Cell-to-cell heterogeneity in ion channel conductance impacts substrate vulnerability to arrhythmia.

Determining whether an ectopic depolarization will lead to a self-perpetuating arrhythmia is of critical importance in determining arrhythmia risk, so it is necessary to understand what factors impact substrate vulnerability. This study sought to explore the impact of cell-to-cell heterogeneity in ion channel conductance on substrate vulnerability to arrhythmia by measuring the duration of the vulnerable window in computational models of one-dimensional cables of ventricular cardiomyocytes. We began by using a population of uniform cable models to determine the mechanisms underlying the vulnerable window phenomenon. We found that in addition to the known importance of GNa, the conductances GCa,L and GKr also play a minor role in determining the vulnerable window duration. We also found that a steeper slope of the repolarizing action potential during the vulnerable window correlated with a shorter vulnerable window duration in uniform cables. We applied our understanding from these initial simulations to an investigation of the vulnerable window in heterogeneous cable models. The heterogeneous cables displayed a great deal of intra-cable variation in vulnerable window duration, highly sensitive to the cardiomyocytes in the local environment of the ectopic stimulus. Coupling strength modulated not only the magnitude of the vulnerable window duration but also the extent of intra-tissue variability in vulnerable window duration.NEW & NOTEWORTHY We investigate the impact of cell-to-cell heterogeneity in ion channel conductance on substrate vulnerability to arrhythmia by measuring the vulnerable window duration in computational cardiomyocyte cable models. We demonstrate a wide range of intra-cable variability in vulnerable window duration (VWD) and show how this is changed by ion channel block and coupling strength perturbations.

Prediction of arrhythmia susceptibility through mathematical modeling and machine learning.

At present, the QT interval on the electrocardiographic (ECG) waveform is the most common metric for assessing an individual's susceptibility to ventricular arrhythmias, with a long QT, or, at the cellular level, a long action potential duration (APD) considered high risk. However, the limitations of this simple approach have long been recognized. Here, we sought to improve prediction of arrhythmia susceptibility by combining mechanistic mathematical modeling with machine learning (ML). Simulations with a model of the ventricular myocyte were performed to develop a large heterogenous population of cardiomyocytes (n = 10,586), and we tested each variant's ability to withstand three arrhythmogenic triggers: 1) block of the rapid delayed rectifier potassium current (IKr Block), 2) augmentation of the L-type calcium current (ICaL Increase), and 3) injection of inward current (Current Injection). Eight ML algorithms were trained to predict, based on simulated AP features in preperturbed cells, whether each cell would develop arrhythmic dynamics in response to each trigger. We found that APD can accurately predict how cells respond to the simple Current Injection trigger but cannot effectively predict the response to IKr Block or ICaL Increase. ML predictive performance could be improved by incorporating additional AP features and simulations of additional experimental protocols. Importantly, we discovered that the most relevant features and experimental protocols were trigger specific, which shed light on the mechanisms that promoted arrhythmia formation in response to the triggers. Overall, our quantitative approach provides a means to understand and predict differences between individuals in arrhythmia susceptibility.

Protein structure-based gene expression signatures.

Gene expression signatures (GES) connect phenotypes to differential messenger RNA (mRNA) expression of genes, providing a powerful approach to define cellular identity, function, and the effects of perturbations. The use of GES has suffered from vague assessment criteria and limited reproducibility. Because the structure of proteins defines the functional capability of genes, we hypothesized that enrichment of structural features could be a generalizable representation of gene sets. We derive structural gene expression signatures (sGES) using features from multiple levels of protein structure (e.g., domain and fold) encoded by the mRNAs in GES. Comprehensive analyses of data from the Genotype-Tissue Expression Project (GTEx), the all RNA-seq and ChIP-seq sample and signature search (ARCHS4) database, and mRNA expression of drug effects on cardiomyocytes show that sGES are useful for characterizing biological phenomena. sGES enable phenotypic characterization across experimental platforms, facilitates interoperability of expression datasets, and describe drug action on cells.

Coupling and heterogeneity modulate pacemaking capability in healthy and diseased two-dimensional sinoatrial node tissue models.

