A semiconductor 96-microplate platform for electrical-imaging based high-throughput phenotypic screening.

High-content imaging for compound and genetic profiling is popular for drug discovery but limited to endpoint images of fixed cells. Conversely, electronic-based devices offer label-free, live cell functional information but suffer from limited spatial resolution or throughput. Here, we introduce a semiconductor 96-microplate platform for high-resolution, real-time impedance imaging. Each well features 4096 electrodes at 25 µm spatial resolution and a miniaturized data interface allows 8× parallel plate operation (768 total wells) for increased throughput. Electric field impedance measurements capture >20 parameter images including cell barrier, attachment, flatness, and motility every 15 min during experiments. We apply this technology to characterize 16 cell types, from primary epithelial to suspension cells, and quantify heterogeneity in mixed co-cultures. Screening 904 compounds across 13 semiconductor microplates reveals 25 distinct responses, demonstrating the platform's potential for mechanism of action profiling. The scalability and translatability of this semiconductor platform expands high-throughput mechanism of action profiling and phenotypic drug discovery applications.

A semiconductor 96-microplate platform for electrical-imaging based high-throughput phenotypic screening.

Profiling compounds and genetic perturbations via high-content imaging has become increasingly popular for drug discovery, but the technique is limited to endpoint images of fixed cells. In contrast, electronic-based devices offer label-free, functional information of live cells, yet current approaches suffer from low-spatial resolution or single-well throughput. Here, we report a semiconductor 96-microplate platform designed for high-resolution real-time impedance "imaging" at scale. Each well features 4,096 electrodes at 25 µm spatial resolution while a miniaturized data interface allows 8× parallel plate operation (768 total wells) within each incubator for enhanced throughputs. New electric field-based, multi-frequency measurement techniques capture >20 parameter images including tissue barrier, cell-surface attachment, cell flatness, and motility every 15 min throughout experiments. Using these real-time readouts, we characterized 16 cell types, ranging from primary epithelial to suspension, and quantified heterogeneity in mixed epithelial and mesenchymal co-cultures. A proof-of-concept screen of 904 diverse compounds using 13 semiconductor microplates demonstrates the platform's capability for mechanism of action (MOA) profiling with 25 distinct responses identified. The scalability of the semiconductor platform combined with the translatability of the high dimensional live-cell functional parameters expands high-throughput MOA profiling and phenotypic drug discovery applications.

Homozygous might be hemizygous: CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls.

The ability to precisely edit the genome of human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 has enabled the development of cellular models that can address genotype to phenotype relationships. While genome editing is becoming an essential tool in iPSC-based disease modeling studies, there is no established quality control workflow for edited cells. Moreover, large on-target deletions and insertions that occur through DNA repair mechanisms have recently been uncovered in CRISPR/Cas9-edited loci. Yet the frequency of these events in human iPSCs remains unclear, as they can be difficult to detect. We examined 27 iPSC clones generated after targeting 9 loci and found that 33% had acquired large, on-target genomic defects, including insertions and loss of heterozygosity. Critically, all defects had escaped standard PCR and Sanger sequencing analysis. We describe a cost-efficient quality control strategy that successfully identified all edited clones with detrimental on-target events and could facilitate the integrity of iPSC-based studies.

Optical Interrogation of Sympathetic Neuronal Effects on Macroscopic Cardiomyocyte Network Dynamics.

Cardiac stimulation via sympathetic neurons can potentially trigger arrhythmias. We present approaches to study neuron-cardiomyocyte interactions involving optogenetic selective probing and all-optical electrophysiology to measure activity in an automated fashion. Here we demonstrate the utility of optical interrogation of sympathetic neurons and their effects on macroscopic cardiomyocyte network dynamics to address research targets such as the effects of adrenergic stimulation via the release of neurotransmitters, the effect of neuronal numbers on cardiac behavior, and the applicability of optogenetics in mechanistic in vitro studies. As arrhythmias are emergent behaviors that involve the coordinated activity of millions of cells, we image at macroscopic scales to capture complex dynamics. We show that neurons can both decrease and increase wave stability and re-entrant activity in culture depending on their induced activity-a finding that may help us understand the often conflicting results seen in experimental and clinical studies.

