Single-cell profiling of transcriptome and histone modifications with EpiDamID.

Recent advances in single-cell sequencing technologies have enabled simultaneous measurement of multiple cellular modalities, but the combined detection of histone post-translational modifications and transcription at single-cell resolution has remained limited. Here, we introduce EpiDamID, an experimental approach to target a diverse set of chromatin types by leveraging the binding specificities of single-chain variable fragment antibodies, engineered chromatin reader domains, and endogenous chromatin-binding proteins. Using these, we render the DamID technology compatible with the genome-wide identification of histone post-translational modifications. Importantly, this includes the possibility to jointly measure chromatin marks and transcription at the single-cell level. We use EpiDamID to profile single-cell Polycomb occupancy in mouse embryoid bodies and provide evidence for hierarchical gene regulatory networks. In addition, we map H3K9me3 in early zebrafish embryogenesis, and detect striking heterochromatic regions specific to notochord. Overall, EpiDamID is a new addition to a vast toolbox to study chromatin states during dynamic cellular processes.

Genome-wide RNA Tomography in the zebrafish embryo.

Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. Although genome-wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes, they are unable to yield spatially resolved information in embryos or tissues. Microscopy-based approaches, using in situ hybridization, for example, can provide spatial information about gene expression, but are limited to analyzing one or a few genes at a time. Here, we present a method where we combine traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. Importantly, our technique enables searching for genes that are expressed in specific spatial patterns without manual image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs.

Ubiad1 is an antioxidant enzyme that regulates eNOS activity by CoQ10 synthesis.

Protection against oxidative damage caused by excessive reactive oxygen species (ROS) by an antioxidant network is essential for the health of tissues, especially in the cardiovascular system. Here, we identified a gene with important antioxidant features by analyzing a null allele of zebrafish ubiad1, called barolo (bar). bar mutants show specific cardiovascular failure due to oxidative stress and ROS-mediated cellular damage. Human UBIAD1 is a nonmitochondrial prenyltransferase that synthesizes CoQ10 in the Golgi membrane compartment. Loss of UBIAD1 reduces the cytosolic pool of the antioxidant CoQ10 and leads to ROS-mediated lipid peroxidation in vascular cells. Surprisingly, inhibition of eNOS prevents Ubiad1-dependent cardiovascular oxidative damage, suggesting a crucial role for this enzyme and nonmitochondrial CoQ10 in NO signaling. These findings identify UBIAD1 as a nonmitochondrial CoQ10-forming enzyme with specific cardiovascular protective function via the modulation of eNOS activity.

Macrophages provide a transient muscle stem cell niche via NAMPT secretion.

Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals1. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages 'dwell' within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.

Spatial transcriptomics reveals asymmetric cellular responses to injury in the regenerating spiny mouse (Acomys) ear.

In contrast to other mammals, the spiny mouse (Acomys) regenerates skin and ear tissue, which includes hair follicles, glands, and cartilage, in a scar-free manner. Ear punch regeneration is asymmetric with only the proximal wound side participating in regeneration. Here, we show that cues originating from the proximal side are required for normal regeneration and use spatially resolved transcriptomics (tomo-seq) to understand the molecular and cellular events underlying this process. Analyzing gene expression across the ear and comparing expression modules between proximal and distal wound sides, we identify asymmetric gene expression patterns and pinpoint regenerative processes in space and time. Moreover, using a comparative approach with nonregenerative rodents (Mus, Meriones), we strengthen a hypothesis in which particularities in the injury-induced immune response may be one of the crucial determinants for why spiny mice regenerate whereas their relatives do not. Our data are available in SpinyMine, an easy-to-use and expandable web-based tool for exploring Acomys regeneration-associated gene expression.

Inherited Ventricular Arrhythmia in Zebrafish: Genetic Models and Phenotyping Tools.

