Cardiac Cellular Electrophysiology Research Articles

High-throughput methods for cardiac cellular electrophysiology studies: the road to personalized medicine.

Personalized medicine refers to the tailored application of medical treatment at an individual level, considering the specific genotype or phenotype of each patient for targeted therapy. In the context of cardiovascular diseases, implementing personalized medicine is challenging due to the high costs involved and the slow pace of identifying the pathogenicity of genetic variants, deciphering molecular mechanisms of disease, and testing treatment approaches. Scalable cellular models such as human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) serve as useful in vitro tools that reflect individual patient genetics and retain clinical phenotypes. High-throughput functional assessment of these constructs is necessary to rapidly assess cardiac pathogenicity and test new therapeutics if personalized medicine is to become a reality. High-throughput photometry recordings of single cells coupled with potentiometric probes offer cost-effective alternatives to traditional patch-clamp assessments of cardiomyocyte action potential characteristics. Importantly, automated patch-clamp (APC) is rapidly emerging in the pharmaceutical industry and academia as a powerful method to assess individual membrane-bound ionic currents and ion channel biophysics over multiple cells in parallel. Now amenable to primary cell and hiPSC-CM measurement, APC represents an exciting leap forward in the characterization of a multitude of molecular mechanisms that underlie clinical cardiac phenotypes. This review provides a summary of state-of-the-art high-throughput electrophysiological techniques to assess cardiac electrophysiology and an overview of recent works that successfully integrate these methods into basic science research that could potentially facilitate future implementation of personalized medicine at a clinical level.

Model-driven optimal experimental design for calibrating cardiac electrophysiology models

Background and Objective: Models of the cardiomyocyte action potential have contributed immensely to the understanding of heart function, pathophysiology, and the origin of heart rhythm disturbances. However, action potential models are highly nonlinear, making them difficult to parameterise and limiting to describing ‘average cell’ dynamics, when cell-specific models would be ideal to uncover inter-cell variability but are too experimentally challenging to be achieved. Here, we focus on automatically designing experimental protocols that allow us to better identify cell-specific maximum conductance values for each major current type.Methods and Results: We developed an approach that applies optimal experimental designs to patch-clamp experiments, including both voltage-clamp and current-clamp experiments. We assessed the models calibrated to these new optimal designs by comparing them to the models calibrated to some of the commonly used designs in the literature. We showed that optimal designs are not only overall shorter in duration but also able to perform better than many of the existing experiment designs in terms of identifying model parameters and hence model predictive power.Conclusions: For cardiac cellular electrophysiology, this approach will allow researchers to define their hypothesis of the dynamics of the system and automatically design experimental protocols that will result in theoretically optimal designs.

Comparison of efficacy and functional consequences of siRNA and CRISPRi gene modulation with different effector domains in post-differentiated human iPSC-CMs.

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.

A unique cardiac electrocardiographic 3D model. Toward interpretable AI diagnosis.

Mathematical models of cardiac electrical activity are one of the most important tools for elucidating information about heart diagnostics. In this paper, we present an efficient mathematical formulation for this modeling simple enough to be easily parameterized and rich enough to provide realistic signals. It relies on a five dipole representation of the cardiac electric source, each one associated with the well-known waves of the electrocardiogram signal. Beyond the physical basis of the model, the parameters are physiologically interpretable as they characterize the wave shape, similar to what a physician would look for in signals, thus making them very useful in diagnosis. The model accurately reproduces the electrocardiogram signals of any diseased or healthy heart. This new discovery represents a significant advance in electrocardiography research. It is especially useful for diagnosis, patient follow-up or decision-making on new therapies; is also a promising tool for well-performing, transparent and interpretable AI approaches.

Dynamical mechanism for generation of arrhythmogenic early afterdepolarizations in cardiac myocytes: Insights from in silico electrophysiological models.

We analyze the dynamical mechanisms underlying the formation of arrhythmogenic early afterdepolarizations (EADs) in two mathematical models of cardiac cellular electrophysiology: the Sato etal. biophysically detailed model of a rabbit ventricular myocyte of dimension 27 and a reduced version of the Luo-Rudy mammalian myocyte model of dimension 3. Based on a comparison of the two models, with detailed bifurcation analysis using spike-counting techniques and continuation methods in the simple model and numerical explorations in the complex model, we locate the point where the first EAD originates in an unstable branch of periodic orbits. These results serve as a basis to propose a conjectured scheme involving a hysteresis mechanism with the creation of alternans and EADs in the unstable branch. This theoretical scheme fits well with electrophysiological experimental data on EAD generation and hysteresis phenomena. Our findings open the door to the development of novel methods for pro-arrhythmia risk prediction related to EAD generation without actual induction of EADs.

