Loss of developmentally derived Irf8+ macrophages promotes hyperinnervation and arrhythmia in the adult zebrafish heart.

Recent developments in cardiac macrophage biology have broadened our understanding of the critical functions of macrophages in the heart. As a result, there is further interest in understanding the independent contributions of distinct subsets of macrophage to cardiac development and function. Here, we demonstrate that genetic loss of interferon regulatory factor 8 (Irf8)-positive embryonic-derived macrophages significantly disrupts cardiac conduction, chamber function, and innervation in adult zebrafish. At 4 months post-fertilization (mpf), homozygous irf8st96/st96 mutants have significantly shortened atrial action potential duration and significant differential expression of genes involved in cardiac contraction. Functional in vivo assessments via electro- and echocardiograms at 12 mpf reveal that irf8 mutants are arrhythmogenic and exhibit diastolic dysfunction and ventricular stiffening. To identify the molecular drivers of the functional disturbances in irf8 null zebrafish, we perform single cell RNA sequencing and immunohistochemistry, which reveal increased leukocyte infiltration, epicardial activation, mesenchymal gene expression, and fibrosis. Irf8 null hearts are also hyperinnervated and have aberrant axonal patterning, a phenotype not previously assessed in the context of cardiac macrophage loss. Gene ontology analysis supports a novel role for activated epicardial-derived cells (EPDCs) in promoting neurogenesis and neuronal remodeling in vivo. Together, these data uncover significant cardiac abnormalities following embryonic macrophage loss and expand our knowledge of critical macrophage functions in heart physiology and governing homeostatic heart health.

Conduction delays across the specialized conduction system of the heart: Revisiting atrioventricular node (AVN) and Purkinje-ventricular junction (PVJ) delays.

Background and significance: The specialized conduction system (SCS) of the heart was extensively studied to understand the synchronization of atrial and ventricular contractions, the large atrial to His bundle (A-H) delay through the atrioventricular node (AVN), and delays between Purkinje (P) and ventricular (V) depolarization at distinct junctions (J), PVJs. Here, we use optical mapping of perfused rabbit hearts to revisit the mechanism that explains A-H delay and the role of a passive electrotonic step-delay at the boundary between atria and the AVN. We further visualize how the P anatomy controls papillary activation and valve closure before ventricular activation. Methods: Rabbit hearts were perfused with a bolus (100-200 µl) of a voltage-sensitive dye (di4ANEPPS), blebbistatin (10-20 µM for 20 min) then the right atrial appendage and ventricular free-wall were cut to expose the AVN, P fibers (PFs), the septum, papillary muscles, and the endocardium. Fluorescence images were focused on a CMOS camera (SciMedia) captured at 1K-5 K frames/s from 100 × 100 pixels. Results: AP propagation across the AVN-His (A-H) exhibits distinct patterns of delay and conduction blocks during S1-S2 stimulation. Refractory periods were 81 ± 9, 90 ± 21, 185 ± 15 ms for Atrial, AVN, and His, respectively. A large delay (>40 ms) occurs between atrial and AVN activation that increased during rapid atrial pacing contributing to the development of Wenckebach periodicity followed by delays within the AVN through slow or blocked conduction. The temporal resolution of the camera allowed us to identify PVJs by detecting doublets of AP upstrokes. PVJ delays were heterogeneous, fastest in PVJ that immediately trigger ventricular APs (3.4 ± 0.8 ms) and slow in regions where PF appear insulated from the neighboring ventricular myocytes (7.8 ± 2.4 ms). Insulated PF along papillary muscles conducted APs (>2 m/s), then triggered papillary muscle APs (<1 m/s), followed by APs firing of septum and endocardium. The anatomy of PFs and PVJs produced activation patterns that control the sequence of contractions ensuring that papillary contractions close the tricuspid valve 2-5 ms before right ventricular contractions. Conclusions: The specialized conduction system can be accessed optically to investigate the electrical properties of the AVN, PVJ and activation patterns in physiological and pathological conditions.

One Billion hiPSC-Cardiomyocytes: Upscaling Engineered Cardiac Tissues to Create High Cell Density Therapies for Clinical Translation in Heart Regeneration.

