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 1

Levels of Cx43 and EB1 at the intercalated disc are reduced with end-stage ischemic cardiomyopathy.

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Levels of Cx43 and EB1 at the intercalated disc are reduced with end-sta...
Immunofluorescence labeling of cryosections from snap-frozen non-failing and end-stage ischemic explanted human heart tissue. (A) Human heart Cx43 immunofluorescence. N-cadherin in red and Cx43 in green, with enlarged overlay images. N-cadherin was used as a marker for the intercalated disc (arrows). (B) Cx43 at intercalated discs. Quantification of Cx43 fluorescence intensity in regions also positive for N-cadherin expression. (C) Tissue fractionation. Triton X-100–based fractionation of soluble (cytoplasmic) and insoluble (junctional) protein from tissue detected by Western blot analysis, quantified in bar charts. (D) EB1 immunofluorescence. N-cadherin in red and EB1 in green with enlarged panels at right displaying comparable intercalated discs. (E) EB1 enrichment at intercalated discs. Quantification of EB1 fluorescence intensity in regions also positive for N-cadherin expression. Original magnification, ×60. Scale bars: 10 μm. Data are representative of 4 non-failing and 4 end-stage ischemic explanted hearts. Statistical analysis was performed using the Student’s unpaired t test. Values represent mean ± SEM. *P < 0.05, **P < 0.01.