Biology subjects

Entcheva, E.

Publications and source records attributed to Entcheva, E..

At least 19 recordsLinked to original sources

bioRxiv2026

Optogenetic and sonogenetic pacing of unmodified cardiac tissue upon quick integration with specialized biological actuators

Electrical stimulation for cardiac pacing has been in clinical use for 100 years and it is the current standard. Alternative scalable pacing methods that are contactless, with good spatial precision and can also be cell-selective are of interest for both in vitro and in vivo applications. Cardiac pacing by light or by ultrasound (US) can be achieved using optogenetic transduction of the tissue and by enhancing responsiveness to ultrasound. In this work we create specialized biological actuators (cell spheroids) out of generic human cells, modified optogenetically (using Channelrhodopsin2) and sonogenetically (using human Piezo1), guide them to a desired location to promote quick integration with unmodified cardiac syncytia within less than two hours. We quantify their ability to confer pacing by light or US and to entrain the native cardiac tissue, modeled here with human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) grown in monolayers, quasi-3D oriented microtissues or cell spheroids. Magnetic guidance (via magnetic nanoparticles) speeds up integration and facilitates positioning to optimize electrotonic load for pacing; size match and three-dimensional growth also accelerate integration. Computational modeling corroborates the possibility to pace by light and by US. Using optogenetically-gated US pulses (0.5 MHz), we show entrainment in voltage and calcium responses and identify the best performing configurations. Experiments reveal the potential role of Piezo1, esp. when activated with Yoda1. The approach is shown to work with physiologically relevant heart cells, such as human cardiac fibroblasts as optimized actuators, and therefore can be considered for in vivo applications as part of heart regeneration efforts. High-throughput cardiotoxicity testing and drug screening in vitro can directly benefit from deployment of such biological actuators.

bioengineering↗

bioRxiv2026

Reinforcement learning discovers new mechanisms of reentry in excitable media

The transition from transient excitation to sustained reentry is a fundamental problem in the physics of excitable media. In cardiac tissue, reentry underlies many life-threatening cardiac arrhythmias, yet the pathway to initiation of reentry remains incompletely understood. Here, we formulate reentry initiation as a reinforcement-learning problem in which an agent applies sequences of spatial stimulation patterns while being rewarded for sustained activity and penalized according to the number of stimuli applied. Using cellular automata in one-, two-, and three-dimensional geometries, the agent discovered several mechanisms for generating unidirectional propagation and reentry. These included a previously described mechanism combining superthreshold and subthreshold stimulation, as well as two new mechanisms based entirely on subthreshold stimuli: a sequential mechanism involving stimuli delivered at different locations and times, and a spatial mechanism in which several individually subthreshold sites collectively initiated reentry. In geometries containing boundaries and branches, the learned protocols additionally exploited structural source-sink asymmetries. Optogenetic experiments in cardiac monolayers further demonstrated reproducible induction of unidirectional propagation using the learned spatial subthreshold patterns, while whole-heart experiments provided preliminary evidence that such patterns can shape early propagation in intact tissue. More broadly, we show that reinforcement learning provides a general framework for discovering mechanisms of reentry in arbitrary geometries and generating testable hypotheses about reentry initiation in excitable systems.

physiology↗

bioRxiv2026

Comparative transcriptomics analysis of histone deacetylases, transcription factors, and ion channel genes in human iPSC-cardiomyocytes vs. the adult human heart

Epigenetic modulators such as histone deacetylases (HDACs) and histone acetyltransferases (HATs) are known master regulators of gene expression that substantially impact cardiac electrophysiology. Novel pharmacological agents, HDAC inhibitors, are rapidly emerging as treatments for cancer and immune diseases, and their effects on cardiac ion channels (ICs) are of great interest. We used small interfering RNAs to individually suppress each of the known HDACs, including sirtuins (SIRTs), in human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs), iCell2. Follow-up deep-sequencing allowed comparison to identically processed and normalized RNA sequencing data from adult human left ventricle (LV) from the GTEx database. The transcriptomics analysis revealed high similarity of gene expression patterns for cardiac ICs (with some differences in calcium influx and calcium buffering related genes), as well as strong co-regulation by cardiac transcription factors (TFs) and HDACs/SIRTs in both hiPSC-CMs and the adult LV. Partial least square regression models helped visualize links between HDACs/HATs, TFs, and cardiac ICs and helped identify potential key regulators of cardiac IC transcription. Powerful TFs, including MEF2A, GATA4, 6 exerted positive effect on IC genes while RUNX1 and SHMT2 were distinct negative regulators in both sample types; TRIM28 was found to serve opposite roles in the two sample types. In functional measurements, HDAC suppression primarily increased excitability, while SIRT suppression decreased excitability, in line with transcriptomic links. Our analysis offers insights about the role of epigenetic modifiers in regulating cardiac electrophysiology and informs the utility of hiPSC-CM as a scalable experimental model for cardiotoxicity testing of HDAC inhibitors.

