Gating kinetics and pharmacological properties of small-conductance Ca2+-activated potassium channels.

Small-conductance (SK) calcium-activated potassium channels are a promising treatment target in atrial fibrillation. However, the functional properties that differentiate SK inhibitors remain poorly understood. The objective of this study was to determine how two unrelated SK channel inhibitors, apamin and AP14145, impact SK channel function in excised inside-out single-channel recordings. Surprisingly, both apamin and AP14145 exert much of their inhibition by inducing a class of very-long-lived channel closures (apamin: τc,vl = 11.8 ± 7.1 s, and AP14145: τc,vl = 10.3 ± 7.2 s), which were never observed under control conditions. Both inhibitors also induced changes to the three closed and two open durations typical of normal SK channel gating. AP14145 shifted the open duration distribution to favor longer open durations, whereas apamin did not alter open-state kinetics. AP14145 also prolonged the two shortest channel closed durations (AP14145: τc,s = 3.50 ± 0.81 ms, and τc,i = 32.0 ± 6.76 ms versus control: τc,s = 1.59 ± 0.19 ms, and τc,i = 13.5 ± 1.17 ms), thus slowing overall gating kinetics within bursts of channel activity. In contrast, apamin accelerated intraburst gating kinetics by shortening the two shortest closed durations (τc,s = 0.75 ± 0.10 ms and τc,i = 5.08 ± 0.49 ms) and inducing periods of flickery activity. Finally, AP14145 introduced a unique form of inhibition by decreasing unitary current amplitude. SK channels exhibited two clearly distinguishable amplitudes (control: Ahigh = 0.76 ± 0.03 pA, and Alow = 0.54 ± 0.03 pA). AP14145 both reduced the fraction of patches exhibiting the higher amplitude (AP14145: 4/9 patches versus control: 16/16 patches) and reduced the mean low amplitude (0.38 ± 0.03 pA). Here, we have demonstrated that both inhibitors introduce very long channel closures but that each also exhibits unique effects on other components of SK gating kinetics and unitary current. The combination of these effects is likely to be critical for understanding the functional differences of each inhibitor in the context of cyclical Ca2+-dependent channel activation in vivo.

Mechanisms of spontaneous Ca2+ release-mediated arrhythmia in a novel 3D human atrial myocyte model: II. Ca2+ -handling protein variation.

Disruption of the transverse-axial tubule system (TATS) in diseases such as heart failure and atrial fibrillation occurs in combination with changes in the expression and distribution of key Ca2+ -handling proteins. Together this ultrastructural and ionic remodelling is associated with aberrant Ca2+ cycling and electrophysiological instabilities that underlie arrhythmic activity. However, due to the concurrent changes in TATs and Ca2+ -handling protein expression and localization that occur in disease it is difficult to distinguish their individual contributions to the arrhythmogenic state. To investigate this, we applied our novel 3D human atrial myocyte model with spatially detailed Ca2+ diffusion and TATS to investigate the isolated and interactive effects of changes in expression and localization of key Ca2+ -handling proteins and variable TATS density on Ca2+ -handling abnormality driven membrane instabilities. We show that modulating the expression and distribution of the sodium-calcium exchanger, ryanodine receptors and the sarcoplasmic reticulum (SR) Ca2+ buffer calsequestrin have varying pro- and anti-arrhythmic effects depending on the balance of opposing influences on SR Ca2+ leak-load and Ca2+ -voltage relationships. Interestingly, the impact of protein remodelling on Ca2+ -driven proarrhythmic behaviour varied dramatically depending on TATS density, with intermediately tubulated cells being more severely affected compared to detubulated and densely tubulated myocytes. This work provides novel mechanistic insight into the distinct and interactive consequences of TATS and Ca2+ -handling protein remodelling that underlies dysfunctional Ca2+ cycling and electrophysiological instability in disease. KEY POINTS: In our companion paper we developed a 3D human atrial myocyte model, coupling electrophysiology and Ca2+ handling with subcellular spatial details governed by the transverse-axial tubule system (TATS). Here we utilize this model to mechanistically examine the impact of TATS loss and changes in the expression and distribution of key Ca2+ -handling proteins known to be remodelled in disease on Ca2+ homeostasis and electrophysiological stability. We demonstrate that varying the expression and localization of these proteins has variable pro- and anti-arrhythmic effects with outcomes displaying dependence on TATS density. Whereas detubulated myocytes typically appear unaffected and densely tubulated cells seem protected, the arrhythmogenic effects of Ca2+ handling protein remodelling are profound in intermediately tubulated cells. Our work shows the interaction between TATS and Ca2+ -handling protein remodelling that underlies the Ca2+ -driven proarrhythmic behaviour observed in atrial fibrillation and may help to predict the effects of antiarrhythmic strategies at varying stages of ultrastructural remodelling.

