Tae Yun Kim

The adenovirus-mediated somatic transfer of the embryonic T-box transcription factor 18 (TBX18) gene can convert chamber cardiomyocytes into induced pacemaker cells. However, the translation of therapeutic TBX18-induced cardiac pacing faces safety challenges. Here we show that the myocardial expression of synthetic TBX18 mRNA in animals generates de novo pacing and limits innate and inflammatory immune responses. In rats, intramyocardially injected mRNA remained localized, whereas direct myocardial injection of an adenovirus carrying a reporter gene resulted in diffuse expression and in substantial spillover to the liver, spleen and lungs. Transient expression of TBX18 mRNA in rats led to de novo automaticity and pacemaker properties and, compared with the injection of adenovirus, to substantial reductions in the expression of inflammatory genes and in activated macrophage populations. In …

Background: Long QT Syndrome Type II (LQT2) is a congenital cause of arrhythmia and sudden cardiac death due to mutations in ion channel hERG responsible for the rapid inward rectifying potassium current (IKr). Progress in gene therapy may result in a viable treatment for LQT2. However, the gene transduction efficacy required to prevent cardiac arrhythmias in LQT2 is unknown. We used computer simulation to examine the minimum gene transduction efficacy necessary to suppress early afterdepolarizations (EADs). Hypothesis: Low transduction efficacy of hERG is sufficient to suppress EADs through electrotonic coupling between transduced IKr(+) and non-transduced IKr(-) cardiomyocytes (Fig 1A). Methods: Computational models of rabbit and human cardiac tissue were implemented in 2D (100 x 100 cells) and 3D (13 x 13 x 13 cells) with a multi-state Markov model for IKr and a dynamic gap junction …

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 myofibroblastcardiomyocyte coupling prolongs action potential duration (APD) and facilitates conduction block permissive of arrhythmias. Aged infarcted human ventricles show levels of senescence …

Long QT syndrome (LQTS) is a congenital disease associated with polymorphic ventricular tachycardia (pVT) and sudden cardiac death. The hallmarks of LQTS are early afterdepolarizations that initiate pVT and dispersion of repolarization allowing reentry formation. Small conductance Ca 2+-activated K+(SK) channels are directly gated by Ca 2+ and provide feedback from Ca 2+ to membrane voltage to accelerate cardiac repolarization, which may help normalizing LQTS. Using optical mapping, we examined the role of I SK in transgenic rabbit models of LQT1, LQT2, and LQT5 and found that enhancing I SK with NS309 consistently shortened APD, reduced APD dispersion, and suppressed pVT genesis universally in the three LQT rabbit models. Enhancement of I SK has a greater impact at longer cycle length, flattening restitution curves at slow heart rate where most pVTs occur in LQTS. We incorporated a six …

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 …

The invention provides a robust in vitro 3D atrial tissue platform made from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. The platform is useful for evaluating atrial-specific chemical responses experimentally and computationally.

BackgroundDynamics and stability of electrical waves in the heart has been attributed to the action potential duration (APD) restitution curve, which is a measure of how the tissue responds to a given cycle length (CL) and the conduction velocity (CV). The restitution curves have been used to investigate how changes in stimulation CL can lead to the induction of alternans and arrhythmias. Only few restitution curves are available from clinical measurements in human hearts, and they were generated using monophasic action potential measurements or from single human cardiomyocytes.ObjectiveTo obtain APD and CV restitution curves from explanted human hearts with optical mapping, and take advantage of the experimental data to refine the dynamics of an ionic model, which can be valuable for arrhythmia simulation studies.MethodsOptical mapping of membrane potentials was performed on explanted human …

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Implantable cardiac pacemaker devices are generally effective for patients with symptomatic bradyarrhythmia. However, device-dependent cardiac pacing is far from ideal and often inadequate, particularly for pediatric patients who need to go through invasive revision of the indwelling hardware. Biological pacemakers have been proposed as device-free alternatives to the current treatment, but sustained, unwavering biological pacing beyond days after the biologic delivery has not been demonstrated. We have previously demonstrated that re-expression of an embryonic transcription factor, TBX18, could reprogram ventricular cardiomyocytes into induced pacemaker myocytes (iPMs). Here, we report that exogenous expression of TBX18 per se leads to severe fibrosis in situ, impairing the iPMs’ ability to pace together. Acute fibrosis is accompanied with proliferation and activation of cardiac fibroblasts via Tgfβ-Smad2/3 pathway. Small molecule inhibition of Tgfβ signaling mitigated the interstitial remodeling, independent from TBX18-induced iPM reprogramming at the single-cell level. Direct and focal gene transfer of TBX18 into the left ventricular myocardium created ventricular pacing in a rat model of chronic atrioventricular block, but such activity began to wane in a week. In contrast, a combination therapy consisting of TBX18 gene transfer and Tgfβ inhibition enabled sustained biological pacing beyond the four-week study period. Our data demonstrate that inhibition of Tgfβ signaling suffices to achieve durable cardiac pacing by TBX18-induced biological pacemakers.