bioengineering↗

bioRxiv2025

Phase resetting in human stem cell derived cardiomyocytes explains complex cardiac arrhythmias

Phase resetting of cardiac oscillators underlies some complex arrhythmias. Here we use optogenetic stimulation to construct phase response curves (PRC) for spheroids of human induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) and a computational cardiomyocyte model to identify ionic mechanisms shaping the PRC. The clinical utility of the human PRCs is demonstrated by adding a patient-based conduction delay to the same equations to explain complex multi-day Holter ECG dynamics and cardiac arrhythmias. Periodic stimulation of these patient-based models and the computational model of human iPSC-CM reveal similar bifurcation patterns and entrainment zones. Cell therapy by injecting iPSC-CM into diseased hearts can induce ectopic foci-based engraftment arrhythmias. The PRC analysis offers a potential strategy to entrain these foci in a parameter space that avoids such arrhythmias.

physiology↗

bioRxiv2025

Response of human iPSC-cardiomyocytes to adrenergic drugs assessed by high-throughput pericellular oxygen measurements

Rate- and contractility-modulating drugs, such as adrenergic agonists and antagonists, are widely used in the treatment of cardiovascular conditions. Preclinical assessment of new modulators of rate, inotropy and metabolism can be aided by high-throughput (HT) methods for chronic measurements, coupled with scalable human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we evaluate the utility of long-term optical (label-free) measurements of pericellular oxygen in a HT format (96-well plates) for the assessment of the effectiveness of adrenergic drugs in hiPSC-CMs. Quantitative oxygen consumption metrics were derived and correlated to measurements performed in the same samples using all-optical electrophysiology. Adrenergic agonists significantly increased oxygen consumption rate (OCR), best seen in the kinetics of initial depletion of pericellular oxygen, i.e. time to reach 5%. Adrenergic antagonists decreased OCR, best quantified using steady-state values for pericellular oxygen after at least 5 hours. OCR-based drug type identification correlated well with the acute spontaneous rate measurements in the same samples. Direct rate modulation with chronic optogenetic pacing sped up OCR in hiPSC-CMs. Blebbistatin, an excitation-contraction uncoupler, significantly reduced OCR. Computational modeling helped interpret our results by capturing the effects of pacing rate, adrenergic stimulation, and blebbistatin on oxygen consumption, thereby highlighting the key contribution of inotropy and mechanical contraction to OCR in hiPSC-CMs. We conclude that HT label-free optical oxygen measurements and the comprehensive in silico hiPSC-CM models, constrained by such measurements, represent valuable human-based approaches for non-invasive assessment of rate- and metabolism-modulating drugs in preclinical studies.

bioengineering↗

bioRxiv2025

Pericellular oxygen dynamics in human cardiac fibroblasts and iPSC-cardiomyocytes in high-throughput plates: insights from experiments and modeling

Adequate oxygen supply is crucial for proper cellular function. The emergence of high-throughput (HT) expansion of human stem-cell-derived cells and HT in vitro cellular assays for drug testing necessitate monitoring and understanding of the oxygenation conditions, yet virtually no data exists for such settings. For metabolically active cells like cardiomyocytes, variations in oxygenation may significantly impact their maturation and function; conversely, electromechanical activity can drive oxygen demands. We used HT label-free optical measurements and computational modeling to gain insights about oxygen availability (peri-cellular oxygen dynamics) in syncytia of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) and human cardiac fibroblasts (cFB) grown in glass-bottom 96-well plates under static conditions. Our experimental results highlight the critical role of cell density and solution height (oxygen delivery path) in peri-cellular oxygen dynamics. The developed 3D reaction-diffusion model with Michaelis-Menten kinetics, trained on the obtained comprehensive data set, revealed that time-variant maximum oxygen consumption rate, Vmax, is needed to faithfully capture the complex peri-cellular oxygen dynamics in the excitable hiPSC-CMs, but not in the cFB. For the latter, accounting for cell proliferation was needed. Interestingly, we found both hypoxic (< 2%) and hyperoxic (> 7%) conditions can easily emerge in these standard HT plates in static culture and that peri-cellular oxygen dynamics evolves with days in culture. Our results and the developed computational model can directly be used to optimize cardiac cell growth in HT plates to achieve desired physiological conditions, important in cellular assays for cardiotoxicity, drug development, personalized medicine and heart regeneration applications.