Mechanisms of spontaneous Ca2+ release-mediated arrhythmia in a novel 3D human atrial myocyte model: I. Transverse-axial tubule variation.

Intracellular calcium (Ca2+ ) cycling is tightly regulated in the healthy heart ensuring effective contraction. This is achieved by transverse (t)-tubule membrane invaginations that facilitate close coupling of key Ca2+ -handling proteins such as the L-type Ca2+ channel and Na+ -Ca2+ exchanger (NCX) on the cell surface with ryanodine receptors (RyRs) on the intracellular Ca2+ store. Although less abundant and regular than in the ventricle, t-tubules also exist in atrial myocytes as a network of transverse invaginations with axial extensions known as the transverse-axial tubule system (TATS). In heart failure and atrial fibrillation, there is TATS remodelling that is associated with aberrant Ca2+ -handling and Ca2+ -induced arrhythmic activity; however, the mechanism underlying this is not fully understood. To address this, we developed a novel 3D human atrial myocyte model that couples electrophysiology and Ca2+ -handling with variable TATS organization and density. We extensively parameterized and validated our model against experimental data to build a robust tool examining TATS regulation of subcellular Ca2+ release. We found that varying TATS density and thus the localization of key Ca2+ -handling proteins has profound effects on Ca2+ handling. Following TATS loss, there is reduced NCX that results in increased cleft Ca2+ concentration through decreased Ca2+ extrusion. This elevated Ca2+ increases RyR open probability causing spontaneous Ca2+ releases and the promotion of arrhythmogenic waves (especially in the cell interior) leading to voltage instabilities through delayed afterdepolarizations. In summary, the present study demonstrates a mechanistic link between TATS remodelling and Ca2+ -driven proarrhythmic behaviour that probably reflects the arrhythmogenic state observed in disease. KEY POINTS: Transverse-axial tubule systems (TATS) modulate Ca2+ handling and excitation-contraction coupling in atrial myocytes, with TATS remodelling in heart failure and atrial fibrillation being associated with altered Ca2+ cycling and subsequent arrhythmogenesis. To investigate the poorly understood mechanisms linking TATS variation and spontaneous Ca2+ release, we built, parameterized and validated a 3D human atrial myocyte model coupling electrophysiology and spatially-detailed subcellular Ca2+ handling governed by the TATS. Simulated TATS loss causes diastolic Ca2+ and voltage instabilities through reduced Na+ -Ca2+ exchanger-mediated Ca2+ removal, cleft Ca2+ accumulation and increased ryanodine receptor open probability, resulting in spontaneous Ca2+ release and promotion of arrhythmogenic waves and delayed afterdepolarizations. At fast electrical rates typical of atrial tachycardia/fibrillation, spontaneous Ca2+ releases are larger and more frequent in the cell interior than at the periphery. Our work provides mechanistic insight into how atrial TATS remodelling can lead to Ca2+ -driven instabilities that may ultimately contribute to the arrhythmogenic state in disease.

Hypokalemia Promotes Arrhythmia by Distinct Mechanisms in Atrial and Ventricular Myocytes.