Both experimental and modeling studies have attempted to determine mechanisms by which a small anatomical region, such as the sinoatrial node (SAN), can robustly drive electrical activity in the human heart. However, despite many advances from prior research, important questions remain unanswered. This study aimed to investigate, through mathematical modeling, the roles of intercellular coupling and cellular heterogeneity in synchronization and pacemaking within the healthy and diseased SAN. In a multicellular computational model of a monolayer of either human or rabbit SAN cells, simulations revealed that heterogenous cells synchronize their discharge frequency into a unique beating rhythm across a wide range of heterogeneity and intercellular coupling values. However, an unanticipated behavior appeared under pathological conditions where perturbation of ionic currents led to reduced excitability. Under these conditions, an intermediate range of intercellular coupling (900-4000 MΩ) was beneficial to SAN automaticity, enabling a very small portion of tissue (3.4%) to drive propagation, with propagation failure occurring at both lower and higher resistances. This protective effect of intercellular coupling and heterogeneity, seen in both human and rabbit tissues, highlights the remarkable resilience of the SAN. Overall, the model presented in this work allowed insight into how spontaneous beating of the SAN tissue may be preserved in the face of perturbations that can cause individual cells to lose automaticity. The simulations suggest that certain degrees of gap junctional coupling protect the SAN from ionic perturbations that can be caused by drugs or mutations.

The role of ß-adrenergic system remodeling in human heart failure: A mechanistic investigation.

ß-adrenergic receptor antagonists (ß-blockers) are extensively used to improve cardiac performance in heart failure (HF), but the electrical improvements with these clinical treatments are not fully understood. The aim of this study was to analyze the electrophysiological effects of ß-adrenergic system remodeling in heart failure with reduced ejection fraction and the underlying mechanisms. We used a combined mathematical model that integrated ß-adrenergic signaling with electrophysiology and calcium cycling in human ventricular myocytes. HF remodeling, both in the electrophysiological and signaling systems, was introduced to quantitatively analyze changes in electrophysiological properties due to the stimulation of ß-adrenergic receptors in failing myocytes. We found that the inotropic effect of ß-adrenergic stimulation was reduced in HF due to the altered Ca2+ dynamics resulting from the combination of structural, electrophysiological and signaling remodeling. Isolated cells showed proarrhythmic risk after sympathetic stimulation because early afterdepolarizations appeared, and the vulnerability was greater in failing myocytes. When analyzing coupled cells, ß-adrenergic stimulation reduced transmural repolarization gradients between endocardium and epicardium in normal tissue, but was less effective at reducing these gradients after HF remodeling. The comparison of the selective activation of ß-adrenergic isoforms revealed that the response to ß2-adrenergic receptors stimulation was blunted in HF while ß1-adrenergic receptors downstream effectors regulated most of the changes observed after sympathetic stimulation. In conclusion, this study was able to reproduce an altered ß-adrenergic activity on failing myocytes and to explain the mechanisms involved. The derived predictions could help in the treatment of HF and guide in the design of future experiments.

Quantitative analysis of variability in an integrated model of human ventricular electrophysiology and ß-adrenergic signaling.