In the last years, the field of inheritable ventricular arrhythmia disease modelling has changed significantly with a push towards the use of novel cellular cardiomyocyte based models. However, there is a growing need for new in vivo models to study the disease pathology at the tissue and organ level. Zebrafish provide an excellent opportunity for in vivo modelling of inheritable ventricular arrhythmia syndromes due to the remarkable similarity between their cardiac electrophysiology and that of humans. Additionally, many state-of-the-art methods in gene editing and electrophysiological phenotyping are available for zebrafish research. In this review, we give a comprehensive overview of the published zebrafish genetic models for primary electrical disorders and arrhythmogenic cardiomyopathy. We summarise and discuss the strengths and weaknesses of the different technical approaches for the generation of genetically modified zebrafish disease models, as well as the electrophysiological approaches in zebrafish phenotyping. By providing this detailed overview, we aim to draw attention to the potential of the zebrafish model for studying arrhythmia syndromes at the organ level and as a platform for personalised medicine and drug testing.

The centriolar satellite protein Cfap53 facilitates formation of the zygotic microtubule organizing center in the zebrafish embryo.

In embryos of most animal species, the zygotic centrosome is assembled by the centriole derived from the sperm cell and pericentriolar proteins present in the oocyte. This zygotic centrosome acts as a microtubule organizing center (MTOC) to assemble the sperm aster and mitotic spindle. As MTOC formation has been studied mainly in adult cells, very little is known about the formation of the zygotic MTOC. Here, we show that zebrafish (Danio rerio) embryos lacking either maternal or paternal Cfap53, a centriolar satellite protein, arrest during the first cell cycle. Although Cfap53 is dispensable for sperm aster function, it aids proper formation of the mitotic spindle. During cell division, Cfap53 colocalizes with γ-tubulin and with other centrosomal and centriolar satellite proteins at the MTOC. Furthermore, we find that γ-tubulin localization at the MTOC is impaired in the absence of Cfap53. Based on these results, we propose a model in which Cfap53 deposited in the oocyte and the sperm participates in the organization of the zygotic MTOC to allow mitotic spindle formation.

Live imaging of adult zebrafish cardiomyocyte proliferation ex vivo.

Zebrafish are excellent at regenerating their heart by reinitiating proliferation in pre-existing cardiomyocytes. Studying how zebrafish achieve this holds great potential in developing new strategies to boost mammalian heart regeneration. Nevertheless, the lack of appropriate live-imaging tools for the adult zebrafish heart has limited detailed studies into the dynamics underlying cardiomyocyte proliferation. Here, we address this by developing a system in which cardiac slices of the injured zebrafish heart are cultured ex vivo for several days while retaining key regenerative characteristics, including cardiomyocyte proliferation. In addition, we show that the cardiac slice culture system is compatible with live timelapse imaging and allows manipulation of regenerating cardiomyocytes with drugs that normally would have toxic effects that prevent their use. Finally, we use the cardiac slices to demonstrate that adult cardiomyocytes with fully assembled sarcomeres can partially disassemble their sarcomeres in a calpain- and proteasome-dependent manner to progress through nuclear division and cytokinesis. In conclusion, we have developed a cardiac slice culture system, which allows imaging of native cardiomyocyte dynamics in real time to discover cellular mechanisms during heart regeneration.

Prrx1b restricts fibrosis and promotes Nrg1-dependent cardiomyocyte proliferation during zebrafish heart regeneration.

Fibroblasts are activated to repair the heart following injury. Fibroblast activation in the mammalian heart leads to a permanent fibrotic scar that impairs cardiac function. In other organisms, such as zebrafish, cardiac injury is followed by transient fibrosis and scar-free regeneration. The mechanisms that drive scarring versus scar-free regeneration are not well understood. Here, we show that the homeobox-containing transcription factor Prrx1b is required for scar-free regeneration of the zebrafish heart as the loss of Prrx1b results in excessive fibrosis and impaired cardiomyocyte proliferation. Through lineage tracing and single-cell RNA sequencing, we find that Prrx1b is activated in epicardial-derived cells where it restricts TGFß ligand expression and collagen production. Furthermore, through combined in vitro experiments in human fetal epicardial-derived cells and in vivo rescue experiments in zebrafish, we conclude that Prrx1 stimulates Nrg1 expression and promotes cardiomyocyte proliferation. Collectively, these results indicate that Prrx1 is a key transcription factor that balances fibrosis and regeneration in the injured zebrafish heart. This article has an associated 'The people behind the papers' interview.

