Die Bedeutung von Plakophilin-2 für die Funktion des spannungsgesteuerten Natriumkanals NaV1.5 im Rahmen der Arrhythmogenen Rechtsventrikulären Kardiomyopathie

Einleitung: Die Arrhythmogene Rechtsventrikuläre Kardiomyopathie (ARVC) stellt eine wichtige Ursache für den plötzlichen Herztod bei jungen, sportlichen Patienten dar. Mutationen in desmosomalen Proteinen werden häufig mit der ARVC assoziiert und für die lebensbedrohlichen Herzrhythmusstörungen sowie die kontraktile Dysfunktion verantwortlich gemacht. Es scheint ein enger funktioneller Zusammenhang zu bestehen zwischen der Integrität der kardialen Glanzstreifen, insbesondere der dort lokalisierten Desmosomen, und der Funktion des spannungsabhängigen Natriumkanals (NaV1.5). Es konnte gezeigt werden, dass eine verminderte Expression des desmosomalen Proteins Plakophilin 2 (PKP2) zu einer Reduktion des spannungsabhängigen Natriumstroms in Maus-Kardiomyozyten führt. Die genauen zellulären Mechanismen der Arrhythmogenese im Rahmen der ARVC sind jedoch noch nicht endgültig verstanden. Fragestellung: Es wurden die Eigenschaften des spannungsabhängigen Natriumstroms (INa) sowie der Aktionspotentiale (APs) in Kardiomyozyten aus induzierten pluripotenten Stammzellen (iPSC) einer jungen ARVC-Patientin untersucht. Methoden und Ergebnisse: Genetische Analysen ergaben eine Loss-of-Function-Mutation im PKP2-Gen in 12 von 19 getesteten Familienmitgliedern, inklusive der Indexpatientin. Nach einer dreimonatigen Reifungszeit wurden funktionelle Messungen an Kardiomyozyten aus iPSCs der Indexpatientin, ihrer klinisch asymptomatischen Schwester und Trägerin der gleichen Mutation sowie einer gesunden weiblichen Kontrollperson durchgeführt. Zur Untersuchung des INa sowie der APs wurden Whole-Cell-Patch-clamp-Messungen eingesetzt. Interessanterweise war der Spitzennatriumstrom (INa,max) in den Kardiomyozyten der Indexpatientin deutlich reduziert, wohingegen die asymptomatische Mutationsträgerin im Vergleich zur Kontrolle sogar eine vergrößerte INa,max-Dichte aufwies. Ursächlich scheint vor allem eine Rechts- (Indexpatientin) bzw. Linksverschiebung (Schwester) der INa-Aktivierung. Die NaV1.5-Expression zeigte sich in den Immunofluoreszenzfärbungen nämlich unverändert. Überraschenderweise war auch der späte Natriumstrom (INa,L), ein bekannter proarrhythmogener Faktor, signifikant vermindert in den Zellen der Indexpatientin, nicht jedoch der asymptomatischen Mutationsträgerin. Darüber hinaus konnten relevante Unterschiede in der AP-Kinetik festgestellt werden, welche sich nur teilweise durch die Veränderungen im INa erklären lassen. Im Vergleich zur Kontrolle war die Aktionspotentialamplitude (APA) signifikant reduziert, das Ruhemembranpotential (RMP) weniger negativ und die Aktionspotentialdauer (APD) deutlich verlängert, und zwar ausschließlich in den Kardiomyozyten der Indexpatientin. Mittels Whole Exome Genome Sequencing wurde eine zusätzliche Mutation im ANK3-Gen (kodierend für das regulatorische Glanzstreifen-Protein Ankyrin G) detektiert, welche nur die Indexpatientin, nicht aber die asymptomatische Schwester trägt. Schlussfolgerung: Unsere Daten zeigen eine Reduktion des Spitzen- (INa,max) sowie des späten Natriumstroms (INa,L) in den Kardiomyozyten aus iPSCs einer symptomatischen ARVC-Patientin, jedoch nicht in denen ihrer asymptomatischen Schwester, welche die gleiche Loss-of-function-Mutation im PKP2-Gen trägt. Ursächlich scheint vor allem eine Verschiebung der Kinetik der INa-Aktivierung; die NaV1.5-Expression hingegen zeigte sich unverändert. Überraschenderweise korrelieren die Ergebnisse der INa-Messungen nur bedingt mit den Veränderungen der Aktionspotentiale. Es zeigten sich neben einer reduzierten AP-Amplitude nämlich ein weniger negatives RMP und eine signifikant verlängerte APD in der Indexpatientin. Dies spricht dafür, dass noch weitere Mechanismen eine Rolle in der Arrhythmogenese bei ARVC-Patienten spielen müssen. Interessanterweise unterschied sich die asymptomatische Mutationsträgerin bis auf einen – möglicherweise kompensatorisch – vergrößerten INa,max nicht von der gesunden Kontrolle. Ursächlich könnte eine Zweit-Mutation im ANK3-Gen sein, welche lediglich die Indexpatientin, nicht jedoch die Schwester trägt

