Bastiaan J. Boukens

Canonical Wnt Signaling Regulates Atrioventricular Junction Programming and Electrophysiological Properties

Rationale: Proper patterning of the atrioventricular canal (AVC) is essential for delay of electrical impulses between atria and ventricles, and defects in AVC maturation can result in congenital heart disease. Objective: To determine the role of canonical Wnt signaling in the myocardium during AVC development. Methods and Results: We used a novel allele of &bgr;-catenin that preserves &bgr;-catenin’s cell adhesive functions but disrupts canonical Wnt signaling, allowing us to probe the effects of Wnt loss of function independently. We show that the loss of canonical Wnt signaling in the myocardium results in tricuspid atresia with hypoplastic right ventricle associated with the loss of AVC myocardium. In contrast, ectopic activation of Wnt signaling was sufficient to induce formation of ectopic AV junction-like tissue as assessed by morphology, gene expression, and electrophysiological criteria. Aberrant AVC development can lead to ventricular pre-excitation, a characteristic feature of Wolff–Parkinson–White syndrome. We demonstrate that postnatal activation of Notch signaling downregulates canonical Wnt targets within the AV junction. Stabilization of &bgr;-catenin protein levels can rescue Notch-mediated ventricular pre-excitation and dysregulated ion channel gene expression. Conclusions: Our data demonstrate that myocardial canonical Wnt signaling is an important regulator of AVC maturation and electric programming upstream of Tbx3. Our data further suggest that ventricular pre-excitation may require both morphological patterning defects, as well as myocardial lineage reprogramming, to allow robust conduction across accessory pathway tissue.

Pitx2 modulates a Tbx5-dependent gene regulatory network to maintain atrial rhythm

A mouse model reveals how seven atrial fibrillation–related risk genes form a network to control heartbeat. The genetic underpinnings of atrial fibrillation The irregular heartbeat of atrial fibrillation puts people in danger of stroke and heart disease; genomic studies have identified gene variants that increase the risk for this abnormality. Nadadur et al. now reveal how these genes influence the beat of the heart’s atrium. In a mouse model of atrial fibrillation, which lacks one of these genes, Tbx5, the authors describe a multitiered transcriptional network that links seven of these atrial fibrillation risk loci. Organized as an incoherent feed-forward loop, this network tightly controls expression of atrial rhythm genes, and its perturbation by the risk loci causes susceptibility to atrial fibrillation. Cardiac rhythm is extremely robust, generating 2 billion contraction cycles during the average human life span. Transcriptional control of cardiac rhythm is poorly understood. We found that removal of the transcription factor gene Tbx5 from the adult mouse caused primary spontaneous and sustained atrial fibrillation (AF). Atrial cardiomyocytes from the Tbx5-mutant mice exhibited action potential abnormalities, including spontaneous depolarizations, which were rescued by chelating free calcium. We identified a multitiered transcriptional network that linked seven previously defined AF risk loci: TBX5 directly activated PITX2, and TBX5 and PITX2 antagonistically regulated membrane effector genes Scn5a, Gja1, Ryr2, Dsp, and Atp2a2. In addition, reduced Tbx5 dose by adult-specific haploinsufficiency caused decreased target gene expression, myocardial automaticity, and AF inducibility, which were all rescued by Pitx2 haploinsufficiency in mice. These results defined a transcriptional architecture for atrial rhythm control organized as an incoherent feed-forward loop, driven by TBX5 and modulated by PITX2. TBX5/PITX2 interplay provides tight control of atrial rhythm effector gene expression, and perturbation of the co-regulated network caused AF susceptibility. This work provides a model for the molecular mechanisms underpinning the genetic implication of multiple AF genome-wide association studies loci and will contribute to future efforts to stratify patients for AF risk by genotype.

Misinterpretation of the mouse ECG: 'musing the waves of Mus musculus'

The ECG is a primary diagnostic tool in patients suffering from heart disease, underscoring the importance of understanding factors contributing to normal and abnormal electrical patterns. Over the past few decades, transgenic mouse models have been increasingly used to study pathophysiological mechanisms of human heart diseases. In order to allow extrapolation of insights gained from murine models to the human condition, knowledge of the similarities and differences between the mouse and human ECG is of crucial importance. In this review, we briefly discuss the physiological mechanisms underlying differences between the baseline ECG of humans and mice, and provide a framework for understanding how these inherent differences are relevant to the interpretation of the mouse ECG during pathology and to the translation of the results from the mouse to man.

