Targeted Inhibition of Connexin 43 Hemichannels Blunts Ca 2+-Induced Intercellular Dyssynchrony and ATP Efflux in Hl-1 Cardiomyocyte Syncitia

Abstract

Gap junctions (GJ) are essential conduits that underpin cell-to-cell coupling between cardiomyocytes and are formed from the coalescence of connexin (Cx) hemichannels from two close opposed cells. However, there is evidence supporting a (patho)physiologic role for ‘unpaired’ Cx hemichannels as they traffic through the plasma membrane (PM) to the GJ. We used beating HL-1 cardiomyocyte monolayers as a model system for GJ intercellular communication (GJIC) and to investigate the functional role of Cx hemichannels in syncitial behaviour. GJIC was quantified as an index of intercellular Ca2+ release synchrony and was reconciled with detailed spatio-temporal analysis of intracellular Ca2+ signalling. Ouabain-evoked Ca2+ perturbation inhibited GJIC in a dose-dependent manner and was associated with reduced cell viability. We hypothesised that the intercellular dyssynchrony and cell death linked to ouabain-induced Ca2+ dysfunction was exacerbated by aberrant Cx hemichannel opening that may also compromise cellular metabolism. Consistent with this concept, the magnitude of intracellular Ca2+ flux dysfunction correlated with ATP release from cells. Trans-PM ATP leak was attenuated by Gap20, a peptide corresponding to an intracellular loop motif in Cx43. Moreover, Gap20 reduced intracellular Ca2+ perturbation, improved intercellular synchrony and reduced cell death in ouabain-treated syncitia. The lack of efficacy of a peptide corresponding to a similar epitope in Cx26, and also following the conjugation of Gap20 to a high molecular weight dextran confirmed i) the specificity of this approach and ii) that peptide bioactivity is dependent on its entry into cells and interaction with the intracellular face of Cx. Our data provides evidence that altered cellular Ca2+ homeostasis opens Cx hemichannels and that this may accelerate the metabolic deterioration of cardiomyocytes and exacerbate cardiac dysfunction in situ.