Electrical Pacing Research Articles

Contractility and Calcium Transient Maturation in the Human iPSC-Derived Cardiac Microfibers.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are considered immature in the sarcomere organization, contractile machinery, calcium transient, and transcriptome profile, which prevent them from further applications in modeling and studying cardiac development and disease. To improve the maturity of hiPSC-CMs, here, we engineered the hiPSC-CMs into cardiac microfibers (iCMFs) by a stencil-based micropatterning method, which enables the hiPSC-CMs to be aligned in an end-to-end connection for prolonged culture on the hydrogel of physiological stiffness. A series of characterization approaches were performed to evaluate the maturation in iCMFs on both structural and functional levels, including immunohistochemistry, calcium transient, reverse-transcription quantitative PCR, cardiac contractility, and electrical pacing analysis. Our results demonstrate an improved cardiac maturation of hiPSC-CMs in iCMFs compared to micropatterned or random single hiPSC-CMs and hiPSC-CMs in a random cluster at the same cell number of iCMFs. We found an increased sarcomere length, better regularity and alignment of sarcomeres, enhanced contractility, matured calcium transient, and T-tubule formation and improved adherens junction and gap junction formation. The hiPSC-CMs in iCMFs showed a robust calcium cycling in response to the programmed and continuous electrical pacing from 0.5 to 7 Hz. Moreover, we generated the iCMFs with hiPSC-CMs with mutations in myosin-binding protein C (MYBPC3) to have a proof-of-concept of iCMFs in modeling cardiac hypertrophic phenotype. These findings suggest that the multipatterned iCMF connection of hiPSC-CMs boosts the cardiac maturation structurally and functionally, which will reveal the full potential of the application of hiPSC-CM models in disease modeling of cardiomyopathy and cardiac regenerative medicine.

Twiddler's Syndrome

A 57-year-old woman with a history of cardiac sarcoidosis, complicated by recent episodes of nonsustained ventricular tachycardia requiring an implantable cardiac defibrillator, presented with 2–3 days of substernal chest wall discomfort and palpitations. Her cardiopulmonary examination was unremarkable, with a regular rhythm and no new murmurs. Initial electrocardiogram (Figure 1) demonstrated a normal sinus rhythm with no ectopic beats, however, supplemental electrical pacing spikes (black arrows) were noted to occur at a regular interval without associated cardiac capture.

Analysis of Cardiac Contractile Dysfunction and Ca<sup>2+</sup> Transients in Rodent Myocytes

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury and/or remodeling. One approach for assessing these functional alterations utilizes unloaded shortening and Ca2+ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca2+ tolerant, and then adhered to laminin-coated coverslips, followed by electrical pacing in serum-free media. The general protocol utilizes adult rat cardiac myocytes but can be readily adjusted for primary myocytes from other species. Functional alterations in myocytes from injured hearts can be compared to sham myocytes and/or to in vitro therapeutic treatments. The methodology includes the essential elements needed for myocyte pacing, along with the cell chamber and platform components. The detailed protocol for this approach incorporates the steps for measuring unloaded shortening by sarcomere length detection and cellular Ca2+ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes.

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury and/or remodeling. One approach for assessing these functional alterations utilizes unloaded shortening and Ca2+ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca2+ tolerant, and then adhered to laminin-coated coverslips, followed by electrical pacing in serum-free media. The general protocol utilizes adult rat cardiac myocytes but can be readily adjusted for primary myocytes from other species. Functional alterations in myocytes from injured hearts can be compared to sham myocytes and/or to in vitro therapeutic treatments. The methodology includes the essential elements needed for myocyte pacing, along with the cell chamber and platform components. The detailed protocol for this approach incorporates the steps for measuring unloaded shortening by sarcomere length detection and cellular Ca2+ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

Fusing spheroids to aligned μ-tissues in a heart-on-chip featuring oxygen sensing and electrical pacing capabilities.