BackgroundVentricular fibrillation (VF) has been the subject of intense experimental studies. However, the main features and mechanisms responsible for the persistence of fibrillation are complex, diverse, and different in various species. There is a paucity of experimental data on human VF for obvious practical considerations.ObjectiveTo map the spatiotemporal organization of human VF as pertinent to the mechanisms of VF.MethodsWe studied three arterially-perfused explanted human hearts (one a recipient heart, two rejected donor hearts) using optical mapping of the transmembrane potential at high spatial and temporal resolution (256 x 256 pixels, 500 Hz) and a large field of view. VF was spontaneously induced (during preparation) and later by rapid pacing. Each∼ 30 sec long recording was processed to identify activation waves and visualized in 2D movies and stack diagrams (one spatial dimension vs …

Most cardiac arrhythmias at the whole heart level result from alteration of cell membrane ionic channels and intracellular calcium concentration ([Ca2+]i) cycling with emerging spatiotemporal behavior through tissue-level coupling. For example, dynamically induced spatial dispersion of action potential duration, QT prolongation, and alternans are clinical markers for arrhythmia susceptibility in regular and heart-failure patients that originate due to changes of the transmembrane voltage (Vm) and [Ca2+]i. We present an optical-mapping methodology that permits simultaneous measurements of the Vm - [Ca2+]i signals using a single-camera without cross-talk, allowing quantitative characterization of favorable/adverse cell and tissue dynamical effects occurring from remodeling and/or drugs in heart failure. We demonstrate theoretically and experimentally in six different species the existence of a family of excitation wavelengths, we termed semasbestic, that give no change in signal for one dye, and thus can be used to record signals from another dye, guaranteeing zero cross-talk.

BackgroundWhile the complex interplay of voltage and calcium dynamics in space and as a function of the pacing cycle length (PCL) has been studied in animal models, it is currently unknown in human hearts.Objective

Background: Induction of VF in Brugada syndrome is thought to be produced by a phase-2 reentry resulting from areas with mismatched action potential duration. While this has been shown numerically and in some experiments with MAP recordings, to date there is no full detailed experimental observation of the phase-2 induction.ObjectiveTo use high-resolution optical mapping in an explanted heart to quantify the induction of ventricular fibrillation by phase-2 reentry in a Brugada-induced model.MethodsA rejected-for-transplant human heart that had high levels of lactic acid during transport was arterially perfused and the endocardial right ventricle was optically mapped with a voltage-sensitive dye and the electrical propagation was measured at high spatial and temporal resolution (256x256 pixels, at 500 Hz) and a large field of view of about 40 cm2.ResultsIn this heart, elevated lactic acid produced action …

Oxidative stress in cardiac disease promotes pro-arrhythmic disturbances in Ca 2+ homeostasis. This includes impaired luminal Ca 2+ regulation of the sarcoplasmic reticulum (SR) Ca 2+ release channel the ryanodine receptor (RyR2), driving increased channel activity. However, exact mechanisms underlying the redox-mediated increase of RyR2 function in cardiac disease remain elusive. Using a rat model of hypertrophy induced by thoracic aorta banding (TAB), we tested whether oxidoreductase proteins that dynamically regulate the SR oxidative environment are involved in this process. The SR-targeted biosensor ERroGFP demonstrated a more oxidized intra-SR redox state in TAB ventricular myocytes (VMs), associated with upregulation of oxidoreductase Ero1α. Pharmacological (EN460) or genetic (shRNA) Ero1α inhibition normalized SR redox state, reversed reduction in Ca 2+ transient amplitude and SR …

BackgroundMuch of dynamics and stability of electrical waves in the heart is attributed to action potential restitution curve. These curves have been used to investigate how changes in periods of stimulation can lead to the induction of alternans and the initiation of electrically driven arrhythmias. To date, there have been only obtained a few restitution curves from clinical measurements in human hearts using MAPs, or from single human cardiac cells.ObjectiveTo obtain the first in tissue action potential duration (APD) restitution curves across large areas of human cardiac tissue.MethodsWe studied three arterially-perfused whole explanted human hearts (two from recipient patient and one from a rejected donor) using simultaneous voltage and calcium optical mapping at high spatial and temporal resolutions (256x256 pixels, at 500 Hz) and a large field of view of 6-10 cm 2. Voltage was measured across the anterior …

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 …

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 …

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 …

The sarcoplasmic reticulum (SR) Ca 2+ release ryanodine receptor channel (RyR2) is susceptible to posttranslational modifications by reactive oxygen species (ROS), impairing luminal Ca 2+ regulation and increasing channel activity. Despite significant research efforts, 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 in the SR are involved in this process. In ventricular myocytes (VMs) from rats with hypertrophy induced by thoracic aortic banding (TAB), expression of SR oxidoreductase Ero1α was significantly increased compared to Sham VMs. Adenoviral expression of intra-SR ROS probe ERroGFP in cultured TAB rat VMs revealed enhanced oxidative stress in the vicinity of RyR2, in parallel with increased RyR2 oxidation, measured with DNP …

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)-1β, increased mitochondrial reactive oxygen species (mitoROS), and oxidation of the sarcoplasmic reticulum (SR) Ca2+ release channel (ryanodine receptor 2 [RyR2]). Inhibiting IL-1β 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-1β mediated the DM-associated arrhythmia through mitoROS generation that enhances SR Ca2+ leak. The mechanistic link between inflammation and arrhythmias provides new therapeutic options.