Fraction of the T-Tubular Membrane as an Important Parameter in Cardiac Cellular Electrophysiology: A New Way of Estimation.

The transverse-axial tubular system (t-tubules) plays an essential role in excitation-contraction coupling in cardiomyocytes. Its remodelling is associated with various cardiac diseases. Numerous attempts were made to analyse characteristics essential for proper understanding of the t-tubules and their impact on cardiac cell function in health and disease. The currently available methodical approaches related to the fraction of the t-tubular membrane area produce diverse data. The widely used detubulation techniques cause irreversible cell impairment, thus, distinct cell samples have to be used for estimation of t-tubular parameters in untreated and detubulated cells. Our proposed alternative method is reversible and allows repetitive estimation of the fraction of t-tubular membrane (f t) in cardiomyocytes using short-term perfusion of the measured cell with a low-conductive isotonic sucrose solution. It results in a substantial increase in the electrical resistance of t-tubular lumen, thus, electrically separating the surface and t-tubular membranes. Using the whole-cell patch-clamp measurement and the new approach in enzymatically isolated rat atrial and ventricular myocytes, a set of data was measured and evaluated. The analysis of the electrical equivalent circuit resulted in the establishment of criteria for excluding measurements in which perfusion with a low conductivity solution did not affect the entire cell surface. As expected, the final average f t in ventricular myocytes (0.337 ± 0.017) was significantly higher than that in atrial myocytes (0.144 ± 0.015). The parameter f t could be estimated repetitively in a particular cell (0.345 ± 0.021 and 0.347 ± 0.023 in ventricular myocytes during the first and second sucrose perfusion, respectively). The new method is fast, simple, and leaves the measured cell intact. It can be applied in the course of experiments for which it is useful to estimate both the surface and t-tubular capacitance/area in a particular cell.

BS-514-03 CRISPRI GENE MODULATION IN PRE-DIFFERENTIATED HIPSC-CMS COMBINED WITH ALL-OPTICAL ELECTROPHYSIOLOGY

Current models for cardiotoxicity screening are limited to only evaluate hERG inhibition for related prediction of QT prolongation and torsadoegnic potential. More comprehensive in vitro cardiotoxicity assays are being proposed using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), aided with experimental and computational approaches. We tested the potential of CRISPRi modulation in pre-differentiated iPSC-CMs combining all-optical cardiac electrophysiology and molecular analysis to characterize functional phenotypes of key ion channels in the human heart. We designed single guide RNAs (sgRNAs) targeting KCNH2, KCNJ2, and GJA1. The sgRNAs were cloned into a lentiviral vector with eGFP reporter. A dox-inducible dCas9-KRAB was inserted into the AAVS1 safe harbor site of pre-differentiated hiPSC-CMs and transduced with lentivirus carrying the sgRNAs. To deploy all-optical electrophysiology, cells were transduced with an adenoviral vector containing ChR2-eYFP (for optical stimulation) and co-labeled with spectrally-compatible voltage and calcium sensors to obtain functional measurements upon 5 days of dox induction. A sgRNA targeting KCNH2 resulted in about 40% knockdown of KCNH2 mRNA and significant but relatively mild prolongation of action potential duration, e.g. spontaneous APD90 (+12%, p<0.05). A sgRNA targeting KCNJ2 caused about 60% downregulation of mRNA levels and exhibited minimal changes in spontaneous beat frequency in high density cell preparations (+7%, n.s.) but more pronounced effects in reduced density cell preparations (+30%, p<0.01). Additionally, sgRNA targeting GJA1 exhibited about 40% knockdown of mRNA and resulted in significant slowing of conduction velocity (-19%, p<0.01) at 1 Hz pacing conditions. Knockdown of key cardiac ion channels in our system yielded mild but specific functional changes. Using this platform for CRISPRi mediated knockdown of disease-associated genes in pre-differentiated cardiomyocytes can improve the assessment of gene function in cellular cardiac electrophysiology. In combination with sgRNA libraries and/or with CRISPRa mediated gene activation, it can allow a more comprehensive evaluation of the mechanisms controlling cardiac electromechanics.