In ventricular myocytes, stimulation of ß-adrenergic receptors activates critical cardiac signaling pathways, leading to shorter action potentials and increased contraction strength during the "fight-or-flight" response. These changes primarily result, at the cellular level, from the coordinated phosphorylation of multiple targets by protein kinase A. Although mathematical models of the intracellular signaling downstream of ß-adrenergic receptor activation have previously been described, only a limited number of studies have explored quantitative interactions between intracellular signaling and electrophysiology in human ventricular myocytes. Accordingly, our objective was to develop an integrative mathematical model of ß-adrenergic receptor signaling, electrophysiology, and intracellular calcium (Ca2+) handling in the healthy human ventricular myocyte. We combined published mathematical models of intracellular signaling and electrophysiology, then calibrated the model results against voltage clamp data and physiological changes occurring after stimulation of ß-adrenergic receptors with isoproterenol. We subsequently: (1) explored how molecular variability in different categories of model parameters translated into phenotypic variability; (2) identified the most important parameters determining physiological cellular outputs in the model before and after ß-adrenergic receptor stimulation; and (3) investigated which molecular level alterations can produce a phenotype indicative of heart failure with preserved ejection fraction (HFpEF). Major results included: (1) variability in parameters that controlled intracellular signaling caused qualitatively different behavior than variability in parameters controlling ion transport pathways; (2) the most important model parameters determining action potential duration and intracellular Ca2+ transient amplitude were generally consistent before and after ß-adrenergic receptor stimulation, except for a shift in the importance of K+ currents in determining action potential duration; and (3) decreased Ca2+ uptake into the sarcoplasmic reticulum, increased Ca2+ extrusion through Na+/Ca2+ exchanger and decreased Ca2+ leak from the sarcoplasmic reticulum may contribute to HFpEF. Overall, this study provided novel insight into the phenotypic consequences of molecular variability, and our integrated model may be useful in the design and interpretation of future experimental studies of interactions between ß-adrenergic signaling and cellular physiology in human ventricular myocytes.

A distinct molecular mechanism by which phenytoin rescues a novel long QT 3 variant.

BACKGROUND: Genetic variants in SCN5A can result in channelopathies such as the long QT syndrome type 3 (LQT3), but the therapeutic response to Na+ channel blockers can vary. We previously reported a case of an infant with malignant LQT3 and a missense Q1475P SCN5A variant, who was effectively treated with phenytoin, but only partially with mexiletine. Here, we functionally characterized this variant and investigated possible mechanisms for the differential drug actions. METHODS: Wild-type or mutant Nav1.5 cDNAs were examined in transfected HEK293 cells with patch clamping and biochemical assays. We used computational modeling to provide insights into altered channel kinetics and to predict effects on the action potential. RESULTS: The Q1475P variant in Nav1.5 reduced the current density and channel surface expression, characteristic of a trafficking defect. The variant also led to positive shifts in the voltage dependence of steady-state activation and inactivation, faster inactivation and recovery from inactivation, and increased the "late" Na+ current. Simulations of Nav1.5 gating with a 9-state Markov model suggested that transitions from inactivated to closed states were accelerated in Q1475P channels, leading to accumulation of channels in non-inactivated closed states. Simulations with a human ventricular myocyte model predicted action potential prolongation with Q1475P, compared with wild type, channels. Patch clamp data showed that mexiletine and phenytoin similarly rescued some of the gating defects. Chronic incubation with mexiletine, but not phenytoin, rescued the Nav1.5-Q1475P trafficking defect, thus increasing mutant channel expression. CONCLUSIONS: The gain-of-function effects of Nav1.5-Q1475P predominate to cause a malignant long QT phenotype. Phenytoin partially corrects the gating defect without restoring surface expression of the mutant channel, whereas mexiletine restores surface expression of the mutant channel, which may explain the lack of efficacy of mexiletine when compared to phenytoin. Our data makes a case for experimental studies before embarking on a one-for-all therapy of arrhythmias.

Holistic optimization of an RNA-seq workflow for multi-threaded environments.

SUMMARY: For many next generation-sequencing pipelines, the most computationally intensive step is the alignment of reads to a reference sequence. As a result, alignment software such as the Burrows-Wheeler Aligner is optimized for speed and is often executed in parallel on the cloud. However, there are other less demanding steps that can also be optimized to significantly increase the speed especially when using many threads. We demonstrate this using a unique molecular identifier RNA-sequencing pipeline consisting of 3 steps: split, align, and merge. Optimization of all three steps yields a 40% increase in speed when executed using a single thread. However, when executed using 16 threads, we observe a 4-fold improvement over the original parallel implementation and more than an 8-fold improvement over the original single-threaded implementation. In contrast, optimizing only the alignment step results in just a 13% improvement over the original parallel workflow using 16 threads. AVAILABILITY AND IMPLEMENTATION: Code (M.I.T. license), supporting scripts and Dockerfiles are available at https://github.com/BioDepot/LINCS_RNAseq_cpp and Docker images at https://hub.docker.com/r/biodepot/rnaseq-umi-cpp/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Experimental and Computational Insight Into Human Mesenchymal Stem Cell Paracrine Signaling and Heterocellular Coupling Effects on Cardiac Contractility and Arrhythmogenicity.

