Interpretation of field and LEAP potentials recorded from cardiomyocyte monolayers.

Multielectrode arrays (MEAs) are the method of choice for electrophysiological characterization of cardiomyocyte monolayers. The field potentials recorded using an MEA are like extracellular electrograms recorded from the myocardium using conventional electrodes. Nevertheless, different criteria are used to interpret field potentials and extracellular electrograms, which hamper correct interpretation and translation to the patient. To validate the criteria for interpretation of field potentials, we used neonatal rat cardiomyocytes to generate monolayers. We recorded field potentials using an MEA and simultaneously recorded action potentials using sharp microelectrodes. In parallel, we recreated our experimental setting in silico and performed simulations. We show that the amplitude of the local RS complex of a field potential correlated with conduction velocity in silico but not in vitro. The peak time of the T wave in field potentials exhibited a strong correlation with APD90 while the steepest upslope correlated well with APD50. However, this relationship only holds when the T wave displayed a biphasic pattern. Next, we simulated local extracellular action potentials (LEAPs). The shape of the LEAP differed markedly from the shape of the local action potential, but the final duration of the LEAP coincided with APD90. Criteria for interpretation of extracellular electrograms should be applied to field potentials. This will provide a strong basis for the analysis of heterogeneity in conduction velocity and repolarization in cultured monolayers of cardiomyocytes. Finally, a LEAP is not a recording of the local action potential but is generated by intracellular current provided by neighboring cardiomyocytes and is superior to field potential duration in estimating APD90.NEW & NOTEWORTHY We present a physiological basis for the interpretation of multielectrode array-derived, extracellular, electrical signals.

Molecular and electrophysiological evaluation of human cardiomyocyte subtypes to facilitate generation of composite cardiac models.

Paucity of physiologically relevant cardiac models has limited the widespread application of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes in drug development. Here, we performed comprehensive characterization of hiPSC-derived cardiomyocyte subtypes from 2D and 3D cultures and established a novel 3D model to study impulse initiation and propagation. Directed differentiation approaches were used to generate sinoatrial nodal (SANCM), atrial (ACM) and ventricular cardiomyocytes (VCM). Single cell RNA sequencing established that the protocols yield distinct cell populations in line with expected identities, which was also confirmed by electrophysiological characterization. In 3D EHT cultures of all subtypes, we observed prominent expression of stretch-responsive genes such as NPPA. Response to rate modulating drugs noradrenaline, carbachol and ivabradine were comparable in single cells and EHTs. Differences in the speed of impulse propagation between the subtypes were more pronounced in EHTs compared with 2D monolayers owing to a progressive increase in conduction velocities in atrial and ventricular cardiomyocytes, in line with a more mature phenotype. In a novel binary EHT model of pacemaker-atrial interface, the SANCM end of the tissue consistently paced the EHTs under baseline conditions, which was inhibited by ivabradine. Taken together, our data provide comprehensive insights into molecular and electrophysiological properties of hiPSC-derived cardiomyocyte subtypes, facilitating the creation of next generation composite cardiac models for drug discovery, disease modeling and cell-based regenerative therapies.

A single cell transcriptional roadmap of human pacemaker cell differentiation.

Each heartbeat is triggered by the sinoatrial node (SAN), the primary pacemaker of the heart. Studies in animal models have revealed that pacemaker cells share a common progenitor with the (pro)epicardium, and that the pacemaker cardiomyocytes further diversify into 'transitional', 'tail', and 'head' subtypes. However, the underlying molecular mechanisms, especially of human pacemaker cell development, are poorly understood. Here, we performed single cell RNA sequencing (scRNA-seq) and trajectory inference on human induced pluripotent stem cells (hiPSCs) differentiating to SAN-like cardiomyocytes (SANCMs) to construct a roadmap of transcriptional changes and lineage decisions. In differentiated SANCM, we identified distinct clusters that closely resemble different subpopulations of the in vivo SAN. Moreover, the presence of a side population of proepicardial cells suggested their shared ontogeny with SANCM, as also reported in vivo. Our results demonstrate that the divergence of SANCM and proepicardial lineages is determined by WNT signaling. Furthermore, we uncovered roles for TGFß and WNT signaling in the branching of transitional and head SANCM subtypes, respectively. These findings provide new insights into the molecular processes involved in human pacemaker cell differentiation, opening new avenues for complex disease modeling in vitro and inform approaches for cell therapy-based regeneration of the SAN.