Edward Carmeliet (1930-2021)-channelling scientific curiosity: a tribute from the ESC Working Group on Cardiac Cellular Electrophysiology†.

Edward Carmeliet (1930-2021)-channelling scientific curiosity: a tribute from the ESC Working Group on Cardiac Cellular Electrophysiology†.

Edward E. Carmeliet, MD, PhD (January 4, 1930–April 5, 2021): A pioneer in cardiac cellular electrophysiology

The passing of Edward E. Carmeliet entrusts to our memories the life and the work of a brilliant cardiac cellular electrophysiologist, indeed, one of the most brilliant of our time. Not only did he have a major innovative impact on our understanding of cardiac bioelectricity in health and disease, he also was a revered mentor who influenced the work of scholars worldwide with his scientific competence and his unique personality.

ESC working group on cardiac cellular electrophysiology position paper: relevance, opportunities, and limitations of experimental models for cardiac electrophysiology research.

Cardiac arrhythmias are a major cause of death and disability. A large number of experimental cell and animal models have been developed to study arrhythmogenic diseases. These models have provided important insights into the underlying arrhythmia mechanisms and translational options for their therapeutic management. This position paper from the ESC Working Group on Cardiac Cellular Electrophysiology provides an overview of (i) currently available in vitro, ex vivo, and in vivo electrophysiological research methodologies, (ii) the most commonly used experimental (cellular and animal) models for cardiac arrhythmias including relevant species differences, (iii) the use of human cardiac tissue, induced pluripotent stem cell (hiPSC)-derived and in silico models to study cardiac arrhythmias, and (iv) the availability, relevance, limitations, and opportunities of these cellular and animal models to recapitulate specific acquired and inherited arrhythmogenic diseases, including atrial fibrillation, heart failure, cardiomyopathy, myocarditis, sinus node, and conduction disorders and channelopathies. By promoting a better understanding of these models and their limitations, this position paper aims to improve the quality of basic research in cardiac electrophysiology, with the ultimate goal to facilitate the clinical translation and application of basic electrophysiological research findings on arrhythmia mechanisms and therapies.

Frontal plane QRS-T angle in the monitoring of intravenous amiodarone infusion for pharmacological cardioversion of acute atrial fibrillation.

Intravenous amiodarone infusion is effective and widely used treatment for pharmacological cardioversion of recent-onset atrial fibrillation (Af). Although amiodarone may trigger various alterations in cardiac electrophysiology and electrocardiography (ECG), the impact of amiodarone treatment on frontal plane QRS-T angle remains unclear. Frontal plane QRS-T angle is the angle between the depolarization and repolarization axes and indicates instability in the cardiac cellular electrophysiology. Therefore, the present study aimed to investigate whether intravenous amiodarone infusion has effect on frontal plane QRS-T angle in patients with acute Af. A total of 179 patients with acute-onset Af who underwent pharmacological cardioversion with intravenous amiodarone infusion were included into the study. Patients with successful and failed pharmacological cardioversion were compared regarding pre- and post-treatment frontal plane QRS-T angle. At the end of the amiodarone infusion, sinus rhythm was restored in 112 (62.6%) patients, whereas Af was persisted in 67 (37.4%) patients. Despite the similar frontal plane QRS-T angle at baseline (59.6°±21.73°vs.60.4°±25.67°, p=0.822), patients with failed pharmacological cardioversion had significantly higher post-treatment frontal plane QRS-T angle compared to patients with successful pharmacological cardioversion (68.8°±21.71°vs.58.6°±25.15° p<0.001). Furthermore, multivariate analysis demonstrated that post-treatment increased frontal plane QRS-T angle was found to be an independent predictor of failure of pharmacological cardioversion with amiodarone infusion (OR:1.233, 95% CI:1.147-1.919, p=0.024). Amiodarone may significantly affect the frontal plane QRS-T angle. As a parameter that can be easily calculated from automated ECG recordings, frontal plane QRS-T angle may be useful in the monitoring of intravenous amiodarone treatment.

Calibration of ionic and cellular cardiac electrophysiology models.