Over the last decade, Organ-on-Chip (OoC) emerged as a promising technology for advanced in vitro models, recapitulating key physiological cues. OoC approaches tailored for cardiac tissue engineering resulted in a variety of platforms, some of which integrate stimulation or probing capabilities. Due to manual handling processes, however, a large-scale standardized and robust tissue generation, applicable in an industrial setting, is still out of reach. Here, we present a novel cell injection and tissue generation concept relying on spheroids, which can be produced in large quantities and uniform size from induced pluripotent stem cell-derived human cardiomyocytes. Hydrostatic flow transports and accumulates spheroids in dogbone-shaped tissue chambers, which subsequently fuse and form aligned, contracting cardiac muscle fibers. Furthermore, we demonstrate electrical stimulation capabilities by utilizing fluidic media connectors as electrodes and provide the blueprint of a low-cost, open-source, scriptable pulse generator. We report on a novel integration strategy of optical O2 sensor spots into resin-based microfluidic systems, enabling in situ determination of O2 partial pressures. Finally, a proof-of-concept demonstrating electrical stimulation combined with in situ monitoring of metabolic activity in cardiac tissues is provided. The developed system thus opens the door for advanced OoCs integrating biophysical stimulation as well as probing capabilities and serves as a blueprint for the facile and robust generation of high density microtissues in microfluidic modules amenable to scaling-up and automation.

Rare case of Torsades de Pointes in severe hypothyroidism: literature review and challenges in management

BackgroundHypothyroidism can manifest as several important cardiac abnormalities. There are few reports of ventricular dysrhythmias (VDs) in hypothyroidism. We described a rare case of VDs in severe hypothyroidism and reviewed the literature behind its management.Case presentationA 67-year-old gentleman, with poor compliance to treatment for Hashimoto’s thyroiditis, presented with palpitations to the Emergency Department. He had runs of non-sustained ventricular tachycardia (NSVT). He was treated with intravenous (IV) amiodarone and admitted to the intensive care unit for observation. He then developed recurrent Torsades de Pointes (Tdp) despite treatment with several anti-arhythmics. He required electrical cardioversion and eventual transvenous overdrive pacing (OP). VT recurred while he was on OP. VT resolved and he was weaned off OP only after adequate thyroid hormone replacement.ConclusionsVDs, including NSVT, Tdp, and VT, are rare and potentially lethal in hypothyroidism. Our case demonstrates important challenges in the management of severe hypothyroidism. Here, VDs are often refractory to treatment with drugs and electrical means. The choice(s) of anti-arrhthymics requires careful consideration and can be difficult before thyroid function tests are known. Amiodarone use should be cautioned as it is associated with thyroid dysfunction and QT interval prolongation.There is no literature to guide thyroid hormone replacement in this disease. Aggressive replacement is associated with adverse cardiovascular effects. Our case showed a fine balance between the risk of rapid thyroid hormone replacement and the urgency to terminate VDs. Its administration should be carefully monitored amidst bridging strategies like electrical cardioversion and OP to manage life-threatening VDs.

Generation and Culture of Cardiac Microtissues in a Microfluidic Chip with a Reversible Open Top Enables Electrical Pacing, Dynamic Drug Dosing and Endothelial Cell Co‐Culture

AbstractCardiovascular disease morbidity has increased worldwide. Organs‐on‐chips and human pluripotent stem cell (hPSC) technologies aid to overcome some of the limitations in cardiac in vitro models. Here, a bi‐compartmental, monolithic heart‐on‐chip device that facilitates porous membrane integration in a single fabrication step is presented. Moreover, the device includes open‐top compartments that allow facile co‐culture of hPSC‐derived cardiomyocytes and human adult cardiac fibroblast into geometrically defined cardiac microtissues. The device can be reversibly closed with a glass seal or a lid with fully customized 3D‐printed pyrolytic carbon electrodes allowing electrical stimulation of cardiac microtissues. A subjacent microfluidic channel allowed localized and dynamic drug administration to the cardiac microtissues, as demonstrated by a chronotropic response to isoprenaline. Moreover, the microfluidic channel can also be populated with human induced pluripotent stem‐derived endothelial cells allowing co‐culture of heterotypic cardiac cells in one device. Overall, this study demonstrates a novel heart‐on‐chip model that systematically integrates an open‐top device with a 3D printed carbon electrode for electrical pacing and culture of cardiac tissues while enabling active perfusion and dynamic drug dosing. Advances in the engineering of human heart‐on‐chip models represent an important step towards making organ‐on‐a‐chip technology a routine aspect of preclinical cardiac drug development.

