Biology subjects

Entcheva, E.

Publications and source records attributed to Entcheva, E..

24 records · Page 2Linked to original sources

bioRxiv2019

Syncytium cell growth increases IK1 contribution in human iPS-cardiomyocytes

Human induced pluripotent stem-cell-derived cardiomyocytes (hiPS-CMs) enable cardiotoxicity testing and personalized medicine. However, their maturity is of concern, including relatively depolarized resting membrane potential and more spontaneous activity compared to adult cardiomyocytes, implicating low or lacking inward-rectifier potassium current (Ik1). Here, protein quantification confirms Ik1 expression in hiPS-CM syncytia, albeit several times lower than in adult heart tissue. We find that hiPS-CM cell culture density influences Ik1 expression and the associated electrophysiology phenotype. All-optical cardiac electrophysiology and pharmacological treatments reveal reduction of spontaneous and irregular activity in denser cultures. Blocking Ik1 with BaCl2 increased spontaneous frequency and blunted action potential upstrokes during pacing in a dose-dependent manner only in the highest-density cultures, in line with Ik1s role in regulating the resting membrane potential. Our results emphasize the importance of syncytial growth of hiPS-CM for more physiologically-relevant phenotype and the power of all-optical electrophysiology to study cardiomyocytes in their multicellular setting.

bioengineering↗

bioRxiv2019

All-optical electrophysiology refines populations of in silico human iPS-CMs for drug evaluation

High-throughput in vitro drug assays have been impacted by recent advances in human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) technology and by contact-free all-optical systems simultaneously measuring action potential (AP) and Ca2+ transient (CaTr). Parallel computational advances have shown that in silico models can predict drug effects with high accuracy. In this work, we combine these in vitro and in silico technologies and demonstrate the utility of high-throughput experimental data to refine in silico hiPS-CM populations, and to predict and explain drug action mechanisms. Optically-obtained hiPS-CM AP and CaTr were used from spontaneous activity and under pacing in control and drug conditions at multiple doses. An updated version of the Paci2018 model was developed to refine the description of hiPS-CM spontaneous electrical activity; a population of in silico hiPS-CMs was constructed and calibrated using the optically-recorded AP and CaTr. We tested five drugs (astemizole, dofetilide, ibutilide, bepridil and diltiazem), and compared simulations against in vitro optical recordings. Our simulations showed that physiologically-accurate population of models can be obtained by integrating AP and CaTr control records. Thus constructed population of models predicted correctly the drug effects and occurrence of adverse episodes, even though the population was optimized only based on control data and in vitro drug testing data were not deployed during its calibration. Furthermore, the in silico investigation yielded mechanistic insights, e.g. through simulations, bepridils more pro-arrhythmic action in adult cardiomyocytes compared to hiPS-CMs could be traced to the different expression of ion currents in the two. Therefore, our work: i) supports the utility of all-optical electrophysiology in providing high-content data to refine experimentally-calibrated populations of in silico hiPS-CMs, ii) offers insights into certain limitations when translating results obtained in hiPS-CMs to humans and shows the strength of combining high-throughput in vitro and population in silico approaches. SignificanceWe demonstrate the integration of human in silico drug trials and optically-recorded simultaneous action potential and calcium transient data from human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) for prediction and mechanistic investigations of drug action. We propose a population of in silico models i) based on a new hiPS-CM model recapitulating the mechanisms underlying hiPS-CM automaticity and ii) calibrated with all-optical measurements. We used our in silico population to predict and evaluate the effects of 5 drugs and the underlying biophysical mechanisms, obtaining results in agreement with our experiments and one independent dataset. This work supports the use of high-content, high-quality all-optical electrophysiology data to develop, calibrate and validate computer models of hiPS-CM for in silico drug trials.

pharmacology and toxicology↗

bioRxiv2019

Optical interrogation of sympathetic neuronal effects on macroscopic cardiac monolayer dynamics

Alterations in autonomic function are known to occur in cardiac conditions including sudden cardiac death. Cardiac stimulation via sympathetic neurons can potentially trigger arrhythmias. Dissecting direct neural-cardiac interactions at the cellular level is technically challenging and understudied due to the lack of experimental model systems and methodologies. Here we demonstrate the utility of optical interrogation of sympathetic neurons and their effects on macroscopic cardiac monolayer dynamics to address research targets such as the effects of adrenergic stimulation via the release of neurotransmitters, the effect of neuronal numbers on cardiac wave behaviour and the applicability of optogenetics in mechanistic in vitro studies. We combine photo-uncaging or optogenetic neural stimulation with imaging of cardiac monolayers to measure electrical activity in an automated fashion, illustrating the power and high throughput capability of such interrogations. The methods described highlight the challenges and benefits of co-cultures as experimental model systems.

biophysics↗

bioRxiv2018

Multimodal on-axis platform for all-optical electrophysiology with near-infrared probes in human stem-cell-derived cardiomyocytes

