Conduction of excitation waves and reentry drift on cardiac tissue with simulated photocontrol-varied excitability.

The development of new approaches to suppressing cardiac arrhythmias requires a deep understanding of spiral wave dynamics. The study of spiral waves is possible in model systems, for example, in a monolayer of cardiomyocytes. A promising way to control cardiac excitability in vitro is the noninvasive photocontrol of cell excitability mediated by light-sensitive azobenzene derivatives, such as azobenzene trimethylammonium bromide (AzoTAB). The trans-isomer of AzoTAB suppresses spontaneous activity and excitation propagation speed, whereas the cis isomer has no detectable effect on the electrical properties of cardiomyocyte monolayers; cis isomerization occurs under the action of near ultraviolet (UV) light, and reverse isomerization occurs when exposed to blue light. Thus, AzoTAB makes it possible to create patterns of excitability in conductive tissue. Here, we investigate the effect of a simulated excitability gradient in cardiac cell culture on the behavior and termination of reentry waves. Experimental data indicate a displacement of the reentry wave, predominantly in the direction of lower excitability. However, both shifts in the direction of higher excitability and shift absence were also observed. To explain this effect, we reproduced these experiments in a computer model. Computer simulations showed that the explanation of the mechanism of observed drift to a lower excitability area requires not only a change in excitability coefficients (ion currents) but also a change in the diffusion coefficient; this may be the effect of the substance on intercellular connections. In addition, it was found that the drift direction depended on the observation time due to the meandering of the spiral wave. Thus, we experimentally proved the possibility of noninvasive photocontrol and termination of spiral waves with a mechanistic explanation in computer models.

Cyclophosphamide arrhythmogenicitytesting using human-induced pluripotent stem cell-derived cardiomyocytes.

Cyclophosphamide (CP) is an anticancer drug, an alkylating agent. Cardiotoxicity of CP is associated with one of its metabolites, acrolein, and clinical cardiotoxicity manifestations are described for cases of taking CP in high doses. Nevertheless, modern arrhythmogenicity prediction assays in vitro include evaluation of beat rhythm and rate as well as suppression of cardiac late markers after acute exposure to CP, but not its metabolites. The mechanism of CP side effects when taken at low doses (i.e., < 100 mg/kg), especially at the cellular level, remains unclear. In this study conduction properties and cytoskeleton structure of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) obtained from a healthy donor under CP were evaluated. Arrhythmogenicity testing including characterization of 3 values: conduction velocity, maximum capture rate (MCR) measurements and number of occasions of re-entry on a standard linear obstacle was conducted and revealed MCR decrease of 25% ± 7% under CP. Also, conductivity area reduced by 34 ± 15%. No effect of CP on voltage-gated ion channels was found. Conduction changes (MCR and conductivity area decrease) are caused by exposure time-dependent alpha-actinin disruption detected both in hiPSC-CMs and neonatal ventricular cardiomyocytes in vitro. Deviation from the external stimulus frequency and appearance of non-conductive areas in cardiac tissue under CP is potentially arrhythmogenic and could develop arrhythmic effects in vivo.

Formation of an electrical coupling between differentiating cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) serve as an indispensable platform for the study of human cardiovascular disease is human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). While the possibility of reproducing rare pathologies, patient-specific selection of drugs, and other issues concerning single cardiomyocytes have been well studied, little attention has been paid to the properties of the whole syncytium of CMs, in which both the functionality of individual cells and the distribution of electrophysiological connections between them are essential. The aim of this work is to directly study the ability of hiPSC-CMs to form a functional syncytium that can stably conduct an excitation wave. For that purpose, syncytium forming hiPSC-CMs were harvested and seeded (transferred) on a new substrate on different days of differentiation. The excitation conduction in a sample was characterized by the stability of the wavefront using optical mapping data. We found that the cells transferred before the 20th day of differentiation were able to organize a functional syncytium capable of further development and stable excitation conduction at high stimulation frequencies, while the cells transferred after 20 days did not form a homogeneous syncytium, and multiple instabilities of the propagating wavefront were observed with the possibility of reentry formation.

The Use of iPSC-Derived Cardiomyocytes and Optical Mapping for Erythromycin Arrhythmogenicity Testing.

Erythromycin is an antibiotic that prolongs the QT-interval and causes Torsade de Pointes (TdP) by blocking the rapid delayed rectifying potassium current (IKr) without affecting either the slow delayed rectifying potassium current (IKs) or inward rectifying potassium current (IK1). Erythromycin exerts this effect in the range of 1.5-100 µM. However, the mechanism of action underlying its cardiotoxic effect and its role in the induction of arrhythmias, especially in multicellular cardiac experimental models, remain unclear. In this study, the re-entry formation, conduction velocity, and maximum capture rate were investigated in a monolayer of human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes from a healthy donor and in a neonatal rat ventricular myocyte (NRVM) monolayer using the optical mapping method under erythromycin concentrations of 15, 30, and 45 µM. In the monolayer of human iPSC-derived cardiomyocytes, the conduction velocity (CV) varied up to 12 ± 9% at concentrations of 15-45 µM as compared with that of the control, whereas the maximum capture rate (MCR) declined substantially up to 28 ± 12% (p < 0.01). In contrast, the tests on the NRVM monolayer showed no significant effect on the MCR. The results of the arrhythmogenicity test provided evidence for a "window" of concentrations of the drug (15-30 µM) at which the probability of re-entry increased.

Applying Patient-Specific Induced Pluripotent Stem Cells to Create a Model of Hypertrophic Cardiomyopathy.

Generation of patient-specific induced pluripotent stem cells (iPSCs) and their subsequent differentiation into cardiomyocytes opened new opportunities for studying pathogenesis of inherited cardiovascular diseases. One of these diseases is hypertrophic cardiomyopathy (HCM) for which no efficient therapy methods have been developed so far. In this study, the approach based on patient-specific iPSCs was applied to create a model of the disease. Genetic analysis of a hypertrophic cardiomyopathy patient revealed R326Q mutation in the MYBPC3 gene. iPSCs of the patient were generated and characterized. The cells were differentiated into cardiomyocytes together with the control iPSCs from a healthy donor. The patient's iPSC-derived cardiomyocytes exhibited early HCM features, such as abnormal calcium handling and increased intracellular calcium concentration. Therefore, cardiomyocytes obtained by directed differentiation of iPSCs from the HCM patient can be used as a model system to study HCM pathogenesis.