Despite the overwhelming use of cellularized therapeutics in cardiac regenerative engineering, approaches to biomanufacture engineered cardiac tissues (ECTs) at clinical scale remain limited. This study aims to evaluate the impact of critical biomanufacturing decisions-namely cell dose, hydrogel composition, and size-on ECT formation and function-through the lens of clinical translation. ECTs were fabricated by mixing human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) and human cardiac fibroblasts into a collagen hydrogel to engineer meso-(3 × 9 mm), macro- (8 × 12 mm), and mega-ECTs (65 × 75 mm). Meso-ECTs exhibited a hiPSC-CM dose-dependent response in structure and mechanics, with high-density ECTs displaying reduced elastic modulus, collagen organization, prestrain development, and active stress generation. Scaling up, cell-dense macro-ECTs were able to follow point stimulation pacing without arrhythmogenesis. Finally, we successfully fabricated a mega-ECT at clinical scale containing 1 billion hiPSC-CMs for implantation in a swine model of chronic myocardial ischemia to demonstrate the technical feasibility of biomanufacturing, surgical implantation, and engraftment. Through this iterative process, we define the impact of manufacturing variables on ECT formation and function as well as identify challenges that must still be overcome to successfully accelerate ECT clinical translation.

Myofibroblast senescence promotes arrhythmogenic remodeling in the aged infarcted rabbit heart.

Progressive tissue remodeling after myocardial infarction (MI) promotes cardiac arrhythmias. This process is well studied in young animals, but little is known about pro-arrhythmic changes in aged animals. Senescent cells accumulate with age and accelerate age-associated diseases. Senescent cells interfere with cardiac function and outcome post-MI with age, but studies have not been performed in larger animals, and the mechanisms are unknown. Specifically, age-associated changes in timecourse of senescence and related changes in inflammation and fibrosis are not well understood. Additionally, the cellular and systemic role of senescence and its inflammatory milieu in influencing arrhythmogenesis with age is not clear, particularly in large animal models with cardiac electrophysiology more similar to humans than previously studied animal models. Here, we investigated the role of senescence in regulating inflammation, fibrosis, and arrhythmogenesis in young and aged infarcted rabbits. Aged rabbits exhibited increased peri-procedural mortality and arrhythmogenic electrophysiological remodeling at the infarct border zone (IBZ) compared to young rabbits. Studies of the aged infarct zone revealed persistent myofibroblast senescence and increased inflammatory signaling over a 12-week timecourse. Senescent IBZ myofibroblasts in aged rabbits appear to be coupled to myocytes, and our computational modeling showed that senescent myofibroblast-cardiomyocyte coupling prolongs action potential duration (APD) and facilitates conduction block permissive of arrhythmias. Aged infarcted human ventricles show levels of senescence consistent with aged rabbits, and senescent myofibroblasts also couple to IBZ myocytes. Our findings suggest that therapeutic interventions targeting senescent cells may mitigate arrhythmias post-MI with age.

Action potential metrics and automated data analysis pipeline for cardiotoxicity testing using optically mapped hiPSC-derived 3D cardiac microtissues.

Recent advances in human induced pluripotent stem cell (hiPSC)-derived cardiac microtissues provide a unique opportunity for cardiotoxic assessment of pharmaceutical and environmental compounds. Here, we developed a series of automated data processing algorithms to assess changes in action potential (AP) properties for cardiotoxicity testing in 3D engineered cardiac microtissues generated from hiPSC-derived cardiomyocytes (hiPSC-CMs). Purified hiPSC-CMs were mixed with 5-25% human cardiac fibroblasts (hCFs) under scaffold-free conditions and allowed to self-assemble into 3D spherical microtissues in 35-microwell agarose gels. Optical mapping was performed to quantify electrophysiological changes. To increase throughput, AP traces from 4x4 cardiac microtissues were simultaneously acquired with a voltage sensitive dye and a CMOS camera. Individual microtissues showing APs were identified using automated thresholding after Fourier transforming traces. An asymmetric least squares method was used to correct non-uniform background and baseline drift, and the fluorescence was normalized (ΔF/F0). Bilateral filtering was applied to preserve the sharpness of the AP upstroke. AP shape changes under selective ion channel block were characterized using AP metrics including stimulation delay, rise time of AP upstroke, APD30, APD50, APD80, APDmxr (maximum rate change of repolarization), and AP triangulation (APDtri = APDmxr-APD50). We also characterized changes in AP metrics under various ion channel block conditions with multi-class logistic regression and feature extraction using principal component analysis of human AP computer simulations. Simulation results were validated experimentally with selective pharmacological ion channel blockers. In conclusion, this simple and robust automated data analysis pipeline for evaluating key AP metrics provides an excellent in vitro cardiotoxicity testing platform for a wide range of environmental and pharmaceutical compounds.