Adeno-Associated Virus Mediated Gene Delivery: Implications for Scalable in vitro and in vivo Cardiac Optogenetic Models.

Adeno-associated viruses (AAVs) provide advantages in long-term, cardiac-specific gene expression. However, AAV serotype specificity data is lacking in experimental models relevant to cardiac electrophysiology and cardiac optogenetics. We aimed to identify the optimal AAV serotype (1, 6, or 9) in pursuit of scalable rodent and human models using genetic modifications in cardiac electrophysiology and optogenetics, in particular, as well as to elucidate the mechanism of virus uptake. In 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. Transgene 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 AAV1/6 (cell surface sialic acid) and AAV9 (37/67 kDa laminin receptor, LamR). In vitro desialylation of NRVMs and hiPSC-CMs with neuraminidase (NM) 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 NM 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 intact heart (neonatal rat hearts as well as adult human and rat hearts), but no expression in vitro in cultured cells (primary rat cells and hiPS-CMs). The dynamic nature of LamR expression and its dependence on environmental factors was further corroborated in intact adult human ventricular tissue. The 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. More broadly, these findings are relevant to future efforts in gene therapy for cardiac electrophysiology abnormalities in vivo as well as for genetic modifications of cardiomyocytes by viral means in vitro applications such as disease modeling or high-throughput drug testing.

OptoDyCE as an automated system for high-throughput all-optical dynamic cardiac electrophysiology.

The improvement of preclinical cardiotoxicity testing, discovery of new ion-channel-targeted drugs, and phenotyping and use of stem cell-derived cardiomyocytes and other biologics all necessitate high-throughput (HT), cellular-level electrophysiological interrogation tools. Optical techniques for actuation and sensing provide instant parallelism, enabling contactless dynamic HT testing of cells and small-tissue constructs, not affordable by other means. Here we show, computationally and experimentally, the limits of all-optical electrophysiology when applied to drug testing, then implement and validate OptoDyCE, a fully automated system for all-optical cardiac electrophysiology. We validate optical actuation by virally introducing optogenetic drivers in rat and human cardiomyocytes or through the modular use of dedicated light-sensitive somatic 'spark' cells. We show that this automated all-optical approach provides HT means of cellular interrogation, that is, allows for dynamic testing of >600 multicellular samples or compounds per hour, and yields high-content information about the action of a drug over time, space and doses.

Optogenetics-enabled assessment of viral gene and cell therapy for restoration of cardiac excitability.

Multiple cardiac pathologies are accompanied by loss of tissue excitability, which leads to a range of heart rhythm disorders (arrhythmias). In addition to electronic device therapy (i.e. implantable pacemakers and cardioverter/defibrillators), biological approaches have recently been explored to restore pacemaking ability and to correct conduction slowing in the heart by delivering excitatory ion channels or ion channel agonists. Using optogenetics as a tool to selectively interrogate only cells transduced to produce an exogenous excitatory ion current, we experimentally and computationally quantify the efficiency of such biological approaches in rescuing cardiac excitability as a function of the mode of application (viral gene delivery or cell delivery) and the geometry of the transduced region (focal or spatially-distributed). We demonstrate that for each configuration (delivery mode and spatial pattern), the optical energy needed to excite can be used to predict therapeutic efficiency of excitability restoration. Taken directly, these results can help guide optogenetic interventions for light-based control of cardiac excitation. More generally, our findings can help optimize gene therapy for restoration of cardiac excitability.

Cardiac Optogenetics: Enhancement by All-trans-Retinal.

