Prevention of connexin-43 remodeling protects against Duchenne muscular dystrophy cardiomyopathy
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
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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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 1

Phosphorylation of S325/S328/S330 in Cx43 is reduced in mouse and human dystrophic hearts.

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Phosphorylation of S325/S328/S330 in Cx43 is reduced in mouse and human ...
(A) Representative Western blot and quantification of WT and mdx ventricular lysates probed for pan-Cx43 (top), pS-Cx43 (middle), and vinculin (loading control, bottom). n = 7 in both groups. ***P < 0.001 versus WT. Note the differential Cx43 migration patterns indicated by the phospho-isoforms P3, P2, P1, and P0 in WT and mdx lysates. WT and mdx samples shown were run on the same gel, but were noncontiguous, as indicated by black lines between samples. (B) Representative immunofluorescence images (magnified images are in insets) of pan-Cx43 (green) and N-cadherin (red) in 4-month-old WT and mdx ventricular cryosections. (C) Representative immunofluorescence images (magnified images are in insets) of pS-Cx43 (green) and N-cadherin (red) in 4-month-old WT and mdx ventricular cryosections. (D) Representative Western blot and quantification of human non-DMD and DMD ventricular lysates probed for pan-Cx43 (top), pS-Cx43 (middle), and vinculin (loading control, bottom). n = 3 in both groups. **P < 0.01 versus non-DMD. p, phosphorylated isoform; np, nonphosphorylated isoform. (E) Representative immunofluorescence images of magnified IDs stained for pan-Cx43 (green) and N-cadherin (red) in human non-DMD and DMD ventricular cryosections. (F) Representative immunofluorescence images of magnified IDs stained for pS-Cx43 (green) and N-cadherin (red) in human non-DMD and DMD ventricular cryosections. White arrows indicate Cx43 localization at the IDs; red arrows indicate lateralized Cx43. Data are presented as mean ± SEM. Statistical significance was determined by 2-sided t test. Scale bars: 20 μm (B and C), original magnification ×60; 5 μm (E and F).