Utilizing human induced pluripotent stem cells to study atrial arrhythmias in the short QT syndrome.

BACKGROUND: Among the monogenic inherited causes of atrial fibrillation is the short QT syndrome (SQTS), a rare channelopathy causing atrial and ventricular arrhythmias. One of the limitations in studying the mechanisms and optimizing treatment of SQTS-related atrial arrhythmias has been the lack of relevant human atrial tissues models. OBJECTIVE: To generate a unique model to study SQTS-related atrial arrhythmias by combining the use of patient-specific human induced pluripotent stem cells (hiPSCs), atrial-specific differentiation schemes, two-dimensional tissue modeling, optical mapping, and drug testing. METHODS AND RESULTS: SQTS (N588K KCNH2 mutation), isogenic-control, and healthy-control hiPSCs were coaxed to differentiate into atrial cardiomyocytes using a retinoic-acid based differentiation protocol. The atrial identity of the cells was confirmed by a distinctive pattern of MLC2v downregulation, connexin 40 upregulation, shorter and triangular-shaped action potentials (APs), and expression of the atrial-specific acetylcholine-sensitive potassium current. In comparison to the healthy- and isogenic control cells, the SQTS-hiPSC atrial cardiomyocytes displayed abbreviated APs and refractory periods along with an augmented rapidly activating delayed-rectifier potassium current (IKr). Optical mapping of a hiPSC-based atrial tissue model of the SQTS displayed shortened APD and altered biophysical properties of spiral waves induced in this model, manifested by accelerated spiral-wave frequency and increased rotor curvature. Both AP shortening and arrhythmia irregularities were reversed by quinidine and vernakalant treatment, but not by sotalol. CONCLUSIONS: Patient-specific hiPSC-based atrial cellular and tissue models of the SQTS were established, which provide examples on how this type of modeling can shed light on the pathogenesis and pharmacological treatment of inherited atrial arrhythmias.

Gene- and variant-specific efficacy of serum/glucocorticoid-regulated kinase 1 inhibition in long QT syndrome types 1 and 2.

AIMS: Current long QT syndrome (LQTS) therapy, largely based on beta-blockade, does not prevent arrhythmias in all patients; therefore, novel therapies are warranted. Pharmacological inhibition of the serum/glucocorticoid-regulated kinase 1 (SGK1-Inh) has been shown to shorten action potential duration (APD) in LQTS type 3. We aimed to investigate whether SGK1-Inh could similarly shorten APD in LQTS types 1 and 2. METHODS AND RESULTS: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and hiPSC-cardiac cell sheets (CCS) were obtained from LQT1 and LQT2 patients; CMs were isolated from transgenic LQT1, LQT2, and wild-type (WT) rabbits. Serum/glucocorticoid-regulated kinase 1 inhibition effects (300 nM-10 µM) on field potential durations (FPD) were investigated in hiPSC-CMs with multielectrode arrays; optical mapping was performed in LQT2 CCS. Whole-cell and perforated patch clamp recordings were performed in isolated LQT1, LQT2, and WT rabbit CMs to investigate SGK1-Inh (3 µM) effects on APD. In all LQT2 models across different species (hiPSC-CMs, hiPSC-CCS, and rabbit CMs) and independent of the disease-causing variant (KCNH2-p.A561V/p.A614V/p.G628S/IVS9-28A/G), SGK1-Inh dose-dependently shortened FPD/APD at 0.3-10 µM (by 20-32%/25-30%/44-45%). Importantly, in LQT2 rabbit CMs, 3 µM SGK1-Inh normalized APD to its WT value. A significant FPD shortening was observed in KCNQ1-p.R594Q hiPSC-CMs at 1/3/10 µM (by 19/26/35%) and in KCNQ1-p.A341V hiPSC-CMs at 10 µM (by 29%). No SGK1-Inh-induced FPD/APD shortening effect was observed in LQT1 KCNQ1-p.A341V hiPSC-CMs or KCNQ1-p.Y315S rabbit CMs at 0.3-3 µM. CONCLUSION: A robust SGK1-Inh-induced APD shortening was observed across different LQT2 models, species, and genetic variants but less consistently in LQT1 models. This suggests a genotype- and variant-specific beneficial effect of this novel therapeutic approach in LQTS.