Mettl3 deficiency leads to the upregulation of Cav1.2 and increases arrhythmia susceptibility in mice.

Methyltransferase-like 3 (Mettl3) is a component of methyltransferase complex that mediates mA modification of RNAs, and participates in multiple biological processes. However, the role of Mettl3 in cardiac electrophysiology remains unknown. This study aims to explore the ventricular arrhythmia susceptibility of Mettl3 mice and the underlying mechanisms. Mice were anesthetized with 2% avertin (0.1 mL/ body weight) for echocardiography and programmed electrical pacing. Whole-cell patch clamp technique was used to examine the electrophysiological property of cardiomyocytes. The expression of Cav1.2 was determined by qRT-PCR and western blot analysis. The mA medication of mRNA was examined by MeRIP-Seq and MeRIP-qPCR. No differences are found in the morphology and function of the hearts between Mettl3 mice and wild-type (WT) controls. The QT and QTc intervals of Mettl3 mice are significantly longer. High-frequency electrical stimulation showed that heterozygous knockout of Mettl3 increases ventricular arrhythmia susceptibility. The whole-cell patch-clamp recordings showed that the APD is prolonged in Mettl3 ventricular myocytes and more EADs were observed. The density of is substantially increased in ventricular myocytes of Mettl3 mice. The pore-forming subunit of L-type calcium channel Cav1.2 is upregulated in Mettl3 mice, while the mRNA of its coding gene does not change. MeRIP-Seq and MeRIP-qPCR showed that the mA methylation of mRNA is decreased in cultured Mettl3-knockdown cardiomyocytes and Mettl3 hearts. Collectively, deficiency of Mettl3 increases ventricular arrhythmia susceptibility due to the upregulation of Cav1.2 by reducing mA modification onmRNA in mice. This study highlights the role of mA modification in the regulation of cardiac electrophysiology.

Permanent electrical cardiac pacing in bradyarrhythmias

Permanent electrical cardiac pacing (ECP) is the most effective method for treatment of patients with bradyarrhythmias, which allows to increase their quality of life, decreases the mortality from trauma and the risk of sudden cardiac death. Lecture provides modern recommendations regarding the ECP in the treatment of different bradyarrhythmias, discusses the letter code, modes of ECP, ECG in different ECP modes and pitfalls in the pacemaker functioning.

Abstract 9103: Electrophysiological Arrhythmia Characterization of a Stress-Induced Idiopathic VF IPS-CMs Using Fluorescent Optical Mapping

Background: Idiopathic ventricular fibrillation (IVF) is a clinically challenging disease entity as phenotypic characteristics are lacking. We studied induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) from a patient with stress-induced IVF to define the cellular arrhythmic behavior and create a path to test therapeutics. Methods: iPS-CMs were generated from control (WT) and stress-induced IVF (si-IVF) patient-specific iPSC lines. Cells were characterized by single-cell optical mapping using calcium sensitive dye (Fluo-4) and electrical pacing (0.5Hz), with β-adrenergic stimulation (1x10 -6 mM isoproterenol (ISO)). Results: si-IVF cells had longer CaT rise time (ms) at baseline (BSL) (p<0.05) (86 +/- 5.45, n=54) and after ISO (83.88 +/- 5.12, n=41) than WT cells (BSL: 64.5 +/- 3.72, n=40) (ISO: 72 +/- 9.92, n=10). si-IVF cells had shorter CaT duration (ms) at BSL (1,069 +/- 36.00, n=54) than WT cells at BSL (1,280 +/- 37.59, n=40), with no significant difference after ISO (IVF: 1,073 +/- 44.51, n= 41 and WT: 1,230 +/- 32.79, n=10). Following ISO, 11.5% of si-IVF cells showed arrhythmic behavior with rapid continuous spontaneous Ca 2+ release (Fig. 1), compared to 0.0% of WT cells. Conclusion: We demonstrate feasibility of differentiating iPS-CMs from patients while investigating arrhythmogenicity of si-IVF cells compared to controls using optical mapping. Insights from CaT duration, rise time, and arrhythmogenic behavior helps establish the arrhythmogenic behavior, and begins to provide mechanistic insights into si-IVF. This methodology can be applied to test therapeutics for IVF patients with β-adrenergic triggers. Figure 1: Patient #1-specific si-IVF iPS-CM. A) Representative optical mapping recordings of iPS-CMs baseline paced at 1Hz (each trace is a separate cell) B) Application of ISO caused arrhythmic behavior and failure to capture paced beats in black, red, blue, and yellow traces but retention of paced response in green and grey traces.

