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.
View: Text | PDF
Research Article Cardiology Cell biology Article has an altmetric score of 49

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

×

Figure 7

The dystrophic MT cytoskeleton contributes to Cx43 phosphorylation and localization.

Options: View larger image (or click on image) Download as PowerPoint
The dystrophic MT cytoskeleton contributes to Cx43 phosphorylation and l...
(A) Representative Western blot (top) and quantification (bottom) for gp91phox in mdx mice treated with either saline or colch. Vinculin was used as a loading control for this and all proceeding blots. (B) Representative Western blots (top) of ox-CaMKII, (top blot), total CaMKII (bottom blots), and vinculin in mdx mice treated with either saline or colch. Ox-CaMKII protein levels were normalized to corresponding total CaMKII levels (both controlled for loading) and quantified (bottom). (C) Representative Western blots (top) for total-Cx43 (top), pS-Cx43 (middle), and vinculin (bottom) in mdx mice treated with either saline or colch. pS-Cx43 protein levels were normalized to corresponding total Cx43 levels (both controlled for loading) and quantified (bottom). (D) Representative Western blots of Cx43 and vinculin (top) from saline- or colch-treated mdx heart tissue subject to Triton X-100 based tissue fractioning. Insoluble Cx43 protein levels were normalized to corresponding total Cx43 levels (both controlled for loading) and then expressed as fold change relative to mdx saline mean value. (E) Left: representative confocal immunofluorescence images of mdx saline (top) and colch (bottom) cardiac IDs and lateral regions of cardiomyocytes stained with pan-Cx43 (green) and N-cadherin (red). Scale bar: 25 μm. White boxes indicate areas magnified in the image; original magnification ×60. Right: quantification of Cx43/N-cadherin colocalization in confocal immunofluorescence images as described for Figure 2, expressed as fold change relative to mdx saline mean value. n = 7 per treatment (A and E); n = 6 per treatment (B–D). *P < 0.05 versus mdx-saline. Data are presented as mean ± SEM. Statistical significance determined by 2-sided t test (all analyses).