All-trans-Retinal (ATR) is a photosensitizer, serving as the chromophore for depolarizing and hyperpolarizing light-sensitive ion channels and pumps (opsins), recently employed as fast optical actuators. In mammalian optogenetic applications (in brain and heart), endogenous ATR availability is not considered a limiting factor, yet it is unclear how ATR modulation may affect the response to optical stimulation. We hypothesized that exogenous ATR may improve light responsiveness of cardiac cells modified by Channelrhodopsin2 (ChR2), hence lowering the optical pacing energy. In virally-transduced (Ad-ChR2(H134R)-eYFP) light-sensitive cardiac syncytium in vitro, ATR supplements ≤2 µM improved cardiomyocyte viability and augmented ChR2 membrane expression several-fold, while >4 µM was toxic. Employing integrated optical actuation (470 nm) and optical mapping, we found that 1-2 µM ATR dramatically reduced optical pacing energy (over 30 times) to several µW/mm(2), lowest values reported to date, but also caused action potential prolongation, minor changes in calcium transients and no change in conduction. Theoretical analysis helped explain ATR-caused reduction of optical excitation threshold in cardiomyocytes. We conclude that cardiomyocytes operate at non-saturating retinal levels, and carefully-dosed exogenous ATR can enhance the performance of ChR2 in cardiac cells and yield energy benefits over orders of magnitude for optogenetic stimulation.

Optical control of excitation waves in cardiac tissue.

In nature, macroscopic excitation waves1,2 are found in a diverse range of settings including chemical reactions, metal rust, yeast, amoeba and the heart and brain. In the case of living biological tissue, the spatiotemporal patterns formed by these excitation waves are different in healthy and diseased states2,3. Current electrical and pharmacological methods for wave modulation lack the spatiotemporal precision needed to control these patterns. Optical methods have the potential to overcome these limitations, but to date have only been demonstrated in simple systems, such as the Belousov-Zhabotinsky chemical reaction4. Here, we combine dye-free optical imaging with optogenetic actuation to achieve dynamic control of cardiac excitation waves. Illumination with patterned light is demonstrated to optically control the direction, speed and spiral chirality of such waves in cardiac tissue. This all-optical approach offers a new experimental platform for the study and control of pattern formation in complex biological excitable systems.

Cardiac applications of optogenetics.

In complex multicellular systems, such as the brain or the heart, the ability to selectively perturb and observe the response of individual components at the cellular level and with millisecond resolution in time, is essential for mechanistic understanding of function. Optogenetics uses genetic encoding of light sensitivity (by the expression of microbial opsins) to provide such capabilities for manipulation, recording, and control by light with cell specificity and high spatiotemporal resolution. As an optical approach, it is inherently scalable for remote and parallel interrogation of biological function at the tissue level; with implantable miniaturized devices, the technique is uniquely suitable for in vivo tracking of function, as illustrated by numerous applications in the brain. Its expansion into the cardiac area has been slow. Here, using examples from published research and original data, we focus on optogenetics applications to cardiac electrophysiology, specifically dealing with the ability to manipulate membrane voltage by light with implications for cardiac pacing, cardioversion, cell communication, and arrhythmia research, in general. We discuss gene and cell delivery methods of inscribing light sensitivity in cardiac tissue, functionality of the light-sensitive ion channels within different types of cardiac cells, utility in probing electrical coupling between different cell types, approaches and design solutions to all-optical electrophysiology by the combination of optogenetic sensors and actuators, and specific challenges in moving towards in vivo cardiac optogenetics.

Optogenetic control of cardiomyocytes via viral delivery.

Optogenetics is an emerging technology for the manipulation and control of excitable tissues, such as the brain and heart. As this technique requires the genetic modification of cells in order to inscribe light sensitivity, for cardiac applications, here we describe the process through which neonatal rat ventricular myocytes are virally infected in vitro with channelrhodopsin-2 (ChR2). We also describe in detail the procedure for quantitatively determining the optimal viral dosage, including instructions for patterning gene expression in multicellular cardiomyocyte preparations (cardiac syncytia) to simulate potential in vivo transgene distributions. Finally, we address optical actuation of ChR2-transduced cells and means to measure their functional response to light.

Computational optogenetics: empirically-derived voltage- and light-sensitive channelrhodopsin-2 model.