Retinoic acid signaling in heart development: Application in the differentiation of cardiovascular lineages from human pluripotent stem cells.

Retinoic acid (RA) signaling plays an important role during heart development in establishing anteroposterior polarity, formation of inflow and outflow tract progenitors, and growth of the ventricular compact wall. RA is also utilized as a key ingredient in protocols designed for generating cardiac cell types from pluripotent stem cells (PSCs). This review discusses the role of RA in cardiogenesis, currently available protocols that employ RA for differentiation of various cardiovascular lineages, and plausible transcriptional mechanisms underlying this fate specification. These insights will inform further development of desired cardiac cell types from human PSCs and their application in preclinical and clinical research.

Sulfonylurea antidiabetics are associated with lower risk of out-of-hospital cardiac arrest: Real-world data from a population-based study.

AIMS: Out-of-hospital cardiac arrest (OHCA) mostly results from ventricular tachycardia/ventricular fibrillation (VT/VF), often triggered by acute myocardial infarction (AMI). Sulfonylurea (SU) antidiabetics can block myocardial ATP-regulated K+ channels (KATP channels), activated during AMI, thereby modulating action potential duration (APD). We studied whether SU drugs impact on OHCA risk, and whether these effects are related to APD changes. METHODS: We conducted a population-based case-control study in 219 VT/VF-documented OHCA cases with diabetes and 697 non-OHCA controls with diabetes. We studied the association of SU drugs (alone or in combination with metformin) with OHCA risk compared to metformin monotherapy, and of individual SU drugs compared to glimepiride, using multivariable logistic regression analysis. We studied the effects of these drugs on APD during simulated ischaemia using patch-clamp studies in human induced pluripotent stem cell-derived cardiomyocytes. RESULTS: Compared to metformin, use of SU drugs alone or in combination with metformin was associated with reduced OHCA risk (ORSUdrugs-alone 0.6 [95% CI 0.4-0.9], ORSUdrugs + metformin 0.6 [95% CI 0.4-0.9]). We found no differences in OHCA risk between SU drug users who suffered OHCA inside or outside the context of AMI. Reduction of OHCA risk compared to glimepiride was found with gliclazide (ORadj 0.5 [95% CI 0.3-0.9]), but not glibenclamide (ORadj 1.3 [95% CI 0.6-2.7]); for tolbutamide, the association with reduced OHCA risk just failed to reach statistical significance (ORadj 0.6 [95% CI 0.3-1.002]). Glibenclamide attenuated simulated ischaemia-induced APD shortening, while the other SU drugs had no effect. CONCLUSIONS: SU drugs were associated with reduced OHCA risk compared to metformin monotherapy, with gliclazide having a lower risk than glimepiride. The differential effects of SU drugs are not explained by differential effects on APD.

Titin Circular RNAs Create a Back-Splice Motif Essential for SRSF10 Splicing.

BACKGROUND: TTN (Titin), the largest protein in humans, forms the molecular spring that spans half of the sarcomere to provide passive elasticity to the cardiomyocyte. Mutations that disrupt the TTN transcript are the most frequent cause of hereditary heart failure. We showed before that TTN produces a class of circular RNAs (circRNAs) that depend on RBM20 to be formed. In this study, we show that the back-splice junction formed by this class of circRNAs creates a unique motif that binds SRSF10 to enable it to regulate splicing. Furthermore, we show that one of these circRNAs (cTTN1) distorts both localization of and splicing by RBM20. METHODS: We calculated genetic constraint of the identified motif in 125 748 exomes collected from the gnomAD database. Furthermore, we focused on the highest expressed RBM20-dependent circRNA in the human heart, which we named cTTN1. We used shRNAs directed to the back-splice junction to induce selective loss of cTTN1 in human induced pluripotent stem cell-derived cardiomyocytes. RESULTS: Human genetics suggests reduced genetic tolerance of the generated motif, indicating that mutations in this motif might lead to disease. RNA immunoprecipitation confirmed binding of circRNAs with this motif to SRSF10. Selective loss of cTTN1 in human induced pluripotent stem cell-derived cardiomyocytes induced structural abnormalities, apoptosis, and reduced contractile force in engineered heart tissue. In line with its SRSF10 binding, loss of cTTN1 caused abnormal splicing of important cardiomyocyte SRSF10 targets such as MEF2A and CASQ2. Strikingly, loss of cTTN1 also caused abnormal splicing of TTN itself. Mechanistically, we show that loss of cTTN1 distorts both localization of and splicing by RBM20. CONCLUSIONS: We demonstrate that circRNAs formed from the TTN transcript are essential for normal splicing of key muscle genes by enabling splice regulators RBM20 and SRSF10. This shows that the TTN transcript also has regulatory roles, besides its well-known signaling and structural function. In addition, we demonstrate that the specific sequence created by the back-splice junction of these circRNAs has important functions. This highlights the existence of functionally important sequences that cannot be recognized as such in the human genome but provides an as-yet unrecognized source for functional sequence variation.

