POPDC1 Variants Cause Atrioventricular Node Dysfunction and Arrhythmogenic Changes in Cardiac Electrophysiology and Intracellular Calcium Handling in Zebrafish.

Popeye domain-containing (POPDC) proteins selectively bind cAMP and mediate cellular responses to sympathetic nervous system (SNS) stimulation. The first discovered human genetic variant (POPDC1S201F) is associated with atrioventricular (AV) block, which is exacerbated by increased SNS activity. Zebrafish carrying the homologous mutation (popdc1S191F) display a similar phenotype to humans. To investigate the impact of POPDC1 dysfunction on cardiac electrophysiology and intracellular calcium handling, homozygous popdc1S191F and popdc1 knock-out (popdc1KO) zebrafish larvae and adult isolated popdc1S191F hearts were studied by functional fluorescent analysis. It was found that in popdc1S191F and popdc1KO larvae, heart rate (HR), AV delay, action potential (AP) and calcium transient (CaT) upstroke speed, and AP duration were less than in wild-type larvae, whereas CaT duration was greater. SNS stress by ß-adrenergic receptor stimulation with isoproterenol increased HR, lengthened AV delay, slowed AP and CaT upstroke speed, and shortened AP and CaT duration, yet did not result in arrhythmias. In adult popdc1S191F zebrafish hearts, there was a higher incidence of AV block, slower AP upstroke speed, and longer AP duration compared to wild-type hearts, with no differences in CaT. SNS stress increased AV delay and led to further AV block in popdc1S191F hearts while decreasing AP and CaT duration. Overall, we have revealed that arrhythmogenic effects of POPDC1 dysfunction on cardiac electrophysiology and intracellular calcium handling in zebrafish are varied, but already present in early development, and that AV node dysfunction may underlie SNS-induced arrhythmogenesis associated with popdc1 mutation in adults.

Suppressors of cGAS-STING are downregulated during fin-limb regeneration and aging in aquatic vertebrates.

During the early stages of limb and fin regeneration in aquatic vertebrates (i.e., fishes and amphibians), blastema undergo transcriptional rewiring of innate immune signaling pathways to promote immune cell recruitment. In mammals, a fundamental component of innate immune signaling is the cytosolic DNA sensing pathway, cGAS-STING. However, to what extent the cGAS-STING pathway influences regeneration in aquatic anamniotes is unknown. In jawed vertebrates, negative regulation of cGAS-STING activity is accomplished by suppressors of cytosolic DNA such as Trex1, Pml, and PML-like exon 9 (Plex9) exonucleases. Here, we examine the expression of these suppressors of cGAS-STING, as well as inflammatory genes and cGAS activity during caudal fin and limb regeneration using the spotted gar (Lepisosteus oculatus) and axolotl (Ambystoma mexicanum) model species, and during age-related senescence in zebrafish (Danio rerio). In the regenerative blastema of wounded gar and axolotl, we observe increased inflammatory gene expression, including interferon genes and interleukins 6 and 8. We also observed a decrease in axolotl Trex1 and gar pml expression during the early phases of wound healing which correlates with a dramatic increase in cGAS activity. In contrast, the plex9.1 gene does not change in expression during wound healing in gar. However, we observed decreased expression of plex9.1 in the senescing cardiac tissue of aged zebrafish, where 2'3'-cGAMP levels are elevated. Finally, we demonstrate a similar pattern of Trex1, pml, and plex9.1 gene regulation across species in response to exogenous 2'3'-cGAMP. Thus, during the early stages of limb-fin regeneration, Pml, Trex1, and Plex9.1 exonucleases are downregulated, presumably to allow an evolutionarily ancient cGAS-STING activity to promote inflammation and the recruitment of immune cells.

The in vivo study of cardiac mechano-electric and mechano-mechanical coupling during heart development in zebrafish.

