Atrial-specific hiPSC-derived cardiomyocytes in drug discovery and disease modeling.

The discovery and application of human-induced pluripotent stem cells (hiPSCs) have been instrumental in the investigation of the pathophysiology of cardiovascular diseases. Patient-specific hiPSCs can now be generated, genome-edited, and subsequently differentiated into various cell types and used for regenerative medicine, disease modeling, drug testing, toxicity screening, and 3D tissue generation. Modulation of the retinoic acid signaling pathway has been shown to direct cardiomyocyte differentiation towards an atrial lineage. A variety of studies have successfully differentiated patient-specific atrial cardiac myocytes (hiPSC-aCM) and atrial engineered heart tissue (aEHT) that express atrial specific genes (e.g., sarcolipin and ANP) and exhibit atrial electrophysiological and contractility profiles. Identification of protocols to differentiate atrial cells from patients with atrial fibrillation and other inherited diseases or creating disease models using genetic mutation studies has shed light on the mechanisms of atrial-specific diseases and identified the efficacy of atrial-selective pharmacological compounds. hiPSC-aCMs and aEHTs can be used in drug discovery and drug screening studies to investigate the efficacy of atrial selective drugs on atrial fibrillation models. Furthermore, hiPSC-aCMs can be effective tools in studying the mechanism, pathophysiology and treatment options of atrial fibrillation and its genetic underpinnings. The main limitation of using hiPSC-CMs is their immature phenotype compared to adult CMs. A wide range of approaches and protocols are used by various laboratories to optimize and enhance CM maturation, including electrical stimulation, culture time, biophysical cues and changes in metabolic factors.

The hERG channel activator, RPR260243, enhances protective IKr current early in the refractory period reducing arrhythmogenicity in zebrafish hearts.

Human ether-à-go-go related gene (hERG) K+ channels are important in cardiac repolarization, and their dysfunction causes prolongation of the ventricular action potential, long QT syndrome, and arrhythmia. As such, approaches to augment hERG channel function, such as activator compounds, have been of significant interest due to their marked therapeutic potential. Activator compounds that hinder channel inactivation abbreviate action potential duration (APD) but carry risk of overcorrection leading to short QT syndrome. Enhanced risk by overcorrection of the APD may be tempered by activator-induced increased refractoriness; however, investigation of the cumulative effect of hERG activator compounds on the balance of these effects in whole organ systems is lacking. Here, we have investigated the antiarrhythmic capability of a hERG activator, RPR260243, which primarily augments channel function by slowing deactivation kinetics in ex vivo zebrafish whole hearts. We show that RPR260243 abbreviates the ventricular APD, reduces triangulation, and steepens the slope of the electrical restitution curve. In addition, RPR260243 increases the post-repolarization refractory period. We provide evidence that this latter effect arises from RPR260243-induced enhancement of hERG channel-protective currents flowing early in the refractory period. Finally, the cumulative effect of RPR260243 on arrhythmogenicity in whole organ zebrafish hearts is demonstrated by the restoration of normal rhythm in hearts presenting dofetilide-induced arrhythmia. These findings in a whole organ model demonstrate the antiarrhythmic benefit of hERG activator compounds that modify both APD and refractoriness. Furthermore, our results demonstrate that targeted slowing of hERG channel deactivation and enhancement of protective currents may provide an effective antiarrhythmic approach.NEW & NOTEWORTHY hERG channel dysfunction causes long QT syndrome and arrhythmia. Activator compounds have been of significant interest due to their therapeutic potential. We used the whole organ zebrafish heart model to demonstrate the antiarrhythmic benefit of the hERG activator, RPR260243. The activator abbreviated APD and increased refractoriness, the combined effect of which rescued induced ventricular arrhythmia. Our findings show that the targeted slowing of hERG channel deactivation and enhancement of protective currents caused by the RPR260243 activator may provide an effective antiarrhythmic approach.

Investigating the utility of adult zebrafish ex vivo whole hearts to pharmacologically screen hERG channel activator compounds.

There is significant interest in the potential utility of small-molecule activator compounds to mitigate cardiac arrhythmia caused by loss of function of hERG1a voltage-gated potassium channels. Zebrafish (Danio rerio) have been proposed as a cost-effective, high-throughput drug-screening model to identify compounds that cause hERG1a dysfunction. However, there are no reports on the effects of hERG1a activator compounds in zebrafish and consequently on the utility of the model to screen for potential gain-of-function therapeutics. Here, we examined the effects of hERG1a blocker and types 1 and 2 activator compounds on isolated zkcnh6a (zERG3) channels in the Xenopus oocyte expression system as well as action potentials recorded from ex vivo adult zebrafish whole hearts using optical mapping. Our functional data from isolated zkcnh6a channels show that under the conditions tested, these channels are blocked by hERG1a channel blockers (dofetilide and terfenadine), and activated by type 1 (RPR260243) and type 2 (NS1643, PD-118057) hERG1a activators with higher affinity than hKCNH2a channels (except NS1643), with differences accounted for by different biophysical properties in the two channels. In ex vivo zebrafish whole hearts, two of the three hERG1a activators examined caused abbreviation of the action potential duration (APD), whereas hERG1a blockers caused APD prolongation. These data represent, to our knowledge, the first pharmacological characterization of isolated zkcnh6a channels and the first assessment of hERG enhancing therapeutics in zebrafish. Our findings lead us to suggest that the zebrafish ex vivo whole heart model serves as a valuable tool in the screening of hKCNH2a blocker and activator compounds.

