Abstract 1510: Oxidative Stress Interferes with Targeted Delivery of Gap Junctions to the Plasma Membrane
Chronically stressed myocardium has decreased gap junction coupling between cardiomyocytes. The mechanisms underlying this decrease are poorly understood. A common component in stressed myocardium is oxidative stress which at a cellular level, can disrupt microtubules. Microtubules are known to transport Connexin43 (Cx43) hemichannels and, together with the microtubule plus-end binding protein EB1, participate in targeted delivery of hemichannels to adherens junctions. We hypothesized that oxidative stress perturbs connexon trafficking through its effect on microtubule based targeting and delivery. Using co-immunoprecipitation and fixed cell immunofluorescence we found to our surprise that EB1 is displaced off microtubules during hydrogen peroxide induced oxidative stress. Moreover, live cell total internal reflection microscopy (TIRFm) studies showed reduced interaction of EB1-tipped microtubules with the plasma membrane. An inducible clonal cell line was used with TIRFm to resolve real-time delivery of nascent Cx43 hemichannels to the cell cortex. Oxidative stress decreased dynamic Cx43 hemichannel delivery. Imaging assays were complimented with biochemistry, including surface biotinylation to quantify Cx43 trafficking to the plasma membrane. We conclude that oxidative stress decreases Cx43 hemichannel delivery to the plasma membrane by displacing EB1 off microtubules. Protection of the microtubule trafficking machinery is a promising approach for therapies aimed at preserving gap junction coupling in the stressed myocardium.