RATIONALE: Myocardial delivery of human mesenchymal stem cells (hMSCs) is an emerging therapy for treating the failing heart. However, the relative effects of hMSC-mediated heterocellular coupling (HC) and paracrine signaling (PS) on human cardiac contractility and arrhythmogenicity remain unresolved. OBJECTIVE: The objective is to better understand hMSC PS and HC effects on human cardiac contractility and arrhythmogenicity by integrating experimental and computational approaches. METHODS AND RESULTS: Extending our previous hMSC-cardiomyocyte HC computational model, we incorporated experimentally calibrated hMSC PS effects on cardiomyocyte L-type calcium channel/sarcoendoplasmic reticulum calcium-ATPase activity and cardiac tissue fibrosis. Excitation-contraction simulations of hMSC PS-only and combined HC+PS effects on human cardiomyocytes were representative of human engineered cardiac tissue (hECT) contractile function measurements under matched experimental treatments. Model simulations and hECTs both demonstrated that hMSC-mediated effects were most pronounced under PS-only conditions, where developed force increased ≈4-fold compared with non-hMSC-supplemented controls during physiological 1-Hz pacing. Simulations predicted contractility of isolated healthy and ischemic adult human cardiomyocytes would be minimally sensitive to hMSC HC, driven primarily by PS. Dominance of hMSC PS was also revealed in simulations of fibrotic cardiac tissue, where hMSC PS protected from potential proarrhythmic effects of HC at various levels of engraftment. Finally, to study the nature of the hMSC paracrine effects on contractility, proteomic analysis of hECT/hMSC conditioned media predicted activation of PI3K/Akt signaling, a recognized target of both soluble and exosomal fractions of the hMSC secretome. Treating hECTs with exosome-enriched, but not exosome-depleted, fractions of the hMSC secretome recapitulated the effects observed with hMSC conditioned media on hECT-developed force and expression of calcium-handling genes (eg, SERCA2a, L-type calcium channel). CONCLUSIONS: Collectively, this integrated experimental and computational study helps unravel relative hMSC PS and HC effects on human cardiac contractility and arrhythmogenicity, and provides novel insight into the role of exosomes in hMSC paracrine-mediated effects on contractility.

A network medicine approach to build a comprehensive atlas for the prognosis of human cancer.

The Cancer Genome Atlas project has generated multi-dimensional and highly integrated genomic data from a large number of patient samples with detailed clinical records across many cancer types, but it remains unclear how to best integrate the massive amount of genomic data into clinical practice. We report here our methodology to build a multi-dimensional subnetwork atlas for cancer prognosis to better investigate the potential impact of multiple genetic and epigenetic (gene expression, copy number variation, microRNA expression and DNA methylation) changes on the molecular states of networks that in turn affects complex cancer survivorship. We uncover an average of 38 novel subnetworks in the protein-protein interaction network that correlate with prognosis across four prominent cancer types. The clinical utility of these subnetwork biomarkers was further evaluated by prognostic impact evaluation, functional enrichment analysis, drug target annotation, tumor stratification and independent validation. Some pathways including the dynactin, cohesion and pyruvate dehydrogenase-related subnetworks are identified as promising new targets for therapy in specific cancer types. In conclusion, this integrative analysis of existing protein interactome and cancer genomics data allows us to systematically dissect the molecular mechanisms that underlie unexpected outcomes for cancer, which could be used to better understand and predict clinical outcomes, optimize treatment and to provide new opportunities for developing therapeutics related to the subnetworks identified.

Differential roles of two delayed rectifier potassium currents in regulation of ventricular action potential duration and arrhythmia susceptibility.