RATIONALE: Hypokalemia occurs in up to 20% of hospitalized patients and is associated with increased incidence of ventricular and atrial fibrillation. It is unclear whether these differing types of arrhythmia result from direct and perhaps distinct effects of hypokalemia on cardiomyocytes. OBJECTIVE: To investigate proarrhythmic mechanisms of hypokalemia in ventricular and atrial myocytes. METHODS AND RESULTS: Experiments were performed in isolated rat myocytes exposed to simulated hypokalemia conditions (reduction of extracellular [K+] from 5.0 to 2.7 mmol/L) and supported by mathematical modeling studies. Ventricular cells subjected to hypokalemia exhibited Ca2+ overload and increased generation of both spontaneous Ca2+ waves and delayed afterdepolarizations. However, similar Ca2+-dependent spontaneous activity during hypokalemia was only observed in a minority of atrial cells that were observed to contain t-tubules. This effect was attributed to close functional pairing of the Na+-K+ ATPase and Na+-Ca2+ exchanger proteins within these structures, as reduction in Na+ pump activity locally inhibited Ca2+ extrusion. Ventricular myocytes and tubulated atrial myocytes additionally exhibited early afterdepolarizations during hypokalemia, associated with Ca2+ overload. However, early afterdepolarizations also occurred in untubulated atrial cells, despite Ca2+ quiescence. These phase-3 early afterdepolarizations were rather linked to reactivation of nonequilibrium Na+ current, as they were rapidly blocked by tetrodotoxin. Na+ current-driven early afterdepolarizations in untubulated atrial cells were enabled by membrane hyperpolarization during hypokalemia and short action potential configurations. Brief action potentials were in turn maintained by ultra-rapid K+ current (IKur); a current which was found to be absent in tubulated atrial myocytes and ventricular myocytes. CONCLUSIONS: Distinct mechanisms underlie hypokalemia-induced arrhythmia in the ventricle and atrium but also vary between atrial myocytes depending on subcellular structure and electrophysiology.

A computational method for identifying an optimal combination of existing drugs to repair the action potentials of SQT1 ventricular myocytes.

Mutations are known to cause perturbations in essential functional features of integral membrane proteins, including ion channels. Even restricted or point mutations can result in substantially changed properties of ion currents. The additive effect of these alterations for a specific ion channel can result in significantly changed properties of the action potential (AP). Both AP shortening and AP prolongation can result from known mutations, and the consequences can be life-threatening. Here, we present a computational method for identifying new drugs utilizing combinations of existing drugs. Based on the knowledge of theoretical effects of existing drugs on individual ion currents, our aim is to compute optimal combinations that can 'repair' the mutant AP waveforms so that the baseline AP-properties are restored. More specifically, we compute optimal, combined, drug concentrations such that the waveforms of the transmembrane potential and the cytosolic calcium concentration of the mutant cardiomyocytes (CMs) becomes as similar as possible to their wild type counterparts after the drug has been applied. In order to demonstrate the utility of this method, we address the question of computing an optimal drug for the short QT syndrome type 1 (SQT1). For the SQT1 mutation N588K, there are available data sets that describe the effect of various drugs on the mutated K+ channel. These published findings are the basis for our computational analysis which can identify optimal compounds in the sense that the AP of the mutant CMs resembles essential biomarkers of the wild type CMs. Using recently developed insights regarding electrophysiological properties among myocytes from different species, we compute optimal drug combinations for hiPSC-CMs, rabbit ventricular CMs and adult human ventricular CMs with the SQT1 mutation. Since the 'composition' of ion channels that form the AP is different for the three types of myocytes under consideration, so is the composition of the optimal drug.

Properties of cardiac conduction in a cell-based computational model.

