Philip Bittihn

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.

Phase-resolved analysis of the susceptibility of pinned spiral waves to far-field pacing in a two-dimensional model of excitable media

Life-threatening cardiac arrhythmias are associated with the existence of stable and unstable spiral waves. Termination of such complex spatio-temporal patterns by local control is substantially limited by anchoring of spiral waves at natural heterogeneities. Far-field pacing (FFP) is a new local control strategy that has been shown to be capable of unpinning waves from obstacles. In this article, we investigate in detail the FFP unpinning mechanism for a single rotating wave pinned to a heterogeneity. We identify qualitatively different phase regimes of the rotating wave showing that the concept of vulnerability is important but not sufficient to explain the failure of unpinning in all cases. Specifically, we find that a reduced excitation threshold can lead to the failure of unpinning, even inside the vulnerable window. The critical value of the excitation threshold (below which no unpinning is possible) decreases for higher electric field strengths and larger obstacles. In contrast, for a high excitation threshold, the success of unpinning is determined solely by vulnerability, allowing for a convenient estimation of the unpinning success rate. In some cases, we also observe phase resetting in discontinuous phase intervals of the spiral wave. This effect is important for the application of multiple stimuli in experiments.

Far field pacing supersedes anti-tachycardia pacing in a generic model of excitable media

Removing anchored spirals from obstacles is an important step in terminating cardiac arrhythmia. Conventional anti-tachycardia pacing (ATP) has this ability, but only under very restrictive conditions. In a generic model of excitable media, we demonstrate that for unpinning spiral waves from obstacles this profound limitation of ATP can be overcome by far field pacing (FFP). More specifically, an argument is presented for why FFP includes and thus can only extend the capabilities of ATP in the configurations considered. By numerical simulations, we show that in the model there exists a parameter region in which unpinning is possible by FFP but not by ATP. The relevance of this result regarding clinical applications is discussed.

A stabilized microbial ecosystem of self-limiting bacteria using synthetic quorum-regulated lysis

Microbial ecologists are increasingly turning to small, synthesized ecosystems1–5 as a reductionist tool to probe the complexity of native microbiomes6,7. Concurrently, synthetic biologists have gone from single-cell gene circuits8–11 to controlling whole populations using intercellular signalling12–16. The intersection of these fields is giving rise to new approaches in waste recycling17, industrial fermentation18, bioremediation19 and human health16,20. These applications share a common challenge7 well-known in classical ecology21,22—stability of an ecosystem cannot arise without mechanisms that prohibit the faster-growing species from eliminating the slower. Here, we combine orthogonal quorum-sensing systems and a population control circuit with diverse self-limiting growth dynamics to engineer two ‘ortholysis’ circuits capable of maintaining a stable co-culture of metabolically competitive Salmonella typhimurium strains in microfluidic devices. Although no successful co-cultures are observed in a two-strain ecology without synthetic population control, the ‘ortholysis’ design dramatically increases the co-culture rate from 0% to approximately 80%. Agent-based and deterministic modelling reveal that our system can be adjusted to yield different dynamics, including phase-shifted, antiphase or synchronized oscillations, as well as stable steady-state population densities. The ‘ortholysis’ approach establishes a paradigm for constructing synthetic ecologies by developing stable communities of competitive microorganisms without the need for engineered co-dependency.

Scanning and resetting the phase of a pinned spiral wave using periodic far field pulses.

Spiral waves in cardiac tissue can pin to tissue heterogeneities and form stable pinned waves. These waves can be unpinned by electric stimuli applied close to the pinning center during the vulnerable window of the spiral. Using a phase transition curve (PTC), we quantify the response of a pinned wave in a cardiac monolayer to secondary excitations generated electric field pulses. The PTC can be used to construct a one-dimensional map that faithfully predicts the pinned waves response to periodic field stimuli. Based on this 1D map, we predict that pacing at a frequency greater than the spiral frequency, over drive pacing, leads to phase locking of the spiral to the stimulus, which hinders unpinning. In contrast, under drive pacing can lead to scanning of the phase window of the spiral, which facilitates unpinning. The predicted mechanisms of phase scanning and phase locking are experimentally tested and confirmed in the same monolayers that were used to obtain the PTC. Our results have the potential to help choose optimal parameters for low energy antifibrillation pacing schemes.

Electromechanical vortex filaments during cardiac fibrillation

The self-organized dynamics of vortex-like rotating waves, which are also known as scroll waves, are the basis of the formation of complex spatiotemporal patterns in many excitable chemical and biological systems. In the heart, filament-like phase singularities that are associated with three-dimensional scroll waves are considered to be the organizing centres of life-threatening cardiac arrhythmias. The mechanisms that underlie the onset, maintenance and control of electromechanical turbulence in the heart are inherently three-dimensional phenomena. However, it has not previously been possible to visualize the three-dimensional spatiotemporal dynamics of scroll waves inside cardiac tissues. Here we show that three-dimensional mechanical scroll waves and filament-like phase singularities can be observed deep inside the contracting heart wall using high-resolution four-dimensional ultrasound-based strain imaging. We found that mechanical phase singularities co-exist with electrical phase singularities during cardiac fibrillation. We investigated the dynamics of electrical and mechanical phase singularities by simultaneously measuring the membrane potential, intracellular calcium concentration and mechanical contractions of the heart. We show that cardiac fibrillation can be characterized using the three-dimensional spatiotemporal dynamics of mechanical phase singularities, which arise inside the fibrillating contracting ventricular wall. We demonstrate that electrical and mechanical phase singularities show complex interactions and we characterize their dynamics in terms of trajectories, topological charge and lifetime. We anticipate that our findings will provide novel perspectives for non-invasive diagnostic imaging and therapeutic applications.

