Limited forward trafficking of connexin 43 reduces cell-cell coupling in stressed human and mouse myocardium
James W. Smyth, … , Neil C. Chi, Robin M. Shaw
James W. Smyth, … , Neil C. Chi, Robin M. Shaw
Published December 28, 2009
Citation Information: J Clin Invest. 2010;120(1):266-279. https://doi.org/10.1172/JCI39740.
View: Text | PDF
Research Article

Limited forward trafficking of connexin 43 reduces cell-cell coupling in stressed human and mouse myocardium

Abstract

Gap junctions form electrical conduits between adjacent myocardial cells, permitting rapid spatial passage of the excitation current essential to each heartbeat. Arrhythmogenic decreases in gap junction coupling are a characteristic of stressed, failing, and aging myocardium, but the mechanisms of decreased coupling are poorly understood. We previously found that microtubules bearing gap junction hemichannels (connexons) can deliver their cargo directly to adherens junctions. The specificity of this delivery requires the microtubule plus-end tracking protein EB1. We performed this study to investigate the hypothesis that the oxidative stress that accompanies acute and chronic ischemic disease perturbs connexon forward trafficking. We found that EB1 was displaced in ischemic human hearts, stressed mouse hearts, and isolated cells subjected to oxidative stress. As a result, we observed limited microtubule interaction with adherens junctions at intercalated discs and reduced connexon delivery and gap junction coupling. A point mutation within the tubulin-binding domain of EB1 reproduced EB1 displacement and diminished connexon delivery, confirming that EB1 displacement can limit gap junction coupling. In zebrafish hearts, oxidative stress also reduced the membrane localization of connexin and slowed the spatial spread of excitation. We anticipate that protecting the microtubule-based forward delivery apparatus of connexons could improve cell-cell coupling and reduce ischemia-related cardiac arrhythmias.

Authors

James W. Smyth, Ting-Ting Hong, Danchen Gao, Jacob M. Vogan, Brian C. Jensen, Tina S. Fong, Paul C. Simpson, Didier Y.R. Stainier, Neil C. Chi, Robin M. Shaw

×

Figure 5

Forward trafficking of Cx43 to the plasma membrane is reduced during oxidative stress.

Options: View larger image (or click on image) Download as PowerPoint
Forward trafficking of Cx43 to the plasma membrane is reduced during oxi...
(A) Timeline of Tet-inducible Cx43-EYFP trafficking assay. A tetracycline-inducible clonal HeLa cell line expressing Cx43-EYFP was induced with 2 μg/ml doxycycline 2.5 hours prior to imaging with TIRFm in the presence or absence of 200 μM H2O2, which was added 30 minutes prior to imaging. (B) TIRFm visualization of Cx43-EYFP delivery. TIRFm detection of Cx43-EYFP at 150, 195, and 240 minutes after the addition of doxycycline in the presence or absence of H2O2. White lines outline cell cortex. (C) Comparison of widefield epifluorescence and TIRFm detection of Cx43-EYFP at 240 minutes after the addition of doxycycline, showing that cells contain comparable levels of Cx43-EYFP. (D) Quantification of TIRFm-detectable Cx43-EYFP surface intensity. Error bars represent SEM. (E) Total and surface N-cadherin and Cx43-EYFP levels following total surface protein biotinylation and pulldown through neutravidin. Input lysates and pulldowns of biotinylated surface proteins were subjected to SDS-PAGE on the same gel. Original magnification, ×100. Scale bars: 10 μm. Values represent mean ± SEM.