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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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 3

MdxS3E mice are protected against Iso-induced arrhythmias and aberrant Cx43 hemichannel opening.

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MdxS3E mice are protected against Iso-induced arrhythmias and aberrant C...
(A) Top row: representative baseline ECG recordings obtained from 4- to 6-month-old mice following anesthesia. Bottom row: representative ECG traces recorded approximately 30 minutes after Iso (5 mg/kg, i.p.) challenge. Scale bars: 100 ms (for all traces). (B) Arrhythmia scores based on predetermined scale where 0 = no arrhythmias, 1 = single PVCs, 2 = double PVCs, 3 = triple PVCs or nonsustained VT, 4 = sustained VT or AV block, 5 = death. ****P < 0.0001 versus WT; ####P < 0.0001 versus mdx. n = 8 (WT); n = 9 (mdx, mdxS3A); n = 10 (mdxS3E). (C) Representative immunofluorescence images of ethidium uptake of heart cryosections after perfusion with ethidium (5 μm) under control conditions (top row) or after Iso treatment (middle row). Cryosections were visualized for ethidium (red) and stained for WGA (green) and nuclei (DAPI, blue). White boxes indicate areas magnified in the bottom row (insets). White arrows indicate nuclei that do not colocalize with ethidium; orange arrows indicate positive ethidium and nuclei colocalization. Scale bars: 20 μm. Original magnification ×20. (D) Quantification of the dye uptake under both control (circles) and Iso (triangles) conditions. **P < 0.005, ***P < 0.001 versus WT control; ###P < 0.001 versus mdx control; §§§§P < 0.0001 versus WT Iso; ††††P < 0.0001 versus mdx Iso; ‡P < 0.05 versus control in each genotype (n = 4 each group per treatment). Data are presented as mean ± SEM. Statistical significance determined by 1-way ANOVA (B) or 2-way ANOVA (D) followed by Tukey’s post hoc test.