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.

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.

Single-Cell Mechanical Analysis of Human Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Testing and Pathophysiological Studies.

Current platforms for studying the mechanical properties of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) as single cells do not measure forces directly, require numerous assumptions, and cannot study cell mechanics at different loading conditions. We present a method for directly measuring the active and passive forces generated by single-cell hPSC-CMs at different stretch levels. Utilizing this technique, single hPSC-CMs exhibited positive length-tension relationship and appropriate inotropic, klinotropic, and lusitropic changes in response to pharmacological treatments (isoproterenol and verapamil). The unique potential of the approach for drug testing and disease modeling was exemplified by doxorubicin and omecamtiv mecarbil drug studies revealing their known actions to suppress (doxorubicin) or augment (omecamtiv mecarbil at low dose) cardiomyocyte contractility, respectively. Finally, mechanistic insights were gained regarding the cellular effects of these drugs as doxorubicin treatment led to cellular mechanical alternans and high doses of omecamtiv mecarbil suppressed contractility and worsened the cellular diastolic properties.

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.

Estimation of fetal weight using Hadlock's formulas: Is head circumference an essential parameter?

OBJECTIVES: To test the equivalence of two fetal weight estimation formulas generated by Hadlock, a formula that includes head circumference parameter (H1), and another (H2) which excludes this parameter. A secondary aim was to identify the patients in which H2 formula is less reliable to use. STUDY DESIGN: This retrospective cohort study included a total of 1220 sonographic fetal weight estimations performed within seven days of delivery and recorded at a single medical center from January 2014 to December 2016. Estimated fetal weight was calculated using H1 and H2 formulas. Their accuracies were compared using percentage error, the proportion of weight estimations falling within ±15% error interval and by Bland-Altman analysis. Multivariate regression was performed to evaluate factors affecting weight estimation by H2 formula. RESULTS: The mean birth weight was 3288.92 ±â€¯641.27gr. The H2 formula presented with statistically significant higher value of systemic mean percent error comparing to H1 (3.19% vs. 1.87%, p < 0.001 respectively). H2 formula had a lower accuracy compared to H1 in predicting fetal weight within ±15% of birth weight (90.49% vs. 93.44%, p < 0.01 respectively). Using Bland-Altman analysis, the 95% limits of agreement between both formulas were (-142.03) to 231.79gr with a mean of 44.88gr. Factors found to influence significantly on H2 formula were long femur length (OR 1.144, p < 0.0001) and low maternal age (OR 0.947, p < 0.01). CONCLUSIONS: H1formula was more accurate than H2 formula in predicting fetal weight at term. However, the accuracy difference was found to be small. Therefore, if ultrasonographic evaluation of HC is technically difficult, Hadlock formula that excludes head circumference can be used with confidence. Caution should be paid with higher values of femur length and lower maternal age.

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.

Engineered heart tissue models from hiPSC-derived cardiomyocytes and cardiac ECM for disease modeling and drug testing applications.

Cardiac tissue engineering provides unique opportunities for cardiovascular disease modeling, drug testing, and regenerative medicine applications. To recapitulate human heart tissue, we combined human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a chitosan-enhanced extracellular-matrix (ECM) hydrogel, derived from decellularized pig hearts. Ultrastructural characterization of the ECM-derived engineered heart tissues (ECM-EHTs) revealed an anisotropic muscle structure, with embedded cardiomyocytes showing more mature properties than 2D-cultured hiPSC-CMs. Force measurements confirmed typical force-length relationships, sensitivity to extracellular calcium, and adequate ionotropic responses to contractility modulators. By combining genetically-encoded calcium and voltage indicators with laser-confocal microscopy and optical mapping, the electrophysiological and calcium-handling properties of the ECM-EHTs could be studied at the cellular and tissue resolutions. This allowed to detect drug-induced changes in contraction rate (isoproterenol, carbamylcholine), optical signal morphology (E-4031, ATX2, isoproterenol, ouabin and quinidine), cellular arrhythmogenicity (E-4031 and ouabin) and alterations in tissue conduction properties (lidocaine, carbenoxolone and quinidine). Similar assays in ECM-EHTs derived from patient-specific hiPSC-CMs recapitulated the abnormal phenotype of the long QT syndrome and catecholaminergic polymorphic ventricular tachycardia. Finally, programmed electrical stimulation and drug-induced pro-arrhythmia led to the development of reentrant arrhythmias in the ECM-EHTs. In conclusion, a novel ECM-EHT model was established, which can be subjected to high-resolution long-term serial functional phenotyping, with important implications for cardiac disease modeling, drug testing and precision medicine. STATEMENT OF SIGNIFICANCE: One of the main objectives of cardiac tissue engineering is to create an in-vitro muscle tissue surrogate of human heart tissue. To this end, we combined a chitosan-enforced cardiac-specific ECM hydrogel derived from decellularized pig hearts with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from healthy-controls and patients with inherited cardiac disorders. We then utilized genetically-encoded calcium and voltage fluorescent indicators coupled with unique optical imaging techniques and force-measurements to study the functional properties of the generated engineered heart tissues (EHTs). These studies demonstrate the unique potential of the new model for physiological and pathophysiological studies (assessing contractility, conduction and reentrant arrhythmias), novel disease modeling strategies ("disease-in-a-dish" approach) for studying inherited arrhythmogenic disorders, and for drug testing applications (safety pharmacology).

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.

Monitoring Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes with Genetically Encoded Calcium and Voltage Fluorescent Reporters.

The advent of the human-induced pluripotent stem cell (hiPSC) technology has transformed biomedical research, providing new tools for human disease modeling, drug development, and regenerative medicine. To fulfill its unique potential in the cardiovascular field, efficient methods should be developed for high-resolution, large-scale, long-term, and serial functional cellular phenotyping of hiPSC-derived cardiomyocytes (hiPSC-CMs). To achieve this goal, we combined the hiPSC technology with genetically encoded voltage (ArcLight) and calcium (GCaMP5G) fluorescent indicators. Expression of ArcLight and GCaMP5G in hiPSC-CMs permitted to reliably follow changes in transmembrane potential and intracellular calcium levels, respectively. This allowed monitoring short- and long-term changes in action-potential and calcium-handling properties and the development of arrhythmias in response to several pharmaceutical agents and in hiPSC-CMs derived from patients with different inherited arrhythmogenic syndromes. Combining genetically encoded fluorescent reporters with hiPSC-CMs may bring a unique value to the study of inherited disorders, developmental biology, and drug development and testing.