Stefan Luther

Toward panoramic in situ mapping of action potential propagation in transgenic hearts to investigate initiation and therapeutic control of arrhythmias

To investigate the dynamics and propensity for arrhythmias in intact transgenic hearts comprehensively, optical strategies for panoramic fluorescence imaging of action potential (AP) propagation are essential. In particular, mechanism-oriented molecular studies usually depend on transgenic mouse hearts of only a few millimeters in size. Furthermore, the temporal scales of the mouse heart remain a challenge for panoramic fluorescence imaging with heart rates ranging from 200 min−1 (e.g., depressed sinus node function) to over 1200 min−1 during fast arrhythmias. To meet these challenging demands, we and others developed physiologically relevant mouse models and characterized their hearts with planar AP mapping. Here, we summarize the progress toward panoramic fluorescence imaging and its prospects for the mouse heart. In general, several high-resolution cameras are synchronized and geometrically arranged for panoramic voltage mapping and the surface and blood vessel anatomy documented through image segmentation and heart surface reconstruction. We expect that panoramic voltage imaging will lead to novel insights about molecular arrhythmia mechanisms through quantitative strategies and organ-representative analysis of intact mouse hearts.

Effects of Pacing Site and Stimulation History on Alternans Dynamics and the Development of Complex Spatiotemporal Patterns in Cardiac Tissue

Alternans of action potential duration has been associated with T wave alternans and the development of arrhythmias because it produces large gradients of repolarization. However, little is known about alternans dynamics in large mammalian hearts. Using optical mapping to record electrical activations simultaneously from the epicardium and endocardium of 9 canine right ventricles, we demonstrate novel arrhythmogenic complex spatiotemporal dynamics. (i) Alternans predominantly develops first on the endocardium. (ii) The postulated simple progression from normal rhythm to concordant to discordant alternans is not always observed; concordant alternans can develop from discordant alternans as the pacing period is decreased. (iii) In contrast to smaller tissue preparations, multiple stationary nodal lines may exist and need not be perpendicular to the pacing site or to each other. (iv) Alternans has fully three-dimensional dynamics and the epicardium and endocardium can show significantly different dynamics: multiple nodal surfaces can be transmural or intramural and can form concave/convex surfaces resulting in islands of discordant alternans. (v) The complex spatiotemporal patterns observed during alternans are very sensitive to both the site of stimulation and the stimulation history. Alternans in canine ventricles not only exhibit larger amplitudes and persist for longer cycle length regimes compared to those found in smaller mammalian hearts, but also show novel dynamics not previously described that enhance dispersion and show high sensitivity to initial conditions. This indicates some underlying predisposition to chaos and can help to guide the design of new drugs and devices controlling and preventing arrhythmic events.

SAP97 and Dystrophin Macromolecular Complexes Determine Two Pools of Cardiac Sodium Channels Nav1.5 in Cardiomyocytes

Rationale: The cardiac sodium channel Nav1.5 plays a key role in excitability and conduction. The 3 last residues of Nav1.5 (Ser-Ile-Val) constitute a PDZ-domain binding motif that interacts with the syntrophin–dystrophin complex. As dystrophin is absent at the intercalated discs, Nav1.5 could potentially interact with other, yet unknown, proteins at this site. Objective: The aim of this study was to determine whether Nav1.5 is part of distinct regulatory complexes at lateral membranes and intercalated discs. Methods and Results: Immunostaining experiments demonstrated that Nav1.5 localizes at lateral membranes of cardiomyocytes with dystrophin and syntrophin. Optical measurements on isolated dystrophin-deficient mdx hearts revealed significantly reduced conduction velocity, accompanied by strong reduction of Nav1.5 at lateral membranes of mdx cardiomyocytes. Pull-down experiments revealed that the MAGUK protein SAP97 also interacts with the SIV motif of Nav1.5, an interaction specific for SAP97 as no pull-down could be detected with other cardiac MAGUK proteins (PSD95 or ZO-1). Furthermore, immunostainings showed that Nav1.5 and SAP97 are both localized at intercalated discs. Silencing of SAP97 expression in HEK293 and rat cardiomyocytes resulted in reduced sodium current (INa) measured by patch-clamp. The INa generated by Nav1.5 channels lacking the SIV motif was also reduced. Finally, surface expression of Nav1.5 was decreased in silenced cells, as well as in cells transfected with SIV-truncated channels. Conclusions: These data support a model with at least 2 coexisting pools of Nav1.5 channels in cardiomyocytes: one targeted at lateral membranes by the syntrophin-dystrophin complex, and one at intercalated discs by SAP97.