Cardiac electrophysiology models are among the most mature and well‐studied mathematical models of biological systems. This maturity is bringing new challenges as models are being used increasingly to make quantitative rather than qualitative predictions. As such, calibrating the parameters within ion current and action potential (AP) models to experimental data sets is a crucial step in constructing a predictive model. This review highlights some of the fundamental concepts in cardiac model calibration and is intended to be readily understood by computational and mathematical modelers working in other fields of biology. We discuss the classic and latest approaches to calibration in the electrophysiology field, at both the ion channel and cellular AP scales. We end with a discussion of the many challenges that work to date has raised and the need for reproducible descriptions of the calibration process to enable models to be recalibrated to new data sets and built upon for new studies.This article is categorized under:Analytical and Computational Methods > Computational MethodsPhysiology > Mammalian Physiology in Health and DiseaseModels of Systems Properties and Processes > Cellular Models

In vitro and in vivo characterization of a synthetic scorpion toxin AmmTx3, a potent inhibitor of cardiac voltage-gated potassium channel Kv4.2

Voltage-gated potassium channel Kv4.2 (encoded by KCND2 gene) contributes to the cardiac transient outward potassium current (Ito1). This current is the main contributor to the repolarisation phase 1 of the cardiac action potential. The toxin AmmTx3, identified from the venom of the scorpion Androctonus mauretanicus , is a blocker of Kv4.x channels, and have interesting therapeutic potential for neurological disorders due to its effect in cerebellar granule neurons. Its effects on cardiac Kv4.2 channels remains unclear. The aim of this study was to: – produce a synthetic AmmTx3 (AmmTx3-NCL); – evaluate in vitro and in vivo, effects of this toxin on Kv4.2 channels compared to native toxin. We did chemical synthesis of AmmTx3 using the native chemical ligation strategy (NCL). We characterized its biological activity compared to native AmmTx3 using an automated patch-clamp system (Syncropatch 384 PE, Nanion) on HEK cells co-expressing Kv4.2 α subunit and two auxiliaries subunits (KChIP and DPP6). Effects of AmmTx3 and AmmTx3-NCL on cardiac cellular electrophysiology were studied by patch clamp on adult rat cardiomyocytes and hiPSC-CMs. Integrated cardiac effects of AmmTx3 were studied on ECG of wild-type mice. In this study, we report the total chemical synthesis of AmmTx3 and 3D structure was also determined. As expected, biological activity of folded AmmTx3-NCL is coherent to native toxin with a similar time course of Kv4.2 current inhibition without any change of activation kinetics. Characterisation of AmmTx3 effects on isolated cardiomyocytes, hiPSC-CMs and in vivo by ECG is ongoing. AmmTx3 toxin can be chemically synthesized and used as a Kv4.2 channel inhibitor to contributed to the better understanding of the exact role of Ito1 in cardiac electrophysiology. Those first results seem to be a promising evidence that AmmTx3 could a potential inhibitor of Ito current in early repolarisation syndrome.

High-speed mechano-active multielectrode array for investigating rapid stretch effects on cardiac tissue

Systematic investigations of the effects of mechano-electric coupling (MEC) on cellular cardiac electrophysiology lack experimental systems suitable to subject tissues to in-vivo like strain patterns while simultaneously reporting changes in electrical activation. Here, we describe a self-contained motor-less device (mechano-active multielectrode-array, MaMEA) that permits the assessment of impulse conduction along bioengineered strands of cardiac tissue in response to dynamic strain cycles. The device is based on polydimethylsiloxane (PDMS) cell culture substrates patterned with dielectric actuators (DEAs) and compliant gold ion-implanted extracellular electrodes. The DEAs induce uniaxial stretch and compression in defined regions of the PDMS substrate at selectable amplitudes and with rates up to 18 s−1. Conduction along cardiomyocyte strands was found to depend linearly on static strain according to cable theory while, unexpectedly, being completely independent on strain rates. Parallel operation of multiple MaMEAs provides for systematic high-throughput investigations of MEC during spatially patterned mechanical perturbations mimicking in-vivo conditions.

Mechanistic insights from targeted molecular profiling of repolarization alternans in the intact human heart.

