Modulating cardiac physiology in engineered heart tissue with the bidirectional optogenetic tool BiPOLES.

Optogenetic actuators are rapidly advancing tools used to control physiology in excitable cells, such as neurons and cardiomyocytes. In neuroscience, these tools have been used to either excite or inhibit neuronal activity. Cell type-targeted actuators have allowed to study the function of distinct cell populations. Whereas the first described cation channelrhodopsins allowed to excite specific neuronal cell populations, anion channelrhodopsins were used to inhibit neuronal activity. To allow for simultaneous excitation and inhibition, opsin combinations with low spectral overlap were introduced. BiPOLES (Bidirectional Pair of Opsins for Light-induced Excitation and Silencing) is a bidirectional optogenetic tool consisting of the anion channel Guillardia theta anion-conducting channelrhodopsin 2 (GtACR2 with a blue excitation spectrum and the red-shifted cation channel Chrimson. Here, we studied the effects of BiPOLES activation in cardiomyocytes. For this, we knocked in BiPOLES into the adeno-associated virus integration site 1 (AAVS1) locus of human-induced pluripotent stem cells (hiPSC), subjected these to cardiac differentiation, and generated BiPOLES expressing engineered heart tissue (EHT) for physiological characterization. Continuous light application activating either GtACR2 or Chrimson resulted in cardiomyocyte depolarization and thus stopped EHT contractility. In contrast, short light pulses, with red as well as with blue light, triggered action potentials (AP) up to a rate of 240 bpm. In summary, we demonstrate that cation, as well as anion channelrhodopsins, can be used to activate stem cell-derived cardiomyocytes with pulsed photostimulation but also to silence cardiac contractility with prolonged photostimulation.

Impulse initiation in engrafted pluripotent stem cell-derived cardiomyocytes can stimulate the recipient heart.

Transplantation of pluripotent stem cell-derived cardiomyocytes is a novel promising cell-based therapeutic approach for patients with heart failure. However, engraftment arrhythmias are a predictable life-threatening complication and represent a major hurdle for clinical translation. Thus, we wanted to experimentally study whether impulse generation by transplanted cardiomyocytes can propagate to the host myocardium and overdrive the recipient rhythm. We transplanted human induced pluripotent stem cell-derived cardiomyocytes expressing the optogenetic actuator Bidirectional Pair of Opsins for Light-induced Excitation and Silencing (BiPOLES) in a guinea pig injury model. Eight weeks after transplantation ex vivo, Langendorff perfusion was used to assess electrical coupling. Pulsed photostimulation was applied to specifically activate the engrafted cardiomyocytes. Photostimulation resulted in ectopic pacemaking that propagated to the host myocardium, caused non-sustained arrhythmia, and stimulated the recipient heart with higher pacing frequency (4/9 hearts). Our study demonstrates that transplanted cardiomyocytes can (1) electrically couple to the host myocardium and (2) stimulate the recipient heart. Thus, our results provide experimental evidence for an important aspect of engraftment-induced arrhythmia induction and thereby support the current hypothesis that cardiomyocyte automaticity can serve as a trigger for ventricular arrhythmias.

Using a qPCR device to screen for modulators of ABC transporter activity: A step-by-step protocol.

INTRODUCTION: Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are transmembrane proteins which actively transport a large variety of substrates across biological membranes. ABC transporter overexpression can be the underlying cause of multidrug resistance in oncology. Moreover, it has been revealed that increased ABCC1 transporter activity can ameliorate behavioural changes and Aß pathology in a rodent model of Alzheimer's disease and it is currently tested in AD patients. METHODS: Finding substances that modulate ABC transporter activity (inhibitors and activators) is of high relevance and thus, different methods have been developed to screen for potential modulators. For this purpose, we have developed a cell-based assay to measure the kinetics of ABCC1-mediated efflux of a fluorescent dye using a common qPCR device (Agilent AriaMx). RESULTS: We validated the specificity of our method with vanadate and benzbromarone controls. Furthermore, we provide a step-by-step protocol including statistical analysis of the resulting data and suggestions how to modify the protocol specifically to screen for activators of ABCC1. DISCUSSION: Our approach is biologically more relevant than cell-free assays. The continuous detection of kinetics allows for a more precise quantification compared with assays with single end-point measurements.