Optogenetic modulation of cardiac action potential properties may prevent arrhythmogenesis in short and long QT syndromes.

Abnormal action potential (AP) properties, as occurs in long or short QT syndromes (LQTS and SQTS, respectively), can cause life-threatening arrhythmias. Optogenetics strategies, utilizing light-sensitive proteins, have emerged as experimental platforms for cardiac pacing, resynchronization, and defibrillation. We tested the hypothesis that similar optogenetic tools can modulate the cardiomyocyte's AP properties, as a potentially novel antiarrhythmic strategy. Healthy control and LQTS/SQTS patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were transduced to express the light-sensitive cationic channel channelrhodopsin-2 (ChR2) or the anionic-selective opsin, ACR2. Detailed patch-clamp, confocal-microscopy, and optical mapping studies evaluated the ability of spatiotemporally defined optogenetic protocols to modulate AP properties and prevent arrhythmogenesis in the hiPSC-CMs cell/tissue models. Depending on illumination timing, light-induced ChR2 activation induced robust prolongation or mild shortening of AP duration (APD), while ACR2 activation allowed effective APD shortening. Fine-tuning these approaches allowed for the normalization of pathological AP properties and suppression of arrhythmogenicity in the LQTS/SQTS hiPSC-CM cellular models. We next established a SQTS-hiPSC-CMs-based tissue model of reentrant-arrhythmias using optogenetic cross-field stimulation. An APD-modulating optogenetic protocol was then designed to dynamically prolong APD of the propagating wavefront, completely preventing arrhythmogenesis in this model. This work highlights the potential of optogenetics in studying repolarization abnormalities and in developing novel antiarrhythmic therapies.

Literature Review and Knowledge Distribution During an Outbreak: A Methodology for Managing Infodemics.

PROBLEM: The COVID-19 pandemic has challenged health care systems in an unprecedented way by imposing new demands on health care resources and scientific knowledge. There has also been an exceedingly fast accumulation of new information on this novel virus. As the traditional peer-review process takes time, there is currently a significant gap between the ability to generate new data and the ability to critically evaluate them. This problem of an excess of mixed-quality data, or infodemic, is echoing throughout the scientific community. APPROACH: The authors aimed to help their colleagues at the Rambam Medical Center, Haifa, Israel, manage the COVID-19 infodemic with a methodologic solution: establishing an in-house mechanism for continuous literature review and knowledge distribution (March-April 2020). Their methodology included the following building blocks: a dedicated literature review team, artificial intelligence-based research algorithms, brief written updates in a graphical format, large-scale webinars and online meetings, and a feedback loop. OUTCOMES: During the first month (April 2020), the project produced 21 graphical updates. After consideration of feedback from colleagues and final editing, 13 graphical updates were uploaded to the center's website; of these, 31% addressed the clinical presentation of the disease and 38% referred to specific treatments. This methodology as well as the graphical updates it generated were adopted by the Israeli Ministry of Health and distributed in a hospital preparation kit. NEXT STEPS: The authors believe they have established a novel methodology that can assist in the battle against COVID-19 by making high-quality scientific data more accessible to clinicians. In the future, they expect this methodology to create a favorable uniform standard for evidence-guided health care during infodemics. Further evolution of the methodology may include evaluation of its long-term sustainability and impact on the day-to-day clinical practice and self-confidence of clinicians who treat COVID-19 patients.

Generating ring-shaped engineered heart tissues from ventricular and atrial human pluripotent stem cell-derived cardiomyocytes.