Abstract 12001: Atrial Fibrillation Induces Ventricular Dysfunction via Altered Ca 2+ Handling and Oxidative CaMKII Activation in the Human Heart

Introduction: The effects of atrial fibrillation (AF) on left ventricular (LV) function are poorly understood. Clinical data indicate that the arrhythmic component of AF may contribute to LV dysfunction. This study investigated the effects of AF on the human LV. Methods and Results: LV myocardium from patients with preserved LV function (EF>50%) with sinus rhythm (SR, n=31) or rate-controlled AF (n=24) obtained during surgery for aortic stenosis was studied. Clinical data were mostly balanced without changes in LV function and remodeling. LV myocardium from SR and AF patients showed no changes in fibrosis. In functional studies, systolic Ca 2+ transient amplitude of human LV cardiomyocytes (CM) was reduced in AF patients, while diastolic Ca 2+ levels and Ca 2+ transient kinetics were unaltered. These results were confirmed in LV CM from non-failing donors with AF (n=4) vs. SR (n=8). In addition, normofrequent AF was simulated in vitro using arrhythmic or rhythmic electrical culture pacing (both at 60 bpm). After 24h of AF-simulation, human LV CM from non-failing donors (n=9) showed also an impaired Ca 2+ transient amplitude. For a standardized chronic AF-simulation, human iPSC-CM (n=22 differentiations/5 individuals) were tested. 7 days of AF-simulation caused reduced systolic Ca 2+ transient amplitude and sarcoplasmic reticulum Ca 2+ load, which could be explained by an increased diastolic Ca 2+ leak. Mechanistically, oxidative stress markers were higher in the LV of AF patients. Ca 2+ /calmodulin-dependent protein kinase IIδ (CaMKII) was found to be more oxidized at Met281/282 in the LV of AF patients leading to an increased CaMKII activity. Thus, RyR2 phosphorylation was elevated, which likely underlies the depression of LV Ca 2+ handling in AF. In translational contractility experiments of human ventricular trabecula from patients with heart failure (n=8), in vitro AF-simulation for 8h worsened systolic and diastolic contractile function. Conclusions: Here we could firstly show that AF impairs human LV function via increased oxidative stress and CaMKII activity in different patient populations and models. Thus, this translational study demonstrates the detrimental effects and mechanisms of AF in the absence of tachycardia on the human LV.

Light-controllable recellularized ventricular and atrial tissues

Abstract Introduction Construction of three-dimensional ventricular and atrial patches using decellularized extracellular matrix could allow the creation of “organ-like” structures for disease modeling, drug testing and regenerative medicine applications. To engineer functional artificial anatomical hearts several obstacles need to be overcome including generation of chamber-specific cells, prevention of immune reaction and donor/host tissue electrical coupling. Purpose In order to tackle these challenges we aimed to combine human induced pluripotent stem cell (hiPSC) technology, developmental biology inspired differentiation system to generate chamber-specific cardiomyocytes, decellularization/recellularization processes, and optogenetics utilizing light-sensitive ion channels to generate light-controllable atrial/ventricular tissue engineered patches. Material and method Atrial and ventricular patches of adult rats were decellularized using 1% SDS, 3% triton-X. The decellularized scaffolds were then recellularized with hiPSC- derived cardiomyocytes (2x107 atrial or ventricular cells). Adenoviral transduction was used to express the light-sensitive cationic channel ChR2 in the tissue engineered constructs. Results and discussion Eight days after cell seeding, we observed the development of spontaneous contraction of the atrial/ventricular tissues. Immunostaining for atrial (Cx40) or ventricular (MLC-2V) markers, gene expression, action-potential morphology, and the response to chamber-specific pharmacology confirmed the atrial/ventricular specific identity of the patches. Histological examination confirmed the preservation of the macro/micro- atrial/ventricular anatomical features in the generated chamber-specific recellularized patches. Optical mapping, using an EM-CCD camera, was used to characterize the conduction and repolarization properties of the generated tissues. The engineered patches could be paced and their electrical activity controlled by either electrical or optogenetic stimulation. Finally, arrhythmogenic reentrant arrhythmias could be induced in the tissue models and terminated by using optogenetic stimulation (“optogenetic-defibrillation”). Conclusions Three-dimensional light-sensitive chamber-specific engineered heart patches could be generated that could be controlled and manipulated through electrical and light pacing. These tissue could be used for several pathophysiological, drug testing, disease modeling and regenerative medicine applications. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): H2020-ERC-COG