AimsAction potential duration (APD) alternans is an established precursor or arrhythmia and sudden cardiac death. Important differences in fundamental electrophysiological properties relevant to arrhythmia exist between experimental models and the diseased in vivo human heart. To investigate mechanisms of APD alternans using a novel approach combining intact heart and cellular cardiac electrophysiology in human in vivo.Methods and resultsWe developed a novel approach combining intact heart electrophysiological mapping during cardiac surgery with rapid on-site data analysis to guide myocardial biopsies for laboratory analysis, thereby linking repolarization dynamics observed at the organ level with underlying ion channel expression. Alternans-susceptible and alternans-resistant regions were identified by an incremental pacing protocol. Biopsies from these sites (n = 13) demonstrated greater RNA expression in Calsequestrin (CSQN) and Ryanodine (RyR) and ion channels underlying IK1 and Ito at alternans-susceptible sites. Electrical restitution properties (n = 7) showed no difference between alternans-susceptible and resistant sites, whereas spatial gradients of repolarization were greater in alternans-susceptible than in alternans-resistant sites (P = 0.001). The degree of histological fibrosis between alternans-susceptible and resistant sites was equivalent. Mathematical modelling of these changes indicated that both CSQN and RyR up-regulation are key determinants of APD alternans.ConclusionCombined intact heart and cellular electrophysiology show that regions of myocardium in the in vivo human heart exhibiting APD alternans are associated with greater expression of CSQN and RyR and show no difference in restitution properties compared to non-alternans regions. In silico modelling identifies up-regulation and interaction of CSQN with RyR as a major mechanism underlying APD alternans.

Use of human induced pluripotent stem cell-derived cardiomyocytes to assess drug cardiotoxicity.

Cardiotoxicity has historically been a major cause of drug removal from the pharmaceutical market. Several chemotherapeutic compounds have been noted for their propensities to induce dangerous cardiac-specific side effects such as arrhythmias or cardiomyocyte apoptosis. However, improved preclinical screening methodologies have enabled cardiotoxic compounds to be identified earlier in the drug development pipeline. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can be used to screen for drug-induced alterations in cardiac cellular contractility, electrophysiology, and viability. We previously established a novel 'cardiac safety index' (CSI) as a metric that can evaluate potential cardiotoxic drugs via high-throughput screening of hiPSC-CMs. This metric quantitatively examines drug-induced alterations in CM function, using several in vitro readouts, and normalizes the resulting toxicity values to the in vivo maximum drug blood plasma concentration seen in preclinical or clinical pharmacokinetic models. In this ~1-month-long protocol, we describe how to differentiate hiPSCs into hiPSC-CMs and subsequently implement contractility and cytotoxicity assays that can evaluate drug-induced cardiotoxicity in hiPSC-CMs. We also describe how to carry out the calculations needed to generate the CSI metric from these quantitative toxicity measurements.

Comparison and assessment of CaV1.2 kinetics described by different in silico cellular cardiac electrophysiology models

Comparison and assessment of CaV1.2 kinetics described by different in silico cellular cardiac electrophysiology models

Automatic Optimization of an in Silico Model of Human iPSC Derived Cardiomyocytes Recapitulating Calcium Handling Abnormalities.

The growing importance of human induced pluripotent stem cell-derived cardiomyoyctes (hiPSC-CMs), as patient-specific and disease-specific models for studying cellular cardiac electrophysiology or for preliminary cardiotoxicity tests, generated better understanding of hiPSC-CM biophysical mechanisms and great amount of action potential and calcium transient data. In this paper, we propose a new hiPSC-CM in silico model, with particular attention to Ca2+ handling. We used (i) the hiPSC-CM Paci2013 model as starting point, (ii) a new dataset of Ca2+ transient measurements to tune the parameters of the inward and outward Ca2+ fluxes of sarcoplasmic reticulum, and (iii) an automatic parameter optimization to fit action potentials and Ca2+ transients. The Paci2018 model simulates, together with the typical hiPSC-CM spontaneous action potentials, more refined Ca2+ transients and delayed afterdepolarizations-like abnormalities, which the old Paci2013 was not able to predict due to its mathematical formulation. The Paci2018 model was validated against (i) the same current blocking experiments used to validate the Paci2013 model, and (ii) recently published data about effects of different extracellular ionic concentrations. In conclusion, we present a new and more versatile in silico model, which will provide a platform for modeling the effects of drugs or mutations that affect Ca2+ handling in hiPSC-CMs.

