximum Na+ current density lowered after LPS treatment (+LPS: −20.6±1.7 pA/pF, n=16, +NaCl 27.1±2.6 pA/pF, n=10, p=0.03). LPS did not affect EPI, CV, upstroke velocity or current density in TLR4−/− mice. Conclusion Herein we report for the first time impaired cardiac depolarisation and conduction after short term activation of TLR4 in vivo. Pro arrhythmogenic mechanisms differ in atria and ventricle: Increased atrial RMP inactivates Na+ current leading to reduction of CV. Ventricular slow CV is caused by reduced current density of Na+ channels. This different TLR4 effect might be important for novel antiarrhythmic and antiinflammatory applications. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): GEROK-grant, University Bonn

Abstract P1084: Mature Ipsc-derived Atrial Cardiomyocytes Show That Saturated Fatty Acids Differentially Modulate Atrial Remodeling In Obesity-induced Atrial Fibrillation Compared To Mono And Poly-unsaturated Fatty Acids.

Background: Increased free fatty acids (FFA) and FA infiltration, both major hallmarks of obesity can individually modulate changes leading to AF. For example, mass spectrometry on obese mouse heart tissues have shown that saturated FAs (SFA), monounsaturated FAs (MUFA), and polyunsaturated FAs (PUFA) are all significantly increased compared to controls. The highest increase was shown to be in linoleic acid (LA), a PUFA followed by palmitic acid (PA, SFA) and oleic acid (OA, MUFA). Although reports suggest that some FAs induce ventricular arrhythmias in mice, the differential electrophysiological effects of SFAs, MUFAs, and PUFAs on the atria remain unclear. Objective: To determine the individual roles of SFAs, MUFAs, and PUFAs in mediating atrial remodeling in obesity-induced AF using mature human induced pluripotent stem-derived atrial cardiomyocytes (iPSC-aCMs). Methods: We compared iPSC-aCMs and HL-1 cells treated with bovine serum albumin (BSA) with palmitic acid (PA), oleic acid (OA) and LA cells (600 uM each) for 24 hours and 1 week. Ion channel function were assessed using optical voltage mapping and whole-cell patch clamping, and we assessed atrial remodeling using qPCR, and western blotting. Results: iPSC-aCMs treated with PA, OA, and LA displayed higher expression of FABP3 and CPT1A suggesting increased FA metabolism (Figure A-C). In HL1 cells, protein expression of Cpt1a was increased in all groups after FA treatment (Figure D). PA differentially modulated expression of Kv7.1 and NCX1 in HL1 cells (Figure E-F) and ion channel mRNA expression in iPSC-aCMs (Figure G-L). After 1 week PA and LA treatment b-adrenergic signaling was significantly increased in iPSC-aCMs while OA decreased it compared to controls (Figure M-O). Lastly, 24-hour PA, OA, LA treatment differentially modulated the atrial action potential (AP). While PA and LA significantly shorten the action potential, there was no significant changes to the AP after OA treatment. Conclusions: Using mature iPSC-aCMs, we showed for the first time that SFA, MUFA, and PUFA not only increased FA metabolism but differentially affected b-adrenergic signaling and ion channel remodeling leading to differential effects on the atrial AP. Our findings may have important implications for the management of obesity-induced AF in patients.

Numerical methods for the detection of phase defect structures in excitable media.

Electrical waves that rotate in the heart organize dangerous cardiac arrhythmias. Finding the region around which such rotation occurs is one of the most important practical questions for arrhythmia management. For many years, the main method for finding such regions was so-called phase mapping, in which a continuous phase was assigned to points in the heart based on their excitation status and defining the rotation region as a point of phase singularity. Recent analysis, however, showed that in many rotation regimes there exist phase discontinuities and the region of rotation must be defined not as a point of phase singularity, but as a phase defect line. In this paper, we use this novel methodology and perform a comparative study of three different phase definitions applied to in silico data and to experimental data obtained from optical voltage mapping experiments on monolayers of human atrial myocytes. We introduce new phase defect detection algorithms and compare them with those that appeared in literature already. We find that the phase definition is more important than the algorithm to identify sudden spatial phase variations. Sharp phase defect lines can be obtained from a phase derived from local activation times observed during one cycle of arrhythmia. Alternatively, similar quality can be obtained from a reparameterization of the classical phase obtained from observation of a single timeframe of transmembrane potential. We found that the phase defect line length was (35.9 ± 6.2)mm in the Fenton-Karma model and (4.01 ± 0.55)mm in cardiac human atrial myocyte monolayers. As local activation times are obtained during standard clinical cardiac mapping, the methods are also suitable to be applied to clinical datasets. All studied methods are publicly available and can be downloaded from an institutional web-server.