bioengineering↗

bioRxiv2024

Control of electromechanical waves in engineered tissue of human iPSC-cardiomyocytes using a Halbach array and magnetic nanoparticles

The Halbach array, originally developed for particle accelerators, is a compact arrangement of permanent magnets to create well-defined magnetic fields without heating. Here, we demonstrate its use for modulating the speed of electromechanical waves in cardiac syncytia of human stem cell-derived cardiomyocytes. At 40-50 mT magnetic field strength, a cylindrical dipolar Halbach array boosted the conduction velocity, CV, of excitation in a directional manner by up to 25% when the magnetic field was co-aligned with the electromechanical wave (but not when perpendicular to it). To observe the effects, a short-term incubation of the cardiac cell constructs with non-targeted magnetic nanoparticles, mNPs, was sufficient. This increased CV anisotropy, and the effects were most pronounced at slower pacing rates. Instantaneous formation and re-arrangement of elongated mNP clusters upon magnetic field rotation was seen, thus creating dynamic structural anisotropy that may have contributed to the directional CV effects. This approach may be useful for anti-arrhythmic control of cardiac waves. One sentence summaryA Halbach array of permanent magnets can modulate the speed of excitation waves in human cardiac cell assemblies with magnetic nanoparticles.

bioengineering↗

bioRxiv2023

OptoDyCE-plate as an affordable high throughput imager for all optical cardiac electrophysiology

We present a simple low-cost system for comprehensive functional characterization of cardiac function under spontaneous and paced conditions, in standard 96 and 384-well plates. This full-plate actuator/imager, OptoDyCE-plate, uses optogenetic stimulation and optical readouts of voltage and calcium from all wells in parallel. The system is validated with syncytia of human induced pluripotent stem cell derived cardiomyocytes, iPSC-CMs, grown as monolayers, or in quasi-3D isotropic and anisotropic constructs using electrospun matrices, in 96 and 394-well format. Genetic modifications, e.g. interference CRISPR (CRISPRi), and nine compounds of acute and chronic action were tested, including five histone deacetylase inhibitors (HDACis). Their effects on voltage and calcium were compared across growth conditions and pacing rates. We also demonstrated deployment of optogenetic cell spheroids for point pacing to study conduction in 96-well format, and the use of temporal multiplexing to register voltage and calcium simultaneously on a single camera in this stand-alone platform. Opto-DyCE-plate showed excellent performance even in the small samples in 384-well plates, in the various configurations. Anisotropic structured constructs may provide some benefits in drug testing, although drug responses were consistent across tested configurations. Differential voltage vs. calcium responses were seen for some drugs, especially for non-traditional modulators of cardiac function, e.g. HDACi, and pacing rate was a powerful modulator of drug response, highlighting the need for comprehensive multiparametric assessment, as offered by OptoDyCE-plate. Increasing throughput and speed and reducing cost of screening can help stratify potential compounds early in the drug development process and accelerate the development of safer drugs.

bioengineering↗

bioRxiv2023

Paradoxical anti-arrhythmic effects of Zim3, a CRISPRi effector, in human iPSC-cardiomyocytes

We show that Zim3, when used as Zim3-KRAB-dCas9 effector in interference CRISPR, without any guide RNAs, paradoxically upregulates key cardiac ion channel genes in human induced pluripotent stem-cell-derived cardiomyocytes, iPSC-CMs, responsible for healthy resting membrane potential, repolarization of the action potential and electrical transmission of signals. These were found to yield expected functional enhancements consistent with a more mature iPSC-CM phenotype, with potentially desirable properties.

molecular biology↗

bioRxiv2023

CRISPRi Gene Modulation and All-Optical Electrophysiology in Post-Differentiated Human iPSC-Cardiomyocytes