The unbinding transition of mixed fluid membranes

A phenomenological model for the unbinding transition of multi-component fluid membranes is proposed, where the unbinding transition is described using a theory analogous to Flory-Huggins theory for polymers. The coupling between the lateral phase separation of inclusion molecules and the membrane-substrate distance explains the phase coexistence between two unbound phases as observed in recent experiments by Marx et al. [Phys. Rev. Lett. 88, 138102 (2002)]. Bellow a critical end-point temperature, we find that the unbinding transition becomes first-order for multi-component membranes.Comment: 7 pages, 3 eps figure

Indirect interactions of membrane-adsorbed cylinders

Biological and biomimetic membranes often contain aggregates of embedded or adsorbed macromolecules. In this article, the indirect interactions of cylindrical objects adhering to a planar membrane are considered theoretically. The adhesion of the cylinders causes a local perturbation of the equilibrium membrane shape, which leads to membrane-mediated interactions. For a planar membrane under lateral tension, the interaction is repulsive for a pair of cylinders adhering to the same side of the membrane, and attractive for cylinders adhering at opposite membrane sides. For a membrane in an external harmonic potential, the interaction of adsorbed cylinders is always attractive and increases if forces perpendicular to the membrane act on the cylinders.Comment: 9 pages, 8 figures; typos correcte

Adhesion-induced phase separation of multiple species of membrane junctions

A theory is presented for the membrane junction separation induced by the adhesion between two biomimetic membranes that contain two different types of anchored junctions (receptor/ligand complexes). The analysis shows that several mechanisms contribute to the membrane junction separation. These mechanisms include (i) the height difference between type-1 and type-2 junctions is the main factor which drives the junction separation, (ii) when type-1 and type-2 junctions have different rigidities against stretch and compression, the ``softer'' junctions are the ``favored'' species, and the aggregation of the softer junction can occur, (iii) the elasticity of the membranes mediates a non-local interaction between the junctions, (iv) the thermally activated shape fluctuations of the membranes also contribute to the junction separation by inducing another non-local interaction between the junctions and renormalizing the binding energy of the junctions. The combined effect of these mechanisms is that when junction separation occurs, the system separates into two domains with different relative and total junction densities.Comment: 23 pages, 6 figure

Electrostatic Interactions of Asymmetrically Charged Membranes

We predict the nature (attractive or repulsive) and range (exponentially screened or long-range power law) of the electrostatic interactions of oppositely charged and planar plates as a function of the salt concentration and surface charge densities (whose absolute magnitudes are not necessarily equal). An analytical expression for the crossover between attractive and repulsive pressure is obtained as a function of the salt concentration. This condition reduces to the high-salt limit of Parsegian and Gingell where the interaction is exponentially screened and to the zero salt limit of Lau and Pincus in which the important length scales are the inter-plate separation and the Gouy-Chapman length. In the regime of low salt and high surface charges we predict - for any ratio of the charges on the surfaces - that the attractive pressure is long-ranged as a function of the spacing. The attractive pressure is related to the decrease in counter-ion concentration as the inter-plate distance is decreased. Our theory predicts several scaling regimes with different scaling expressions for the pressure as function of salinity and surface charge densities. The pressure predictions can be related to surface force experiments of oppositely charged surfaces that are prepared by coating one of the mica surfaces with an oppositely charged polyelectrolyte