Ultrarapid Delayed Rectifier K+ Channelopathies in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. About 5-15% of AF patients have a mutation in a cardiac gene, including mutations in KCNA5, encoding the Kv1.5 α-subunit of the ion channel carrying the atrial-specific ultrarapid delayed rectifier K+ current (IKur). Both loss-of-function and gain-of-function AF-related mutations in KCNA5 are known, but their effects on action potentials (APs) of human cardiomyocytes have been poorly studied. Here, we assessed the effects of wild-type and mutant IKur on APs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We found that atrial-like hiPSC-CMs, generated by a retinoic acid-based differentiation protocol, have APs with faster repolarization compared to ventricular-like hiPSC-CMs, resulting in shorter APs with a lower AP plateau. Native IKur, measured as current sensitive to 50 µM 4-aminopyridine, was 1.88 ± 0.49 (mean ± SEM, n = 17) and 0.26 ± 0.26 pA/pF (n = 17) in atrial- and ventricular-like hiPSC-CMs, respectively. In both atrial- and ventricular-like hiPSC-CMs, IKur blockade had minimal effects on AP parameters. Next, we used dynamic clamp to inject various amounts of a virtual IKur, with characteristics as in freshly isolated human atrial myocytes, into 11 atrial-like and 10 ventricular-like hiPSC-CMs, in which native IKur was blocked. Injection of IKur with 100% density shortened the APs, with its effect being strongest on the AP duration at 20% repolarization (APD20) of atrial-like hiPSC-CMs. At IKur densities < 100% (compared to 100%), simulating loss-of-function mutations, significant AP prolongation and raise of plateau were observed. At IKur densities > 100%, simulating gain-of-function mutations, APD20 was decreased in both atrial- and ventricular-like hiPSC-CMs, but only upon a strong increase in IKur. In ventricular-like hiPSC-CMs, lowering of the plateau resulted in AP shortening. We conclude that a decrease in IKur, mimicking loss-of-function mutations, has a stronger effect on the AP of hiPSC-CMs than an increase, mimicking gain-of-function mutations, whereas in ventricular-like hiPSC-CMs such increase results in AP shortening, causing their AP morphology to become more atrial-like. Effects of native IKur modulation on atrial-like hiPSC-CMs are less pronounced than effects of virtual IKur injection because IKur density of atrial-like hiPSC-CMs is substantially smaller than that of freshly isolated human atrial myocytes.

Toward Biological Pacing by Cellular Delivery of Hcn2/SkM1.