Combined optogenetic stimulation and optical imaging permits scalable, high-throughput probing of cellular electrophysiology and optimization of stem-cell derived excitable cells, such as neurons and muscle cells. We report a new \"on-axis\" configuration of OptoDyCE, our all-optical platform for studying human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) and other cell types, optically driven by Channelrhodopsin2 (ChR2). This solid-state system integrates optogenetic stimulation with temporally-multiplexed simultaneous recording of membrane voltage (Vm) and intracellular calcium ([Ca2+]i) dynamics using a single photodetector. We demonstrate the capacity for combining multiple spectrally-compatible actuators and sensors, including newer high-performance near-infrared (NIR) voltage probes BeRST1 and Di-4-ANBDQBS, to record complex spatiotemporal responses of hiPSC-CMs to drugs in a high-throughput manner.

bioengineering↗

bioRxiv2017

Adeno-associated virus mediated gene delivery: Implications for scalable in vitro and in vivo cardiac optogenetic models

AimsAdeno-associated viruses (AAVs) provide advantages in long-term, cardiac-specific gene expression. However, AAV serotype specificity data is lacking in cardiac models relevant to optogenetics. We aimed to identify the optimal AAV serotype (1, 6, or 9) in pursuit of scalable rodent and human models for cardiac optogenetics and elucidate the mechanism of virus uptake.\n\nMethodsIn vitro syncytia of primary neonatal rat ventricular cardiomyocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were infected with AAVs 1, 6, and 9 containing the transgene for eGFP or channelrhodopsin-2 (ChR2) fused to mCherry. In vivo adult rats were intravenously injected with AAV1 and 9 containing ChR2-mCherry.\n\nResultsTransgene expression profiles of rat and human cells in vitro revealed that AAV1 and 6 significantly outperformed AAV9. In contrast, systemic delivery of AAV9 in adult rat hearts yielded significantly higher levels of ChR2-mCherry expression and optogenetic responsiveness. We tracked the mechanism of virus uptake to purported receptor-mediators for AAV 1/6 (cell surface sialic acid) and AAV9(37/67kDa laminin receptor, LamR). In vitro desialylation of NRVMs and hiPSC-CMs with neuraminidase significantly decreased AAV1,6-mediated gene expression, but interestingly, desialylation of hiPSC-CMs increased AAV9-mediated expression. In fact, only very high viral doses of AAV9-ChR2-mCherry, combined with neuraminidase treatment yielded consistent optogenetic responsiveness in hiPSC-CMs. Differences between the in vitro and in vivo performance of AAV9 could be correlated to robust LamR expression in the adult and neonatal rat hearts, but no expression in vitro in cultured cells. The dynamic nature of LamR expression and its dependence on environmental factors was further corroborated in intact adult human ventricular tissue slices.\n\nConclusionThe combined transgene expression and cell surface receptor data may explain the preferential efficiency of AAV1/6 in vitro and AAV9 in vivo for cardiac delivery and mechanistic knowledge of their action can help guide cardiac optogenetic efforts.

bioengineering↗

bioRxiv2017

OptoGap: an optogenetics-enabled assay for quantification of cell-cell coupling in multicellular cardiac tissue

Intercellular electrical coupling is an essential means of communication between cells. It is important to obtain quantitative knowledge of such coupling between cardiomyocytes and nonexcitable cells when, for example, pathological electrical coupling between myofibroblasts and cardiomyocytes yields increased arrhythmia risk or during the integration of donor (e.g. cardiac progenitor) cells with native cardiomyocytes in cell-therapy approaches. Currently, there is no direct method for assessing heterocellular coupling within multicellular tissue. Here we demonstrate experimentally and computationally a new contactless assay for electrical coupling, OptoGap, based on selective illumination of inexcitable cells that express optogenetic actuators and optical sensing of the response of coupled excitable cells, e.g. cardiomyocytes, that are light-insensitive. Cell-cell coupling is quantified by the energy required to elicit an action potential via junctional current from the light-stimulated cell(s). The proposed technique is experimentally validated against the standard indirect approach, GapFRAP, using light-sensitive cardiac fibroblasts and non-transformed cardiomyocytes in a two-dimensional setting. Its potential applicability to the complex three-dimensional setting of the native heart is corroborated by computational modeling and proper calibration.\n\nIntercellular coupling is a fundamental form of communication between cells, essential for the synchronization of physiological processes in different organs. Pathologically altered coupling or the emergence of de novo coupling between native and donor cells are problems of interest in many cardiac applications, e.g. during cell delivery and cell integration for cardiac repair therapy1,2. In particular, interactions between cardiomyocytes and fibroblasts are of interest, especially the pro-arrhythmic increase in coupling as the latter transition to myofibroblasts3-6.\n\nElectrical coupling in cardiac tissue is mediated primarily by low-resistance paths formed by gap-junctional proteins (connexins), that can link cardiomyocytes (CMs) to each other and to non-cardiomyocytes (nCMs), such as fibroblasts. Qualitative and quantitative methods, e.g. immunofluorescence, messenger RNA and Western blots, are often used to assay connexin expression levels as a surrogate measure of coupling, but they do not provide functional information. A method for direct quantification of cell-cell coupling within the multicellular tissue context is highly desirable.

bioengineering↗