The zebrafish grime mutant uncovers an evolutionarily conserved role for Tmem161b in the control of cardiac rhythm.

The establishment of cardiac function in the developing embryo is essential to ensure blood flow and, therefore, growth and survival of the animal. The molecular mechanisms controlling normal cardiac rhythm remain to be fully elucidated. From a forward genetic screen, we identified a unique mutant, grime, that displayed a specific cardiac arrhythmia phenotype. We show that loss-of-function mutations in tmem161b are responsible for the phenotype, identifying Tmem161b as a regulator of cardiac rhythm in zebrafish. To examine the evolutionary conservation of this function, we generated knockout mice for Tmem161b. Tmem161b knockout mice are neonatal lethal and cardiomyocytes exhibit arrhythmic calcium oscillations. Mechanistically, we find that Tmem161b is expressed at the cell membrane of excitable cells and live imaging shows it is required for action potential repolarization in the developing heart. Electrophysiology on isolated cardiomyocytes demonstrates that Tmem161b is essential to inhibit Ca2+ and K+ currents in cardiomyocytes. Importantly, Tmem161b haploinsufficiency leads to cardiac rhythm phenotypes, implicating it as a candidate gene in heritable cardiac arrhythmia. Overall, these data describe Tmem161b as a highly conserved regulator of cardiac rhythm that functions to modulate ion channel activity in zebrafish and mice.

T-box transcription factor 3 governs a transcriptional program for the function of the mouse atrioventricular conduction system.

Genome-wide association studies have identified noncoding variants near TBX3 that are associated with PR interval and QRS duration, suggesting that subtle changes in TBX3 expression affect atrioventricular conduction system function. To explore whether and to what extent the atrioventricular conduction system is affected by Tbx3 dose reduction, we first characterized electrophysiological properties and morphology of heterozygous Tbx3 mutant (Tbx3+/-) mouse hearts. We found PR interval shortening and prolonged QRS duration, as well as atrioventricular bundle hypoplasia after birth in heterozygous mice. The atrioventricular node size was unaffected. Transcriptomic analysis of atrioventricular nodes isolated by laser capture microdissection revealed hundreds of deregulated genes in Tbx3+/- mutants. Notably, Tbx3+/- atrioventricular nodes showed increased expression of working myocardial gene programs (mitochondrial and metabolic processes, muscle contractility) and reduced expression of pacemaker gene programs (neuronal, Wnt signaling, calcium/ion channel activity). By integrating chromatin accessibility profiles (ATAC sequencing) of atrioventricular tissue and other epigenetic data, we identified Tbx3-dependent atrioventricular regulatory DNA elements (REs) on a genome-wide scale. We used transgenic reporter assays to determine the functionality of candidate REs near Ryr2, an up-regulated chamber-enriched gene, and in Cacna1g, a down-regulated conduction system-specific gene. Using genome editing to delete candidate REs, we showed that a strong intronic bipartite RE selectively governs Cacna1g expression in the conduction system in vivo. Our data provide insights into the multifactorial Tbx3-dependent transcriptional network that regulates the structure and function of the cardiac conduction system, which may underlie the differences in PR duration and QRS interval between individuals carrying variants in the TBX3 locus.

The zebrafish cohesin protein Sgo1 is required for cardiac function and eye development.

BACKGROUND: Cohesinopathies is a term that refers to/covers rare genetic diseases caused by mutations in the cohesin complex proteins. The cohesin complex is a multiprotein complex that facilitates different aspects of cell division, gene transcription, DNA damage repair, and chromosome architecture. Shugoshin proteins prevent the cohesin complex from premature dissociation from chromatids during cell division. Patients with a homozygous missense mutation in SGO1, which encodes for Shugoshin1, have problems with normal pacing of the heart and gut. RESULTS: To study the role of shugoshin during embryo development, we mutated the zebrafish sgo1 gene. Homozygous sgo1 mutant embryos display various phenotypes related to different organs, including a reduced heart rate accompanied by reduced cardiac function. In addition, sgo1 mutants are vision-impaired as a consequence of structurally defective and partially non-functional photoreceptor cells. Furthermore, the sgo1 mutants display reduced food intake and early lethality. CONCLUSION: We have generated a zebrafish model of Sgo1 that showed its importance during organ development and function.