Electronic pacemakers still face major shortcomings that are largely intrinsic to their hardware-based design. Radical improvements can potentially be generated by gene or cell therapy-based biological pacemakers. Our previous work identified adenoviral gene transfer of Hcn2 and SkM1, encoding a "funny current" and skeletal fast sodium current, respectively, as a potent combination to induce short-term biological pacing in dogs with atrioventricular block. To achieve long-term biological pacemaker activity, alternative delivery platforms need to be explored and optimized. The aim of the present study was therefore to investigate the functional delivery of Hcn2/SkM1 via human cardiomyocyte progenitor cells (CPCs). Nucleofection of Hcn2 and SkM1 in CPCs was optimized and gene transfer was determined for Hcn2 and SkM1 in vitro. The modified CPCs were analyzed using patch-clamp for validation and characterization of functional transgene expression. In addition, biophysical properties of Hcn2 and SkM1 were further investigated in lentivirally transduced CPCs by patch-clamp analysis. To compare both modification methods in vivo, CPCs were nucleofected or lentivirally transduced with GFP and injected in the left ventricle of male NOD-SCID mice. After 1 week, hearts were collected and analyzed for GFP expression and cell engraftment. Subsequent functional studies were carried out by computational modeling. Both nucleofection and lentiviral transduction of CPCs resulted in functional gene transfer of Hcn2 and SkM1 channels. However, lentiviral transduction was more efficient than nucleofection-mediated gene transfer and the virally transduced cells survived better in vivo. These data support future use of lentiviral transduction over nucleofection, concerning CPC-based cardiac gene delivery. Detailed patch-clamp studies revealed Hcn2 and Skm1 current kinetics within the range of previously reported values of other cell systems. Finally, computational modeling indicated that CPC-mediated delivery of Hcn2/SkM1 can generate stable pacemaker function in human ventricular myocytes. These modeling studies further illustrated that SkM1 plays an essential role in the final stage of diastolic depolarization, thereby enhancing biological pacemaker functioning delivered by Hcn2. Altogether these studies support further development of CPC-mediated delivery of Hcn2/SkM1 and functional testing in bradycardia models.

Transcriptional regulation of the cardiac conduction system.

The rate and rhythm of heart muscle contractions are coordinated by the cardiac conduction system (CCS), a generic term for a collection of different specialized muscular tissues within the heart. The CCS components initiate the electrical impulse at the sinoatrial node, propagate it from atria to ventricles via the atrioventricular node and bundle branches, and distribute it to the ventricular muscle mass via the Purkinje fibre network. The CCS thereby controls the rate and rhythm of alternating contractions of the atria and ventricles. CCS function is well conserved across vertebrates from fish to mammals, although particular specialized aspects of CCS function are found only in endotherms (mammals and birds). The development and homeostasis of the CCS involves transcriptional and regulatory networks that act in an embryonic-stage-dependent, tissue-dependent, and dose-dependent manner. This Review describes emerging data from animal studies, stem cell models, and genome-wide association studies that have provided novel insights into the transcriptional networks underlying CCS formation and function. How these insights can be applied to develop disease models and therapies is also discussed.

Cardiac differentiation of pluripotent stem cells and implications for modeling the heart in health and disease.

Cellular models comprising cardiac cell types derived from human pluripotent stem cells are valuable for studying heart development and disease. We discuss transcriptional differences that define cellular identity in the heart, current methods for generating different cardiomyocyte subtypes, and implications for disease modeling, tissue engineering, and regenerative medicine.

A COUP-TFII Human Embryonic Stem Cell Reporter Line to Identify and Select Atrial Cardiomyocytes.

Reporter cell lines have already proven valuable in identifying, tracking, and purifying cardiac subtypes and progenitors during differentiation of human pluripotent stem cells (hPSCs). We previously showed that chick ovalbumin upstream promoter transcription factor II (COUP-TFII) is highly enriched in human atrial cardiomyocytes (CMs), but not ventricular. Here, we targeted mCherry to the COUP-TFII genomic locus in hPSCs expressing GFP from the NKX2.5 locus. This dual atrial NKX2.5EGFP/+-COUP-TFIImCherry/+ reporter line allowed identification and selection of GFP+ (G+)/mCherry+ (M+) CMs following cardiac differentiation. These cells exhibited transcriptional and functional properties of atrial CMs, whereas G+/M- CMs displayed ventricular characteristics. Via CRISPR/Cas9-mediated knockout, we demonstrated that COUP-TFII is not required for atrial specification in hPSCs. This new tool allowed selection of human atrial and ventricular CMs from mixed populations, of relevance for studying cardiac specification, developing human atrial disease models, and examining distinct effects of drugs on the atrium versus ventricle.

TECRL, a new life-threatening inherited arrhythmia gene associated with overlapping clinical features of both LQTS and CPVT.