KEY POINTS: Arrhythmias result from disruptions to cardiac electrical activity, although the factors that control cellular action potentials are incompletely understood. We combined mathematical modelling with experiments in heart cells from guinea pigs to determine how cellular electrical activity is regulated. A mismatch between modelling predictions and the experimental results allowed us to construct an improved, more predictive mathematical model. The balance between two particular potassium currents dictates how heart cells respond to perturbations and their susceptibility to arrhythmias. ABSTRACT: Imbalances of ionic currents can destabilize the cardiac action potential and potentially trigger lethal cardiac arrhythmias. In the present study, we combined mathematical modelling with information-rich dynamic clamp experiments to determine the regulation of action potential morphology in guinea pig ventricular myocytes. Parameter sensitivity analysis was used to predict how changes in ionic currents alter action potential duration, and these were tested experimentally using dynamic clamp, a technique that allows for multiple perturbations to be tested in each cell. Surprisingly, we found that a leading mathematical model, developed with traditional approaches, systematically underestimated experimental responses to dynamic clamp perturbations. We then re-parameterized the model using a genetic algorithm, which allowed us to estimate ionic current levels in each of the cells studied. This unbiased model adjustment consistently predicted an increase in the rapid delayed rectifier K+ current and a drastic decrease in the slow delayed rectifier K+ current, and this prediction was validated experimentally. Subsequent simulations with the adjusted model generated the clinically relevant prediction that the slow delayed rectifier is better able to stabilize the action potential and suppress pro-arrhythmic events than the rapid delayed rectifier. In summary, iterative coupling of simulations and experiments enabled novel insight into how the balance between cardiac K+ currents influences ventricular arrhythmia susceptibility.

Cross-regulation of Phosphodiesterase 1 and Phosphodiesterase 2 Activities Controls Dopamine-mediated Striatal α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Trafficking.

Dopamine, a key striatal neuromodulator, increases synaptic strength by promoting surface insertion and/or retention of AMPA receptors (AMPARs). This process is mediated by the phosphorylation of the GluA1 subunit of AMPAR by cyclic nucleotide-dependent kinases, making cyclic nucleotide phosphodiesterases (PDEs) potential regulators of synaptic strength. In this study, we examined the role of phosphodiesterase 2 (PDE2), a medium spiny neuron-enriched and cGMP-activated PDE, in AMPAR trafficking. We found that inhibiting PDE2 resulted in enhancement of dopamine-induced surface GluA1 expression in dopamine receptor 1-expressing medium spiny neurons. Using pharmacological and genetic approaches, we found that inhibition of PDE1 resulted in a decrease in surface AMPAR levels because of the allosteric activation of PDE2. The cross-regulation of PDE1 and PDE2 activities results in counterintuitive control of surface AMPAR expression, making it possible to regulate the directionality and magnitude of AMPAR trafficking.

Modeling Electrophysiological Coupling and Fusion between Human Mesenchymal Stem Cells and Cardiomyocytes.

Human mesenchymal stem cell (hMSC) delivery has demonstrated promise in preclinical and clinical trials for myocardial infarction therapy; however, broad acceptance is hindered by limited understanding of hMSC-human cardiomyocyte (hCM) interactions. To better understand the electrophysiological consequences of direct heterocellular connections between hMSCs and hCMs, three original mathematical models were developed, representing an experimentally verified triad of hMSC families with distinct functional ion channel currents. The arrhythmogenic risk of such direct electrical interactions in the setting of healthy adult myocardium was predicted by coupling and fusing these hMSC models to the published ten Tusscher midcardial hCM model. Substantial variations in action potential waveform-such as decreased action potential duration (APD) and plateau height-were found when hCMs were coupled to the two hMSC models expressing functional delayed rectifier-like human ether à-go-go K+ channel 1 (hEAG1); the effects were exacerbated for fused hMSC-hCM hybrid cells. The third family of hMSCs (Type C), absent of hEAG1 activity, led to smaller single-cell action potential alterations during coupling and fusion, translating to longer tissue-level mean action potential wavelength. In a simulated 2-D monolayer of cardiac tissue, re-entry vulnerability with low (5%) hMSC insertion was approximately eight-fold lower with Type C hMSCs compared to hEAG1-functional hMSCs. A 20% decrease in APD dispersion by Type C hMSCs compared to hEAG1-active hMSCs supports the claim of reduced arrhythmogenic potential of this cell type with low hMSC insertion. However, at moderate (15%) and high (25%) hMSC insertion, the vulnerable window increased independent of hMSC type. In summary, this study provides novel electrophysiological models of hMSCs, predicts possible arrhythmogenic effects of hMSCs when directly coupled to healthy hCMs, and proposes that isolating a subset of hMSCs absent of hEAG1 activity may offer increased safety as a cell delivery cardiotherapy at low levels of hMSC-hCM coupling.