In the adult heart, acute adaptation of electrical and mechanical activity to changes in mechanical load occurs via feedback processes known as "mechano-electric coupling" and "mechano-mechanical coupling." Whether this occurs during cardiac development is ill-defined, as acutely altering the heart's mechanical load while measuring functional responses in traditional experimental models is difficult, as embryogenesis occurs in utero, making the heart inaccessible. These limitations can be overcome with zebrafish, as larvae develop in a dish and are nearly transparent, allowing for in vivo manipulation and measurement of cardiac structure and function. Here we present a novel approach for the in vivo study of mechano-electric and mechano-mechanical coupling in the developing zebrafish heart. This innovative methodology involves acute in vivo atrial dilation (i.e., increased atrial preload) in larval zebrafish by injection of a controlled volume into the venous circulation immediately upstream of the heart, combined with optical measurement of the acute electrical (change in heart rate) and mechanical (change in stroke area) response. In proof-of-concept experiments, we applied our new method to 48 h post-fertilisation zebrafish, which revealed differences between the electrical and mechanical response to atrial dilation. In response to an acute increase in atrial preload there is a large increase in atrial stroke area but no change in heart rate, demonstrating that in contrast to the fully developed heart, during early cardiac development mechano-mechanical coupling alone drives the adaptive increase in atrial output. Overall, in this methodological paper we present our new experimental approach for the study of mechano-electric and mechano-mechanical coupling during cardiac development and demonstrate its potential for understanding the essential adaptation of heart function to acute changes in mechanical load.

Cell-contact-mediated assembly of contractile airway smooth muscle rings.

Microtissues in the shape of toroidal rings provide an ideal geometry to better represent the structure and function of the airway smooth muscle present in the small airways, and to better understand diseases such as asthma. Here, polydimethylsiloxane devices consisting of a series of circular channels surrounding central mandrels are used to form microtissues in the shape of toroidal rings by way of the self-aggregation and -assembly of airway smooth muscle cell (ASMC) suspensions. Over time, the ASMCs present in the rings become spindle-shaped and axially align along the ring circumference. Ring strength and elastic modulus increase over 14 d in culture, without significant changes in ring size. Gene expression analysis indicates stable expression of mRNA for extracellular matrix-associated proteins, including collagen I and lamininsα1 andα4 over 21 d in culture. Cells within the rings respond to TGF-ß1 treatment, leading to dramatic decreases in ring circumference, with increases in mRNA and protein levels for extracellular matrix and contraction-associated markers. These data demonstrate the utility of ASMC rings as a platform for modeling diseases of the small airways such as asthma.

Guidelines for assessment of cardiac electrophysiology and arrhythmias in small animals.

Cardiac arrhythmias are a major cause of morbidity and mortality worldwide. Although recent advances in cell-based models, including human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM), are contributing to our understanding of electrophysiology and arrhythmia mechanisms, preclinical animal studies of cardiovascular disease remain a mainstay. Over the past several decades, animal models of cardiovascular disease have advanced our understanding of pathological remodeling, arrhythmia mechanisms, and drug effects and have led to major improvements in pacing and defibrillation therapies. There exist a variety of methodological approaches for the assessment of cardiac electrophysiology and a plethora of parameters may be assessed with each approach. This guidelines article will provide an overview of the strengths and limitations of several common techniques used to assess electrophysiology and arrhythmia mechanisms at the whole animal, whole heart, and tissue level with a focus on small animal models. We also define key electrophysiological parameters that should be assessed, along with their physiological underpinnings, and the best methods with which to assess these parameters.

Drivers of Sinoatrial Node Automaticity in Zebrafish: Comparison With Mechanisms of Mammalian Pacemaker Function.

Cardiac excitation originates in the sinoatrial node (SAN), due to the automaticity of this distinct region of the heart. SAN automaticity is the result of a gradual depolarisation of the membrane potential in diastole, driven by a coupled system of transarcolemmal ion currents and intracellular Ca2+ cycling. The frequency of SAN excitation determines heart rate and is under the control of extra- and intracardiac (extrinsic and intrinsic) factors, including neural inputs and responses to tissue stretch. While the structure, function, and control of the SAN have been extensively studied in mammals, and some critical aspects have been shown to be similar in zebrafish, the specific drivers of zebrafish SAN automaticity and the response of its excitation to vagal nerve stimulation and mechanical preload remain incompletely understood. As the zebrafish represents an important alternative experimental model for the study of cardiac (patho-) physiology, we sought to determine its drivers of SAN automaticity and the response to nerve stimulation and baseline stretch. Using a pharmacological approach mirroring classic mammalian experiments, along with electrical stimulation of intact cardiac vagal nerves and the application of mechanical preload to the SAN, we demonstrate that the principal components of the coupled membrane- Ca2+ pacemaker system that drives automaticity in mammals are also active in the zebrafish, and that the effects of extra- and intracardiac control of heart rate seen in mammals are also present. Overall, these results, combined with previously published work, support the utility of the zebrafish as a novel experimental model for studies of SAN (patho-) physiological function.