Beyond pharmaceuticals: Fit-for-purpose new approach methodologies for environmental cardiotoxicity testing.

Environmental factors play a substantial role in determining cardiovascular health, but data informing the risks presented by environmental toxicants is insufficient. In vitro new approach methodologies (NAMs) offer a promising approach with which to address the limitations of traditional in vivo and in vitro assays for assessing cardiotoxicity. Driven largely by the needs of pharmaceutical toxicity testing, considerable progress in developing NAMs for cardiotoxicity analysis has already been made. As the scientific and regulatory interest in NAMs for environmental chemicals continues to grow, a thorough understanding of the unique features of environmental cardiotoxicants and their associated cardiotoxicities is needed. Here, we review the key characteristics of as well as important regulatory and biological considerations for fit-for-purpose NAMs for environmental cardiotoxicity. By emphasizing the challenges and opportunities presented by NAMs for environmental cardiotoxicity we hope to accelerate their development, acceptance, and application.

Arrhythmia Assessment in Heterotypic Human Cardiac Myocyte-Fibroblast Microtissues.

Risk assessment assays for chemically induced arrhythmia are critical, but significant limitations exist with current cardiotoxicity testing, including a focus on single select ion channels, the use of non-human species in vitro and in vivo, and limited direct physiological translation. To be predictive of actual adverse clinical arrhythmic risk, arrhythmia assessment models for chemicals and drugs should be fit-for-purpose and suited for evaluating compounds in which the mechanism of action may not be entirely known. Here, we describe methods for efficient and reliable screening for arrhythmogenic cardiotoxicity with a 3D human cardiac microtissue model using purified human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and human cardiac fibroblasts. Applying optical mapping of voltage and calcium-sensitive dyes-an established approach to evaluate cardiac action potentials and calcium transients-to 3D heterotypic cardiac myocyte-fibroblast tissues allows for the generation and functional analysis of a large number of individual microtissues to provide greater throughput and high statistical power in analyses. Hundreds of microtissues in standard cell culture plates can be produced with low variability beat-to-beat, microtissue-to-microtissue, and across hiPSC-cardiomyocyte differentiation batches, reducing the number of microtissues required per condition for predictive outputs. The platform described here can be used as a sensitive, efficient, and predictive preclinical model validated for the purpose of assessing human pro-arrhythmic risk.

Ero1α-Dependent ERp44 Dissociation From RyR2 Contributes to Cardiac Arrhythmia.

BACKGROUND: Oxidative stress in cardiac disease promotes proarrhythmic disturbances in Ca2+ homeostasis, impairing luminal Ca2+ regulation of the sarcoplasmic reticulum (SR) Ca2+ release channel, the RyR2 (ryanodine receptor), and increasing channel activity. However, exact mechanisms underlying redox-mediated increase of RyR2 function in cardiac disease remain elusive. We tested whether the oxidoreductase family of proteins that dynamically regulate the oxidative environment within the SR are involved in this process. METHODS: A rat model of hypertrophy induced by thoracic aortic banding (TAB) was used for ex vivo whole heart optical mapping and for Ca2+ and reactive oxygen species imaging in isolated ventricular myocytes (VMs). RESULTS: The SR-targeted reactive oxygen species biosensor ERroGFP showed increased intra-SR oxidation in TAB VMs that was associated with increased expression of Ero1α (endoplasmic reticulum oxidoreductase 1 alpha). Pharmacological (EN460) or genetic Ero1α inhibition normalized SR redox state, increased Ca2+ transient amplitude and SR Ca2+ content, and reduced proarrhythmic spontaneous Ca2+ waves in TAB VMs under ß-adrenergic stimulation (isoproterenol). Ero1α overexpression in Sham VMs had opposite effects. Ero1α inhibition attenuated Ca2+-dependent ventricular tachyarrhythmias in TAB hearts challenged with isoproterenol. Experiments in TAB VMs and human embryonic kidney 293 cells expressing human RyR2 revealed that an Ero1α-mediated increase in SR Ca2+-channel activity involves dissociation of intraluminal protein ERp44 (endoplasmic reticulum protein 44) from the RyR2 complex. Site-directed mutagenesis and molecular dynamics simulations demonstrated a novel redox-sensitive association of ERp44 with RyR2 mediated by intraluminal cysteine 4806. ERp44-RyR2 association in TAB VMs was restored by Ero1α inhibition, but not by reducing agent dithiothreitol, as hypo-oxidation precludes formation of covalent bond between RyR2 and ERp44. CONCLUSIONS: A novel axis of intraluminal interaction between RyR2, ERp44, and Ero1α has been identified. Ero1α inhibition exhibits promising therapeutic potential by stabilizing RyR2-ERp44 complex, thereby reducing spontaneous Ca2+ release and Ca2+-dependent tachyarrhythmias in hypertrophic hearts, without causing hypo-oxidative stress in the SR.