There and back again: Iterating between population-based modeling and experiments reveals surprising regulation of calcium transients in rat cardiac myocytes.

While many ion channels and transporters involved in cardiac cellular physiology have been identified and described, the relative importance of each in determining emergent cellular behaviors remains unclear. Here we address this issue with a novel approach that combines population-based mathematical modeling with experimental tests to systematically quantify the relative contributions of different ion channels and transporters to the amplitude of the cellular Ca(2+) transient. Sensitivity analysis of a mathematical model of the rat ventricular cardiomyocyte quantified the response of cell behaviors to changes in the level of each ion channel and transporter, and experimental tests of these predictions were performed to validate or invalidate the predictions. The model analysis found that partial inhibition of the transient outward current in rat ventricular epicardial myocytes was predicted to have a greater impact on Ca(2+) transient amplitude than either: (1) inhibition of the same current in endocardial myocytes, or (2) comparable inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA). Experimental tests confirmed the model predictions qualitatively but showed some quantitative disagreement. This guided us to recalibrate the model by adjusting the relative importance of several Ca(2+) fluxes, thereby improving the consistency with experimental data and producing a more predictive model. Analysis of human cardiomyocyte models suggests that the relative importance of outward currents to Ca(2+) transporters is generalizable to human atrial cardiomyocytes, but not ventricular cardiomyocytes. Overall, our novel approach of combining population-based mathematical modeling with experimental tests has yielded new insight into the relative importance of different determinants of cell behavior.

Improving cardiomyocyte model fidelity and utility via dynamic electrophysiology protocols and optimization algorithms.

Mathematical models of cardiac electrophysiology are instrumental in determining mechanisms of cardiac arrhythmias. However, the foundation of a realistic multiscale heart model is only as strong as the underlying cell model. While there have been myriad advances in the improvement of cellular-level models, the identification of model parameters, such as ion channel conductances and rate constants, remains a challenging problem. The primary limitations to this process include: (1) such parameters are usually estimated from data recorded using standard electrophysiology voltage-clamp protocols that have not been developed with model building in mind, and (2) model parameters are typically tuned manually to subjectively match a desired output. Over the last decade, methods aimed at overcoming these disadvantages have emerged. These approaches include the use of optimization or fitting tools for parameter estimation and incorporating more extensive data for output matching. Here, we review recent advances in parameter estimation for cardiomyocyte models, focusing on the use of more complex electrophysiology protocols and global search heuristics. We also discuss future applications of such parameter identification, including development of cell-specific and patient-specific mathematical models to investigate arrhythmia mechanisms and predict therapy strategies.

Induction of autoimmune response to the extracellular loop of the HERG channel pore induces QTc prolongation in guinea-pigs.