Binding cooperativity of membrane adhesion receptors

The adhesion of cells is mediated by receptors and ligands anchored in apposing membranes. A central question is how to characterize the binding affinity of these membrane-anchored molecules. For soluble molecules, the binding affinity is typically quantified by the binding equilibrium constant K3D in the linear relation [RL] = K3D [R][L] between the volume concentration [RL] of bound complexes and the volume concentrations [R] and [L] of unbound molecules. For membrane-anchored molecules, it is often assumed by analogy that the area concentration of bound complexes [RL] is proportional to the product [R][L] of the area concentrations for the unbound receptor and ligand molecules. We show here (i) that this analogy is only valid for two planar membranes immobilized on rigid surfaces, and (ii) that the thermal roughness of flexible membranes leads to cooperative binding of receptors and ligands. In the case of flexible membranes, the area concentration [RL] of receptor-ligand bonds is proportional to the square of [R][L] for typical lengths and concentrations of receptors and ligands in cell adhesion zones. The cooperative binding helps to understand why different experimental methods for measuring the binding affinity of membrane-anchored molecules have led to values differing by several orders of magnitude.Comment: 9 pages, 4 figures; to appear in Soft Matte

Rupture of multiple parallel molecular bonds under dynamic loading

Biological adhesion often involves several pairs of specific receptor-ligand molecules. Using rate equations, we study theoretically the rupture of such multiple parallel bonds under dynamic loading assisted by thermal activation. For a simple generic type of cooperativity, both the rupture time and force exhibit several different scaling regimes. The dependence of the rupture force on the number of bonds is predicted to be either linear, like a square root or logarithmic.Comment: 8 pages, 2 figure

Adhesion of membranes via receptor-ligand complexes: Domain formation, binding cooperativity, and active processes

Cell membranes interact via anchored receptor and ligand molecules. Central questions on cell adhesion concern the binding affinity of these membrane-anchored molecules, the mechanisms leading to the receptor-ligand domains observed during adhesion, and the role of cytoskeletal and other active processes. In this review, these questions are addressed from a theoretical perspective. We focus on models in which the membranes are described as elastic sheets, and the receptors and ligands as anchored molecules. In these models, the thermal membrane roughness on the nanometer scale leads to a cooperative binding of anchored receptor and ligand molecules, since the receptor-ligand binding smoothens out the membranes and facilitates the formation of additional bonds. Patterns of receptor domains observed in Monte Carlo simulations point towards a joint role of spontaneous and active processes in cell adhesion. The interactions mediated by the receptors and ligand molecules can be characterized by effective membrane adhesion potentials that depend on the concentrations and binding energies of the molecules.Comment: Review article, 13 pages, 9 figures, to appear in Soft Matte

Dynamic phase separation of fluid membranes with rigid inclusions

Membrane shape fluctuations induce attractive interactions between rigid inclusions. Previous analytical studies showed that the fluctuation-induced pair interactions are rather small compared to thermal energies, but also that multi-body interactions cannot be neglected. In this article, it is shown numerically that shape fluctuations indeed lead to the dynamic separation of the membrane into phases with different inclusion concentrations. The tendency of lateral phase separation strongly increases with the inclusion size. Large inclusions aggregate at very small inclusion concentrations and for relatively small values of the inclusions' elastic modulus.Comment: 6 pages, 6 figure

Swelling behavior and viscoelasticity of ultrathin grafted hyaluronic acid films

PACS. 36.20.Ey Conformation (statistics and dynamics) - 61.25.Hq Macromolecular and polymer solutions; polymer melts; swelling - 83.80.Lz Biological materials: blood, collagen, wood, food, etc.,