Uncovering gene-phenotype relationships can be enabled by precise gene modulation in human induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs) and follow up phenotyping using scalable all- optical electrophysiology platforms. Such efforts towards human functional genomics can be aided by recent CRISPR-derived technologies for reversible gene inhibition or activation (CRISPRi/a). We set out to characterize the performance of CRISPRi in post-differentiated iPSC-CMs, targeting key cardiac ion channel genes, KCNH2, KCNJ2, and GJA1, and providing a multiparametric quantification of the effects on cardiac repolarization, stability of the resting membrane potential and conduction properties using all- optical tools. More potent CRISPRi effectors, e.g. Zim3, and optimized viral delivery led to improved performance on par with the use of CRISPRi iPSC lines. Confirmed mild yet specific phenotype changes when CRISPRi is deployed in non-dividing differentiated heart cells is an important step towards more holistic pre-clinical cardiotoxicity testing and for future therapeutic use in vivo.

cell biology↗

bioRxiv2023

High-throughput optical sensing of peri-cellular oxygen in cardiac cells: system characterization, calibration, and testing

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a scalable experimental model relevant to human physiology. Oxygen consumption of hiPSC-CMs has not been studied in high-throughput (HT) format plates used in pre-clinical studies. Here, we provide comprehensive characterization and validation of a system for HT long-term optical measurements of peri-cellular oxygen in cardiac syncytia (human iPSC-CM and human cardiac fibroblasts), grown in glass-bottom 96-well plates. Laser-cut oxygen sensors having a ruthenium dye and an oxygen-insensitive reference dye were used. Ratiometric measurements (409nm excitation) reflected dynamic changes in oxygen, as validated with simultaneous Clark electrode measurements. Emission ratios (653nm vs. 510nm) were calibrated for percent oxygen using two-point calibration. Time-dependent changes in the Stern-Volmer parameter, Ksv, were observed during the initial 40 min of incubation, likely temperature-related. Effects of pH on oxygen measurements were negligible in the pH range of 4 to 8, with a small ratio reduction for pH>10. Time-dependent calibration was implemented, and light exposure time was optimized (0.6 to 0.8s) for oxygen measurements inside an incubator. Peri-cellular oxygen dropped to levels < 5% within 3 -10 hours for densely-plated hiPSC-CMs in glass-bottom 96-well plates. After the initial oxygen decrease, samples either settled to low steady-state or exhibited intermittent peri-cellular oxygen dynamics. Cardiac fibroblasts showed slower oxygen depletion and higher steady-state levels without oscillations, compared to hiPSC-CMs. Overall, the system has great utility for long-term HT monitoring of peri-cellular oxygen dynamics in vitro for tracking cellular oxygen consumption, metabolic perturbations, and characterization of the maturation of hiPSC-CMs.

bioengineering↗

bioRxiv2022

Simultaneous widefield voltage and interferometric dye-free optical mapping quantifies electromechanical waves in human iPSC-cardiomyocytes.

Coupled electro-mechanical waves define hearts function in health and disease. Genetic abnormalities, drug-triggered or acquired pathologies can disrupt and uncouple these waves with potentially lethal consequences. Optical mapping of electrical waves using fluorescent dyes or genetically-encoded sensors in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) offers mechanistic insights into cardiac conduction abnormalities. Interferometric dye-free/label-free wave mapping (without specific sensors) presents an alternative, likely capturing the mechanical aspects of cardiac conduction. Because of its non-invasive nature and spectral flexibility (not restricted to a specific excitation wavelength), it is an attractive chronic imaging tool in iPSC-CMs, as part of all-optical high-throughput platforms. In this study, we developed simultaneous widefield voltage and interferometric dye-free optical imaging methodology that was used: 1) to validate dye-free optical mapping for quantification of cardiac wave properties in human iPSC-CMs; 2) to demonstrate low-cost optical mapping of electromechanical waves in hiPSC-CMs using recent near-infrared (NIR) voltage sensors and orders of magnitude cheaper miniature CMOS cameras; 3) to uncover previously underexplored frequency- and space-varying parameters of cardiac electromechanical waves in hiPSC-CMs. We find similarity in the frequency-dependent responses of electrical (NIR fluorescence imaged) and mechanical (dye-free imaged) waves, with the latter being more sensitive to faster rates and showing steeper restitution and earlier appearance of wave-front tortuosity. During regular pacing, the dye-free imaged conduction velocity and the electrical wave velocity are correlated; both modalities being sensitive to pharmacological uncoupling and both dependent on gap-junctional protein (connexins) determinants of wave propagation. We uncover strong frequency-dependence of the electromechanical delay (EMD) locally and globally in hiPSC-CMs on a rigid substrate. The presented framework and results offer new means to track the functional responses of hiPSC-CM inexpensively and non-invasively for counteracting heart disease and aiding cardiotoxicity testing and drug development.