What keeps us ticking? Sinoatrial node mechano-sensitivity: the grandfather clock of cardiac rhythm.

The rhythmic and spontaneously generated electrical excitation that triggers the heartbeat originates in the sinoatrial node (SAN). SAN automaticity has been thoroughly investigated, which has uncovered fundamental mechanisms involved in cardiac pacemaking that are generally categorised into two interacting and overlapping systems: the 'membrane' and 'Ca2+ clock'. The principal focus of research has been on these two systems of oscillators, which have been studied primarily in single cells and isolated tissue, experimental preparations that do not consider mechanical factors present in the whole heart. SAN mechano-sensitivity has long been known to be a contributor to SAN pacemaking-both as a driver and regulator of automaticity-but its essential nature has been underappreciated. In this review, following a description of the traditional 'clocks' of SAN automaticity, we describe mechanisms of SAN mechano-sensitivity and its vital role for SAN function, making the argument that the 'mechanics oscillator' is, in fact, the 'grandfather clock' of cardiac rhythm.

From Mice to Mainframes: Experimental Models for Investigation of the Intracardiac Nervous System.

The intracardiac nervous system (IcNS), sometimes referred to as the "little brain" of the heart, is involved in modulating many aspects of cardiac physiology. In recent years our fundamental understanding of autonomic control of the heart has drastically improved, and the IcNS is increasingly being viewed as a therapeutic target in cardiovascular disease. However, investigations of the physiology and specific roles of intracardiac neurons within the neural circuitry mediating cardiac control has been hampered by an incomplete knowledge of the anatomical organisation of the IcNS. A more thorough understanding of the IcNS is hoped to promote the development of new, highly targeted therapies to modulate IcNS activity in cardiovascular disease. In this paper, we first provide an overview of IcNS anatomy and function derived from experiments in mammals. We then provide descriptions of alternate experimental models for investigation of the IcNS, focusing on a non-mammalian model (zebrafish), neuron-cardiomyocyte co-cultures, and computational models to demonstrate how the similarity of the relevant processes in each model can help to further our understanding of the IcNS in health and disease.

Genomic and physiological analyses of the zebrafish atrioventricular canal reveal molecular building blocks of the secondary pacemaker region.

The atrioventricular canal (AVC) is the site where key structures responsible for functional division between heart regions are established, most importantly, the atrioventricular (AV) conduction system and cardiac valves. To elucidate the mechanism underlying AVC development and function, we utilized transgenic zebrafish line sqet31Et expressing EGFP in the AVC to isolate this cell population and profile its transcriptome at 48 and 72 hpf. The zebrafish AVC transcriptome exhibits hallmarks of mammalian AV node, including the expression of genes implicated in its development and those encoding connexins forming low conductance gap junctions. Transcriptome analysis uncovered protein-coding and noncoding transcripts enriched in AVC, which have not been previously associated with this structure, as well as dynamic expression of epithelial-to-mesenchymal transition markers and components of TGF-ß, Notch, and Wnt signaling pathways likely reflecting ongoing AVC and valve development. Using transgenic line Tg(myl7:mermaid) encoding voltage-sensitive fluorescent protein, we show that abolishing the pacemaker-containing sinoatrial ring (SAR) through Isl1 loss of function resulted in spontaneous activation in the AVC region, suggesting that it possesses inherent automaticity although insufficient to replace the SAR. The SAR and AVC transcriptomes express partially overlapping species of ion channels and gap junction proteins, reflecting their distinct roles. Besides identifying conserved aspects between zebrafish and mammalian conduction systems, our results established molecular hallmarks of the developing AVC which underlies its role in structural and electrophysiological separation between heart chambers. This data constitutes a valuable resource for studying AVC development and function, and identification of novel candidate genes implicated in these processes.

Cardiac Mechano-Electric Coupling: Acute Effects of Mechanical Stimulation on Heart Rate and Rhythm.

The heart is vital for biological function in almost all chordates, including humans. It beats continually throughout our life, supplying the body with oxygen and nutrients while removing waste products. If it stops, so does life. The heartbeat involves precise coordination of the activity of billions of individual cells, as well as their swift and well-coordinated adaption to changes in physiological demand. Much of the vital control of cardiac function occurs at the level of individual cardiac muscle cells, including acute beat-by-beat feedback from the local mechanical environment to electrical activity (as opposed to longer term changes in gene expression and functional or structural remodeling). This process is known as mechano-electric coupling (MEC). In the current review, we present evidence for, and implications of, MEC in health and disease in human; summarize our understanding of MEC effects gained from whole animal, organ, tissue, and cell studies; identify potential molecular mediators of MEC responses; and demonstrate the power of computational modeling in developing a more comprehensive understanding of ?what makes the heart tick.Ë®.