Genetic causes of many familial arrhythmia syndromes remain elusive. In this study, whole-exome sequencing (WES) was carried out on patients from three different families that presented with life-threatening arrhythmias and high risk of sudden cardiac death (SCD). Two French Canadian probands carried identical homozygous rare variant in TECRL gene (p.Arg196Gln), which encodes the trans-2,3-enoyl-CoA reductase-like protein. Both patients had cardiac arrest, stress-induced atrial and ventricular tachycardia, and QT prolongation on adrenergic stimulation. A third patient from a consanguineous Sudanese family diagnosed with catecholaminergic polymorphic ventricular tachycardia (CPVT) had a homozygous splice site mutation (c.331+1G>A) in TECRL Analysis of intracellular calcium ([Ca2+]i) dynamics in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated from this individual (TECRLHom-hiPSCs), his heterozygous but clinically asymptomatic father (TECRLHet-hiPSCs), and a healthy individual (CTRL-hiPSCs) from the same Sudanese family, revealed smaller [Ca2+]i transient amplitudes as well as elevated diastolic [Ca2+]i in TECRLHom-hiPSC-CMs compared with CTRL-hiPSC-CMs. The [Ca2+]i transient also rose markedly slower and contained lower sarcoplasmic reticulum (SR) calcium stores, evidenced by the decreased magnitude of caffeine-induced [Ca2+]i transients. In addition, the decay phase of the [Ca2+]i transient was slower in TECRLHom-hiPSC-CMs due to decreased SERCA and NCX activities. Furthermore, TECRLHom-hiPSC-CMs showed prolonged action potentials (APs) compared with CTRL-hiPSC-CMs. TECRL knockdown in control human embryonic stem cell-derived CMs (hESC-CMs) also resulted in significantly longer APs. Moreover, stimulation by noradrenaline (NA) significantly increased the propensity for triggered activity based on delayed afterdepolarizations (DADs) in TECRLHom-hiPSC-CMs and treatment with flecainide, a class Ic antiarrhythmic drug, significantly reduced the triggered activity in these cells. In summary, we report that mutations in TECRL are associated with inherited arrhythmias characterized by clinical features of both LQTS and CPVT Patient-specific hiPSC-CMs recapitulated salient features of the clinical phenotype and provide a platform for drug screening evidenced by initial identification of flecainide as a potential therapeutic. These findings have implications for diagnosis and treatment of inherited cardiac arrhythmias.

Contractile Defect Caused by Mutation in MYBPC3 Revealed under Conditions Optimized for Human PSC-Cardiomyocyte Function.

Maximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed to identify factors that would promote an adequate level of function to permit robust single-cell contractility measurements in a human induced pluripotent stem cell (hiPSC) model of hypertrophic cardiomyopathy (HCM). A simple screen revealed the collaborative effects of thyroid hormone, IGF-1 and the glucocorticoid analog dexamethasone on the electrophysiology, bioenergetics, and contractile force generation of hPSC-CMs. In this optimized condition, hiPSC-CMs with mutations in MYBPC3, a gene encoding myosin-binding protein C, which, when mutated, causes HCM, showed significantly lower contractile force generation than controls. This was recapitulated by direct knockdown of MYBPC3 in control hPSC-CMs, supporting a mechanism of haploinsufficiency. Modeling this disease in vitro using human cells is an important step toward identifying therapeutic interventions for HCM.

Expansion and patterning of cardiovascular progenitors derived from human pluripotent stem cells.

The inability of multipotent cardiovascular progenitor cells (CPCs) to undergo multiple divisions in culture has precluded stable expansion of precursors of cardiomyocytes and vascular cells. This contrasts with neural progenitors, which can be expanded robustly and are a renewable source of their derivatives. Here we use human pluripotent stem cells bearing a cardiac lineage reporter to show that regulated MYC expression enables robust expansion of CPCs with insulin-like growth factor-1 (IGF-1) and a hedgehog pathway agonist. The CPCs can be patterned with morphogens, recreating features of heart field assignment, and controllably differentiated to relatively pure populations of pacemaker-like or ventricular-like cardiomyocytes. The cells are clonogenic and can be expanded for >40 population doublings while retaining the ability to differentiate into cardiomyocytes and vascular cells. Access to CPCs will allow precise recreation of elements of heart development in vitro and facilitate investigation of the molecular basis of cardiac fate determination. This technology is applicable for cardiac disease modeling, toxicology studies and tissue engineering.