bioengineering↗

bioRxiv2022

Portable low-cost macroscopic mapping system for all-optical cardiac electrophysiology

SignificanceAll-optical cardiac electrophysiology enables the visualization and control of key parameters relevant to the detection of cardiac arrhythmias. Mapping such responses in human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) is of great interest for cardiotoxicity and personalized medicine applications. AimWe introduce and validate a very low-cost compact mapping system for macroscopic all-optical electrophysiology in layers of hiPSC-CMs. ApproachThe system uses oblique trans-illumination, low-cost cameras, light-emitting diodes and off-the-shelf components (total < $15,000) to capture voltage, calcium and mechanical waves under electrical or optical stimulation. ResultsOur results corroborate the equivalency of electrical and optogenetic stimulation of hiPSC-CMs, and Vm - [Ca2+]i similarity in conduction under pacing. Green-excitable optical sensors are combinable with blue optogenetic actuators (Chanelrhodopsin2) only under very low green light (< 0.05mW/mm2). Measurements in warmer culture medium yield larger spread of action potential duration and higher conduction velocities compared to Tyrodes solution at room temperature. ConclusionsAs multiple optical sensors and actuators are combined, our results can help handle the "spectral congestion" and avoid parameter distortion. We illustrate the utility of the system for uncovering the action of cellular uncoupling agents and show extensibility to an epi-illumination mode for future imaging of thicker native or engineered tissues.

bioengineering↗

bioRxiv2022

Sex-dependent transcriptional control of cardiac electrophysiology by histone acetylation modifiers based on the GTEx database

Development of safer drugs based on epigenetic modifiers, e.g. histone deacetylase inhibitors (HDACi), requires better understanding of their effects on cardiac electrophysiology. Using RNAseq data from the genotype-tissue-expression database (GTEx), we created models that link the abundance of chromatin modifiers, such as histone acetylation enzymes (HDAC/SIRT/HATs), and the gene expression of ion channels (IC) via select cardiac transcription factors (TFs) in male and female adult human hearts (left ventricle, LV). Gene expression data (transcripts per million, TPM) from GTEx donors (21 to 70 y.o.) were filtered, normalized and transformed to Euclidian space to allow quantitative comparisons in 84 female and 158 male LVs. Sex-specific partial least-square (PLS) regression models, linking gene expression data for HDAC/SIRT/HATs to TFs and to ICs gene expression, revealed tight co-regulation of cardiac ion channels by HDAC/SIRT/HATs, with stronger clustering in the male LV. Co-regulation of genes encoding excitatory and inhibitory processes in cardiac tissue by the histone modifiers may help their predominantly net-neutral effects on cardiac electrophysiology. ATP1A1, encoding for the Na/K pump, represented an outlier - with orthogonal regulation by the histone modifiers to most of the ICs. The HDAC/SIRT/HAT effects were mediated by strong (+) TF regulators of ICs, e.g. MEF2A and TBX5, in both sexes. Furthermore, for male hearts, PLS models revealed a stronger (+)/(-) mediatory role on ICs for NKX25 and TGF1B/KLF4, respectively, while RUNX1 exhibited larger (-) TF effects on ICs in females. Male-trained PLS models of HDAC/SIRT/HAT effects on ICs underestimated the effects on some ICs in females. Insights from the GTEx dataset about the co-expression and transcriptional co-regulation of histone-modifying enzymes, transcription factors and key cardiac ion channels in a sex-specific manner can help inform safer drug design.