Channelrhodospin-2 (ChR2), a light-sensitive ion channel, and its variants have emerged as new excitatory optogenetic tools not only in neuroscience, but also in other areas, including cardiac electrophysiology. An accurate quantitative model of ChR2 is necessary for in silico prediction of the response to optical stimulation in realistic tissue/organ settings. Such a model can guide the rational design of new ion channel functionality tailored to different cell types/tissues. Focusing on one of the most widely used ChR2 mutants (H134R) with enhanced current, we collected a comprehensive experimental data set of the response of this ion channel to different irradiances and voltages, and used these data to develop a model of ChR2 with empirically-derived voltage- and irradiance- dependence, where parameters were fine-tuned via simulated annealing optimization. This ChR2 model offers: 1) accurate inward rectification in the current-voltage response across irradiances; 2) empirically-derived voltage- and light-dependent kinetics (activation, deactivation and recovery from inactivation); and 3) accurate amplitude and morphology of the response across voltage and irradiance settings. Temperature-scaling factors (Q10) were derived and model kinetics was adjusted to physiological temperatures. Using optical action potential clamp, we experimentally validated model-predicted ChR2 behavior in guinea pig ventricular myocytes. The model was then incorporated in a variety of cardiac myocytes, including human ventricular, atrial and Purkinje cell models. We demonstrate the ability of ChR2 to trigger action potentials in human cardiomyocytes at relatively low light levels, as well as the differential response of these cells to light, with the Purkinje cells being most easily excitable and ventricular cells requiring the highest irradiance at all pulse durations. This new experimentally-validated ChR2 model will facilitate virtual experimentation in neural and cardiac optogenetics at the cell and organ level and provide guidance for the development of in vivo tools.

A comprehensive multiscale framework for simulating optogenetics in the heart.

Optogenetics has emerged as an alternative method for electrical control of the heart, where illumination is used to elicit a bioelectric response in tissue modified to express photosensitive proteins (opsins). This technology promises to enable evocation of spatiotemporally precise responses in targeted cells or tissues, thus creating new possibilities for safe and effective therapeutic approaches to ameliorate cardiac function. Here we present a comprehensive framework for multiscale modelling of cardiac optogenetics, allowing both mechanistic examination of optical control and exploration of potential therapeutic applications. The framework incorporates accurate representations of opsin channel kinetics and delivery modes, spatial distribution of photosensitive cells, and tissue illumination constraints, making possible the prediction of emergent behaviour resulting from interactions at sub-organ scales. We apply this framework to explore how optogenetic delivery characteristics determine energy requirements for optical stimulation and to identify cardiac structures that are potential pacemaking targets with low optical excitation thresholds.

Gender differences in electrophysiological gene expression in failing and non-failing human hearts.

The increasing availability of human cardiac tissues for study are critically important in increasing our understanding of the impact of gender, age, and other parameters, such as medications and cardiac disease, on arrhythmia susceptibility. In this study, we aimed to compare the mRNA expression of 89 ion channel subunits, calcium handling proteins, and transcription factors important in cardiac conduction and arrhythmogenesis in the left atria (LA) and ventricles (LV) of failing and nonfailing human hearts of both genders. Total RNA samples, prepared from failing male (n = 9) and female (n = 7), and from nonfailing male (n = 9) and female (n = 9) hearts, were probed using custom-designed Taqman gene arrays. Analyses were performed to explore the relationships between gender, failure state, and chamber expression. Hierarchical cluster analysis revealed chamber specific expression patterns, but failed to identify disease- or gender-dependent clustering. Gender-specific analysis showed lower expression levels in transcripts encoding for K(v)4.3, KChIP2, K(v)1.5, and K(ir)3.1 in the failing female as compared with the male LA. Analysis of LV transcripts, however, did not reveal significant differences based on gender. Overall, our data highlight the differential expression and transcriptional remodeling of ion channel subunits in the human heart as a function of gender and cardiac disease. Furthermore, the availability of such data sets will allow for the development of disease-, gender-, and, most importantly, patient-specific cardiac models, with the ability to utilize such information as mRNA expression to predict cardiac phenotype.

Right ventricular arrhythmogenesis in failing human heart: the role of conduction and repolarization remodeling.

