A comprehensive framework for evaluation of high pacing frequency and arrhythmic optical mapping signals

Introduction: High pacing frequency or irregular activity due to arrhythmia produces complex optical mapping signals and challenges for processing. The objective is to establish an automated activation time-based analytical framework applicable to optical mapping images of complex electrical behavior.Methods: Optical mapping signals with varying complexity from sheep (N = 7) ventricular preparations were examined. Windows of activation centered on each action potential upstroke were derived using Hilbert transform phase. Upstroke morphology was evaluated for potential multiple activation components and peaks of upstroke signal derivatives defined activation time. Spatially and temporally clustered activation time points were grouped in to wave fronts for individual processing. Each activation time point was evaluated for corresponding repolarization times. Each wave front was subsequently classified based on repetitive or non-repetitive events. Wave fronts were evaluated for activation time minima defining sites of wave front origin. A visualization tool was further developed to probe dynamically the ensemble activation sequence.Results: Our framework facilitated activation time mapping during complex dynamic events including transitions to rotor-like reentry and ventricular fibrillation. We showed that using fixed AT windows to extract AT maps can impair interpretation of the activation sequence. However, the phase windowing of action potential upstrokes enabled accurate recapitulation of repetitive behavior, providing spatially coherent activation patterns. We further demonstrate that grouping the spatio-temporal distribution of AT points in to coherent wave fronts, facilitated interpretation of isolated conduction events, such as conduction slowing, and to derive dynamic changes in repolarization properties. Focal origins precisely detected sites of stimulation origin and breakthrough for individual wave fronts. Furthermore, a visualization tool to dynamically probe activation time windows during reentry revealed a critical single static line of conduction slowing associated with the rotation core.Conclusion: This comprehensive analytical framework enables detailed quantitative assessment and visualization of complex electrical behavior

The Urokinase Receptor (uPAR) Facilitates Clearance of Borrelia burgdorferi

The causative agent of Lyme borreliosis, the spirochete Borrelia burgdorferi, has been shown to induce expression of the urokinase receptor (uPAR); however, the role of uPAR in the immune response against Borrelia has never been investigated. uPAR not only acts as a proteinase receptor, but can also, dependently or independently of ligation to uPA, directly affect leukocyte function. We here demonstrate that uPAR is upregulated on murine and human leukocytes upon exposure to B. burgdorferi both in vitro as well as in vivo. Notably, B. burgdorferi-inoculated C57BL/6 uPAR knock-out mice harbored significantly higher Borrelia numbers compared to WT controls. This was associated with impaired phagocytotic capacity of B. burgdorferi by uPAR knock-out leukocytes in vitro. B. burgdorferi numbers in vivo, and phagocytotic capacity in vitro, were unaltered in uPA, tPA (low fibrinolytic activity) and PAI-1 (high fibrinolytic activity) knock-out mice compared to WT controls. Strikingly, in uPAR knock-out mice partially backcrossed to a B. burgdorferi susceptible C3H/HeN background, higher B. burgdorferi numbers were associated with more severe carditis and increased local TLR2 and IL-1β mRNA expression. In conclusion, in B. burgdorferi infection, uPAR is required for phagocytosis and adequate eradication of the spirochete from the heart by a mechanism that is independent of binding of uPAR to uPA or its role in the fibrinolytic system

Embryology, anatomy, and pathology of ventricular outflow tracts related to cardiac mapping and arrhythmias

Ventricular arrhythmias, especially those of non-ischemic heart diseases, arise mostly from the outflows. Differences in embryonic origin and phenotype may account for arrhythmogenic propensity of right ventricular outflow tract (RVOT). The anatomy of the ventricles may be divided into inflow, apex, and outflow. The RVOT and left VOT (LVOT) have a common origin, which may point to a common mechanism underlying outflow tract arrhythmias. During the fetal stage of development, the embryonic outflow tract (OFT) is fully incorporated into the RV myocardium and forms the RVOT and LVOT. Most of the tachyarrhythmias arising from the ventricular outflow have a substrate, accounting for triggered activity or reentry circuits, and are mostly non-ischemic. Arrhythmogenic cardiomyopathy is heredo-familial disorder due to mutations of genes encoding cell junction proteins, characterized by a pathognomonic substrate, namely fibro-fatty replacement of the ventricular myocardium. RVOT or LVOT tachyarrhythmias may occur in the absence of structural myocardial disease

Developmental basis for electrophysiological heterogeneity in the ventricular and outflow tract myocardium as a substrate for life-threatening ventricular arrhythmias

Reentry is the main mechanism of life-threatening ventricular arrhythmias, including ventricular fibrillation and tachycardia. Its occurrence depends on the simultaneous presence of an arrhythmogenic substrate (a preexisting condition) and a "trigger," and is favored by electrophysiological heterogeneities. In the adult heart, electrophysiological heterogeneities of the ventricle exist along the apicobasal, left-right, and transmural axes. Also, conduction is preferentially slowed in the right ventricular outflow tract, especially during pharmacological sodium channel blockade. We propose that the origin of electrophysiological heterogeneities of the adult heart lies in early heart development. The heart is formed from several progenitor regions: the first heart field predominantly forms the left ventricle, whereas the second heart field forms the right ventricle and outflow tract. Furthermore, the embryonic outflow tract consists of slowly conducting tissue until it is incorporated into the ventricles and develops rapidly conducting properties. The subepicardial myocytes and subendocardial myocytes run distinctive gene programs from their formation onwards. This review discusses the hypothesis that electrophysiological heterogeneities in the adult heart result from persisting patterns in gene expression and function along the craniocaudal and epicardial-endocardial axes of the developing heart. Understanding the developmental origins of electrophysiological heterogeneity contributing to ventricular arrhythmias may give rise to new therapie