KeyGenes, a Tool to Probe Tissue Differentiation Using a Human Fetal Transcriptional Atlas.

Differentiated derivatives of human pluripotent stem cells in culture are generally phenotypically immature compared to their adult counterparts. Their identity is often difficult to determine with certainty because little is known about their human fetal equivalents in vivo. Cellular identity and signaling pathways directing differentiation are usually determined by extrapolating information from either human adult tissue or model organisms, assuming conservation with humans. To resolve this, we generated a collection of human fetal transcriptional profiles at different developmental stages. Moreover, we developed an algorithm, KeyGenes, which uses this dataset to quantify the extent to which next-generation sequencing or microarray data resemble specific cell or tissue types in the human fetus. Using KeyGenes combined with the human fetal atlas, we identified multiple cell and tissue samples unambiguously on a limited set of features. We thus provide a flexible and expandable platform to monitor and evaluate the efficiency of differentiation in vitro.

Atrial-like cardiomyocytes from human pluripotent stem cells are a robust preclinical model for assessing atrial-selective pharmacology.

Drugs targeting atrial-specific ion channels, Kv1.5 or Kir3.1/3.4, are being developed as new therapeutic strategies for atrial fibrillation. However, current preclinical studies carried out in non-cardiac cell lines or animal models may not accurately represent the physiology of a human cardiomyocyte (CM). In the current study, we tested whether human embryonic stem cell (hESC)-derived atrial CMs could predict atrial selectivity of pharmacological compounds. By modulating retinoic acid signaling during hESC differentiation, we generated atrial-like (hESC-atrial) and ventricular-like (hESC-ventricular) CMs. We found the expression of atrial-specific ion channel genes, KCNA5 (encoding Kv1.5) and KCNJ3 (encoding Kir 3.1), in hESC-atrial CMs and further demonstrated that these ion channel genes are regulated by COUP-TF transcription factors. Moreover, in response to multiple ion channel blocker, vernakalant, and Kv1.5 blocker, XEN-D0101, hESC-atrial but not hESC-ventricular CMs showed action potential (AP) prolongation due to a reduction in early repolarization. In hESC-atrial CMs, XEN-R0703, a novel Kir3.1/3.4 blocker restored the AP shortening caused by CCh. Neither CCh nor XEN-R0703 had an effect on hESC-ventricular CMs. In summary, we demonstrate that hESC-atrial CMs are a robust model for pre-clinical testing to assess atrial selectivity of novel antiarrhythmic drugs.

Molecular analysis of patterning of conduction tissues in the developing human heart.

BACKGROUND: Recent studies in experimental animals have revealed some molecular mechanisms underlying the differentiation of the myocardium making up the conduction system. To date, lack of gene expression data for the developing human conduction system has precluded valid extrapolations from experimental studies to the human situation. METHODS AND RESULTS: We performed immunohistochemical analyses of the expression of key transcription factors, such as ISL1, TBX3, TBX18, and NKX2-5, ion channel HCN4, and connexins in the human embryonic heart. We supplemented our molecular analyses with 3-dimensional reconstructions of myocardial TBX3 expression. TBX3 is expressed in the developing conduction system and in the right venous valve, atrioventricular ring bundles, and retro-aortic nodal region. TBX3-positive myocardium, with exception of the top of the ventricular septum, is devoid of fast-conducting connexin40 and connexin43 and hence identifies slowly conducting pathways. In the early embryonic heart, we found wide expression of the pacemaker channel HCN4 at the venous pole, including the atrial chambers. HCN4 expression becomes confined during later developmental stages to the components of the conduction system. Patterns of expression of transcription factors, known from experimental studies to regulate the development of the sinus node and atrioventricular conduction system, are similar in the human and mouse developing hearts. CONCLUSIONS: Our findings point to the comparability of mechanisms governing the development of the cardiac conduction patterning in human and mouse, which provide a molecular basis for understanding the functioning of the human developing heart before formation of a discrete conduction system.