Report on the Ion Channel Symposium : Organized by the German Cardiac Society Working Group on Cellular Electrophysiology (AG18).

To support scientific exchange and activity in the field of cardiac cellular electrophysiology, the German Cardiac Society Working Group on Cellular Electrophysiology (AG18) established atwo-day symposium to be held every 2years. The second Ion Channel Symposium entitled "Göttingen Channels 2017-Of Benches and Beds" took place in Göttingen from September22nd to23rd under the auspices of the German Cardiac Society. Agroup of national and international experts presented scientific advances in cardiac electrophysiology and rhythmology. The symposium's primary focus was the significance of cellular electrophysiology findings for the optimization of diagnostic and therapeutic strategies against cardiac arrhythmias. To this end, speakers, chairpersons and attendees discussed the contribution of specific molecular alterations to the initiation and perpetuation of atrial and ventricular arrhythmias. Furthermore, the meeting highlighted how discoveries in electrophysiological research may lead to novel therapeutic targets. The interdisciplinary assessment of mechanisms and therapeutic strategies of cardiac arrhythmias represented akey feature of the meeting. Aunique combination of topics and speakers representing both basic science and clinical electrophysiology ensured the scientific success of the "Göttingen Channels 2017" symposium. The next Ion Channel Symposium is planned to be hosted by the incoming co-chair of the German Cardiac Society Working Group on Cellular Electrophysiology in fall 2019.

Optogenetic sensors in the zebrafish heart: a novel in vivo electrophysiological tool to study cardiac arrhythmogenesis

Cardiac arrhythmias are among the most challenging human disorders to diagnose and treat due to their complex underlying pathophysiology. Suitable experimental animal models are needed to study the mechanisms causative for cardiac arrhythmogenesis. To enable in vivo analysis of cardiac cellular electrophysiology with a high spatial and temporal resolution, we generated and carefully validated two zebrafish models, one expressing an optogenetic voltage indicator (chimeric VSFP-butterfly CY) and the other a genetically encoded calcium indicator (GCaMP6f) in the heart.Methods: High-speed epifluorescence microscopy was used to image chimeric VSFP-butterfly CY and GCaMP6f in the embryonic zebrafish heart, providing information about the spatiotemporal patterning of electrical activation, action potential configuration and intracellular Ca2+ dynamics. Plotting VSFP or GCaMP6f signals on a line along the myocardial wall over time facilitated the visualization and analysis of electrical impulse propagation throughout the heart. Administration of drugs targeting the sympathetic nervous system or cardiac ion channels was used to validate sensitivity and kinetics of both zebrafish sensor lines. Using the same microscope setup, we imaged transparent juvenile casper fish expressing GCaMP6f, demonstrating the feasibility of imaging cardiac optogenetic sensors at later stages of development.Results: Isoproterenol slightly increased heart rate, diastolic Ca2+ levels and Ca2+ transient amplitudes, whereas propranolol caused a profound decrease in heart rate and Ca2+ transient parameters in VSFP-Butterfly and GCaMP6f embryonic fish. Ikr blocker E-4031 decreased heart rate and increased action potential duration in VSFP-Butterfly fish. ICa,L blocker nifedipine caused total blockade of Ca2+ transients in GCaMP6f fish and a reduced heart rate, altered ventricular action potential duration and disrupted atrial-ventricular electrical conduction in VSFP-Butterfly fish. Imaging of juvenile animals demonstrated the possibility of employing an older zebrafish model for in vivo cardiac electrophysiology studies. We observed differences in atrial and ventricular Ca2+ recovery dynamics between 3 dpf and 14 dpf casper fish, but not in Ca2+ upstroke dynamics.Conclusion: By introducing the optogenetic sensors chimeric VSFP-butterfly CY and GCaMP6f into the zebrafish we successfully generated an in vivo cellular electrophysiological readout tool for the zebrafish heart. Complementary use of both sensor lines demonstrated the ability to study heart rate, cardiac action potential configuration, spatiotemporal patterning of electrical activation and intracellular Ca2+ homeostasis in embryonic zebrafish. In addition, we demonstrated the first successful use of an optogenetic sensor to study cardiac function in older zebrafish. These models present a promising new research tool to study the underlying mechanisms of cardiac arrhythmogenesis.