The Zebrafish Heart as a Model of Mammalian Cardiac Function.

Zebrafish (Danio rerio) are widely used as vertebrate model in developmental genetics and functional genomics as well as in cardiac structure-function studies. The zebrafish heart has been increasingly used as a model of human cardiac function, in part, due to the similarities in heart rate and action potential duration and morphology with respect to humans. The teleostian zebrafish is in many ways a compelling model of human cardiac function due to the clarity afforded by its ease of genetic manipulation, the wealth of developmental biological information, and inherent suitability to a variety of experimental techniques. However, in addition to the numerous advantages of the zebrafish system are also caveats related to gene duplication (resulting in paralogs not present in human or other mammals) and fundamental differences in how zebrafish hearts function. In this review, we discuss the use of zebrafish as a cardiac function model through the use of techniques such as echocardiography, optical mapping, electrocardiography, molecular investigations of excitation-contraction coupling, and their physiological implications relative to that of the human heart. While some of these techniques (e.g., echocardiography) are particularly challenging in the zebrafish because of diminutive size of the heart (~1.5 mm in diameter) critical information can be derived from these approaches and are discussed in detail in this article.

Defining cellular complexity in human autosomal dominant polycystic kidney disease by multimodal single cell analysis.

Autosomal dominant polycystic kidney disease (ADPKD) is the leading genetic cause of end stage renal disease characterized by progressive expansion of kidney cysts. To better understand the cell types and states driving ADPKD progression, we analyze eight ADPKD and five healthy human kidney samples, generating single cell multiomic atlas consisting of ~100,000 single nucleus transcriptomes and ~50,000 single nucleus epigenomes. Activation of proinflammatory, profibrotic signaling pathways are driven by proximal tubular cells with a failed repair transcriptomic signature, proinflammatory fibroblasts and collecting duct cells. We identify GPRC5A as a marker for cyst-lining collecting duct cells that exhibits increased transcription factor binding motif availability for NF-κB, TEAD, CREB and retinoic acid receptors. We identify and validate a distal enhancer regulating GPRC5A expression containing these motifs. This single cell multiomic analysis of human ADPKD reveals previously unrecognized cellular heterogeneity and provides a foundation to develop better diagnostic and therapeutic approaches.

Drug screening platform using human induced pluripotent stem cell-derived atrial cardiomyocytes and optical mapping.

Current drug development efforts for the treatment of atrial fibrillation are hampered by the fact that many preclinical models have been unsuccessful in reproducing human cardiac physiology and its response to medications. In this study, we demonstrated an approach using human induced pluripotent stem cell-derived atrial and ventricular cardiomyocytes (hiPSC-aCMs and hiPSC-vCMs, respectively) coupled with a sophisticated optical mapping system for drug screening of atrial-selective compounds in vitro. We optimized differentiation of hiPSC-aCMs by modulating the WNT and retinoid signaling pathways. Characterization of the transcriptome and proteome revealed that retinoic acid pushes the differentiation process into the atrial lineage and generated hiPSC-aCMs. Functional characterization using optical mapping showed that hiPSC-aCMs have shorter action potential durations and faster Ca2+ handling dynamics compared with hiPSC-vCMs. Furthermore, pharmacological investigation of hiPSC-aCMs captured atrial-selective effects by displaying greater sensitivity to atrial-selective compounds 4-aminopyridine, AVE0118, UCL1684, and vernakalant when compared with hiPSC-vCMs. These results established that a model system incorporating hiPSC-aCMs combined with optical mapping is well-suited for preclinical drug screening of novel and targeted atrial selective compounds.

Mechanisms of Arrhythmogenicity of Hypertrophic Cardiomyopathy-Associated Troponin T (TNNT2) Variant I79N.

Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiovascular disease and often results in cardiac remodeling and an increased incidence of sudden cardiac arrest (SCA) and death, especially in youth and young adults. Among thousands of different variants found in HCM patients, variants of TNNT2 (cardiac troponin T-TNNT2) are linked to increased risk of ventricular arrhythmogenesis and sudden death despite causing little to no cardiac hypertrophy. Therefore, studying the effect of TNNT2 variants on cardiac propensity for arrhythmogenesis can pave the way for characterizing HCM in susceptible patients before sudden cardiac arrest occurs. In this study, a TNNT2 variant, I79N, was generated in human cardiac recombinant/reconstituted thin filaments (hcRTF) to investigate the effect of the mutation on myofilament Ca2+ sensitivity and Ca2+ dissociation rate using steady-state and stopped-flow fluorescence techniques. The results revealed that the I79N variant significantly increases myofilament Ca2+ sensitivity and decreases the Ca2+ off-rate constant (k off). To investigate further, a heterozygous I79N+/- TNNT2 variant was introduced into human-induced pluripotent stem cells using CRISPR/Cas9 and subsequently differentiated into ventricular cardiomyocytes (hiPSC-CMs). To study the arrhythmogenic properties, monolayers of I79N+/- hiPSC-CMs were studied in comparison to their isogenic controls. Arrhythmogenesis was investigated by measuring voltage (V m) and cytosolic Ca2+ transients over a range of stimulation frequencies. An increasing stimulation frequency was applied to the cells, from 55 to 75 bpm. The results of this protocol showed that the TnT-I79N cells had reduced intracellular Ca2+ transients due to the enhanced cytosolic Ca2+ buffering. These changes in Ca2+ handling resulted in beat-to-beat instability and triangulation of the cardiac action potential, which are predictors of arrhythmia risk. While wild-type (WT) hiPSC-CMs were accurately entrained to frequencies of at least 150 bpm, the I79N hiPSC-CMs demonstrated clear patterns of alternans for both V m and Ca2+ transients at frequencies >75 bpm. Lastly, a transcriptomic analysis was conducted on WT vs. I79N+/- TNNT2 hiPSC-CMs using a custom NanoString codeset. The results showed a significant upregulation of NPPA (atrial natriuretic peptide), NPPB (brain natriuretic peptide), Notch signaling pathway components, and other extracellular matrix (ECM) remodeling components in I79N+/- vs. the isogenic control. This significant shift demonstrates that this missense in the TNNT2 transcript likely causes a biophysical trigger, which initiates this significant alteration in the transcriptome. This TnT-I79N hiPSC-CM model not only reproduces key cellular features of HCM-linked mutations but also suggests that this variant causes uncharted pro-arrhythmic changes to the human action potential and gene expression.

Physiological phenotyping of the adult zebrafish heart.

The zebrafish has proven to be an excellent organism for manipulation of its genome from a long history of transcript down-regulation using morpholino oligimers to more recent genome editing tools such as CRISPR-Cas9. Early forward and reverse genetic screens significantly benefited from the transparency of zebrafish embryos, allowing cardiac development as a function of genetics to be directly observed. However, gradual loss of transparency with subsequent maturation limited many of these approaches to the first several days post-fertilization. As many genes are developmentally regulated, the immature phenotype is not entirely indicative of that of the mature zebrafish. For accurate phenotyping, subsequent developmental stages including full maturation must also be considered. In adult zebrafish, cardiac function can now be studied in great detail due both to the size of the hearts as well as recent technological improvements. Because of their small size, zebrafish are particularly amenable to high frequency echocardiography for detailed functional recordings. Although relatively small, the hearts are easily excised and contractile parameters can be measured from whole hearts, heart slices, individual cardiomyocytes and even single myofibrils. Similarly, electrical activity can also be measured using a variety of techniques, including in vivo and ex vivo electrocardiograms, optical mapping and traditional microelectrode techniques. In this report, the major advantages and technical considerations of these physiological tools are discussed.

Zebrafish as a model of mammalian cardiac function: Optically mapping the interplay of temperature and rate on voltage and calcium dynamics.

The zebrafish (Danio rerio) heart is a viable model of mammalian cardiovascular function due to similarities in heart rate, ultrastructure, and action potential morphology. Zebrafish are able to tolerate a wide range of naturally occurring temperatures through altering chronotropic and inotropic properties of the heart. Optical mapping of cannulated zebrafish hearts can be used to assess the effect of temperature on excitation-contraction (EC) coupling and to explore the mechanisms underlying voltage (Vm) and calcium (Ca2+) transients. Applicability of zebrafish as a model of mammalian cardiac physiology should be understood in the context of numerous subtle differences in structure, ion channel expression, and Ca2+ handling. In contrast to mammalian systems, Ca2+ release from the sarcoplasmic reticulum (SR) plays a relatively small role in activating the contractile apparatus in teleosts, which may contribute to differences in restitution. The contractile function of the zebrafish heart is closely tied to extracellular Ca2+ which enters cardiomyocytes through L-type Ca2+ channel (LTCC), T-type Ca2+ channel (TTCC), and the sodium-calcium exchanger (NCX). Novel data found that despite large temperature effects on heart rate, Vm, and Ca2+ durations, the relationship between Vm and Ca2+ signals was only minimally altered in the face of acute temperature change. This suggests that zebrafish Vm and Ca2+ kinetics are largely rate-independent. In comparison to mammalian systems, zebrafish Ca2+ cycling is inherently more dependent on transsarcolemmal Ca2+ transport and less reliant on SR Ca2+ release. However, the compensatory actions of various components of the Ca2+ cycling machinery of the zebrafish cardiomyocytes, allow for maintenance of EC coupling over a wide range of environmental temperatures.