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.
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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

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Figure 3

EB1 is displaced off of microtubule plus ends and interacts less with the plasma membrane during oxidative stress.

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EB1 is displaced off of microtubule plus ends and interacts less with th...
(A) HeLa cells exposed to 200 μM H2O2 for 4 hours were fixed in ice-cold methanol, and confocal immunofluorescence detection of EB1 (green) and α-tubulin (red) was performed with nuclei counterstained using TO-PRO-3 (blue). Enlargement of a cell-cell border region (scale bar: 10 μm) shows a reduction in EB1 at the microtubule plus end. Original magnification, ×60. (B) Quantification of EB1 comet length. (C) Schematic representation of TIRFm detection of EB1-EGFP at dynamic microtubule plus ends interacting with the plasma membrane. (D) EB1 movement visualized by TIRFm. HeLa cells were transfected with EB1-EGFP 24 hours prior to imaging. Maximum-intensity projections of compiled images from 2-minute TIRFm acquisitions before, after 45 minutes of exposure to 200 μM H2O2, and 45 minutes following end of exposure to 200 μM H2O2. Original magnification, ×100. Scale bar: 10 μm. (E) Density of EB1 comet events detectable by TIRFm. Data are representative of 3 separate experiments. Statistical analysis was performed using the Student’s unpaired t test (B) and a 1-way ANOVA with Bonferroni post-hoc correction (E). Data represent mean ± SEM. ***P < 0.001 compared with control.