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 h. 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.

The heart's contractions are triggered by action potential waves, which propagate through the cardiac muscle and exhibit diverse spatio-temporal dynamics during different heart rhythms. The dynamics are modeled with partial differential equations (PDEs) in cardiac electrophysiology simulations. However, fitting such models to measurement data to develop digital twins or patient-specific computer models is challenging. Here, we introduce differentiable cardiac electrophysiology simulations that can be fitted automatically to spatio-temporal measurement data of action potential waves in cardiac tissue. By comparing the simulated dynamics with the observation data, we define a loss function that is minimized via gradient-based optimization. Backpropagating the loss gradient through the differentiable PDE solver enables us to learn the parameters and recover the full dynamics, even with sparse, noisy, or partial observations. Implemented using both the finite-difference and smoothed particle hydrodynamics methods, our simulation framework can be applied to pixel-, voxel-, or point-based data, such as 2D or 3D slabs, or arbitrary shapes, such as the heart's ventricles. Using this methodology, we locate early activation sites inside a 3D bi-ventricular simulation geometry and fit a phenomenological model to imaging data of a voltage spiral wave in a cardiac monolayer cell culture. With experimental data, we employed a perceptual loss based on the Video Joint-Embedding Predictive Architecture, which enables fitting to noisy imaging data, and a generative diffusion model to estimate initial conditions and constrain solutions. Differentiable cardiac electrophysiology simulations could improve the diagnosis of rhythm abnormalities in patients and facilitate the development of personalized models or digital twins of the heart.

Significance:Cardiac panoramic optical mapping is a powerful approach for studying action potential dispersion and mapping arrhythmia triggers and propagation pathways over the entire surface of the heart. However, tissue type (muscle, connective tissue, and infarct scar) is also important for interpreting mapping data and is difficult to identify using optical mapping data alone. Aim:Panoramically map transmembrane potential and tissue type from the surface of infarcted hearts for correlative analysis of cardiac structure and function. Approach:We developed a multimodal panoramic imaging system to map epicardial tissue type (determined by collagen content) using a line-scan hyperspectral camera and a precision stage to translate and rotate the heart while illuminating the epicardial surface with UV light. Transmembrane potential was subsequently optically mapped by imaging a potentiometric probe with four high speed CMOS cameras position around the heart. The epicardial surface was reconstructed for each heart using images acquired every 3.6 deg of rotation, onto which hyperspectral and optical mapping data were texture mapped. All cameras were registered to one coordinate frame using a calibration procedure. Results:This system combines, for the first time, high-resolution hyperspectral imaging with optical mapping for quantitative correlative tissue structure-function analyses. It was used to study excitation wave propagation and action potentials across the surface of perfused rat hearts having a four-week-old infarct. The spectral band of collagen fluorescence (400 to 520 nm) revealed infarcted and border zone tissue. PVCs and reentrant activity were observed in 3 of 4 hearts at S1-S2 pacing intervals between 80 and 65 msec (S1 = 150 msec). PVCs originated near the infarct border and propagated around the infarct. Using the integral of spectral intensity from 400 to 435 nm, a k-means clustering algorithm classified each mapped site as either healthy, border zone, or infarcted tissue. Average action potential duration within those tissue types was longest for infarcted tissue, shorter for border zone tissue, and shortest for healthy tissue, a preliminary result that is consistent with the effect of an infarct on ventricular electrophysiology. Conclusions:This work demonstrates that panoramic hyperspectral mapping of tissue type and transmembrane potential is a powerful approach that enables functional mapping data to be analyzed within the context of local tissue type (healthy, infarct, and border) in living hearts.

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 a positive effect on IC genes while RUNX1 and SHMT2 were distinct negative regulators in both sample types; TRIM28 was found to serve opposite roles regulating ICs in the hiPSC-CM compared to the adult LV. In functional measurements, HDAC suppression primarily increased excitability, while SIRT suppression decreased excitability, in line with transcriptomic links and in qualitative agreement with predictions by a computational ionic model of hiPSC-CMs. 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.

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 (hiPSC-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.

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. We used HT label-free optical measurements and computational modeling to gain insights about oxygen availability (pericellular 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 pericellular oxygen dynamics. The developed computational model, trained on the obtained comprehensive data set, revealed that time-variant maximum oxygen consumption rate, Vmax, is needed to faithfully capture the complex pericellular oxygen dynamics in the excitable hiPSC-CMs, but not in the cFBs. Interestingly, hypoxia (<2 % pericellular oxygen) developed within hours in the dense iPSC-CM cultures when the solution volume was sufficiently large. Conversely, hiPSC-CMs grown at low cell density or in smaller solution volume, as well as cFB under all studied conditions, were found to operate in hyperoxic (>7 %) conditions. Pericellular oxygen dynamics of the differentiated hiPSC-CMs evolved over days in culture, with the best improvement in respiration seen in samples operating close to normoxia. Our results and the developed computational model can be used directly to optimize cardiac cell growth in HT plates and achieve desired physiological conditions, which is important in cellular assays for cardiotoxicity, drug development, personalized medicine and heart regeneration applications.

