Investigating inherited arrhythmias using hiPSC-derived cardiomyocytes.

Fundamental to the functional behavior of cardiac muscle is that the cardiomyocytes are integrated as a functional syncytium. Disrupted electrical activity in the cardiac tissue can lead to serious complications including cardiac arrhythmias. Therefore, it is important to study electrophysiological properties of the cardiac tissue. With advancements in stem cell research, protocols for the production of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been established, providing great potential in modelling cardiac arrhythmias and drug testing. The hiPSC-CM model can be used in conjunction with electrophysiology-based platforms to examine the electrical activity of the cardiac tissue. Techniques for determining the myocardial electrical activity include multielectrode arrays (MEAs), optical mapping (OM), and patch clamping. These techniques provide critical approaches to investigate cardiac electrical abnormalities that underlie arrhythmias.

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.

Binding of calcium and magnesium to human cardiac troponin C.

Cardiac muscle thin filaments are composed of actin, tropomyosin, and troponin that change conformation in response to Ca2+ binding, triggering muscle contraction. Human cardiac troponin C (cTnC) is the Ca2+-sensing component of the thin filament. It contains structural sites (III/IV) that bind both Ca2+ and Mg2+ and a regulatory site (II) that has been thought to bind only Ca2+. Binding of Ca2+ at this site initiates a series of conformational changes that culminate in force production. However, the mechanisms that underpin the regulation of binding at site II remain unclear. Here, we have quantified the interaction between site II and Ca2+/Mg2+ through isothermal titration calorimetry and thermodynamic integration simulations. Direct and competitive binding titrations with WT N-terminal cTnC and full-length cTnC indicate that physiologically relevant concentrations of both Ca2+/Mg2+ interacted with the same locus. Moreover, the D67A/D73A N-terminal cTnC construct in which two coordinating residues within site II were removed was found to have significantly reduced affinity for both cations. In addition, 1 mM Mg2+ caused a 1.4-fold lower affinity for Ca2+. These experiments strongly suggest that cytosolic-free Mg2+ occupies a significant population of the available site II. Interaction of Mg2+ with site II of cTnC likely has important functional consequences for the heart both at baseline as well as in diseased states that decrease or increase the availability of Mg2+, such as secondary hyperparathyroidism or ischemia, respectively.

In vitro analyses of suspected arrhythmogenic thin filament variants as a cause of sudden cardiac death in infants.

Sudden unexpected death of an infant (SUDI) is a devastating occurrence for families. To investigate the genetic pathogenesis of SUDI, we sequenced >70 genes from 191 autopsy-negative SUDI victims. Ten infants sharing a previously unknown variant in troponin I (TnI) were identified. The mutation (TNNI1 R37C+/-) is in the fetal/neonatal paralog of TnI, a gene thought to be expressed in the heart up to the first 24 months of life. Using phylogenetic analysis and molecular dynamics simulations, it was determined that arginine at residue 37 in TNNI1 may play a critical functional role, suggesting that the variant may be pathogenic. We investigated the biophysical properties of the TNNI1 R37C mutation in human reconstituted thin filaments (RTFs) using fluorometry. RTFs reconstituted with the mutant R37C TnI exhibited reduced Ca2+-binding sensitivity due to an increased Ca2+ off-rate constant. Furthermore, we generated TNNI1 R37C+/- mutants in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) using CRISPR-Cas9. In monolayers of hiPSC-CMs, we simultaneously monitored voltage and Ca2+ transients through optical mapping and compared them to their isogenic controls. We observed normal intrinsic beating patterns under control conditions in TNNI1 R37C+/- at stimulation frequencies of 55 beats/min (bpm), but these cells showed no restitution with increased stimulation frequency to 65 bpm and exhibited alternans at >75 bpm. The WT hiPSC-CMs did not exhibit any sign of arrhythmogenicity even at stimulation frequencies of 120 bpm. The approach used in this study provides critical physiological and mechanistic bases to investigate sarcomeric mutations in the pathogenesis of SUDI.