KEY POINTS: Channelopathies of autoimmune origin are novel and are associated with corrected QT (QTc) prolongation and complex ventricular arrhythmias. We have recently demonstrated that anti-SSA/Ro antibodies from patients with autoimmune diseases and with QTc prolongation on the ECG target the human ether-à-go-go-related gene (HERG) K+ channel by inhibiting the corresponding current, IKr , at the pore region. Immunization of guinea-pigs with a peptide (E-pore peptide) corresponding to the extracellular loop region connecting the S5 and S6 segments of the HERG channel induces high titres of antibodies that inhibit IKr , lengthen the action potential and cause QTc prolongation on the surface ECG. In addition, anti-SSA/Ro-positive sera from patients with connective tissue diseases showed high reactivity to the E-pore peptide. The translational impact is the development of a peptide-based approach for the diagnosis and treatment of autoimmune-associated long QT syndrome. ABSTRACT: We recently demonstrated that anti-SSA/52 kDa Ro antibodies (Abs) from patients with autoimmune diseases and corrected QT (QTc) prolongation directly target and inhibit the human ether-à-go-go-related gene (HERG) K+ channel at the extracellular pore (E-pore) region, where homology with SSA/52 kDa Ro antigen was demonstrated. We tested the hypothesis that immunization of guinea-pigs with a peptide corresponding to the E-pore region (E-pore peptide) will generate pathogenic inhibitory Abs and cause QTc prolongation. Guinea-pigs were immunized with a 31-amino-acid peptide corresponding to the E-pore region of HERG. On days 10-62 after immunization, ECGs were recorded and blood was sampled for the detection of E-pore peptide Abs. Serum samples from patients with autoimmune diseases were evaluated for reactivity to E-pore peptide by enzyme-linked immunosorbent assay (ELISA), and histology was performed on hearts using Masson's Trichrome. Inhibition of the HERG channel was assessed by electrophysiology and by computational modelling of the human ventricular action potential. The ELISA results revealed the presence of high titres of E-pore peptide Abs and significant QTc prolongation after immunization. High reactivity to E-pore peptide was found using anti-SSA/Ro Ab-positive sera from patients with QTc prolongation. Histological data showed no evidence of fibrosis in immunized hearts. Simulations of simultaneous inhibition of repolarizing currents by anti-SSA/Ro Ab-positive sera showed the predominance of the HERG channel in controlling action potential duration and the QT interval. These results are the first to demonstrate that inhibitory Abs to the HERG E-pore region induce QTc prolongation in immunized guinea-pigs by targeting the HERG channel independently from fibrosis. The reactivity of anti-SSA/Ro Ab-positive sera from patients with connective tissue diseases with the E-pore peptide opens novel pharmacotherapeutic avenues in the diagnosis and management of autoimmune-associated QTc prolongation.

Pathogenesis of the Novel Autoimmune-Associated Long-QT Syndrome.

BACKGROUND: Emerging clinical evidence demonstrates high prevalence of QTc prolongation and complex ventricular arrhythmias in patients with anti-Ro antibody (anti-Ro Ab)-positive autoimmune diseases. We tested the hypothesis that anti-Ro Abs target the HERG (human ether-a-go-go-related gene) K(+) channel, which conducts the rapidly activating delayed K(+) current, IKr, thereby causing delayed repolarization seen as QT interval prolongation on the ECG. METHODS AND RESULTS: Anti-Ro Ab-positive sera, purified IgG, and affinity-purified anti-52kDa Ro Abs from patients with autoimmune diseases and QTc prolongation were tested on IKr using HEK293 cells expressing HERG channel and native cardiac myocytes. Electrophysiological and biochemical data demonstrate that anti-Ro Abs inhibit IKr to prolong action potential duration by directly binding to the HERG channel protein. The 52-kDa Ro antigen-immunized guinea pigs showed QTc prolongation on ECG after developing high titers of anti-Ro Abs, which inhibited native IKr and cross-reacted with guinea pig ERG channel. CONCLUSIONS: The data establish that anti-Ro Abs from patients with autoimmune diseases inhibit IKr by cross-reacting with the HERG channel likely at the pore region where homology between anti-52-kDa Ro antigen and HERG channel is present. The animal model of autoimmune-associated QTc prolongation is the first to provide strong evidence for a pathogenic role of anti-Ro Abs in the development of QTc prolongation. It is proposed that adult patients with anti-Ro Abs may benefit from routine ECG screening and that those with QTc prolongation should receive counseling about drugs that may increase the risk for life-threatening arrhythmias.

Maximal acceleration of Ca2+ release refractoriness by ß-adrenergic stimulation requires dual activation of kinases PKA and CaMKII in mouse ventricular myocytes.