Seeing the Light: The Use of Zebrafish for Optogenetic Studies of the Heart.

Optogenetics, involving the optical measurement and manipulation of cellular activity with genetically encoded light-sensitive proteins ("reporters" and "actuators"), is a powerful experimental technique for probing (patho-)physiological function. Originally developed as a tool for neuroscience, it has now been utilized in cardiac research for over a decade, providing novel insight into the electrophysiology of the healthy and diseased heart. Among the pioneering cardiac applications of optogenetic actuators were studies in zebrafish, which first demonstrated their use for precise spatiotemporal control of cardiac activity. Zebrafish were also adopted early as an experimental model for the use of optogenetic reporters, including genetically encoded voltage- and calcium-sensitive indicators. Beyond optogenetic studies, zebrafish are becoming an increasingly important tool for cardiac research, as they combine many of the advantages of integrative and reduced experimental models. The zebrafish has striking genetic and functional cardiac similarities to that of mammals, its genome is fully sequenced and can be modified using standard techniques, it has been used to recapitulate a variety of cardiac diseases, and it allows for high-throughput investigations. For optogenetic studies, zebrafish provide additional advantages, as the whole zebrafish heart can be visualized and interrogated in vivo in the transparent, externally developing embryo, and the relatively small adult heart allows for in situ cell-specific observation and control not possible in mammals. With the advent of increasingly sophisticated fluorescence imaging approaches and methods for spatially-resolved light stimulation in the heart, the zebrafish represents an experimental model with unrealized potential for cardiac optogenetic studies. In this review we summarize the use of zebrafish for optogenetic investigations in the heart, highlighting their specific advantages and limitations, and their potential for future cardiac research.

Sinoatrial Node Structure, Mechanics, Electrophysiology and the Chronotropic Response to Stretch in Rabbit and Mouse.

The rhythmic electrical activity of the heart's natural pacemaker, the sinoatrial node (SAN), determines cardiac beating rate (BR). SAN electrical activity is tightly controlled by multiple factors, including tissue stretch, which may contribute to adaptation of BR to changes in venous return. In most animals, including human, there is a robust increase in BR when the SAN is stretched. However, the chronotropic response to sustained stretch differs in mouse SAN, where it causes variable responses, including decreased BR. The reasons for this species difference are unclear. They are thought to relate to dissimilarities in SAN electrophysiology (particularly action potential morphology) between mouse and other species and to how these interact with subcellular stretch-activated mechanisms. Furthermore, species-related differences in structural and mechanical properties of the SAN may influence the chronotropic response to SAN stretch. Here we assess (i) how the BR response to sustained stretch of rabbit and mouse isolated SAN relates to tissue stiffness, (ii) whether structural differences could account for observed differences in BR responsiveness to stretch, and (iii) whether pharmacological modification of mouse SAN electrophysiology alters stretch-induced chronotropy. We found disparities in the relationship between SAN stiffness and the magnitude of the chronotropic response to stretch between rabbit and mouse along with differences in SAN collagen structure, alignment, and changes with stretch. We further observed that pharmacological modification to prolong mouse SAN action potential plateau duration rectified the direction of BR changes during sustained stretch, resulting in a positive chronotropic response akin to that of other species. Overall, our results suggest that structural, mechanical, and background electrophysiological properties of the SAN influence the chronotropic response to stretch. Improved insight into the biophysical determinants of stretch effects on SAN pacemaking is essential for a comprehensive understanding of SAN regulation with important implications for studies of SAN physiology and its dysfunction, such as in the aging and fibrotic heart.

Ischemia Enhances the Acute Stretch-Induced Increase in Calcium Spark Rate in Ventricular Myocytes.