bioengineering↗

bioRxiv2021

INTEGRATION OF ENGINEERED "SPARK-CELL" SPHEROIDS FOR OPTICAL PACING OF CARDIAC TISSUE

Optogenetic methods for pacing of cardiac tissue can be realized by direct genetic modification of the cardiomyocytes to express light-sensitive actuators, such as channelrhodopsin-2, ChR2, or by introduction of light-sensitized non-myocytes that couple to the cardiac cells and yield responsiveness to optical pacing. In this study, we engineer three-dimensional "spark cells" spheroids, composed of ChR2-expressing human embryonic kidney cells, and characterize their morphology as function of cell density and time. These "spark-cell" spheroids are then deployed to demonstrate site-specific optical pacing of human stem-cell-derived cardiomyocytes (hiPSC-CMs) in 96-well format using non-localized light application and all-optical electrophysiology. We show that the spheroids can be handled using liquid pipetting and can confer optical responsiveness of cardiac tissue earlier than direct viral or liposomal genetic modification of the cardiomyocytes, with 24% providing reliable stimulation of the iPSC-CMs within 6 hours and >80% within 24 hours. Our results demonstrate a scalable, cost-effective method to achieve contactless optical stimulation of cardiac cell constructs that can be integrated in a robotics-amenable workflow for high-throughput drug testing. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/428177v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@156108org.highwire.dtl.DTLVardef@1956092org.highwire.dtl.DTLVardef@1f7237eorg.highwire.dtl.DTLVardef@3b37a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Optogenetic currents in myofibroblasts acutely alter electrophysiology and conduction of co-cultured cardiomyocytes

Interactions between cardiac myofibroblasts and myocytes may slow conduction after cardiac injury, increasing the chance of life-threatening arrhythmia. While co-culture studies have shown that myofibroblasts can affect cardiomyocyte electrophysiology in vitro, the mechanism(s) remain debatable. In this study, primary neonatal rat cardiac myofibroblasts were transduced with the light-activated ion channel Channelrhodopsin-2, which allowed acute and selective modulation of myofibroblast currents in co-cultures with cardiomyocytes. Optical mapping revealed that myofibroblast-specific optogenetically induced inward currents decreased conduction velocity in the co-cultures by 27{+/-}6% (baseline = 17.7{+/-}5.3 cm/s), and shortened the cardiac action potential duration by 14{+/-}7% (baseline = 161{+/-}11 ms) when 0.017 mW/mm2 light was applied. When light irradiance was increased to 0.057 mW/mm2, the myofibroblast currents led to spontaneous beating in 6/7 co-cultures. Experiments showed that optogenetic perturbation did not lead to changes in myofibroblast strain and force generation, suggesting purely electrical effects in this model. In silico modeling of optogenetically modified myofibroblast-cardiomyocyte co-cultures largely reproduced these results and enabled a comprehensive study of relevant parameters. These results clearly demonstrate that myofibroblasts are sufficiently electrically connected to cardiomyocytes to effectively alter macroscopic electrophysiological properties in this model of cardiac tissue.

physiology↗

bioRxiv2020

Microfluidics-enabled 96-well perfusion system for high-throughput tissue engineering and long-term all-optical electrophysiology

This work demonstrates a novel high-throughput (HT) microfluidics-enabled uninterrupted perfusion system (HT-UPS) and validates its use with chronic all-optical electrophysiology in human excitable cells. HT-UPS consists of a soft multichannel microfluidic plate cover which could button on a commercial HT 96-well plate. Herein, we demonstrate the manufacturing process of the system and its usages in acute and chronic all-optical electrophysiological studies of human induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CM) and engineered excitable (Spiking HEK) cells. HT-UPS perfusion maintained functional voltage and calcium responses in iPSC-CM and Spiking HEK cells under spontaneous conditions and under optogenetic pacing. Long-term culture with HT-UPS improved cell viability and optogenetically-tracked calcium responses in Spiking HEK cells. The scalability and simplicity of this design and its compatibility with HT all-optical electrophysiology can empower cell-based assays for personalized medicine using patient-derived cells.

bioengineering↗

bioRxiv2020

Hysteresis in calcium rhythm patterns during electric field stimulation of cardiac tissue

Cardiac tissue subjected to fast pacing via electric field stimulation revealed hysteresis in calcium instability patterns (stimulus:response patterns) beyond departure and return to 1:1 response. The pacing frequency at which the first appearance of instabilities occurred (Fa) was higher than the frequency of ultimate disappearance (Fd) upon rate deceleration. Furthermore, hysteresis was observed in multiple pattern transitions. In the spatially extended system studied here, 2:2 alternans were the preferred starting point (Fa) in calcium instability development, while 2:1 blocks were more common in the return to Fa from higher pacing rates. Recovery of 1:1 patterns was preceded mostly by 2:2 alternans at Fd. In addition to previously reported hysteresis in action potential duration during 1:1 rhythm and alternans magnitude hysteresis (in 2:2 rhythm), our data reveal hysteresis in rhythm pattern transitions not just away from and return to 1:1, but also between different instability patterns, and thus provide insight into the rules of such transitions in electrically stimulated cardiac tissue.

bioengineering↗