Prevention of connexin-43 remodeling protects against Duchenne muscular dystrophy cardiomyopathy
Eric Himelman, … , Jorge E. Contreras, Diego Fraidenraich
Eric Himelman, … , Jorge E. Contreras, Diego Fraidenraich
Published January 7, 2020
Citation Information: J Clin Invest. 2020;130(4):1713-1727. https://doi.org/10.1172/JCI128190.
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Research Article Cardiology Cell biology

Prevention of connexin-43 remodeling protects against Duchenne muscular dystrophy cardiomyopathy

Abstract

Aberrant expression of the cardiac gap junction protein connexin-43 (Cx43) has been suggested as playing a role in the development of cardiac disease in the mdx mouse model of Duchenne muscular dystrophy (DMD); however, a mechanistic understanding of this association is lacking. Here, we identified a reduction of phosphorylation of Cx43 serines S325/S328/S330 in human and mouse DMD hearts. We hypothesized that hypophosphorylation of Cx43 serine-triplet triggers pathological Cx43 redistribution to the lateral sides of cardiomyocytes (remodeling). Therefore, we generated knockin mdx mice in which the Cx43 serine-triplet was replaced with either phospho-mimicking glutamic acids (mdxS3E) or nonphosphorylatable alanines (mdxS3A). The mdxS3E, but not mdxS3A, mice were resistant to Cx43 remodeling, with a corresponding reduction of Cx43 hemichannel activity. MdxS3E cardiomyocytes displayed improved intracellular Ca2+ signaling and a reduction of NADPH oxidase 2 (NOX2)/ROS production. Furthermore, mdxS3E mice were protected against inducible arrhythmias, related lethality, and the development of cardiomyopathy. Inhibition of microtubule polymerization by colchicine reduced both NOX2/ROS and oxidized CaMKII, increased S325/S328/S330 phosphorylation, and prevented Cx43 remodeling in mdx hearts. Together, these results demonstrate a mechanism of dystrophic Cx43 remodeling and suggest that targeting Cx43 may be a therapeutic strategy for preventing heart dysfunction and arrhythmias in DMD patients.

Authors

Eric Himelman, Mauricio A. Lillo, Julie Nouet, J. Patrick Gonzalez, Qingshi Zhao, Lai-Hua Xie, Hong Li, Tong Liu, Xander H.T. Wehrens, Paul D. Lampe, Glenn I. Fishman, Natalia Shirokova, Jorge E. Contreras, Diego Fraidenraich

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Figure 4

Normalization of intracellular Ca2+ response to hypo-osmotic shock and reduction of ROS production in mdxS3E cardiomyocytes.

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Normalization of intracellular Ca2+ response to hypo-osmotic shock and r...
(A) Representative images of mdx (top left) and mdxS3E (top right) cardiomyocytes imaged for intracellular Ca2+ indicator Fluo-4AM (green, top row) and transmitted light (BF, bottom row of top panel) at times in isotonic solution (0 seconds), at the end of hypo-osmotic shock (80 seconds), and after return to isotonic solution (120 seconds). Bottom left panel represents time course of normalized Fluo-4 fluorescence in WT (red), mdx (gray), and mdxS3E (blue) cells. Bottom right panel shows pooled data of mean values of normalized fluorescence during 60 seconds after the osmotic shock. n = 3 animals; n = 13 myocytes for all genotypes. (B) Representative images of DCF fluorescence in mdx (top left) and mdxS3E (top right) cardiomyocytes at the beginning (0 s) and end (120 s) of exposure. Bottom left graph illustrates changes in average DCF signals in WT (red), mdx (gray), and mdxS3E (blue) myocytes over 120 seconds. Bottom right graph illustrates the rate of oxidation × 1000. n = 4; n = 43 (WT); n = 4; n = 19 (mdx); n = 4; n = 30 (mdxS3E). Scale bars: 20 μm. ****P < 0.0001 versus WT; ####P < 0.0001 versus mdx (both analyses). Data are presented as mean ± SEM. Statistical significance determined by 1-way ANOVA followed by Tukey’s post hoc test.