The conduction of electrical signals through cardiac tissue is essential for maintaining the function of the heart, and conduction abnormalities are known to potentially lead to life-threatening arrhythmias. The properties of cardiac conduction have therefore been the topic of intense study for decades, but a number of questions related to the mechanisms of conduction still remain unresolved. In this paper, we demonstrate how the so-called EMI model may be used to study some of these open questions. In the EMI model, the extracellular space, the cell membrane, the intracellular space and the cell connections are all represented as separate parts of the computational domain, and the model therefore allows for study of local properties that are hard to represent in the classical homogenized bidomain or monodomain models commonly used to study cardiac conduction. We conclude that a non-uniform sodium channel distribution increases the conduction velocity and decreases the time delays over gap junctions of reduced coupling in the EMI model simulations. We also present a theoretical optimal cell length with respect to conduction velocity and consider the possibility of ephaptic coupling (i.e. cell-to-cell coupling through the extracellular potential) acting as an alternative or supporting mechanism to gap junction coupling. We conclude that for a non-uniform distribution of sodium channels and a sufficiently small intercellular distance, ephaptic coupling can influence the dynamics of the sodium channels and potentially provide cell-to-cell coupling when the gap junction connection is absent.

Alterations in Schizophrenia-Associated Genes Can Lead to Increased Power in Delta Oscillations.

Genome-wide association studies have implicated many ion channels in schizophrenia pathophysiology. Although the functions of these channels are relatively well characterized by single-cell studies, the contributions of common variation in these channels to neurophysiological biomarkers and symptoms of schizophrenia remain elusive. Here, using computational modeling, we show that a common biomarker of schizophrenia, namely, an increase in delta-oscillation power, may be a direct consequence of altered expression or kinetics of voltage-gated ion channels or calcium transporters. Our model of a circuit of layer V pyramidal cells highlights multiple types of schizophrenia-related variants that contribute to altered dynamics in the delta-frequency band. Moreover, our model predicts that the same membrane mechanisms that increase the layer V pyramidal cell network gain and response to delta-frequency oscillations may also cause a deficit in a single-cell correlate of the prepulse inhibition, which is a behavioral biomarker highly associated with schizophrenia.

Arrhythmogenic Current Generation by Myofilament-Triggered Ca2+ Release and Sarcomere Heterogeneity.

Heterogeneous mechanical dyskinesis has been implicated in many arrhythmogenic phenotypes. Strain-dependent perturbations to cardiomyocyte electrophysiology may contribute to this arrhythmogenesis through processes referred to as mechanoelectric feedback. Although the role of stretch-activated ion currents has been investigated using computational models, experimental studies suggest that mechanical strain may also promote arrhythmia by facilitating calcium wave propagation. To investigate whether strain-dependent changes in calcium affinity to the myofilament may promote arrhythmogenic intracellular calcium waves, we modified a mathematical model of rabbit excitation-contraction coupling coupled to a model of myofilament activation and force development. In a one-dimensional compartmental analysis, we bidirectionally coupled 50 sarcomere models in series to model calcium diffusion and stress transfer between adjacent sarcomeres. These considerations enabled the model to capture 1) the effects of mechanical feedback on calcium homeostasis at the sarcomeric level and 2) the combined effects of mechanical and calcium heterogeneities at the cellular level. The results suggest that in conditions of calcium overload, the vulnerable window of stretch-release to trigger suprathreshold delayed afterdepolarizations can be affected by heterogeneity in sarcomere length. Furthermore, stretch and sarcomere heterogeneity may modulate the susceptibility threshold for delayed afterdepolarizations and the aftercontraction wave propagation velocity.

3D dSTORM imaging reveals novel detail of ryanodine receptor localization in rat cardiac myocytes.