Abstract P359: Electroconductive Scaffolds To Mature Induced Pluripotent Stem Cell-derived Cardiomyocytes For Cardiac Tissue Engineering

Heart disease is the leading cause of death worldwide. Cardiac tissue engineering (CTE) aims to repair and replace heart tissue, offering a solution. Induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) could revolutionize CTE due to their theoretical ability to supply limitless patient-specific CMs. However, iPSC-CMs are electrophysiologically immature compared to functional adult CMs, and therefore incapable of sustaining a heartbeat. Thus, a scaffold capable of electrophysiologically maturing iPSC-CMs is needed. My research increases the electroconductivity of electrospun (ES) scaffolds by incorporating carbon nanotubes (CNTs), which I hypothesize will mature iPSC-CMs seeded onto them due to their excellent electroconductive properties. Morphological, biocompatibility, and electrical analyses have been performed on ES polycaprolactone (PCL) and gelatin scaffolds with CNTs incorporated via a ‘sandwich’ and dual deposition method in order to increase electroconductivity. Morphological analyses were performed via ImageJ on SEM images. Fiber diameter and pore size quantification confirmed the ability to exert morphological control by modifying solution properties and ES parameters, which is crucial to achieve biomimicry of the cardiac extracellular matrix. Live/dead assays and immunofluorescence revealed the CNT scaffolds offer high biocompatibility for NIH 3T3 fibroblasts, which attach, proliferate, and migrate well. Electrical analysis performed with a multimeter and two-probe resistance measurement confirms that inclusion of CNTs significantly increases scaffold conductivity, moreso for dual deposition scaffolds than ‘sandwich’ ones, and moreso parallel to the CNT arrays than orthogonally. These results prove the feasibility of using such scaffolds as a method for in vitro electrophysiological iPSC-CM maturation. Next steps include optimization of scaffolds, analysis of iPSC-CM biocompatibility and response, and recapitulation and manipulation of the electrophysiology of cardiac tissue, including quantification of markers for cardiac function and maturity, and assessment of iPSC-CM + scaffold response to electrical pacing.

Evaluation of acute and long-term drug effects on contractility at 1 Hz electrical pacing with human induced pluripotent stem cell-derived cardiac cell sheet-tissues

Evaluation of acute and long-term drug effects on contractility at 1 Hz electrical pacing with human induced pluripotent stem cell-derived cardiac cell sheet-tissues

Pharmacological and simulated exercise cardiac stress tests produce different ischemic signatures in high-resolution experimental mapping studies