Complex Structure and Dynamics of the Heart

Excitable media are a class of spatially extended biological and chemical systems, which display a characteristic type of pattern formation by supporting the propagation of nonlinear excitation waves. In the heart, plane waves are associated with the normal heartbeat. They spread from specialized pacemaker cells and travel through the muscle tissue to trigger the coordinated contraction of the four chambers of the heart. Self-excited activation patterns, such as spiral waves and high-dimensional spatio-temporal chaos, underlie cardiac arrhythmias, such as tachycardia and life-threatening ventricular fibrillation. For lack of a better strategy, high-energy electrical shocks remain the only reliable way to terminate fibrillation, despite severe side effects including tissue damage and intolerable pain. To enable low-energy approaches, it is necessary to quantitatively characterize the dynamics of arrhythmias and to identify the mechanisms governing the interaction of electric fields with the cardiac muscle. This work addresses both aspects and is focused on the effect of structural substrate heterogeneity. This heterogeneity is inherent to the cardiac muscle and results from, e.g., spatially varying cell properties, different cell types, lesions and complex cardiac anatomy. The thesis consists of three parts. The aim of the first is to establish a method of nonlinear dynamics as a new tool for the characterization of activation patterns in excitable media. It adopts the generic view of cardiac tissue as a heterogeneous excitable medium and utilizes Lyapunov stability analysis to assess, in a numerical model, the stability and complexity of undesired activation patterns under the influence of spatially varying substrate properties. The results show that heterogeneity can both stabilize and destabilize spiral wave dynamics. Furthermore, it can lead to emergent effects on the complexity of spatio-temporal chaos, which may prove to be significant in the attempt to control fibrillation. Methodologically, Lyapunov stability analysis is shown to provide information that is not otherwise accessible: symmetry breaks in the system can be detected, spatial domains controlled by different spiral waves can be objectively defined, (de-)stabilizing effects of parameter changes and heterogeneity can be identified before they result in qualitatively altered dynamics. In the second part of the thesis, a theory of the fundamental mechanisms governing the interaction of weak electric fields with complex anatomical shape of cardiac tissue is developed. The devised mathematical framework indicates that the curvature and shape of tissue boundaries resulting from cardiac anatomy determine the locations that are most sensitive to electric-field stimulation. In particular, electric fields are shown to cause strong tissue depolarization near convex outer tissue boundaries, an effect that is confirmed in cell-culture experiments, though not expected from previous theories. Other effects of boundary curvature are also discussed, e.g. on the time scale of tissue depolarization. The identified mechanisms determine where in the tissue the local excitation threshold can be overcome by stimulation with a global electric field. The theory accordingly explains the emergence of virtual electrodes that induce localized wave sources in response to weak electric fields during low-energy control approaches. One promising control strategy is low-energy anti-fibrillation pacing (LEAP), which uses a series of weak electric-field pulses to terminate fibrillation and achieves an energy reduction of more than 80% compared to standard, high-energy defibrillation. The physical mechanisms underlying this control strategy are investigated in the third part of this work. It is shown that wave patterns in canine hearts, triggered by pulsed weak electric fields and observed on the cardiac surface in optical mapping experiments, result from a field strength tunable number of wave nucleation sites in the tissue. The heterogeneities that give rise to these virtual electrodes are hypothesized to stem from the branches of coronary vessels, which penetrate the tissue, create internal tissue boundaries and serve as wave nucleation sites at different field strengths depending on their size. To test this hypothesis, a model is constructed that predicts the characteristics of activation patterns from the size distribution of coronary vessels, which is found to follow a power law. Outstanding quantitative agreement is found between the model prediction and the experimentally observed activation patterns for different field strengths. The inherent heterogeneity of the muscle therefore enables multi-site control of chaos in the heart. In summary, the results presented in this work show that, while structural heterogeneity can stabilize malignant activity and may lead to unexpected emergent effects on the complexity of chaotic activity, it also provides the substrate for promising low-energy control methods.

Grid-based spectral fiber clustering

We introduce novel data structures and algorithms for clustering white matter fiber tracts to improve accuracy and robustness of existing techniques. Our novel fiber grid combined with a new randomized soft-division algorithm allows for defining the fiber similarity more precisely and efficiently than a feature space. A fine-tuning of several parameters to a particular fiber set - as it is often required if using a feature space - becomes obsolete. The idea is to utilize a 3D grid where each fiber point is assigned to cells with a certain weight. From this grid, an affinity matrix representing the fiber similarity can be calculated very efficiently in time O(n) in the average case, where n denotes the number of fibers. This is superior to feature space methods which need O(n2) time. Our novel eigenvalue regression is capable of determining a reasonable number of clusters as it accounts for inter-cluster connectivity. It performs a linear regression of the eigenvalues of the affinity matrix to find the point of maximum curvature in a list of descending order. This allows for identifying inner clusters within coarse structures, which automatically and drastically reduces the a-priori knowledge required for achieving plausible clustering results. Our extended multiple eigenvector clustering exhibits a drastically improved robustness compared to the well-known elongated clustering, which also includes an automatic detection of the number of clusters. We present several examples of artificial and real fiber sets clustered by our approach to support the clinical suitability and robustness of the proposed techniques.

Suppression of Beneficial Mutations in Dynamic Microbial Populations

Quantitative predictions for the spread of mutations in bacterial populations are essential to interpret evolution experiments and to improve the stability of synthetic gene circuits. We derive analytical expressions for the suppression factor for beneficial mutations in populations that undergo periodic dilutions, covering arbitrary population sizes, dilution factors, and growth advantages in a single stochastic model. We find that the suppression factor grows with the dilution factor and depends nontrivially on the growth advantage, resulting in the preferential elimination of mutations with certain growth advantages. We confirm our results by extensive numerical simulations.