KEY POINTS: Using 3D direct stochastic optical reconstruction microscopy (dSTORM), we developed novel approaches to quantitatively describe the nanoscale, 3D organization of ryanodine receptors (RyRs) in cardiomyocytes. Complex arrangements of RyR clusters were observed in 3D space, both at the cell surface and within the cell interior, with allocation to dyadic and non-dyadic pools. 3D imaging importantly allowed discernment of clusters overlapping in the z-axis, for which detection was obscured by conventional 2D imaging techniques. Thus, RyR clusters were found to be significantly smaller than previous 2D estimates. Ca2+ release units (CRUs), i.e. functional groupings of neighbouring RyR clusters, were similarly observed to be smaller than earlier reports. Internal CRUs contained more RyRs in more clusters than CRUs on the cell surface, and yielded longer duration Ca2+ sparks. ABSTRACT: Cardiomyocyte contraction is dependent on Ca2+ release from ryanodine receptors (RyRs). However, the precise localization of RyRs remains unknown, due to shortcomings of imaging techniques which are diffraction limited or restricted to 2D. We aimed to determine the 3D nanoscale organization of RyRs in rat cardiomyocytes by employing direct stochastic optical reconstruction microscopy (dSTORM) with phase ramp technology. Initial observations at the cell surface showed an undulating organization of RyR clusters, resulting in their frequent overlap in the z-axis and obscured detection by 2D techniques. Non-overlapping clusters were imaged to create a calibration curve for estimating RyR number based on recorded fluorescence blinks. Employing this method at the cell surface and interior revealed smaller RyR clusters than 2D estimates, as erroneous merging of axially aligned RyRs was circumvented. Functional groupings of RyR clusters (Ca2+ release units, CRUs), contained an average of 18 and 23 RyRs at the surface and interior, respectively, although half of all CRUs contained only a single 'rogue' RyR. Internal CRUs were more tightly packed along z-lines than surface CRUs, contained larger and more numerous RyR clusters, and constituted ∼75% of the roughly 1 million RyRs present in an average cardiomyocyte. This complex internal 3D geometry was underscored by correlative imaging of RyRs and t-tubules, which enabled quantification of dyadic and non-dyadic RyR populations. Mirroring differences in CRU size and complexity, Ca2+ sparks originating from internal CRUs were of longer duration than those at the surface. These data provide novel, nanoscale insight into RyR organization and function across cardiomyocytes.

Atrial-selective targeting of arrhythmogenic phase-3 early afterdepolarizations in human myocytes.

BACKGROUND: We have previously shown that non-equilibrium Na(+) current (INa) reactivation drives isoproterenol-induced phase-3 early afterdepolarizations (EADs) in mouse ventricular myocytes. In these cells, EAD initiation occurs secondary to potentiated sarcoplasmic reticulum Ca(2+) release and enhanced Na(+)/Ca(2+) exchange (NCX). This can be abolished by tetrodotoxin-blockade of INa, but not ranolazine, which selectively inhibits ventricular late INa. AIM: Since repolarization of human atrial myocytes is similar to mouse ventricular myocytes in that it is relatively rapid and potently modulated by Ca(2+), we investigated whether similar mechanisms can evoke EADs in human atrium. Indeed, phase-3 EADs have been shown to re-initiate atrial fibrillation (AF) during autonomic stimulation, which is a well-recognized initiator of AF. METHODS: We integrated a Markov model of INa gating in our human atrial myocyte model. To simulate experimental results, we rapidly paced this cell model at 10Hz in the presence of 0.1µM acetylcholine and 1µM isoproterenol, and assessed EAD occurrence upon return to sinus rhythm (1Hz). RESULTS: Cellular Ca(2+) loading during fast pacing results in a transient period of hypercontractility after return to sinus rhythm. Here, fast repolarization and enhanced NCX facilitate INa reactivation via the canonical gating mode (i.e., not late INa burst mode), which drives EAD initiation. Simulating ranolazine administration reduces atrial peak INa and leads to faster repolarization, during which INa fails to reactivate and EADs are prevented. CONCLUSIONS: Non-equilibrium INa reactivation can critically contribute to arrhythmias, specifically in human atrial myocytes. Ranolazine might be beneficial in this context by blocking peak (not late) atrial INa.

Balancing Patient Access to Fetoscopic Laser Photocoagulation for Twin-to-Twin Transfusion Syndrome With Maintaining Procedural Competence: Are Collaborative Services Part of the Solution?