Human Atrial Cardiac Microtissues for Chamber-Specific Arrhythmic Risk Assessment.

INTRODUCTION: Although atrial fibrillation is the most prevalent disorder of electrical conduction, the mechanisms behind atrial arrhythmias remain elusive. To address this challenge, we developed a robust in vitro model of 3D atrial microtissue from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and evaluated chamber-specific chemical responses experimentally and computationally. METHODS: We differentiated atrial and ventricular cardiomyocytes (aCMs/vCMs) from GCaMP6f-expressing hiPSCs and assessed spontaneous AP activity using fluorescence imaging. Self-assembling 3D microtissues were formed with lactate purified CMs and 5% human cardiac fibroblasts and electrically stimulated for one week before high resolution action potential (AP) optical mapping. AP responses to the atrial-specific potassium repolarizing current I Kur-blocker 4-Aminopyridine (4-AP) and funny current I f-blocker Ivabradine were characterized within their therapeutic window. Finally, we expanded upon a published hiPSC-CM computational model by incorporating the atrial-specific I Kur current, modifying ion channel conductances to match the AP waveforms of our microtissues, and employing the updated model to reinforce our experimental findings. RESULTS: High purity CMs (> 75% cTnT+) demonstrated subtype specification by MLC2v expression. Spontaneous beating rates significantly decreased following 3D microtissue formation, with atrial microtissues characterized by their faster spontaneous beating rate, slower AP rise time, and shorter AP duration (APD) compared to ventricular microtissues. We measured atrial-specific responses, including dose-dependent APD prolongation with 4-AP treatment and dose-dependent reduction in spontaneous activity post-Ivabradine treatment. CONCLUSION: The presented in vitro platform for screening atrial-specific responses is both robust and sensitive, with high throughput, enabling studies focused at elucidating the mechanisms underlying atrial arrhythmias. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00703-x.

A predictive in vitro risk assessment platform for pro-arrhythmic toxicity using human 3D cardiac microtissues.

Cardiotoxicity of pharmaceutical drugs, industrial chemicals, and environmental toxicants can be severe, even life threatening, which necessitates a thorough evaluation of the human response to chemical compounds. Predicting risks for arrhythmia and sudden cardiac death accurately is critical for defining safety profiles. Currently available approaches have limitations including a focus on single select ion channels, the use of non-human species in vitro and in vivo, and limited direct physiological translation. We have advanced the robustness and reproducibility of in vitro platforms for assessing pro-arrhythmic cardiotoxicity using human induced pluripotent stem cell-derived cardiomyocytes and human cardiac fibroblasts in 3-dimensional microtissues. Using automated algorithms and statistical analyses of eight comprehensive evaluation metrics of cardiac action potentials, we demonstrate that tissue-engineered human cardiac microtissues respond appropriately to physiological stimuli and effectively differentiate between high-risk and low-risk compounds exhibiting blockade of the hERG channel (E4031 and ranolazine, respectively). Further, we show that the environmental endocrine disrupting chemical bisphenol-A (BPA) causes acute and sensitive disruption of human action potentials in the nanomolar range. Thus, this novel human 3D in vitro pro-arrhythmic risk assessment platform addresses critical needs in cardiotoxicity testing for both environmental and pharmaceutical compounds and can be leveraged to establish safe human exposure levels.