Neonatal Deletion of Hand1 and Hand2 within Murine Cardiac Conduction System Reveals a Novel Role for HAND2 in Rhythm Homeostasis.

The cardiac conduction system, a network of specialized cells, is required for the functioning of the heart. The basic helix loop helix factors Hand1 and Hand2 are required for cardiac morphogenesis and have been implicated in cardiac conduction system development and maintenance. Here we use embryonic and post-natal specific Cre lines to interrogate the role of Hand1 and Hand2 in the function of the murine cardiac conduction system. Results demonstrate that loss of HAND1 in the post-natal conduction system does not result in any change in electrocardiogram parameters or within the ventricular conduction system as determined by optical voltage mapping. Deletion of Hand2 within the post-natal conduction system results in sex-dependent reduction in PR interval duration in these mice, suggesting a novel role for HAND2 in regulating the atrioventricular conduction. Surprisingly, results show that loss of both HAND factors within the post-natal conduction system does not cause any consistent changes in cardiac conduction system function. Deletion of Hand2 in the embryonic left ventricle results in inconsistent prolongation of PR interval and susceptibility to atrial arrhythmias. Thus, these results suggest a novel role for HAND2 in homeostasis of the murine cardiac conduction system and that HAND1 loss potentially rescues the shortened HAND2 PR phenotype.

Centres of spiral waves can be detected as phase defect lines in optical voltage mapping data and numerical simulations

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): KU Leuven and FWO-Flanders Introduction To study the excitation patterns of spiral waves in two spatial dimensions over time, it is useful to abstract them to much simpler mathematical structures. Previously, those waves have been abstracted to phase singularities: single points around which spiral moves [1,2]. Recently, it was proposed to upgrade these points to phase defect lines [3,4]. The dynamics of either of these structures can then be studied in place of the spiral waves. Purpose To work with phase defects, firstly, we need a reliable method of locating them. Methods We recorded the transmembrane voltage over space and time in a numerical simulation of the mono-domain model as well as by using optical voltage mapping data of 10 cm² monolayers of conditionally immortalised human atrial myocytes [5]. Optical voltage mapping was performed with a sampling time of 6 ms and 100×100 square pixels of 0.25 mm width. The recorded intensity were smoothed and rescaled to get an estimate of the transmembrane voltages. Detailed protocols can be found in [5]. For the numerical simulations, we have implemented the linear-core tissue model by Bueno-Orovio, Cherry, and Fenton (2008) [6] with finite differences on a 1200×1200 rectangular grid with a grid size of 0.25 mm and a time step of 9 μs. We normalised the transmembrane voltage u such that zero corresponds to the resting state and one to the plateau of the action potential. A phase φ is an angle in radians which monotonously increases from zero to 2π during one action potential. Phases are defined in such a manner that, even for the sharp wave front of a cardiac action potential, this increase happens in a slow, gradual way. A phase can for instance be defined by using the local arrival time and the characteristic action potential duration [4]. At the centre of spiral waves, neighbouring parts of tissue have vastly different phase values φ. A phase singularity is a point where all phases meet. Phase defects are large jumps across space in phase. Phase defect lines can then be detected as lines in the phase defect field ρ with a value much larger than zero [7]. Results For the simulation, we can clearly see that the linear core of the Bueno-Orovio, Cherry, and Fenton (2008) [6] model is identified as a phase defect line of up to 100 mm in length (Fig.1). The spiral wave moves along it and turns at the end points. The same behaviour can be seen in vitro in the optical voltage mapping data (Fig.2). Multiple spirals can be observed, all of which revolve around phase defect lines of lengths of up to 4 mm. Conclusion In this study, we have shown that phase defect lines can be detected both in silico and in vitro. Their properties such as length, rotational frequency, and their dynamics can be used to gain additional insight into spiral waves in arrhythmias. We have introduced multiple methods to localise phase defects for which the code is publicly available.