The functions of the heart are achieved through coordination of different cardiac cell subtypes (e.g., ventricular, atrial, conduction-tissue cardiomyocytes). Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer unique opportunities for cardiac research. Traditional studies using these cells focused on single-cells and utilized mixed cell populations. Our goal was to develop clinically-relevant engineered heart tissues (EHTs) comprised of chamber-specific hPSC-CMs. Here we show that such EHTs can be generated by directing hPSCs to differentiate into ventricular or atrial cardiomyocytes, and then embedding these cardiomyocytes in a collagen-hydrogel to create chamber-specific, ring-shaped, EHTs. The chamber-specific EHTs display distinct atrial versus ventricular phenotypes as revealed by immunostaining, gene-expression, optical assessment of action-potentials and conduction velocity, pharmacology, and mechanical force measurements. We also establish an atrial EHT-based arrhythmia model and confirm its usefulness by applying relevant pharmacological interventions. Thus, our chamber-specific EHT models can be used for cardiac disease modeling, pathophysiological studies and drug testing.

Modeling Reentry in the Short QT Syndrome With Human-Induced Pluripotent Stem Cell-Derived Cardiac Cell Sheets.

BACKGROUND: The short QT syndrome (SQTS) is an inherited arrhythmogenic syndrome characterized by abnormal ion channel function, life-threatening arrhythmias, and sudden cardiac death. OBJECTIVES: The purpose of this study was to establish a patient-specific human-induced pluripotent stem cell (hiPSC) model of the SQTS, and to provide mechanistic insights into its pathophysiology and therapy. METHODS: Patient-specific hiPSCs were generated from a symptomatic SQTS patient carrying the N588K mutation in the KCNH2 gene, differentiated into cardiomyocytes, and compared with healthy and isogenic (established by CRISPR/Cas9-based mutation correction) control hiPSC-derived cardiomyocytes (hiPSC-CMs). Patch-clamp was used to evaluate action-potential (AP) and IKr current properties at the cellular level. Conduction and arrhythmogenesis were studied at the tissue level using confluent 2-dimensional hiPSC-derived cardiac cell sheets (hiPSC-CCSs) and optical mapping. RESULTS: Intracellular recordings demonstrated shortened action-potential duration (APD) and abbreviated refractory period in the SQTS-hiPSC-CMs. Similarly, voltage- and AP-clamp recordings revealed increased IKr current density due to attenuated inactivation, primarily in the AP plateau phase. Optical mapping of the SQTS-hiPSC-CCSs revealed shortened APD, impaired APD-rate adaptation, abbreviated wavelength of excitation, and increased inducibility of sustained spiral waves. Phase-mapping analysis revealed accelerated and stabilized rotors manifested by increased rotor rotation frequency, increased rotor curvature, decreased core meandering, and increased rotor complexity. Application of quinidine and disopyramide, but not sotalol, normalized APD and suppressed arrhythmia induction. CONCLUSIONS: A novel hiPSC-based model of the SQTS was established at both the cellular and tissue levels. This model recapitulated the disease phenotype in the culture dish and provided important mechanistic insights into arrhythmia mechanisms in the SQTS and its treatment.

Human Induced Pluripotent Stem Cell-Derived Cardiac Cell Sheets Expressing Genetically Encoded Voltage Indicator for Pharmacological and Arrhythmia Studies.

Fulfilling the potential of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes for studying conduction and arrhythmogenesis requires development of multicellular models and methods for long-term repeated tissue phenotyping. We generated confluent hiPSC-derived cardiac cell sheets (hiPSC-CCSs), expressing the genetically encoded voltage indicator ArcLight. ArcLight-based optical mapping allowed generation of activation and action-potential duration (APD) maps, which were validated by mapping the same hiPSC-CCSs with the voltage-sensitive dye, Di-4-ANBDQBS. ArcLight mapping allowed long-term assessment of electrical remodeling in the hiPSC-CCSs and evaluation of drug-induced conduction slowing (carbenoxolone, lidocaine, and quinidine) and APD prolongation (quinidine and dofetilide). The latter studies also enabled step-by-step depiction of drug-induced arrhythmogenesis ("torsades de pointes in the culture dish") and its prevention by MgSO4 and rapid pacing. Phase-mapping analysis allowed biophysical characterization of spiral waves induced in the hiPSC-CCSs and their termination by electrical cardioversion and overdrive pacing. In conclusion, ArcLight mapping of hiPSC-CCSs provides a powerful tool for drug testing and arrhythmia investigation.