Pediatric Catecholaminergic Polymorphic Ventricular Tachycardia: A Translational Perspective for the Clinician-Scientist.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare and potentially lethal inherited arrhythmia disease characterized by exercise or emotion-induced bidirectional or polymorphic ventricular tachyarrhythmias. The median age of disease onset is reported to be approximately 10 years of age. The majority of CPVT patients have pathogenic variants in the gene encoding the cardiac ryanodine receptor, or calsequestrin 2. These lead to mishandling of calcium in cardiomyocytes resulting in after-depolarizations, and ventricular arrhythmias. Disease severity is particularly pronounced in younger individuals who usually present with cardiac arrest and arrhythmic syncope. Risk stratification is imprecise and long-term prognosis on therapy is unknown despite decades of research focused on pediatric CPVT populations. The purpose of this review is to summarize contemporary data on pediatric CPVT, highlight knowledge gaps and present future research directions for the clinician-scientist to address.

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.

The mitochondrial metallochaperone SCO1 maintains CTR1 at the plasma membrane to preserve copper homeostasis in the murine heart.

SCO1 is a ubiquitously expressed, mitochondrial protein with essential roles in cytochrome c oxidase (COX) assembly and the regulation of copper homeostasis. SCO1 patients present with severe forms of early onset disease, and ultimately succumb from liver, heart or brain failure. However, the inherent susceptibility of these tissues to SCO1 mutations and the clinical heterogeneity observed across SCO1 pedigrees remain poorly understood phenomena. To further address this issue, we generated Sco1hrt/hrt and Sco1stm/stm mice in which Sco1 was specifically deleted in heart and striated muscle, respectively. Lethality was observed in both models due to a combined COX and copper deficiency that resulted in a dilated cardiomyopathy. Left ventricular dilation and loss of heart function was preceded by a temporal decrease in COX activity and copper levels in the longer-lived Sco1stm/stm mice. Interestingly, the reduction in copper content of Sco1stm/stm cardiomyocytes was due to the mislocalisation of CTR1, the high affinity transporter that imports copper into the cell. CTR1 was similarly mislocalized to the cytosol in the heart of knockin mice carrying a homozygous G115S substitution in Sco1, which in humans causes a hypertrophic cardiomyopathy. Our current findings in the heart are in marked contrast to our prior observations in the liver, where Sco1 deletion results in a near complete absence of CTR1 protein. These data collectively argue that mutations perturbing SCO1 function have tissue-specific consequences for the machinery that ultimately governs copper homeostasis, and further establish the importance of aberrant mitochondrial signaling to the etiology of copper handling disorders.

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.

Changes in the dynamics of the cardiac troponin C molecule explain the effects of Ca2+-sensitizing mutations.

Cardiac troponin C (cTnC) is the regulatory protein that initiates cardiac contraction in response to Ca2+ TnC binding Ca2+ initiates a cascade of protein-protein interactions that begins with the opening of the N-terminal domain of cTnC, followed by cTnC binding the troponin I switch peptide (TnISW). We have evaluated, through isothermal titration calorimetry and molecular-dynamics simulation, the effect of several clinically relevant mutations (A8V, L29Q, A31S, L48Q, Q50R, and C84Y) on the Ca2+ affinity, structural dynamics, and calculated interaction strengths between cTnC and each of Ca2+ and TnISW Surprisingly the Ca2+ affinity measured by isothermal titration calorimetry was only significantly affected by half of these mutations including L48Q, which had a 10-fold higher affinity than WT, and the Q50R and C84Y mutants, each of which had affinities 3-fold higher than wild type. This suggests that Ca2+ affinity of the N-terminal domain of cTnC in isolation is insufficient to explain the pathogenicity of these mutations. Molecular-dynamics simulation was used to evaluate the effects of these mutations on Ca2+ binding, structural dynamics, and TnI interaction independently. Many of the mutations had a pronounced effect on the balance between the open and closed conformations of the TnC molecule, which provides an indirect mechanism for their pathogenic properties. Our data demonstrate that the structural dynamics of the cTnC molecule are key in determining myofilament Ca2+ sensitivity. Our data further suggest that modulation of the structural dynamics is the underlying molecular mechanism for many disease mutations that are far from the regulatory Ca2+-binding site of cTnC.

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.