Increased dispersion of repolarization has been suggested to underlie increased arrhythmogenesis in human heart failure (HF). However, no detailed repolarization mapping data were available to support the presence of increased dispersion of repolarization in failing human heart. In the present study, we aimed to determine the existence of enhanced repolarization dispersion in the right ventricular (RV) endocardium from failing human heart and examine its association with arrhythmia inducibility. RV free wall preparations were dissected from five failing and five nonfailing human hearts, cannulated and coronary perfused. RV endocardium was optically mapped from an ∼6.3 × 6.3 cm(2) field of view. Action potential duration (APD), dispersion of APD, and conduction velocity (CV) were quantified for basic cycle lengths (BCL) ranging from 2,000 ms to the functional refractory period. We found that RV APD was significantly prolonged within the failing group compared with the nonfailing group (560 ± 44 vs. 448 ± 39 ms, at BCL = 2,000 ms, P < 0.05). Dispersion of APD was increased in three failing hearts (161 ± 5 vs. 86 ± 19 ms, at BCL = 2,000 ms). APD alternans were induced by rapid pacing in these same three failing hearts. CV was significantly reduced in the failing group compared with the nonfailing group (81 ± 11 vs. 98 ± 8 cm/s, at BCL = 2,000 ms). Arrhythmias could be induced in two failing hearts exhibiting an abnormally steep CV restitution and increased dispersion of repolarization due to APD alternans. Dispersion of repolarization is enhanced across the RV endocardium in the failing human heart. This dispersion, together with APD alternans and abnormal CV restitution, could be responsible for the arrhythmia susceptibility in human HF.

Quantification of fiber orientation in the canine atrial pacemaker complex using optical coherence tomography.

The atrial pacemaker complex is responsible for the initiation and early propagation of cardiac impulses. Optical coherence tomography (OCT), a nondestructive imaging modality with spatial resolutions of ∼1 to 15 µm, can be used to identify unique fiber orientation patterns in this region of the heart. Functionally characterized canine sinoatrial nodes (SAN) (n=7) were imaged using OCT up to ∼1 mm below the endocardial tissue surface. OCT images were directly compared to their corresponding histological sections. Fiber orientation patterns unique to the crista terminalis (CT), SAN, and surrounding atrial myocardium were identified with dominant average fiber angles of 89 ± 12 deg, 110 ± 16 deg, and 95 ± 35 deg, respectively. Both the CT and surrounding atrial myocardium displayed predominantly unidirectionally based fiber orientation patterns within each specimen, whereas the SAN displayed an increased amount of fiber disarray manifested quantitatively as a significantly greater standard deviation in fiber angle distribution within specimens [33 ± 7 deg versus 23 ± 5 deg, atrium (p=0.02); 18 ± 3 deg, CT (p=0.0003)]. We also identified unique, local patterns of fiber orientation specific to the functionally characterized block zone. We demonstrate the ability of OCT in detecting components of the atrial pacemaker complex which are intimately involved in both normal and abnormal cardiac conduction.

Anatomic localization and autonomic modulation of atrioventricular junctional rhythm in failing human hearts.

BACKGROUND: The structure-function relationship in the atrioventricular junction (AVJ) of various animal species has been investigated in detail; however, less is known about the human AVJ. In this study, we performed high-resolution optical mapping of the human AVJ (n = 6) to define its pacemaker properties and response to autonomic stimulation. METHODS AND RESULTS: Isolated, coronary-perfused AVJ preparations from failing human hearts (n = 6, 53 ± 6 years) were optically mapped using the near-infrared, voltage-sensitive dye, di-4-ANBDQBS, with isoproterenol (1 µmol/L) and acetylcholine (1 µmol/L). An algorithm detecting multiple components of optical action potentials was used to reconstruct multilayered intramural AVJ activation and to identify specialized slow and fast conduction pathways (SP and FP). The anatomic origin and propagation of pacemaker activity was verified by histology. Spontaneous AVJ rhythms of 29 ± 11 bpm (n = 6) originated in the nodal-His region (n = 3) and/or the proximal His bundle (n = 4). Isoproterenol accelerated the AVJ rhythm to 69 ± 12 bpm (n = 5); shifted the leading pacemaker to the transitional cell regions near the FP and SP (n = 4) and/or coronary sinus (n = 2); and triggered reentrant arrhythmias (n = 2). Acetylcholine (n = 4) decreased the AVJ rhythm to 18 ± 4 bpm; slowed FP/SP conduction leading to block between the AVJ and atrium; and shifted the pacemaker to either the transitional cell region or the nodal-His region (bifocal activation). CONCLUSIONS: We have demonstrated that the AVJ pacemaker in failing human hearts is located in the nodal-His region or His bundle regions and can be modified with autonomic stimulation. Moreover, we found that both the FP and SP are involved in anterograde and retrograde conduction.