Interleukin-1ß, Oxidative Stress, and Abnormal Calcium Handling Mediate Diabetic Arrhythmic Risk.

Diabetes mellitus (DM) is associated with increased arrhythmia. Type 2 DM (T2DM) mice showed prolonged QT interval and increased ventricular arrhythmic inducibility, accompanied by elevated cardiac interleukin (IL)-1ß, increased mitochondrial reactive oxygen species (mitoROS), and oxidation of the sarcoplasmic reticulum (SR) Ca2+ release channel (ryanodine receptor 2 [RyR2]). Inhibiting IL-1ß and mitoROS reduced RyR2 oxidation and the ventricular arrhythmia in DM. Inhibiting SR Ca2+ leak by stabilizing the oxidized RyR2 channel reversed the diabetic arrhythmic risk. In conclusion, cardiac IL-1ß mediated the DM-associated arrhythmia through mitoROS generation that enhances SR Ca2+ leak. The mechanistic link between inflammation and arrhythmias provides new therapeutic options.

IL-18 mediates sickle cell cardiomyopathy and ventricular arrhythmias.

Previous reports indicate that IL18 is a novel candidate gene for diastolic dysfunction in sickle cell disease (SCD)-related cardiomyopathy. We hypothesize that interleukin-18 (IL-18) mediates the development of cardiomyopathy and ventricular tachycardia (VT) in SCD. Compared with control mice, a humanized mouse model of SCD exhibited increased cardiac fibrosis, prolonged duration of action potential, higher VT inducibility in vivo, higher cardiac NF-κB phosphorylation, and higher circulating IL-18 levels, as well as reduced voltage-gated potassium channel expression, which translates to reduced transient outward potassium current (Ito) in isolated cardiomyocytes. Administering IL-18 to isolated mouse hearts resulted in VT originating from the right ventricle and further reduced Ito in SCD mouse cardiomyocytes. Sustained IL-18 inhibition via IL-18-binding protein resulted in decreased cardiac fibrosis and NF-κB phosphorylation, improved diastolic function, normalized electrical remodeling, and attenuated IL-18-mediated VT in SCD mice. Patients with SCD and either myocardial fibrosis or increased QTc displayed greater IL18 gene expression in peripheral blood mononuclear cells (PBMCs), and QTc was strongly correlated with plasma IL-18 levels. PBMC-derived IL18 gene expression was increased in patients who did not survive compared with those who did. IL-18 is a mediator of sickle cell cardiomyopathy and VT in mice and a novel therapeutic target in patients at risk for sudden death.

Human Cardiac Fibroblast Number and Activation State Modulate Electromechanical Function of hiPSC-Cardiomyocytes in Engineered Myocardium.

Cardiac tissue engineering using hiPSC-derived cardiomyocytes is a promising avenue for cardiovascular regeneration, pharmaceutical drug development, cardiotoxicity evaluation, and disease modeling. Limitations to these applications still exist due in part to the need for more robust structural support, organization, and electromechanical function of engineered cardiac tissues. It is well accepted that heterotypic cellular interactions impact the phenotype of cardiomyocytes. The current study evaluates the functional effects of coculturing adult human cardiac fibroblasts (hCFs) in 3D engineered tissues on excitation and contraction with the goal of recapitulating healthy, nonarrhythmogenic myocardium in vitro. A small population (5% of total cell number) of hCFs in tissues improves tissue formation, material properties, and contractile function. However, two perturbations to the hCF population create disease-like phenotypes in engineered cardiac tissues. First, increasing the percentage of hCFs to 15% resulted in tissues with increased ectopic activity and spontaneous excitation rate. Second, hCFs undergo myofibroblast activation in traditional two-dimensional culture, and this altered phenotype ablated the functional benefits of hCFs when incorporated into engineered cardiac tissues. Taken together, the results of this study demonstrate that human cardiac fibroblast number and activation state modulate electromechanical function of hiPSC-cardiomyocytes and that a low percentage of quiescent hCFs are a valuable cell source to advance a healthy electromechanical response of engineered cardiac tissue for regenerative medicine applications.