ObjectiveTest the hypothesis that exercise and pharmacological cardiac stressors create different electrical ischemic signatures. IntroductionCurrent clinical stress tests for detecting ischemia lack sensitivity and specificity. One unexplored source of the poor detection is whether pharmacological stimulation and regulated exercise produce identical cardiac stress. MethodsWe used a porcine model of acute myocardial ischemia in which animals were instrumented with transmural plunge-needle electrodes, an epicardial sock array, and torso arrays to simultaneously measure cardiac electrical signals within the heart wall, the epicardial surface, and the torso surface, respectively. Ischemic stress via simulated exercise and pharmacological stimulation were created with rapid electrical pacing and dobutamine infusion, respectively, and mimicked clinical stress tests of five 3-minute stages. Perfusion to the myocardium was regulated by a hydraulic occluder around the left anterior descending coronary artery. Ischemia was measured as deflections to the ST-segment on ECGs and electrograms. ResultsAcross eight experiments with 30 (14 simulated exercise and 16 dobutamine) ischemic interventions, the spatial correlations between exercise and pharmacological stress diverged at stage three or four during interventions (p<0.05). We found more detectable ST-segment changes on the epicardial surface during simulated exercise than with dobutamine (p<0.05). The intramyocardial ischemia formed during simulated exercise had larger ST40 potential gradient magnitudes (p<0.05). ConclusionWe found significant differences on the epicardium between cardiac stress types using our experimental model, which became more pronounced at the end stages of each test. A possible mechanism for these differences was the larger ST40 potential gradient magnitudes within the myocardium during exercise. The presence of microvascular dysfunction during exercise and its absence during dobutamine stress may explain these differences.

Ca2+ handling in induced-pluripotent cardiomyocytes from female CPVT patient, and comparison to male

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agence Nationale de la Recherche Introduction Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is an inherited disease manifested as syncope or sudden death in apparently healthy children or young adults, in the absence of structural cardiac disease. The ryanodine receptor (RyR2) R420Q mutation was identified in a Spanish 14-year-old boy who died suddenly due to emotional stress. We generated human induced-pluripotent (h-iPS) derived cardiomyocytes from two brothers (see Yin’s poster in this meeting). As most of sudden death occurs in males, we had the hypothesis that there might be some sex-dependent differences. Purpose and methods We aim to elucidate the key role of [Ca2+]i in the genesis of cardiac arrhythmia in female RyR2R420Q CPVT patients . We generated here hiPSC-CM from two sisters of the same family, one exhibiting the mutation RyR2R420Q and the other without the mutation used as control. The experiments were conducted at 30-35 days after differentiation into cardiomyocytes. Using confocal microscopy, we assessed intracellular [Ca2+]i handling both spontaneous and during electrical pacing. To record [Ca2+]i transients, h-iPS-CMs were electrically field stimulated by two Pt electrodes at 1Hz. Spontaneous Ca2+ sparks and Ca2+ waves were recorded in quiescent cells, after [Ca2+]i transients recordings. For SR Ca2+ load quantification, h-iPS-CMs were rapidly perfused with 10 mmol/L caffeine. To induce β-adrenergic activation, 100nM isoproterenol (ISO) was added in some experiments. All confocal Ca2+ images analyses were performed by home-made routines using IDL software. Results First, we verify by immunofluorescence the expression of alpha-actinin in the differentiated h-iPS-CMs. Then, by confocal microscopy we found that woman CPVT h-iPS-CMs presented longer cycle length than wt h-iPS-CMs, correlating with the bradycardia observed in CPVT patients. The amplitude of the Ca2+ transients were significantly higher for CPVT h-iPS-CMs compared to wt; however, after ISO perfusion Ca2+ transients did not show a significant increase in CPVT h-iPS-CMs compared to CPVT h-iPS-CMs before ISO. SR Ca2+ load presented no differences between groups but the time decay constant of the caffeine-evoked Ca2+ transients were significantly faster in CPVT h-iPS-CMs whereas it became slower after ISO perfusion. In addition, analysis of Ca2+ pro-arrhythmogenic events were significantly augmented in CPVT h-iPS-CMs compared to wt h-iPS-CMs. After ISO perfusion, pro-arrhythmogenic events in CPVT cells presented no significant difference compared to CPVT cell before ISO perfusion. Although proarrhythmogenic events were similarly higher in both male and female hiPSC-CM, the Ca2+ handling characteristics were slightly different. Conclusion The RyR2R420Q mutation in woman CPVT h-iPS-CMs provides a reliable model to study CPVT in human context.