A balance of cholinergic and catecholaminergic activation is necessary to maintain heart health. Interrogating the interaction between these pathways can be done using optogenetics through selective expression of channelrhodopsin-2 (ChR2) in cardiac autonomic neurons. Such cardiac applications of optogenetics allow for the study of the intrinsic release of neurotransmitters in a spatiotemporal manner. This method illustrates an ex vivo approach for specific optogenetic stimulation of cardiac neurons in perfused mouse hearts. Transgenic mice were bred to express ChR2 in either choline acetyltransferase (ChAT) or tyrosine hydroxylase (TH) neurons throughout the body. A micro-LED (465 nm) encased in a silicone elastomer was prepared for stimulating the neurons of the right atrium that innervate the sinoatrial node. The micro-LED was connected to a function generator set to pulse waves at 10 Hz with a 30 ms pulse width. Hearts with confirmed expression were excised and retrogradely perfused on a Langendorff system circulating Krebs-Henseleit solution. Electrocardiogram (ECG), temperature, and coronary flow rate were recorded using the LabChart software. Once the heart stabilized, the micro-LED was placed on the right atrium and tested for optimal heart rate response. An application of this approach combines the intrinsic release of cholinergic neurotransmitter (acetylcholine) during optogenetic activation of a ChAT-ChR2 mouse heart simultaneously with increasing exogenous catecholaminergic neurotransmitter (norepinephrine) added to the perfusate. The resulting changes in heart rate during the simultaneous cholinergic and catecholaminergic activation are presented. This method describes a valuable experimental approach for investigating the kinetics of sudden intrinsic autonomic neuron activation in perfused hearts and the interactions between cardiac cholinergic and catecholaminergic activity.

The Halbach array, originally developed for particle accelerators, is a compact arrangement of permanent magnets that creates 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, 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 led to increased CV anisotropy, and effects were most pronounced at slower pacing rates. Instantaneous formation and re-arrangement of elongated mNP clusters upon magnetic field rotation was seen, 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.

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. ### Competing Interest Statement The authors have declared no competing interest.

We show that zinc finger imprinted 3 (Zim3), when used as Zim3-KRAB-dCas9 effector in interference CRISPR, without any guide RNAs, paradoxically up-regulates 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.

Current models for cardiotoxicity screening are limited in their ability to only evaluate hERG inhibition and related prediction of QT prolongation and torsadoegnic potential. More comprehensive cardiotoxicity assays are being developed using in vitro platforms with human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), which are further aided by experimental and computational approaches. CRISPRi gene perturbation is a powerful source for human functional genomics in the cardiac field as it can link genes to electrophysiological phenotype. We developed a high-throughput system involving all-optical cardiac electrophysiology in 96-well format and CRISPRi gene modulation in post-differentiated iPSC-CMs. Single guide RNAs (sgRNAs) were tested targeting around the transcription start sites of KCNH2, an ion channel responsible for the repolarization of the cardiac action potential. Optogenetic pacing and spectrally-compatible voltage and calcium sensors were used to obtain functional measurements for voltage and calcium responses. The efficacy and functional consequences of gene modulation by siRNA and CRISPRi with two effector domains (dCas9-KRAB and dCas9-Zim3) were compared. We found that adenoviral transduction with a recently developed dCas9-Zim3, induced the most robust APD prolongation (+20%, p<0.0001) in the hiPSC-CMs upon KCNH2 knockdown when compared to using siRNA (+11%, p=0.0002) and transfection of an inducible-dCas9-KRAB (+<10%, p=0.02). Knockdown of hERG in our system yielded mild but specific functional changes which can be combined with computational approaches using dimension reduction approaches to help visualize and quantify electrophysiological changes in the heart. Usage of this platform for CRISPRi mediated knockdown of diseases-associated genes in pre-differentiated cardiomyocytes can improve the assessment of gene function in cellular 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 (parallel recordings from up to 100 wells in 384-well plates are demonstrated). 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 384-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 optogenetic point pacing via cell spheroids to study conduction in 96-well format, as well as temporal multiplexing to register voltage and calcium simultaneously on a single camera. Opto-DyCE-plate showed excellent performance even in the small samples in 384-well plates. 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.

Precise control of gene expression (knock-out, knock-in, knockdown or overexpression) is at the heart of functional genomics - an approach to dissect the contribution of a gene/protein to the system's function. The development of a human in vitro system that can be patient-specific, induced pluripotent stem cells, iPSC, and the ability to obtain various cell types of interest, have empowered human disease modeling and therapeutic development. Scalable tools have been deployed for gene modulation in these cells and derivatives, including pharmacological means, DNA-based RNA interference and standard RNA interference (shRNA/siRNA). The CRISPR/Cas9 gene editing system, borrowed from bacteria and adopted for use in mammalian cells a decade ago, offers cell-specific genetic targeting and versatility. Outside genome editing, more subtle, time-resolved gene modulation is possible by using a catalytically "dead" Cas9 enzyme linked to an effector of gene transcription in combination with a guide RNA. The CRISPRi / CRISPRa (interference/activation) system evolved over the last decade as a scalable technology for performing functional genomics with libraries of gRNAs. Here, we review key developments of these approaches and their deployment in cardiovascular research. We discuss specific use with iPSC-cardiomyocytes and the challenges in further translation of these techniques.

Significance All-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. Aim We introduce and validate a very low-cost compact mapping system for macroscopic all-optical electrophysiology in layers of hiPSC-CMs. Approach The 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. Results Our results corroborate the equivalency of electrical and optogenetic stimulation of hiPSC-CMs, and Vm – [Ca 2+ ]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/mm 2 ). Measurements in warmer culture medium yield larger spread of action potential duration and higher conduction velocities compared to Tyrode’s solution at room temperature. Conclusions As 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.