Late INa Blocker GS967 Supresses Polymorphic Ventricular Tachycardia in a Transgenic Rabbit Model of Long QT Type 2.

BACKGROUND: Long QT syndrome has been associated with sudden cardiac death likely caused by early afterdepolarizations (EADs) and polymorphic ventricular tachycardias (PVTs). Suppressing the late sodium current (INaL) may counterbalance the reduced repolarization reserve in long QT syndrome and prevent EADs and PVTs. METHODS: We tested the effects of the selective INaL blocker GS967 on PVT induction in a transgenic rabbit model of long QT syndrome type 2 using intact heart optical mapping, cellular electrophysiology and confocal Ca2+ imaging, and computer modeling. RESULTS: GS967 reduced ventricular fibrillation induction under a rapid pacing protocol (n=7/14 hearts in control versus 1/14 hearts at 100 nmol/L) without altering action potential duration or restitution and dispersion. GS967 suppressed PVT incidences by reducing Ca2+-mediated EADs and focal activity during isoproterenol perfusion (at 30 nmol/L, n=7/12 and 100 nmol/L n=8/12 hearts without EADs and PVTs). Confocal Ca2+ imaging of long QT syndrome type 2 myocytes revealed that GS967 shortened Ca2+ transient duration via accelerating Na+/Ca2+ exchanger (INCX)-mediated Ca2+ efflux from cytosol, thereby reducing EADs. Computer modeling revealed that INaL potentiates EADs in the long QT syndrome type 2 setting through (1) providing additional depolarizing currents during action potential plateau phase, (2) increasing intracellular Na+ (Nai) that decreases the depolarizing INCX thereby suppressing the action potential plateau and delaying the activation of slowly activating delayed rectifier K+ channels (IKs), suggesting important roles of INaL in regulating Nai. CONCLUSIONS: Selective INaL blockade by GS967 prevents EADs and abolishes PVT in long QT syndrome type 2 rabbits by counterbalancing the reduced repolarization reserve and normalizing Nai. Graphic Abstract: A graphic abstract is available for this article.

Impact of ISK Voltage and Ca2+/Mg2+-Dependent Rectification on Cardiac Repolarization.

Cardiac small conductance Ca2+-activated K+ (SK) channels are activated solely by Ca2+, but the SK current (ISK) is inwardly rectified. However, the impact of inward rectification in shaping action potentials (APs) in ventricular cardiomyocytes under ß-adrenergic stimulation or in disease states remains undefined. Two processes underlie this inward rectification: an intrinsic rectification caused by an electrostatic energy barrier from positively charged amino acids at the inner pore and a voltage-dependent Ca2+/Mg2+ block. Thus, Ca2+ has a biphasic effect on ISK, activating at low [Ca2+] yet inhibiting ISK at high [Ca2+]. We examined the effect of ISK rectification on APs in rat cardiomyocytes by simultaneously recording whole-cell apamin-sensitive currents and Ca2+ transients during an AP waveform and developed a computer model of SK channels with rectification features. The typical profile of ISK during AP clamp included an initial peak (mean 1.6 pA/pF) followed by decay to the point that submembrane [Ca2+] reached ∼10 µM. During the rest of the AP stimulus, ISK either plateaued or gradually increased as the cell repolarized and submembrane [Ca2+] decreased further. We used a six-state gating model combined with intrinsic and Ca2+/Mg2+-dependent rectification to simulate ISK and investigated the relative contributions of each type of rectification to AP shape. This SK channel model replicates key features of ISK recording during AP clamp showing that intrinsic rectification limits ISK at high Vm during the early and plateau phase of APs. Furthermore, the initial rise of Ca2+ transients activates, but higher [Ca2+] blocks SK channels, yielding a transient outward-like ISK trajectory. During the decay phase of Ca2+, the Ca2+-dependent block is released, causing ISK to rise again and contribute to repolarization. Therefore, ISK is an important repolarizing current, and the rectification characteristics of an SK channel determine its impact on early, plateau, and repolarization phases of APs.

PKA phosphorylation underlies functional recruitment of sarcolemmal SK2 channels in ventricular myocytes from hypertrophic hearts.