Transmural APD gradient synchronizes repolarization in the human left ventricular wall

AIMSnThe duration and morphology of the T wave predict risk for ventricular fibrillation. A transmural gradient in action potential duration (APD) in the ventricular wall has been suggested to underlie the T wave in humans. We hypothesize that the transmural gradient in APD compensates for the normal endocardium-to-epicardium activation sequence and synchronizes repolarization in the human ventricular wall.nnnMETHODS AND RESULTSnWe made left ventricular wedge preparations from 10 human donor hearts and measured transmural activation and repolarization patterns by optical mapping, while simultaneously recording a pseudo-ECG. We also studied the relation between local timings of repolarization with the T wave in silico. During endocardial pacing (1 Hz), APD was longer at the subendocardium than at the subepicardium (360 ± 17 vs. 317 ± 20 ms, P < 0.05). The transmural activation time was 32 ± 4 ms and resulted in final repolarization of the subepicardium at 349 ± 18 ms. The overall transmural dispersion in repolarization time was smaller than that of APD. During epicardial pacing, the dispersion in repolarization time increased, whereas that of APD remained similar. The morphology of the T wave did not differ between endocardial and epicardial stimulation. Simulations explained the constant T wave morphology without transmural APD gradients.nnnCONCLUSIONnThe intrinsic transmural difference in APD compensates for the normal cardiac activation sequence, resulting in more homogeneous repolarization of the left ventricular wall. Our data suggest that the transmural repolarization differences do not fully explain the genesis of the T wave.

An activation-repolarization time metric to predict localized regions of high susceptibility to reentry

BACKGROUNDnInitiation of reentrant ventricular tachycardia (VT) involves complex interactions between front and tail of the activation wave. Recent experimental work has identified the time interval between S2 repolarization proximal to a line of functional block and S2 activation at the adjacent distal side as a critical determinant of reentry.nnnOBJECTIVESnWe hypothesized that (1) an algorithm could be developed to generate a spatial map of this interval (reentry vulnerability index [RVI]), (2) this would accurately identify a site of reentry without the need to actually induce the arrhythmia, and (3) it would be possible to generate an RVI map in patients during routine clinical procedures.nnnMETHODSnAn algorithm was developed that calculated RVI between all pairs of electrodes within a given radius.nnnRESULTSnThe algorithm successfully identified the region with increased susceptibility to reentry in an established Langendorff pig heart model and the site of reentry and rotor formation in an optically mapped sheep ventricular preparation and computational simulations. The feasibility of RVI mapping was evaluated during a clinical procedure by coregistering with cardiac anatomy and physiology of a patient undergoing VT ablation.nnnCONCLUSIONnWe developed an algorithm to calculate a reentry vulnerability index from intervals between local repolarization and activation. The algorithm accurately identified the region of reentry in 2 animal models of functional reentry. The clinical application was demonstrated in a patient with VT and identified the area of reentry without the need of inducing the arrhythmia.

Alk3 mediated Bmp signaling controls the contribution of epicardially derived cells to the tissues of the atrioventricular junction

Recent studies using mouse models for cell fate tracing of epicardial derived cells (EPDCs) have demonstrated that at the atrioventricular (AV) junction EPDCs contribute to the mesenchyme of the AV sulcus, the annulus fibrosus, and the parietal leaflets of the AV valves. There is little insight, however, into the mechanisms that govern the contribution of EPDCs to these tissues. While it has been demonstrated that bone morphogenetic protein (Bmp) signaling is required for AV cushion formation, its role in regulating EPDC contribution to the AV junction remains unexplored. To determine the role of Bmp signaling in the contribution of EPDCs to the AV junction, the Bmp receptor activin-like kinase 3 (Alk3; or Bmpr1a) was conditionally deleted in the epicardium and EPDCs using the mWt1/IRES/GFP-Cre (Wt1(Cre)) mouse. Embryonic Wt1(Cre);Alk3(fl/fl) specimens showed a significantly smaller AV sulcus and a severely underdeveloped annulus fibrosus. Electrophysiological analysis of adult Wt1(Cre);Alk3(fl/fl) mice showed, unexpectedly, no ventricular pre-excitation. Cell fate tracing revealed a significant decrease in the number of EPDCs within the parietal leaflets of the AV valves. Postnatal Wt1(Cre);Alk3(fl/fl) specimens showed myxomatous changes in the leaflets of the mitral valve. Together these observations indicate that Alk3 mediated Bmp signaling is important in the cascade of events that regulate the contribution of EPDCs to the AV sulcus, annulus fibrosus, and the parietal leaflets of the AV valves. Furthermore, this study shows that EPDCs do not only play a critical role in early developmental events at the AV junction, but that they also are important in the normal maturation of the AV valves.