KEY POINTS: Refractoriness of calcium release in heart cells is altered in several disease states, but the physiological mechanisms that regulate this process are incompletely understood. We examined refractoriness of calcium release in mouse ventricular myocytes and investigated how activation of different intracellular signalling pathways influenced this process. We found that refractoriness of calcium release is abbreviated by stimulation of the 'fight-or-flight' response, and that simultaneous activation of multiple intracellular signalling pathways contributes to this response. Data obtained under several conditions at the subcellular, microscopic level were consistent with results obtained at the cellular level. The results provide insight into regulation of cardiac calcium release and how alterations to this process may increase arrhythmia risk under different conditions. ABSTRACT: Time-dependent refractoriness of calcium (Ca(2+)) release in cardiac myocytes is an important factor in determining whether pro-arrhythmic release patterns develop. At the subcellular level of the Ca(2+) spark, recent studies have suggested that recovery of spark amplitude is controlled by local sarcoplasmic reticulum (SR) refilling whereas refractoriness of spark triggering depends on both refilling and the sensitivity of the ryanodine receptor (RyR) release channels that produce sparks. Here we studied regulation of Ca(2+) spark refractoriness in mouse ventricular myocytes by examining how ß-adrenergic stimulation influenced sequences of Ca(2+) sparks originating from individual RyR clusters. Our protocol allowed us to separately measure recovery of spark amplitude and delays between successive sparks, and data were interpreted quantitatively through simulations with a stochastic mathematical model. We found that, compared with spark sequences measured under control conditions: (1) ß-adrenergic stimulation with isoproterenol (isoprenaline) accelerated spark amplitude recovery and decreased spark-to-spark delays; (2) activating protein kinase A (PKA) with forskolin accelerated amplitude recovery but did not affect spark-to-spark delays; (3) inhibiting PKA with H89 retarded amplitude recovery and increased spark-to-spark delays; (4) preventing phosphorylation of the RyR at serine 2808 with a knock-in mouse prevented the decrease in spark-to-spark delays seen with ß-adrenergic stimulation; (5) inhibiting either PKA or Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) during ß-adrenergic stimulation prevented the decrease in spark-to-spark delays seen without inhibition. The results suggest that activation of either PKA or CaMKII is sufficient to speed SR refilling, but activation of both kinases appears necessary to observe increased RyR sensitivity. The data provide novel insight into ß-adrenergic regulation of Ca(2+) release refractoriness in mouse myocytes.

Na+ channel function, regulation, structure, trafficking and sequestration.

This paper is the second of a series of three reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation-contraction coupling and arrhythmias: Na(+) channel and Na(+) transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on Na(+) channel function and regulation, Na(+) channel structure and function, and Na(+) channel trafficking, sequestration and complexing.

Calcium homeostasis in a local/global whole cell model of permeabilized ventricular myocytes with a Langevin description of stochastic calcium release.

Population density approaches to modeling local control of Ca(2+)-induced Ca(2+) release in cardiac myocytes can be used to construct minimal whole cell models that accurately represent heterogeneous local Ca(2+) signals. Unfortunately, the computational complexity of such "local/global" whole cell models scales with the number of Ca(2+) release unit (CaRU) states, which is a rapidly increasing function of the number of ryanodine receptors (RyRs) per CaRU. Here we present an alternative approach based on a Langevin description of the collective gating of RyRs coupled by local Ca(2+) concentration ([Ca(2+)]). The computational efficiency of this approach no longer depends on the number of RyRs per CaRU. When the RyR model is minimal, Langevin equations may be replaced by a single Fokker-Planck equation, yielding an extremely compact and efficient local/global whole cell model that reproduces and helps interpret recent experiments that investigate Ca(2+) homeostasis in permeabilized ventricular myocytes. Our calculations show that elevated myoplasmic [Ca(2+)] promotes elevated network sarcoplasmic reticulum (SR) [Ca(2+)] via SR Ca(2+)-ATPase-mediated Ca(2+) uptake. However, elevated myoplasmic [Ca(2+)] may also activate RyRs and promote stochastic SR Ca(2+) release, which can in turn decrease SR [Ca(2+)]. Increasing myoplasmic [Ca(2+)] results in an exponential increase in spark-mediated release and a linear increase in nonspark-mediated release, consistent with recent experiments. The model exhibits two steady-state release fluxes for the same network SR [Ca(2+)] depending on whether myoplasmic [Ca(2+)] is low or high. In the later case, spontaneous release decreases SR [Ca(2+)] in a manner that maintains robust Ca(2+) sparks.