PO-615-03 MUTANT ATRIAL NATRIURETIC PEPTIDE INDUCES MITOCHONDRIAL DEFECTS AND ION CHANNEL REMODELING IN A HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED ATRIAL FIBRILLATION MODEL

We identified a mutation in NPPA, encoding atrial natriuretic peptide (ANP), as the first non-ion channel gene causing familial atrial fibrillation (AF). However, lack of a mature and comprehensive model prevents a deeper understanding of the underlying cellular mechanisms. Induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) are ideally suited for modeling heritable AF and personalized pharmacological therapy, but there is limited data on the maturation of iPSC-atrial (a) CMs. We hypothesized that an electro-metabolic maturation (EMM) approach using biochemical cues (T3, IGF-1, dexamethasone), bioenergetic supplement (fatty acids), and electrical stimulation, synergistically promotes metabolic, structural, and electrophysiological maturity of iPSC-aCMs to a level comparable to human atrial cardiomyocytes (haCMs) and will reveal the novel underlying cellular mechanisms of the NPPA mutation. We applied our EMM approach to iPSC-aCMs and compared the metabolic (Seahorse Analyzer, western blots [WB], RT-PCR), structural (immunofluorescence, WB), electrophysiological (patch clamping, optical voltage mapping), and transcriptomic (RNA-seq) maturity with immature iPSC-aCMs and adult haCMs from the same patient. EMM matured iPSC-aCMs from a family carrying the NPPA-S64R mutation, and an isogenic control using CRISPR-Cas9, elucidated the novel underlying mechanism by which the mutation causes AF. EMM iPSC-aCMs displayed improved sarcomeric organization (Fig. 1a) and oxidative capacity (Fig. 1b), as well as more hyperpolarized RMP and increased APA (Fig. 1c,d). Only mature NPPA-S64R iPSC-aCMs demonstrated that the NPPA mutation caused mitochondrial defects resulting in reduced oxidative capacity and electron transport chain dysfunction (Fig. 2a), as well as electrophysiological remodeling causing prolonged APD (Fig. 2b,c) via increased IKs density (Fig. 2d-g). We established a combinatorial EMM approach that promoted comprehensive maturation of iPSC-aCMs. Using this maturation approach, we unmasked the novel electrophysiological remodeling and mitochondrial dysfunction underlying an AF-causing NPPA mutation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Arrhythmia Assessment in Heterotypic Human Cardiac Myocyte-Fibroblast Microtissues.

Risk assessment assays for chemically induced arrhythmia are critical, but significant limitations exist with current cardiotoxicity testing, including a focus on single select ion channels, the use of non-human species in vitro and in vivo, and limited direct physiological translation. To be predictive of actual adverse clinical arrhythmic risk, arrhythmia assessment models for chemicals and drugs should be fit-for-purpose and suited for evaluating compounds in which the mechanism of action may not be entirely known. Here, we describe methods for efficient and reliable screening for arrhythmogenic cardiotoxicity with a 3D human cardiac microtissue model using purified human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and human cardiac fibroblasts. Applying optical mapping of voltage and calcium-sensitive dyes-an established approach to evaluate cardiac action potentials and calcium transients-to 3D heterotypic cardiac myocyte-fibroblast tissues allows for the generation and functional analysis of a large number of individual microtissues to provide greater throughput and high statistical power in analyses. Hundreds of microtissues in standard cell culture plates can be produced with low variability beat-to-beat, microtissue-to-microtissue, and across hiPSC-cardiomyocyte differentiation batches, reducing the number of microtissues required per condition for predictive outputs. The platform described here can be used as a sensitive, efficient, and predictive preclinical model validated for the purpose of assessing human pro-arrhythmic risk.

A dataset of dual calcium and voltage optical mapping in healthy and hypertrophied murine hearts

Pathological hypertrophy underlies sudden cardiac death due to its high incidence of occurrence of ventricular arrhythmias. The alteration of transmural electrophysiological properties in hypertrophic cardiac murine tissue has never been explored previously. In this dataset, we have for the first time conducted high-throughput simultaneous optical imaging of transmembrane potential and calcium transients (CaT) throughout the entire hypertrophic murine hearts at high temporal and spatial resolution. Using ElectroMap, we have conducted multiple parameters analysis including action potential duration/calcium transient duration, conduction velocity, alternans and diastolic interval. Voltage-calcium latency was measured as time difference between action potential and CaT peak. The dataset therefore provides the first high spatial resolution transmural electrophysiological profiling of the murine heart, allowing interrogation of mechanisms driving ventricular arrhythmias associated with pathological hypertrophy. The dataset allows for further reuse and detailed analyses of geometrical, topological and functional analyses and reconstruction of 2-dimensional and 3-dimentional models.