Chronic electrical performance of a new ultra-thin left ventricular quadripolar pacing lead

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): H2020-FTI [Fast Track to Innovation] Pilot-2016 and MicroPort CRM Background Left ventricular (LV) lead positioning is an important contributor to cardiac resynchronization therapy (CRT) response. Multiple additional LV sites could be stimulated by passing a new ultra-thin (1.2 Fr, 0.4 mm) quadripolar microlead from one LV vein into another via venous collaterals. Purpose Study the acute and chronic stability and electrical pacing performance of a novel 1.2 Fr quadripolar microlead ("Axone 4LV"). Methods Seven healthy adult dogs underwent CRT defibrillator implantation, including a right ventricular lead and the microlead. The microlead was advanced into the coronary sinus network using a dedicated microguide catheter. The animals were followed up at 1, 15, 30 and 90 days post-implant to evaluate chronic stability and electrical pacing performance. Results Successful uncomplicated implantation was achieved in all cases. Despite the small diameter of the distal veins and collaterals, placement of the pacing electrodes in two different LV veins was feasible via available collateral passages. Lead position was stable in all cases and over the entire study period. Capture threshold and pacing impedance at 90 days post-implant were 1.7 ± 0.5 V with 1323 ± 245 Ω, respectively, at 0.5 ms pulse width. The mean pacing energy to get capture was 1.1 ± 0.5 µJ and less than 2 µJ in all cases (2 µJ ≈ 1.4 V @500 Ω, 0.5 ms). No phrenic nerve stimulation was observed during pacing. Conclusions The novel 1.2 Fr quadripolar microlead demonstrated adequate stability and good electrical performance allied to low energy consumption. This quadripolar microlead may extend pacing options while increasing device longevity in CRT. Changes in pacing parameters over time Post-implant Follow-up Pacing Threshold Pacing Impedance Pacing Energy 1 day 1.4 ± 0.7 V 1294 ± 270 Ω 0.8 ± 0.7 µJ 15 days 1.9 ± 0.9 V 1336 ± 274 Ω 1.8 ± 1.8 µJ 30 days 1.8 ± 0.7 V 1187 ± 303 Ω 1.5 ± 1.2 µJ 90 days 1.7 ± 0.5 V 1323 ± 245 Ω 1.1 ± 0.5 µJ

Modelling of atrial fibrillation at physiologically relevant scales enabled by massive expansion of native human atrial cardiomyocytes

Abstract Funding Acknowledgements Type of funding sources: Public hospital(s). Main funding source(s): LUMC Background Current in vitro models of atrial fibrillation have limited translational potential due to a lack of relevant human physiology or the inability to reach the high activation frequencies present in human atrial fibrillation. Absence of relevant models is the result of a general deficit of readily available and standardized sources of well-differentiated human atrial cardiomyocytes. Therefore, we aimed to immortalize native human atrial cardiomyocytes to produce natural and standardized lines of these cells. Methods Human fetal atrial cardiomyocytes were transduced with a lentiviral vector directing myocyte-specific and doxycycline-inducible expression of simian virus 40 large T antigen. Addition of doxycycline to the culture medium pushed cardiomyocytes towards a highly proliferative phenotype (proliferation up to 10^12 cells). These cells were labelled hiAMs (human immortalised Atrial Myocytes). After differentiation upon doxycycline removal, hiAM cells were characterized using various molecular, biological and electrophysiological assays. Results Following cardiomyogenic differentiation, hiAMs no longer expressed the proliferation marker Ki67, revealed striated α-actinin and troponin T staining patterns and displayed synchronous contractions. Optical voltage mapping of hiAM monolayers revealed excitable cells showing homogeneous spreading of action potentials at 22.5 ± 3.1 cm/s with a mean APD80 of 139 ± 22 ms. Addition of flecainide (10 µM) to hiAM monolayers decreased the conduction velocity by 35% and increased the APD80 by 107%. Dofetilide (10 nM) addition had no effect on the conduction velocity, but did increase the APD80 by 81%. Due to their scalability, monolayers of hiAMs as big as 10 cm2 showing homogenous action potential propagation could easily be created. Following high-frequency electrical pacing, rotors could be induced with an average activation frequency of 7.5 ± 0.9 Hz. Infusion of flecainide during arrhythmic activity resulted in termination of the rotor in 18 of 24 attempts (75%), whereas addition of 0.1% DMSO (vehicle control) did not result in termination in any of the attempts. Dofetilide infusion did not result in termination. However, it did lower the average activation frequency to 2.1 ± 0.7 Hz. Conclusion We have generated first-of-a-kind lines of human atrial cardiomyocytes, allowing massive cell expansion under proliferation conditions and robust formation of cross-striated, contractile and excitable cardiomyocytes after differentiation. These characteristics allow, for the first time, the modelling, at a large-scale, of human atrial arrhythmias with frequencies similar to human atrial fibrillation. With the generation of hiAMs, a user-friendly, clinically-relevant and much-anticipated human atrial research model has been produced. Abstract Figure. hiAM AF Model