The benefits of fetoscopic laser photocoagulation (FLP) for treatment of twin-to-twin transfusion syndrome (TTTS) have been recognized for over a decade, yet access to FLP remains limited in many settings. This means at a population level, the potential benefits of FLP for TTTS are far from being fully realized. In part, this is because there are many centers where the case volume is relatively low. This creates an inevitable tension; on one hand, wanting FLP to be readily accessible to all women who may need it, yet on the other, needing to ensure that a high degree of procedural competence is maintained. Some of the solutions to these apparently competing priorities may be found in novel training solutions to achieve, and maintain, procedural proficiency, and with the increased utilization of 'competence based' assessment and credentialing frameworks. We suggest an under-utilized approach is the development of collaborative surgical services, where pooling of personnel and resources can improve timely access to surgery, improve standardized assessment and management of TTTS, minimize the impact of the surgical learning curve, and facilitate audit, education, and research. When deciding which centers should offer laser for TTTS and how we decide, we propose some solutions from a collaborative model.

Modelling cardiac calcium sparks in a three-dimensional reconstruction of a calcium release unit.

Triggered release of Ca2+ from an individual sarcoplasmic reticulum (SR) Ca(2+) release unit (CRU) is the fundamental event of cardiac excitation­contraction coupling, and spontaneous release events (sparks) are the major contributor to diastolic Ca(2+) leak in cardiomyocytes. Previous model studies have predicted that the duration and magnitude of the spark is determined by the local CRU geometry, as well as the localization and density of Ca(2+) handling proteins. We have created a detailed computational model of a CRU, and developed novel tools to generate the computational geometry from electron tomographic images. Ca(2+) diffusion was modelled within the SR and the cytosol to examine the effects of localization and density of the Na(+)/Ca(2+) exchanger, sarco/endoplasmic reticulum Ca(2+)-ATPase 2 (SERCA), and calsequestrin on spark dynamics. We reconcile previous model predictions of approximately 90% local Ca(2+) depletion in junctional SR, with experimental reports of about 40%. This analysis supports the hypothesis that dye kinetics and optical averaging effects can have a significant impact on measures of spark dynamics. Our model also predicts that distributing calsequestrin within non-junctional Z-disc SR compartments, in addition to the junctional compartment, prolongs spark release time as reported by Fluo5. By pumping Ca(2+) back into the SR during a release, SERCA is able to prolong a Ca(2+) spark, and this may contribute to SERCA-dependent changes in Ca(2+) wave speed. Finally, we show that including the Na(+)/Ca(2+) exchanger inside the dyadic cleft does not alter local [Ca(2+)] during a spark.

Image-Driven Modeling of Nanoscopic Cardiac Function: Where Have We Come From, and Where Are We Going?

Complementary developments in microscopy and mathematical modeling have been critical to our understanding of cardiac excitation-contraction coupling. Historically, limitations imposed by the spatial or temporal resolution of imaging methods have been addressed through careful mathematical interrogation. Similarly, limitations imposed by computational power have been addressed by imaging macroscopic function in large subcellular domains or in whole myocytes. As both imaging resolution and computational tractability have improved, the two approaches have nearly merged in terms of the scales that they can each be used to interrogate. With this review we will provide an overview of these advances and their contribution to understanding ventricular myocyte function, including exciting developments over the last decade. We specifically focus on experimental methods that have pushed back limits of either spatial or temporal resolution of nanoscale imaging (e.g., DNA-PAINT), or have permitted high resolution imaging on large cellular volumes (e.g., serial scanning electron microscopy). We also review the progression of computational approaches used to integrate and interrogate these new experimental data sources, and comment on near-term advances that may unify understanding of the underlying biology. Finally, we comment on several outstanding questions in cardiac physiology that stand to benefit from a concerted and complementary application of these new experimental and computational methods.

Arrhythmogenic influence of mutations in a myocyte-based computational model of the pulmonary vein sleeve.