Is zebrafish heart regeneration "complete"? Lineage-restricted cardiomyocytes proliferate to pre-injury numbers but some fail to differentiate in fibrotic hearts.

Adult zebrafish are frequently described to be able to "completely" regenerate the heart. Yet, the extent to which cardiomyocytes lost to injury are replaced is unknown, since existing evidence for cardiomyocyte proliferation is indirect or non-quantitative. We established stereological methods to quantify the number of cardiomyocytes at several time-points post cryoinjury. Intriguingly, after cryoinjuries that killed about 1/3 of the ventricular cardiomyocytes, pre-injury cardiomyocyte numbers were restored already within 30 days. Yet, many hearts retained small residual scars, and a subset of cardiomyocytes bordering these fibrotic areas remained smaller, lacked differentiated sarcomeric structures, and displayed defective calcium signaling. Thus, a subset of regenerated cardiomyocytes failed to fully mature. While lineage-tracing experiments have shown that regenerating cardiomyocytes are derived from differentiated cardiomyocytes, technical limitations have previously made it impossible to test whether cardiomyocyte trans-differentiation contributes to regeneration of non-myocyte cell lineages. Using Cre responder lines that are expressed in all major cell types of the heart, we found no evidence for cardiomyocyte transdifferentiation into endothelial, epicardial, fibroblast or immune cell lineages. Overall, our results imply a refined answer to the question whether zebrafish can completely regenerate the heart: in response to cryoinjury, preinjury cardiomyocyte numbers are indeed completely regenerated by proliferation of lineage-restricted cardiomyocytes, while restoration of cardiomyocyte differentiation and function, as well as resorption of scar tissue, is less robustly achieved.

Genome-Wide Analysis Identifies an Essential Human TBX3 Pacemaker Enhancer.

RATIONALE: The development and function of the pacemaker cardiomyocytes of the sinoatrial node (SAN), the leading pacemaker of the heart, are tightly controlled by a conserved network of transcription factors, including TBX3 (T-box transcription factor 3), ISL1 (ISL LIM homeobox 1), and SHOX2 (short stature homeobox 2). Yet, the regulatory DNA elements (REs) controlling target gene expression in the SAN pacemaker cells have remained undefined. OBJECTIVE: Identification of the regulatory landscape of human SAN-like pacemaker cells and functional assessment of SAN-specific REs potentially involved in pacemaker cell gene regulation. METHODS AND RESULTS: We performed Assay for Transposase-Accessible Chromatin using sequencing on human pluripotent stem cell-derived SAN-like pacemaker cells and ventricle-like cells and identified thousands of putative REs specific for either human cell type. We validated pacemaker cell-specific elements in the SHOX2 and TBX3 loci. CRISPR-mediated homozygous deletion of the mouse ortholog of a noncoding region with candidate pacemaker-specific REs in the SHOX2 locus resulted in selective loss of Shox2 expression from the developing SAN and embryonic lethality. Putative pacemaker-specific REs were identified up to 1 Mbp upstream of TBX3 in a region close to MED13L harboring variants associated with heart rate recovery after exercise. The orthologous region was deleted in mice, which resulted in selective loss of expression of Tbx3 from the SAN and (cardiac) ganglia and in neonatal lethality. Expression of Tbx3 was maintained in other tissues including the atrioventricular conduction system, lungs, and liver. Heterozygous adult mice showed increased SAN recovery times after pacing. The human REs harboring the associated variants robustly drove expression in the SAN of transgenic mouse embryos. CONCLUSIONS: We provided a genome-wide collection of candidate human pacemaker-specific REs, including the loci of SHOX2, TBX3, and ISL1, and identified a link between human genetic variants influencing heart rate recovery after exercise and a variant RE with highly conserved function, driving SAN expression of TBX3.