Introduction: In ventricular myocytes, spontaneous release of calcium (Ca2+) from the sarcoplasmic reticulum via ryanodine receptors ("Ca2+ sparks") is acutely increased by stretch, due to a stretch-induced increase of reactive oxygen species (ROS). In acute regional ischemia there is stretch of ischemic tissue, along with an increase in Ca2+ spark rate and ROS production, each of which has been implicated in arrhythmogenesis. Yet, whether there is an impact of ischemia on the stretch-induced increase in Ca2+ sparks and ROS has not been investigated. We hypothesized that ischemia would enhance the increase of Ca2+ sparks and ROS that occurs with stretch. Methods: Isolated ventricular myocytes from mice (male, C57BL/6J) were loaded with fluorescent dye to detect Ca2+ sparks (4.6 µM Fluo-4, 10 min) or ROS (1 µM DCF, 20 min), exposed to normal Tyrode (NT) or simulated ischemia (SI) solution (hyperkalemia [15 mM potassium], acidosis [6.5 pH], and metabolic inhibition [1 mM sodium cyanide, 20 mM 2-deoxyglucose]), and subjected to sustained stretch by the carbon fiber technique (~10% increase in sarcomere length, 15 s). Ca2+ spark rate and rate of ROS production were measured by confocal microscopy. Results: Baseline Ca2+ spark rate was greater in SI (2.54 ± 0.11 sparks·s-1·100 µm-2; n = 103 cells, N = 10 mice) than NT (0.29 ± 0.05 sparks·s-1·100 µm-2; n = 33 cells, N = 9 mice; p < 0.0001). Stretch resulted in an acute increase in Ca2+ spark rate in both SI (3.03 ± 0.13 sparks·s-1·100 µm-2; p < 0.0001) and NT (0.49 ± 0.07 sparks·s-1·100 µm-2; p < 0.0001), with the increase in SI being greater than NT (+0.49 ± 0.04 vs. +0.20 ± 0.04 sparks·s-1·100 µm-2; p < 0.0001). Baseline rate of ROS production was also greater in SI (1.01 ± 0.01 normalized slope; n = 11, N = 8 mice) than NT (0.98 ± 0.01 normalized slope; n = 12, N = 4 mice; p < 0.05), but there was an acute increase with stretch only in SI (+12.5 ± 2.6%; p < 0.001). Conclusion: Ischemia enhances the stretch-induced increase of Ca2+ sparks in ventricular myocytes, with an associated enhancement of stretch-induced ROS production. This effect may be important for premature excitation and/or in the development of an arrhythmogenic substrate in acute regional ischemia.

Neurohumoral Control of Sinoatrial Node Activity and Heart Rate: Insight From Experimental Models and Findings From Humans.

The sinoatrial node is perhaps one of the most important tissues in the entire body: it is the natural pacemaker of the heart, making it responsible for initiating each-and-every normal heartbeat. As such, its activity is heavily controlled, allowing heart rate to rapidly adapt to changes in physiological demand. Control of sinoatrial node activity, however, is complex, occurring through the autonomic nervous system and various circulating and locally released factors. In this review we discuss the coupled-clock pacemaker system and how its manipulation by neurohumoral signaling alters heart rate, considering the multitude of canonical and non-canonical agents that are known to modulate sinoatrial node activity. For each, we discuss the principal receptors involved and known intracellular signaling and protein targets, highlighting gaps in our knowledge and understanding from experimental models and human studies that represent areas for future research.

Age-associated changes in electrical function of the zebrafish heart.

Age-associated changes in cardiac structure and function have been observed from the molecular to whole organ level in humans and mammalian models. Understanding the mechanisms involved is important for explaining the development of cardiac disease with age and developing novel strategies for its treatment and prevention. The zebrafish represents a powerful model for cardiovascular research, as various cardiac pathologies have been recapitulated, it is easily genetically modified, and it is a relatively low cost and high-throughput option. In aged zebrafish, myocyte hypertrophy, increased ventricular density and fibrosis, valvular lesions, and reductions in coronary vasculature have been described. The functional consequences of these structural changes however, are relatively unknown. In the current study, we investigated age-related changes in cardiac function in the isolated zebrafish heart. In older animals, heart rate was less stable, sinoatrial node recovery time was increased, and the heart rate response to vagal nerve stimulation was reduced, suggesting an age-dependent change in sinoatrial node function. These changes were accompanied by age-related differences in intracardiac innervation, with the total number and proportion of cholinergic neurons being higher in older animals. In contrast, calcium transient duration was highly variable in young animals, while baseline heart rate and rates of contraction and relaxation were also highly variable, but on average did not change with age. These results suggest that age-associated changes in both myocyte and intracardiac neuronal structure and function exist in the zebrafish heart, offering a new model for studies of cardiac ageing.

A beginner's guide to understanding and implementing the genetic modification of zebrafish.