Abstract 10530: Stretch-Induced Arrhythmias Are Increased by Microtubule Detyrosination in a TRPA1 Dependent Manner in the Rabbit Heart

Introduction: Clinical evidence indicates that in hypertension, acute fluctuations in blood pressure can trigger ventricular arrhythmias. Single cell rabbit studies suggest that these stretch-induced arrhythmias (SIA) are related to increased microtubule (MT) density or detyrosination and depend on transient receptor potential ankyrin 1 channels (TRPA1). However, whether these mechanisms of SIA apply to the whole heart is unknown. Hypothesis: An increase in MT density or detyrosination will reduce the threshold for SIA in the whole heart and be dependent on TRPA1. Methods: Isolated rabbit hearts were instrumented with surface electrodes to monitor electrical activity and an intraventricular balloon to alter LV load. Transient changes in LV volume were applied to induce SIA (50-500μL, 10mL/s, 20 repetitions of each volume separated by 30s). SIA incidence vs volume was used to calculate the volume that resulted in a 50% SIA incidence (V 50 ). Paclitaxel (5μM) was applied alone to increase MT density and detyrosination or in combination with parthenolide (10μM) to prevent the increase in detyrosination or HC-030031 (10μM) to block TRPA1. Voltage optical mapping was used to determine the nature of SIA. MT density was assessed by immunofluorescence in isolated LV myocytes. Results: Paclitaxel reduced the threshold for SIA (decreased V 50 versus control: 133±16 vs 173±11μL; n =8 , p =0.02 by paired t-test) and resulted in the conversion of some stretch-induced ectopy to sustained activity. These effects were prevented by reduced detyrosination with parthenolide (262±27 vs 256±26μL; n =8 , p =0.75). Paclitaxel also increased MT density versus control, however this was not prevented by parthenolide (63±3 and 60±3 vs 50±4%; n =15, p =0.04 by one-way ANOVA). The decreased V 50 and occurrence of sustained arrhythmias with paclitaxel was also prevented by TRPA1 block with HC-030031 (249±26 vs 237±27μL; n =8, p =0.60). Optical mapping revealed stretch-induced ectopic excitation originating from the LV freewall, which resulted in re-entrant activity with paclitaxel. Conclusions: An increase in MT detyrosination leads to a reduced threshold for SIA and the triggering of sustained arrhythmias in the whole heart in a TRPA1 dependent manner.

Formation of human cardiomyocytes is impaired in a fibrotic environment: unravelling human cardiac regeneration

Abstract Loss of myocardial tissue remains a leading cause of disease and death, as the adult heart has insufficient regenerative potential. The (pre-)clinical effects of inducing cardiac regeneration by cardiac cell therapy have been disappointing. This lack of success may result from the fact that it remains largely unclear how the receiving pathological microenvironment affects this process of cardiomyogenic differentiation of implanted cells, and thereby may (negatively) influence the therapeutic outcome. However, the tools to address this lacuna in a proper manner are lacking, as this requires tightly controllable and quantitative models of cardiomyogenic differentiation. Therefore, we have recently generated lines of conditionally immortalized human CMCs (ciCMCs) through doxycycline-dependent expression of SV40 large T antigen after genetic modification and subsequent clonal expansion. In these cells, proliferation and differentiation can be tightly controlled, allowing cardiomyogenic differentiation to be i) induced on cue, ii) precisely monitored and quantified, and iii) manipulated. The aim of this study is to improve our understanding of cardiomyogenic differentiation of guest (transplanted) cells in the context of the host (receiving) microenvironment. To create pathological microenvironments and study the effects on cardiac differentiation, co-culture experiments with human ciCMCs and cardiac fibroblasts were conducted in different ratios (10%, 30%, 60% and 90% ciCMCs). Cardiomyogenic differentiation was determined by immunostaining for cardiac specific markers and electrophysiological analysis by optical voltage mapping. After 12 days of co-culture with cardiac fibroblasts, the amount of ciCMCs that expressed the sarcomeric protein cardiac troponin T was significantly and increasingly reduced (P&amp;lt;0.01) in the groups with higher amounts of cardiac fibroblasts (39.4±3.9, 33±3.4, 25±1.9, 20.3±2.6, 5±1.7 for 100%, 90%, 60%, 30% and 10% ciCMCs respectively (%, mean±SD). Electrophysiological analysis showed a significantly reduced (P&amp;lt;0.01) conduction velocity in the co-cultures compared to the pure cultures of ciCMCs (19.1±2.1 vs 16.0±0.5, 15.8±0.9, 8.6±0.6, 5±1.67 for 100% vs 90%, 60%, 30% and 10% ciCMCs respectively (cm/s, mean±SD). However, no significant difference in conduction velocity was present between the groups with 10% and 30% ciCMCs and 30% and 60% ciCMCs present. In conclusion, a fibrotic environment has a negative effect on the formation of human cardiomyocytes as revealed by the use of ciCMCs. This not only emphasizes the need to consider the interaction between the guest (transplanted) cells and the host (receiving) microenvironment in cardiac regenerative medicine, but also offers new leads to increase the therapeutic potential of this strategy. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Leiden University Fund