Cardiac regenerative capacity: an evolutionary afterthought?

Cardiac regeneration is the outcome of the highly regulated interplay of multiple processes, including the inflammatory response, cardiomyocyte dedifferentiation and proliferation, neovascularization and extracellular matrix turnover. Species-specific traits affect these injury-induced processes, resulting in a wide variety of cardiac regenerative potential between species. Indeed, while mammals are generally considered poor regenerators, certain amphibian and fish species like the zebrafish display robust regenerative capacity post heart injury. The species-specific traits underlying these differential injury responses are poorly understood. In this review, we will compare the injury induced processes of the mammalian and zebrafish heart, describing where these processes overlap and diverge. Additionally, by examining multiple species across the animal kingdom, we will highlight particular traits that either positively or negatively affect heart regeneration. Last, we will discuss the possibility of overcoming regeneration-limiting traits to induce heart regeneration in mammals.

GLS hyperactivity causes glutamate excess, infantile cataract and profound developmental delay.

Loss-of-function mutations in glutaminase (GLS), the enzyme converting glutamine into glutamate, and the counteracting enzyme glutamine synthetase (GS) cause disturbed glutamate homeostasis and severe neonatal encephalopathy. We report a de novo Ser482Cys gain-of-function variant in GLS encoding GLS associated with profound developmental delay and infantile cataract. Functional analysis demonstrated that this variant causes hyperactivity and compensatory downregulation of GLS expression combined with upregulation of the counteracting enzyme GS, supporting pathogenicity. Ser482Cys-GLS likely improves the electrostatic environment of the GLS catalytic site, thereby intrinsically inducing hyperactivity. Alignment of +/-12.000 GLS protein sequences from >1000 genera revealed extreme conservation of Ser482 to the same degree as catalytic residues. Together with the hyperactivity, this indicates that Ser482 is evolutionarily preserved to achieve optimal-but submaximal-GLS activity. In line with GLS hyperactivity, increased glutamate and decreased glutamine concentrations were measured in urine and fibroblasts. In the brain (both grey and white matter), glutamate was also extremely high and glutamine was almost undetectable, demonstrated with magnetic resonance spectroscopic imaging at clinical field strength and subsequently supported at ultra-high field strength. Considering the neurotoxicity of glutamate when present in excess, the strikingly high glutamate concentrations measured in the brain provide an explanation for the developmental delay. Cataract, a known consequence of oxidative stress, was evoked in zebrafish expressing the hypermorphic Ser482Cys-GLS and could be alleviated by inhibition of GLS. The capacity to detoxify reactive oxygen species was reduced upon Ser482Cys-GLS expression, providing an explanation for cataract formation. In conclusion, we describe an inborn error of glutamate metabolism caused by a GLS hyperactivity variant, illustrating the importance of balanced GLS activity.

Notch and Bmp signaling pathways act coordinately during the formation of the proepicardium.

BACKGROUND: The epicardium is the outer mesothelial layer of the heart. It encloses the myocardium and plays key roles in heart development and regeneration. It derives from the proepicardium (PE), cell clusters that appear in the dorsal pericardium (DP) close to the atrioventricular canal and the venous pole of the heart, and are released into the pericardial cavity. PE cells are advected around the beating heart until they attach to the myocardium. Bmp and Notch signaling influence PE formation, but it is unclear how both signaling pathways interact during this process in the zebrafish. RESULTS: Here, we show that the developing PE is influenced by Notch signaling derived from the endothelium. Overexpression of the intracellular receptor of notch in the endothelium enhances bmp expression, increases the number of pSmad1/5 positive cells in the DP and PE, and enhances PE formation. On the contrary, pharmacological inhibition of Notch1 impairs PE formation. bmp2b overexpression can rescue loss of PE formation in the presence of a Notch1 inhibitor, but Notch gain-of-function could not recover PE formation in the absence of Bmp signaling. CONCLUSIONS: Endothelial Notch signaling activates bmp expression in the heart tube, which in turn induces PE cluster formation from the DP layer.

Conserved NPPB+ Border Zone Switches From MEF2- to AP-1-Driven Gene Program.