KEY POINTS: Small-conductance Ca2+ -activated K+ (SK) channels expressed in ventricular myocytes are dormant in health, yet become functional in cardiac disease. SK channels are voltage independent and their gating is controlled by intracellular [Ca2+ ] in a biphasic manner. Submicromolar [Ca2+ ] activates the channel via constitutively-bound calmodulin, whereas higher [Ca2+ ] exerts inhibitory effect during depolarization. Using a rat model of cardiac hypertrophy induced by thoracic aortic banding, we found that functional upregulation of SK2 channels in hypertrophic rat ventricular cardiomyocytes is driven by protein kinase A (PKA) phosphorylation. Using site-directed mutagenesis, we identified serine-465 as the site conferring PKA-dependent effects on SK2 channel function. PKA phosphorylation attenuates ISK rectification by reducing the Ca2+ /voltage-dependent inhibition of SK channels without changing their sensitivity to activating submicromolar [Ca2+ ]i . This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive. ABSTRACT: Small-conductance Ca2+ -activated K+ (SK) channels expressed in ventricular myocytes (VMs) are dormant in health, yet become functional in cardiac disease. We aimed to test the hypothesis that post-translational modification of SK channels under conditions accompanied by enhanced adrenergic drive plays a central role in disease-related activation of the channels. We investigated this phenomenon using a rat model of hypertrophy induced by thoracic aortic banding (TAB). Western blot analysis using anti-pan-serine/threonine antibodies demonstrated enhanced phosphorylation of immunoprecipitated SK2 channels in VMs from TAB rats vs. Shams, which was reversible by incubation of the VMs with PKA inhibitor H89 (1 µmol L-1 ). Patch clamped VMs under basal conditions from TABs but not Shams exhibited outward current sensitive to the specific SK inhibitor apamin (100 nmol L-1 ), which was eliminated by inhibition of PKA (1 µmol L-1 ). Beta-adrenergic stimulation (isoproterenol, 100 nmol L-1 ) evoked ISK in VMs from Shams, resulting in shortening of action potentials in VMs and ex vivo optically mapped Sham hearts. Using adenoviral gene transfer, wild-type and mutant SK2 channels were overexpressed in adult rat VMs, revealing serine-465 as the site that elicits PKA-dependent phosphorylation effects on SK2 channel function. Concurrent confocal Ca2+ imaging experiments established that PKA phosphorylation lessens rectification of ISK via reduction Ca2+ /voltage-dependent inhibition of the channels at high [Ca2+ ] without affecting their sensitivity to activation by Ca2+ in the submicromolar range. In conclusion, upregulation of SK channels in diseased VMs is mediated by hyperadrenergic drive in cardiac hypertrophy, with functional effects on the channel conferred by PKA-dependent phosphorylation at serine-465.

Short-Long Heart Rate Variation Increases Dispersion of Action Potential Duration in Long QT Type 2 Transgenic Rabbit Model.

The initiation of polymorphic ventricular tachycardia in long QT syndrome type 2 (LQT2) has been associated with a characteristic ECG pattern of short-long RR intervals. We hypothesize that this characteristic pattern increases APD dispersion in LQT2, thereby promoting arrhythmia. We investigated APD dispersion and its dependence on two previous cycle lengths (CLs) in transgenic rabbit models of LQT2, LQT1, and their littermate controls (LMC) using random stimulation protocols. The results show that the short-long RR pattern was associated with a larger APD dispersion in LQT2 but not in LQT1 rabbits. The multivariate analyses of APD as a function of two previous CLs (APDn = C + α1CLn-1 + α2CLn-2) showed that α1 (APD restitution slope) is largest and heterogeneous in LQT2 but uniform in LQT1, enhancing APD dispersion under long CLn-1 in LQT2. The α2 (short-term memory) was negative in LQT2 while positive in LQT1, and the spatial pattern of α1 was inversely correlated to α2 in LQT2, which explains why a short-long combination causes a larger APD dispersion in LQT2 but not in LQT1 rabbits. In conclusion, short-long RR pattern increased APD dispersion only in LQT2 rabbits through heterogeneous APD restitution and the short-term memory, underscoring the genotype-specific triggering of arrhythmias in LQT syndrome.