Cardiotoxicity assessment using 3D vascularized cardiac tissue consisting of human iPSC-derived cardiomyocytes and fibroblasts

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for cardiac safety assessment but have limitations for the evaluation of drug-induced contractility. Three-dimensional (3D) cardiac tissues are similar to native tissue and valuable for the assessment of contractility. However, a longer time and specialized equipment are required to generate 3D tissues. We previously developed a simple method to generate 3D tissue in a short period by coating the cell surfaces with extracellular matrix proteins. We hypothesized that this 3D cardiac tissue could be used for simultaneous evaluation of drug-induced repolarization and contractility. In the present work, we examined the effects of several compounds with different mechanisms of action by cell motion imaging. Consequently, human ether-a-go-go-related gene (HERG) channel blockers with high arrhythmogenic risk caused prolongation of contraction-relaxation duration and arrhythmia-like waveforms. Positive inotropic drugs, which increase intracellular Ca2+ levels or myocardial Ca2+ sensitivity, caused an increase in maximum contraction speed (MCS) or average deformation distance (ADD) (ouabain, 138% for MCS at 300 nM; pimobendane, 132% for ADD at 3 μM). For negative inotropic drugs, verapamil reduced both MCS and ADD (61% at 100 nM). Thus, this 3D cardiac tissue detected the expected effects of various cardiovascular drugs, suggesting its usefulness for cardiotoxicity evaluation.

Induced pluripotent cardiomyocytes from female CPVT patient present different Ca2+ mishandling than men

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disease manifested as syncope or sudden death in children and young adults. The ryanodine receptor (RyR2) R420Q mutation was identified in a 14-year-old boy who died suddenly. We generated human induced pluripotent (h-iPS) derived cardiomyocytes from two sisters. As most of sudden death occurs in males, we had the hypothesis that there might be some sex-dependent differences. We aim to elucidate the key role of [Ca 2+ ] i in the genesis of cardiac arrhythmia in female RyR2 R420Q CPVT patients. We generated hiPSC-CM from two sisters, one carrier and the other non-carrier. We assessed intracellular [Ca 2+ ] i handling both spontaneous and during electrical pacing at 1 Hz by confocal microscopy. For SR Ca 2+ load quantification, h-iPS-CMs were rapidly perfused with 10 mmol/L caffeine. To induce β-adrenergic activation, 100 nM isoproterenol (ISO) was added in some experiments. All confocal Ca 2+ images were analysed by homemade routines using IDL software. First, we found that woman CPVT h-iPS-CMs presented longer cycle length than wt cells, correlating with the bradycardia observed in CPVT patients. The amplitude of the Ca 2+ transients were significantly higher for CPVT h-iPS-CMs compared to wt; however Ca 2+ transients did not change in CPVT h-iPS-CMs, after ISO perfusion. SR Ca 2+ load presented no differences between groups but the time decay constant of the caffeine-evoked Ca 2+ transients were faster in CPVT h-iPS-CMs whereas it became slower after ISO perfusion. Moreover, Ca 2+ pro-arrhythmogenic events were significantly higher in CPVT h-iPS-CMs compared to wt h-iPS-CMs whereas similar values were observed in CPVT cells after ISO perfusion. Although pro-arrythmogenic events were similarly higher in both male and female hiPSC-CM, the Ca 2+ handling characteristics were slightly different. The RyR2R420Q mutation in woman CPVT h-iPS-CMs provides a reliable model to study CPVT in human context.