Single-site neural tube closure in human embryos revisited

Since the multi-site closure theory was first proposed in 1991 as explanation for the preferential localizations of neural tube defects, the closure of the neural tube has been debated. Although the multi-site closure theory is much cited in clinical literature, single-site closure is most apparent in literature concerning embryology. Inspired by Victor Hamburgers (1900-2001) statement that "our real teacher has been and still is the embryo, who is, incidentally, the only teacher who is always right", we decided to critically review both theories of neural tube closure. To verify the theories of closure, we studied serial histological sections of 10 mouse embryos between 8.5 and 9.5 days of gestation and 18 human embryos of the Carnegie collection between Carnegie stage 9 (19-21 days) and 13 (28-32 days). Neural tube closure was histologically defined by the neuroepithelial remodeling of the two adjoining neural fold tips in the midline. We did not observe multiple fusion sites in neither mouse nor human embryos. A meta-analysis of case reports on neural tube defects showed that defects can occur at any level of the neural axis. Our data indicate that the human neural tube fuses at a single site and, therefore, we propose to reinstate the single-site closure theory for neural tube closure. We showed that neural tube defects are not restricted to a specific location, thereby refuting the reasoning underlying the multi-site closure theory. Clin. Anat. 30:988-999, 2017. © 2017 Wiley Periodicals, In

The sodium channel NaV1.5 impacts on early murine embryonic cardiac development, structure and function in a non-electrogenic manner

Aim: The voltage-gated sodium channel NaV1.5, encoded by SCN5A, is essential for cardiac excitability and ensures proper electrical conduction. Early embryonic death has been observed in several murine models carrying homozygous Scn5amutations. We investigated when sodium current (INa) becomes functionally relevant in the murine embryonic heart and how Scn5a/NaV1.5 dysfunction impacts on cardiac development. Methods: Involvement of NaV1.5-generated INa in murine cardiac electrical function was assessed by optical mapping in wild type (WT) embryos (embryonic day (E)9.5 and E10.5) in the absence and presence of the sodium channel blocker tetrodotoxin (30 µmol/L). INa was assessed by patch-clamp analysis in cardiomyocytes isolated from WT embryos (E9.5-17.5). In addition, cardiac morphology and electrical function was assessed in Scn5a-1798insD−/− embryos (E9.5-10.5) and their WT littermates. Results: In WT embryos, tetrodotoxin did not affect cardiac activation at E9.5, but slowed activation at E10.5. Accordingly, patch-clamp measurements revealed that INa was virtually absent at E9.5 but robustly present at E10.5. Scn5a-1798insD−/− embryos died in utero around E10.5, displaying severely affected cardiac activation and morphology. Strikingly, altered ventricular activation was observed in Scn5a-1798insD−/− E9.5 embryos before the onset of INa, in addition to reduced cardiac tissue volume compared to WT littermates. Conclusion: We here demonstrate that NaV1.5 is involved in cardiac electrical function from E10.5 onwards. Scn5a-1798insD−/− embryos displayed cardiac structural abnormalities at E9.5, indicating that NaV1.5 dysfunction impacts on embryonic cardiac development in a non-electrogenic manner. These findings are potentially relevant for understanding structural defects observed in relation to NaV1.5 dysfunction

Molecular and electrophysiological evaluation of human cardiomyocyte subtypes to facilitate generation of composite cardiac models

Paucity of physiologically relevant cardiac models has limited the widespread application of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes in drug development. Here, we performed comprehensive characterization of hiPSC-derived cardiomyocyte subtypes from 2D and 3D cultures and established a novel 3D model to study impulse initiation and propagation. Directed differentiation approaches were used to generate sinoatrial nodal (SANCM), atrial (ACM) and ventricular cardiomyocytes (VCM). Single cell RNA sequencing established that the protocols yield distinct cell populations in line with expected identities, which was also confirmed by electrophysiological characterization. In 3D EHT cultures of all subtypes, we observed prominent expression of stretch-responsive genes such as NPPA. Response to rate modulating drugs noradrenaline, carbachol and ivabradine were comparable in single cells and EHTs. Differences in the speed of impulse propagation between the subtypes were more pronounced in EHTs compared with 2D monolayers owing to a progressive increase in conduction velocities in atrial and ventricular cardiomyocytes, in line with a more mature phenotype. In a novel binary EHT model of pacemaker-atrial interface, the SANCM end of the tissue consistently paced the EHTs under baseline conditions, which was inhibited by ivabradine. Taken together, our data provide comprehensive insights into molecular and electrophysiological properties of hiPSC-derived cardiomyocyte subtypes, facilitating the creation of next generation composite cardiac models for drug discovery, disease modeling and cell-based regenerative therapies

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

BACKGROUND Initiation 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. OBJECTIVES We 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. METHODS An algorithm was developed that calculated RVI between all pairs of electrodes within a given radius. RESULTS The 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. CONCLUSION We 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