BACKGROUND: Surviving cells in the postinfarction border zone are subjected to intense fluctuations of their microenvironment. Recently, border zone cardiomyocytes have been specifically implicated in cardiac regeneration. Here, we defined their unique transcriptional and regulatory properties, and comprehensively validated new molecular markers, including Nppb, encoding B-type natriuretic peptide, after infarction. METHODS: Transgenic reporter mice were used to identify the Nppb-positive border zone after myocardial infarction. Transcriptome analysis of remote, border, and infarct zones and of purified cardiomyocyte nuclei was performed using RNA-sequencing. Top candidate genes displaying border zone spatial specificity were histologically validated in ischemic human hearts. Mice in which Nppb was deleted by genome editing were subjected to myocardial infarction. Chromatin accessibility landscapes of border zone and control cardiomyocyte nuclei were assessed by using assay for transposase-accessible chromatin using sequencing. RESULTS: We identified the border zone as a spatially confined region transcriptionally distinct from the remote myocardium. The transcriptional response of the border zone was much stronger than that of the remote ventricular wall, involving acute downregulation of mitochondrial oxidative phosphorylation, fatty acid metabolism, calcium handling, and sarcomere function, and the activation of a stress-response program. Analysis of infarcted human hearts revealed that the transcriptionally discrete border zone is conserved in humans, and led to the identification of novel conserved border zone markers including NPPB, ANKRD1, DES, UCHL1, JUN, and FOXP1. Homozygous Nppb mutant mice developed acute and lethal heart failure after myocardial infarction, indicating that B-type natriuretic peptide is required to preserve postinfarct heart function. Assay for transposase-accessible chromatin using sequencing revealed thousands of cardiomyocyte lineage-specific MEF2-occupied regulatory elements that lost accessibility in the border zone. Putative injury-responsive enhancers that gained accessibility were highly associated with AP-1 (activator protein 1) binding sites. Nuclear c-Jun, a component of AP-1, was observed specifically in border zone cardiomyocytes. CONCLUSIONS: Cardiomyocytes in a discrete zone bordering the infarct switch from a MEF2-driven homeostatic lineage-specific to an AP-1-driven injury-induced gene expression program. This program is conserved between mouse and human, and includes Nppb expression, which is required to prevent acute heart failure after infarction.

MUSCLEMOTION: A Versatile Open Software Tool to Quantify Cardiomyocyte and Cardiac Muscle Contraction In Vitro and In Vivo.

RATIONALE: There are several methods to measure cardiomyocyte and muscle contraction, but these require customized hardware, expensive apparatus, and advanced informatics or can only be used in single experimental models. Consequently, data and techniques have been difficult to reproduce across models and laboratories, analysis is time consuming, and only specialist researchers can quantify data. OBJECTIVE: Here, we describe and validate an automated, open-source software tool (MUSCLEMOTION) adaptable for use with standard laboratory and clinical imaging equipment that enables quantitative analysis of normal cardiac contraction, disease phenotypes, and pharmacological responses. METHODS AND RESULTS: MUSCLEMOTION allowed rapid and easy measurement of movement from high-speed movies in (1) 1-dimensional in vitro models, such as isolated adult and human pluripotent stem cell-derived cardiomyocytes; (2) 2-dimensional in vitro models, such as beating cardiomyocyte monolayers or small clusters of human pluripotent stem cell-derived cardiomyocytes; (3) 3-dimensional multicellular in vitro or in vivo contractile tissues, such as cardiac "organoids," engineered heart tissues, and zebrafish and human hearts. MUSCLEMOTION was effective under different recording conditions (bright-field microscopy with simultaneous patch-clamp recording, phase contrast microscopy, and traction force microscopy). Outcomes were virtually identical to the current gold standards for contraction measurement, such as optical flow, post deflection, edge-detection systems, or manual analyses. Finally, we used the algorithm to quantify contraction in in vitro and in vivo arrhythmia models and to measure pharmacological responses. CONCLUSIONS: Using a single open-source method for processing video recordings, we obtained reliable pharmacological data and measures of cardiac disease phenotype in experimental cell, animal, and human models.