Arrhythmogenic cardiomyopathy-related cadherin variants affect desmosomal binding kinetics.

Cadherins are calcium dependent adhesion proteins that establish and maintain the intercellular mechanical contact by bridging the gap between adjacent cells. Desmoglein-2 (Dsg2) and desmocollin-2 (Dsc2) are tissue specific cadherin isoforms of the cell-cell contact in cardiac desmosomes. Mutations in the DSG2-gene and in the DSC2-gene are related to arrhythmogenic right ventricular cardiomyopathy (ARVC) a rare but severe heart muscle disease. Here, several possible homophilic and heterophilic binding interactions of wild-type Dsg2, wild-type Dsc2, as well as one Dsg2- and two Dsc2-variants, each associated with ARVC, are investigated. Using single molecule force spectroscopy (SMFS) with atomic force microscopy (AFM) and applying Jarzynski's equality the kinetics and thermodynamics of Dsg2/Dsc2 interaction can be determined. The free energy landscape of Dsg2/Dsc2 dimerization exposes a high activation energy barrier, which is in line with the proposed strand-swapping binding motif. Although the binding motif is not affected by any of the mutations, the binding kinetics of the interactions differ significantly from the wild-type. While wild-type cadherins exhibit an average complex lifetime of approx. 0.3 s interactions involving a variant consistently show - lifetimes that are substantially larger. The lifetimes of the wild-type interactions give rise to the picture of a dynamic adhesion interface consisting of continuously dissociating and (re)associating molecular bonds, while the delayed binding kinetics of interactions involving an ARVC-associated variant might be part of the pathogenesis. Our data provide a comprehensive and consistent thermodynamic and kinetic description of cardiac cadherin binding, allowing detailed insight into the molecular mechanisms of cell adhesion.

Cardiac desmosomal adhesion relies on ideal-, slip- and catch bonds.

The cardiac muscle consists of individual cardiomyocytes that are mechanically linked by desmosomes. Desmosomal adhesion is mediated by densely packed and organized cadherins which, in presence of Ca2+, stretch out their extracellular domains (EC) and dimerize with opposing binding partners by exchanging an N-terminal tryptophan. The strand-swap binding motif of cardiac cadherins like desmocollin 2 (Dsc2) (and desmoglein2 alike) is highly specific but of low affinity with average bond lifetimes in the range of approximately 0.3 s. Notably, despite this comparatively weak interaction, desmosomes mediate a stable, tensile-resistant bond. In addition, force mediated dissociation of strand-swap dimers exhibit a reduced bond lifetime as external forces increase (slip bond). Using atomic force microscopy based single molecule force spectroscopy (AFM-SMFS), we demonstrate that Dsc2 has two further binding modes that, in addition to strand-swap dimers, most likely play a significant role in the integrity of the cardiac muscle. At short interaction times, the Dsc2 monomers associate only loosely, as can be seen from short-lived force-independent bonds. These ideal bonds are a precursor state and probably stabilize the formation of the self-inhibiting strand-swap dimer. The addition of tryptophan in the measurement buffer acts as a competitive inhibitor, preventing the N-terminal strand exchange. Here, Dsc2 dimerizes as X-dimer which clearly shows a tri-phasic slip-catch-slip type of dissociation. Within the force-mediated transition (catch) regime, Dsc2 dimers switch between a rather brittle low force and a strengthened high force adhesion state. As a result, we can assume that desmosomal adhesion is mediated not only by strand-swap dimers (slip) but also by their precursor states (ideal bond) and force-activated X-dimers (catch bond).