In the heart, electrophysiological dysregulation arises from defects at many biological levels (from point mutations in ion channel proteins to gross structural abnormalities). These defects disrupt the normal pattern of electrical activation, producing ectopic activity and reentrant arrhythmia. To interrogate mechanisms that link these primary biological defects to macroscopic electrophysiologic dysregulation most prior computational studies have utilized either (i) detailed models of myocyte ion channel dynamics at limited spatial scales, or (ii) homogenized models of action potential conduction that reproduce arrhythmic activity at tissue and organ levels. Here we apply our recent model (EMI), which integrates electrical activation and propagation across these scales, to study human atrial arrhythmias originating in the pulmonary vein (PV) sleeves. These small structures initiate most supraventricular arrhythmias and include pronounced myocyte-to-myocyte heterogeneities in ion channel expression and intercellular coupling. To test EMI's cell-based architecture in this physiological context we asked whether ion channel mutations known to underlie atrial fibrillation are capable of initiating arrhythmogenic behavior via increased excitability or reentry in a schematic PV sleeve geometry. Our results illustrate that EMI's improved spatial resolution can directly interrogate how electrophysiological changes at the individual myocyte level manifest in tissue and as arrhythmia in the PV sleeve.

Validating the Arrhythmogenic Potential of High-, Intermediate-, and Low-Risk Drugs in a Human-Induced Pluripotent Stem Cell-Derived Cardiac Microphysiological System.

Evaluation of arrhythmogenic drugs is required by regulatory agencies before any new compound can obtain market approval. Despite rigorous review, cardiac disorders remain the second most common cause for safety-related market withdrawal. On the other hand, false-positive preclinical findings prohibit potentially beneficial candidates from moving forward in the development pipeline. Complex in vitro models using cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) have been identified as a useful tool that allows for rapid and cost-efficient screening of proarrhythmic drug risk. Currently available hiPSC-CM models employ simple two-dimensional (2D) culture formats with limited structural and functional relevance to the human heart muscle. Here, we present the use of our 3D cardiac microphysiological system (MPS), composed of a hiPSC-derived heart micromuscle, as a platform for arrhythmia risk assessment. We employed two different hiPSC lines and tested seven drugs with known ion channel effects and known clinical risk: dofetilide and bepridil (high risk); amiodarone and terfenadine (intermediate risk); and nifedipine, mexiletine, and lidocaine (low risk). The cardiac MPS successfully predicted drug cardiotoxicity risks based on changes in action potential duration, beat waveform (i.e., shape), and occurrence of proarrhythmic events of healthy patient hiPSC lines in the absence of risk cofactors. We showcase examples where the cardiac MPS outperformed existing hiPSC-CM 2D models.

Prolonged ß-adrenergic stimulation disperses ryanodine receptor clusters in cardiomyocytes and has implications for heart failure.

Ryanodine receptors (RyRs) exhibit dynamic arrangements in cardiomyocytes, and we previously showed that 'dispersion' of RyR clusters disrupts Ca2+ homeostasis during heart failure (HF) (Kolstad et al., eLife, 2018). Here, we investigated whether prolonged ß-adrenergic stimulation, a hallmark of HF, promotes RyR cluster dispersion and examined the underlying mechanisms. We observed that treatment of healthy rat cardiomyocytes with isoproterenol for 1 hr triggered progressive fragmentation of RyR clusters. Pharmacological inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) reversed these effects, while cluster dispersion was reproduced by specific activation of CaMKII, and in mice with constitutively active Ser2814-RyR. A similar role of protein kinase A (PKA) in promoting RyR cluster fragmentation was established by employing PKA activation or inhibition. Progressive cluster dispersion was linked to declining Ca2+ spark fidelity and magnitude, and slowed release kinetics from Ca2+ propagation between more numerous RyR clusters. In healthy cells, this served to dampen the stimulatory actions of ß-adrenergic stimulation over the longer term and protect against pro-arrhythmic Ca2+ waves. However, during HF, RyR dispersion was linked to impaired Ca2+ release. Thus, RyR localization and function are intimately linked via channel phosphorylation by both CaMKII and PKA, which, while finely tuned in healthy cardiomyocytes, underlies impaired cardiac function during pathology.

Metabolically driven maturation of human-induced-pluripotent-stem-cell-derived cardiac microtissues on microfluidic chips.