Low-energy control of electrical turbulence in the heart

Controlling the complex spatio-temporal dynamics underlying life-threatening cardiac arrhythmias such as fibrillation is extremely difficult, because of the nonlinear interaction of excitation waves in a heterogeneous anatomical substrate. In the absence of a better strategy, strong, globally resetting electrical shocks remain the only reliable treatment for cardiac fibrillation. Here we establish the relationship between the response of the tissue to an electric field and the spatial distribution of heterogeneities in the scale-free coronary vascular structure. We show that in response to a pulsed electric field, E, these heterogeneities serve as nucleation sites for the generation of intramural electrical waves with a source density ρ(E) and a characteristic time, τ, for tissue depolarization that obeys the power law τ ∝ Eα. These intramural wave sources permit targeting of electrical turbulence near the cores of the vortices of electrical activity that drive complex fibrillatory dynamics. We show in vitro that simultaneous and direct access to multiple vortex cores results in rapid synchronization of cardiac tissue and therefore, efficient termination of fibrillation. Using this control strategy, we demonstrate low-energy termination of fibrillation in vivo. Our results give new insights into the mechanisms and dynamics underlying the control of spatio-temporal chaos in heterogeneous excitable media and provide new research perspectives towards alternative, life-saving low-energy defibrillation techniques.

Stimulated Emission Depletion Live-Cell Super-Resolution Imaging Shows Proliferative Remodeling of T-Tubule Membrane Structures After Myocardial Infarction

Rationale: Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca2+ release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF). Objectives: Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction. Methods and Results: Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca2+ release and action potential prolongation. Conclusions: This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca2+ release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing.

STED Live Cell Super-Resolution Imaging Shows Proliferative Remodeling of T-Tubule Membrane Structures After Myocardial Infarction

Rationale: Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca2+ release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF). Objectives: Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction. Methods and Results: Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca2+ release and action potential prolongation. Conclusions: This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca2+ release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing.

Termination of Atrial Fibrillation Using Pulsed Low-Energy Far-Field Stimulation

Background— Electrically based therapies for terminating atrial fibrillation (AF) currently fall into 2 categories: antitachycardia pacing and cardioversion. Antitachycardia pacing uses low-intensity pacing stimuli delivered via a single electrode and is effective for terminating slower tachycardias but is less effective for treating AF. In contrast, cardioversion uses a single high-voltage shock to terminate AF reliably, but the voltages required produce undesirable side effects, including tissue damage and pain. We propose a new method to terminate AF called far-field antifibrillation pacing, which delivers a short train of low-intensity electric pulses at the frequency of antitachycardia pacing but from field electrodes. Prior theoretical work has suggested that this approach can create a large number of activation sites (“virtual” electrodes) that emit propagating waves within the tissue without implanting physical electrodes and thereby may be more effective than point-source stimulation. Methods and Results— Using optical mapping in isolated perfused canine atrial preparations, we show that a series of pulses at low field strength (0.9 to 1.4 V/cm) is sufficient to entrain and subsequently extinguish AF with a success rate of 93% (69 of 74 trials in 8 preparations). We further demonstrate that the mechanism behind far-field antifibrillation pacing success is the generation of wave emission sites within the tissue by the applied electric field, which entrains the tissue as the field is pulsed. Conclusions— AF in our model can be terminated by far-field antifibrillation pacing with only 13% of the energy required for cardioversion. Further studies are needed to determine whether this marked reduction in energy can increase the effectiveness and safety of terminating atrial tachyarrhythmias clinically.

Spatio–temporal dynamics of acoustic cavitation bubble clouds

Bubble clouds forming in an extended volume of liquid in acoustic cavitation show a slowly varying filamentary structure, whose origin is still not completely understood. Experimental observations are reported that provide some characteristics of the phenomenon, such as bubble distributions and sound–field measurements. A discussion of relevant physical interactions in bubbly liquids is comprised of wave dynamics, Bjerknes and drag forces, nucleation and coalescence. For describing the structure formation process, continuum and particle approaches are employed. In the framework of the continuum model it is shown that homogeneous bubble distributions are unstable, and regions with high bubble concentration emerge in the course of a self–concentration process. In the particle model, all bubbles are treated as interacting objects that move in the liquid. This approach is complementary to the continuum model. It allows the inclusion of some particular features, for instance Bjerknes forces based on nonlinear bubble oscillations. Both models are discussed and results are compared with experimentally observed patterns.

Acoustic cavitation structures and simulations by a particle model

Cavitation bubbles in acoustic resonators are observed to arrange in branch-like patterns. We give a brief review of the anatomy of such structures and outline an approach for simulation by individual, moving bubbles. This particle model can reproduce an experimentally observed transition between different structure types in a rectangular resonator cell.

Classifying cardiac biosignals using ordinal pattern statistics and symbolic dynamics

The performance of (bio-)signal classification strongly depends on the choice of suitable features (also called parameters or biomarkers). In this article we evaluate the discriminative power of ordinal pattern statistics and symbolic dynamics in comparison with established heart rate variability parameters applied to beat-to-beat intervals. As an illustrative example we distinguish patients suffering from congestive heart failure from a (healthy) control group using beat-to-beat time series. We assess the discriminative power of individual features as well as pairs of features. These comparisons show that ordinal patterns sampled with an additional time lag are promising features for efficient classification.