Zebrafish are a relevant and useful vertebrate model species to study normal- and patho-physiology, including that of the heart, due to conservation of protein-coding genes, organ system organisation and function, and efficient breeding and housing. Their amenability to genetic modification, particularly compared to other vertebrate species, is another great advantage, and is the focus of this review. A vast number of genetically engineered zebrafish lines and methods for their creation exist, but their incorporation into research programs is hindered by the overwhelming amount of technical details. The purpose of this paper is to provide a simplified guide to the fundamental information required by the uninitiated researcher for the thorough understanding, critical evaluation, and effective implementation of genetic approaches in the zebrafish. First, an overview of existing zebrafish lines generated through large scale chemical mutagenesis, retroviral insertional mutagenesis, and gene and enhancer trap screens is presented. Second, descriptions of commonly-used genetic modification methods are provided including Tol2 transposon, TALENs (transcription activator-like effector nucleases), and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9). Lastly, design features of genetic modification strategies such as promoters, fluorescent reporters, and conditional transgenesis, are summarised. As a comprehensive resource containing both background information and technical notes of how to obtain or generate zebrafish, this review compliments existing resources to facilitate the use of genetically-modified zebrafish by researchers who are new to the field.

Cardiac Electrophysiological Effects of Light-Activated Chloride Channels.

During the last decade, optogenetics has emerged as a paradigm-shifting technique to monitor and steer the behavior of specific cell types in excitable tissues, including the heart. Activation of cation-conducting channelrhodopsins (ChR) leads to membrane depolarization, allowing one to effectively trigger action potentials (AP) in cardiomyocytes. In contrast, the quest for optogenetic tools for hyperpolarization-induced inhibition of AP generation has remained challenging. The green-light activated ChR from Guillardia theta (GtACR1) mediates Cl--driven photocurrents that have been shown to silence AP generation in different types of neurons. It has been suggested, therefore, to be a suitable tool for inhibition of cardiomyocyte activity. Using single-cell electrophysiological recordings and contraction tracking, as well as intracellular microelectrode recordings and in vivo optical recordings of whole hearts, we find that GtACR1 activation by prolonged illumination arrests cardiac cells in a depolarized state, thus inhibiting re-excitation. In line with this, GtACR1 activation by transient light pulses elicits AP in rabbit isolated cardiomyocytes and in spontaneously beating intact hearts of zebrafish. Our results show that GtACR1 inhibition of AP generation is caused by cell depolarization. While this does not address the need for optogenetic silencing through physiological means (i.e., hyperpolarization), GtACR1 is a potentially attractive tool for activating cardiomyocytes by transient light-induced depolarization.

Mechanically Induced Ectopy via Stretch-Activated Cation-Nonselective Channels Is Caused by Local Tissue Deformation and Results in Ventricular Fibrillation if Triggered on the Repolarization Wave Edge (Commotio Cordis).

BACKGROUND: External chest impacts (commotio cordis) can cause mechanically induced premature ventricular excitation (PVEM) and, rarely, ventricular fibrillation (VF). Because block of stretch-sensitive ATP-inactivated potassium channels curtailed VF occurrence in a porcine model of commotio cordis, VF has been suggested to arise from abnormal repolarization caused by stretch activation of potassium channels. Alternatively, VF could result from abnormal excitation by PVEM, overlapping with normal repolarization-related electric heterogeneity. Here, we investigate mechanisms and determinants of PVEM induction and its potential role in commotio cordis-induced VF. METHODS AND RESULTS: Subcontusional mechanical stimuli were applied to isolated rabbit hearts during optical voltage mapping, combined with pharmacological block of ATP-inactivated potassium or stretch-activated cation-nonselective channels. We demonstrate that local mechanical stimulation reliably triggers PVEM at the contact site, with inducibility predicted by local tissue indentation. PVEM induction is diminished by pharmacological block of stretch-activated cation-nonselective channels. In hearts where electrocardiogram T waves involve a well-defined repolarization edge traversing the epicardium, PVEM can reliably provoke VF if, and only if, the mechanical stimulation site overlaps the repolarization wave edge. In contrast, application of short-lived intraventricular pressure surges neither triggers PVEM nor changes repolarization. ATP-inactivated potassium channel block has no effect on PVEM inducibility per se, but shifts it to later time points by delaying repolarization and prolonging refractoriness. CONCLUSIONS: Local mechanical tissue deformation determines PVEM induction via stretch-activation of cation-nonselective channels, with VF induction requiring PVEM overlap with the trailing edge of a normal repolarization wave. This defines a narrow, subject-specific vulnerable window for commotio cordis-induced VF that exists both in time and in space.