The immature physiology of cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) limits their utility for drug screening and disease modelling. Here we show that suitable combinations of mechanical stimuli and metabolic cues can enhance the maturation of hiPSC-derived cardiomyocytes, and that the maturation-inducing cues have phenotype-dependent effects on the cells' action-potential morphology and calcium handling. By using microfluidic chips that enhanced the alignment and extracellular-matrix production of cardiac microtissues derived from genetically distinct sources of hiPSC-derived cardiomyocytes, we identified fatty-acid-enriched maturation media that improved the cells' mitochondrial structure and calcium handling, and observed divergent cell-source-dependent effects on action-potential duration (APD). Specifically, in the presence of maturation media, tissues with abnormally prolonged APDs exhibited shorter APDs, and tissues with aberrantly short APDs displayed prolonged APDs. Regardless of cell source, tissue maturation reduced variabilities in spontaneous beat rate and in APD, and led to converging cell phenotypes (with APDs within the 300-450 ms range characteristic of human left ventricular cardiomyocytes) that improved the modelling of the effects of pro-arrhythmic drugs on cardiac tissue.

From Millimeters to Micrometers; Re-introducing Myocytes in Models of Cardiac Electrophysiology.

Computational modeling has contributed significantly to present understanding of cardiac electrophysiology including cardiac conduction, excitation-contraction coupling, and the effects and side-effects of drugs. However, the accuracy of in silico analysis of electrochemical wave dynamics in cardiac tissue is limited by the homogenization procedure (spatial averaging) intrinsic to standard continuum models of conduction. Averaged models cannot resolve the intricate dynamics in the vicinity of individual cardiomyocytes simply because the myocytes are not present in these models. Here we demonstrate how recently developed mathematical models based on representing every myocyte can significantly increase the accuracy, and thus the utility of modeling electrophysiological function and dysfunction in collections of coupled cardiomyocytes. The present gold standard of numerical simulation for cardiac electrophysiology is based on the bidomain model. In the bidomain model, the extracellular (E) space, the cell membrane (M) and the intracellular (I) space are all assumed to be present everywhere in the tissue. Consequently, it is impossible to study biophysical processes taking place close to individual myocytes. The bidomain model represents the tissue by averaging over several hundred myocytes and this inherently limits the accuracy of the model. In our alternative approach both E, M, and I are represented in the model which is therefore referred to as the EMI model. The EMI model approach allows for detailed analysis of the biophysical processes going on in functionally important spaces very close to individual myocytes, although at the cost of significantly increased CPU-requirements.

In vitro safety "clinical trial" of the cardiac liability of drug polytherapy.

Only a handful of US Food and Drug Administration (FDA) Emergency Use Authorizations exist for drug and biologic therapeutics that treat severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection. Potential therapeutics include repurposed drugs, some with cardiac liabilities. We report on a chronic preclinical drug screening platform, a cardiac microphysiological system (MPS), to assess cardiotoxicity associated with repurposed hydroxychloroquine (HCQ) and azithromycin (AZM) polytherapy in a mock phase I safety clinical trial. The MPS contained human heart muscle derived from induced pluripotent stem cells. The effect of drug response was measured using outputs that correlate with clinical measurements, such as QT interval (action potential duration) and drug-biomarker pairing. Chronic exposure (10 days) of heart muscle to HCQ alone elicited early afterdepolarizations and increased QT interval past 5 days. AZM alone elicited an increase in QT interval from day 7 onward, and arrhythmias were observed at days 8 and 10. Monotherapy results mimicked clinical trial outcomes. Upon chronic exposure to HCQ and AZM polytherapy, we observed an increase in QT interval on days 4-8. Interestingly, a decrease in arrhythmias and instabilities was observed in polytherapy relative to monotherapy, in concordance with published clinical trials. Biomarkers, most of them measurable in patients' serum, were identified for negative effects of monotherapy or polytherapy on tissue contractile function, morphology, and antioxidant protection. The cardiac MPS correctly predicted clinical arrhythmias associated with QT prolongation and rhythm instabilities. This high content system can help clinicians design their trials, rapidly project cardiac outcomes, and define new monitoring biomarkers to accelerate access of patients to safe coronavirus disease 2019 (COVID-19) therapeutics.