LITAF (Lipopolysaccharide-Induced Tumor Necrosis Factor) Regulates Cardiac L-Type Calcium Channels by Modulating NEDD (Neural Precursor Cell Expressed Developmentally Downregulated Protein) 4-1 Ubiquitin Ligase.

BACKGROUND: The turnover of cardiac ion channels underlying action potential duration is regulated by ubiquitination. Genome-wide association studies of QT interval identified several single-nucleotide polymorphisms located in or near genes involved in protein ubiquitination. A genetic variant upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor) gene prompted us to determine its role in modulating cardiac excitation. METHODS: Optical mapping was performed in zebrafish hearts to determine Ca2+ transients. Live-cell confocal calcium imaging was performed on adult rabbit cardiomyocytes to determine intracellular Ca2+handling. L-type calcium channel (LTCC) current (ICa,L) was measured using whole-cell recording. To study the effect of LITAF on Cav1.2 (L-type voltage-gated calcium channel 1.2) channel expression, surface biotinylation, and Westerns were performed. LITAF interactions were studied using coimmunoprecipitation and in situ proximity ligation assay. RESULTS: LITAF knockdown in zebrafish resulted in a robust increase in calcium transients. Overexpressed LITAF in 3-week-old rabbit cardiomyocytes resulted in a decrease in ICa,L and Cavα1c abundance, whereas LITAF knockdown increased ICa,L and Cavα1c protein. LITAF-overexpressing decreases calcium transients in adult rabbit cardiomyocytes, which was associated with lower Cavα1c levels. In tsA201 cells, overexpressed LITAF downregulated total and surface pools of Cavα1c via increased Cavα1c ubiquitination and its subsequent lysosomal degradation. We observed colocalization between LITAF and LTCC in tsA201 and cardiomyocytes. In tsA201, NEDD (neural precursor cell expressed developmentally downregulated protein) 4-1, but not its catalytically inactive form NEDD4-1-C867A, increased Cavα1c ubiquitination. Cavα1c ubiquitination was further increased by coexpressed LITAF and NEDD4-1 but not NEDD4-1-C867A. NEDD4-1 knockdown abolished the negative effect of LITAF on ICa,L and Cavα1c levels in 3-week-old rabbit cardiomyocytes. Computer simulations demonstrated that a decrease of ICa,L current associated with LITAF overexpression simultaneously shortened action potential duration and decreased calcium transients in rabbit cardiomyocytes. CONCLUSIONS: LITAF acts as an adaptor protein promoting NEDD4-1-mediated ubiquitination and subsequent degradation of LTCC, thereby controlling LTCC membrane levels and function and thus cardiac excitation.

NCX-Mediated Subcellular Ca2+ Dynamics Underlying Early Afterdepolarizations in LQT2 Cardiomyocytes.

Long QT syndrome type 2 (LQT2) is a congenital disease characterized by loss of function mutations in hERG potassium channels (IKr). LQT2 is associated with fatal ventricular arrhythmias promoted by triggered activity in the form of early afterdepolarizations (EADs). We previously demonstrated that intracellular Ca2+ handling is remodeled in LQT2 myocytes. Remodeling leads to aberrant late RyR-mediated Ca2+ releases that drive forward-mode Na+-Ca2+ exchanger (NCX) current and slow repolarization to promote reopening of L-type calcium channels and EADs. Forward-mode NCX was found to be enhanced despite the fact that these late releases do not significantly alter the whole-cell cytosolic calcium concentration during a vulnerable period of phase 2 of the action potential corresponding to the onset of EADs. Here, we use a multiscale ventricular myocyte model to explain this finding. We show that because the local NCX current is a saturating nonlinear function of the local submembrane calcium concentration, a larger number of smaller-amplitude discrete Ca2+ release events can produce a large increase in whole-cell forward-mode NCX current without increasing significantly the whole-cell cytosolic calcium concentration. Furthermore, we develop novel insights, to our knowledge, into how alterations of stochastic RyR activity at the single-channel level cause late aberrant Ca2+ release events. Experimental measurements in transgenic LTQ2 rabbits confirm the critical arrhythmogenic role of NCX and identify this current as a potential target for antiarrhythmic therapies in LQT2.