Specialized impulse conduction pathway in the alligator heart.

Mammals and birds have a specialized cardiac atrioventricular conduction system enabling rapid activation of both ventricles. This system may have evolved together with high heart rates to support their endothermic state (warm-bloodedness) and is seemingly lacking in ectothermic vertebrates from which first mammals then birds independently evolved. Here, we studied the conduction system in crocodiles (Alligator mississippiensis), the only ectothermic vertebrates with a full ventricular septum. We identified homologues of mammalian conduction system markers (Tbx3-Tbx5, Scn5a, Gja5, Nppa-Nppb) and show the presence of a functional atrioventricular bundle. The ventricular Purkinje network, however, was absent and slow ventricular conduction relied on trabecular myocardium, as it does in other ectothermic vertebrates. We propose the evolution of the atrioventricular bundle followed full ventricular septum formation prior to the development of high heart rates and endothermy. In contrast, the evolution of the ventricular Purkinje network is strongly associated with high heart rates and endothermy.

Mitochondrial depolarization and electrophysiological changes during ischemia in the rabbit and human heart

Instability of the inner mitochondrial membrane potential (ΔΨm) has been implicated in electrical dysfunction, including arrhythmogenesis during ischemia-reperfusion. Monitoring ΔΨm has led to conflicting results, where depolarization has been reported as sporadic and as a propagating wave. The present study was designed to resolve the aforementioned difference and determine the unknown relationship between ΔΨm and electrophysiology. We developed a novel imaging modality for simultaneous optical mapping of ΔΨm and transmembrane potential (Vm). Optical mapping was performed using potentiometric dyes on preparations from 4 mouse hearts, 14 rabbit hearts, and 7 human hearts. Our data showed that during ischemia, ΔΨm depolarization is sporadic and changes asynchronously with electrophysiological changes. Spatially, ΔΨm depolarization was associated with action potential duration shortening but not conduction slowing. Analysis of focal activity indicated that ΔΨm is not different within the myocardium where the focus originates compared with normal ventricular tissue. Overall, our data suggest that during ischemia, mitochondria maintain their function at the expense of sarcolemmal electrophysiology, but ΔΨm depolarization does not have a direct association to ischemia-induced arrhythmias.

Embryonic development of the right ventricular outflow tract and arrhythmias

Bastiaan J. Boukens, PhD, Ruben Coronel, MD, PhD, FHRS, Vincent M. Christoffels, PhD From the Department of Biomedical Engineering, George Washington University, Washington, DC, Department of Anatomy, Embryology and Physiology, Department of Clinical and Experiment Cardiology, Academic Medical Center, Amsterdam, The Netherlands, IHU Institut de Rythmologie et Modelisation Cardiaque, Fondation Bordeaux Universite, Bordeaux, France, and Inserm U1045 CRCTB, Universite de Bordeaux, Bordeaux, France.

Feasibility of a semi-automated method for cardiac conduction velocity analysis of high-resolution activation maps

Myocardial conduction velocity is important for the genesis of arrhythmias. In the normal heart, conduction is primarily dependent on fiber direction (anisotropy) and may be discontinuous at sites with tissue heterogeneities (trabeculated or fibrotic tissue). We present a semi-automated method for the accurate measurement of conduction velocity based on high-resolution activation mapping following central stimulation. The method was applied to activation maps created from myocardium from man, sheep and mouse with anisotropic and discontinuous conduction. Advantages of the presented method over existing methods are discussed.