Effects of KATP channel openers diazoxide and pinacidil in coronary-perfused atria and ventricles from failing and non-failing human hearts.

This study compared the effects of ATP-regulated potassium channel (K(ATP)) openers, diazoxide and pinacidil, on diseased and normal human atria and ventricles. We optically mapped the endocardium of coronary-perfused right (n=11) or left (n=2) posterior atrial-ventricular free wall preparations from human hearts with congestive heart failure (CHF, n=8) and non-failing human hearts without (NF, n=3) or with (INF, n=2) infarction. We also analyzed the mRNA expression of the K(ATP) targets K(ir)6.1, K(ir)6.2, SUR1, and SUR2 in the left atria and ventricles of NF (n=8) and CHF (n=4) hearts. In both CHF and INF hearts, diazoxide significantly decreased action potential durations (APDs) in atria (by -21±3% and -27±13%, p<0.01) and ventricles (by -28±7% and -28±4%, p<0.01). Diazoxide did not change APD (0±5%) in NF atria. Pinacidil significantly decreased APDs in both atria (-46 to -80%, p<0.01) and ventricles (-65 to -93%, p<0.01) in all hearts studied. The effect of pinacidil on APD was significantly higher than that of diazoxide in both atria and ventricles of all groups (p<0.05). During pinacidil perfusion, burst pacing induced flutter/fibrillation in all atrial and ventricular preparations with dominant frequencies of 14.4±6.1 Hz and 17.5±5.1 Hz, respectively. Glibenclamide (10 µM) terminated these arrhythmias and restored APDs to control values. Relative mRNA expression levels of K(ATP) targets were correlated to functional observations. Remodeling in response to CHF and/or previous infarct potentiated diazoxide-induced APD shortening. The activation of atrial and ventricular K(ATP) channels enhances arrhythmogenicity, suggesting that such activation may contribute to reentrant arrhythmias in ischemic hearts.

Termination of sustained atrial flutter and fibrillation using low-voltage multiple-shock therapy.

BACKGROUND: Defibrillation therapy for atrial fibrillation (AF) and flutter (AFl) is limited by pain induced by high-energy shocks. Thus, lowering the defibrillation energy for AFl/AF is desirable. OBJECTIVE: In this study we applied low-voltage multiple-shock defibrillation therapy in a rabbit model of atrial tachyarrhythmias comparing its efficacy to single shocks and antitachycardia pacing (ATP). METHODS: Optical mapping was performed in Langendorff-perfused rabbit hearts (n = 18). Acetylcholine (7 ± 5 to 17 ± 16 µM) was administered to promote sustained AFl and AF, respectively. Single and multiple monophasic shocks were applied within 1 or 2 cycle lengths (CLs) of the arrhythmia. RESULTS: We observed AFl (CL = 83 ± 15 ms, n = 17) and AF (CL = 50 ± 8 ms, n = 11). ATP had a success rate of 66.7% in the case of AFl, but no success with AF (n = 9). Low-voltage multiple shocks had 100% success for both arrhythmias. Multiple low-voltage shocks terminated AFl at 0.86 ± 0.73 V/cm (within 1 CL) and 0.28 ± 0.13 V/cm (within 2 CLs), as compared with single shocks at 2.12 ± 1.31 V/cm (P < .001) and AF at 3.46 ± 3 V/cm (within 1 CL), as compared with single shocks at 6.83 ± 3.12 V/cm (P =.06). No ventricular arrhythmias were induced. Optical mapping revealed that termination of AFl was achieved by a properly timed, local shock-induced wave that collides with the arrhythmia wavefront, whereas AF required the majority of atrial tissue to be excited and reset for termination. CONCLUSION: Low-voltage multiple-shock therapy terminates AFl and AF with different mechanisms and thresholds based on spatiotemporal characteristics of the arrhythmias.