S-nitrosylation of connexin43 hemichannels elicits cardiac stress–induced arrhythmias in Duchenne muscular dystrophy mice
Mauricio A. Lillo, … , Diego Fraidenraich, Jorge E. Contreras
Mauricio A. Lillo, … , Diego Fraidenraich, Jorge E. Contreras
Published December 19, 2019; First published November 12, 2019
Citation Information: JCI Insight. 2019;4(24):e130091. https://doi.org/10.1172/jci.insight.130091.
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Research Article Muscle biology

S-nitrosylation of connexin43 hemichannels elicits cardiac stress–induced arrhythmias in Duchenne muscular dystrophy mice

Abstract

Patients with Duchenne muscular dystrophy (DMD) commonly present with severe ventricular arrhythmias that contribute to heart failure. Arrhythmias and lethality are also consistently observed in adult Dmdmdx mice, a mouse model of DMD, after acute β-adrenergic stimulation. These pathological features were previously linked to aberrant expression and remodeling of the cardiac gap junction protein connexin43 (Cx43). Here, we report that remodeled Cx43 protein forms Cx43 hemichannels in the lateral membrane of Dmdmdx cardiomyocytes and that the β-adrenergic agonist isoproterenol (Iso) aberrantly activates these hemichannels. Block of Cx43 hemichannels or a reduction in Cx43 levels (using Dmdmdx Cx43+/– mice) prevents the abnormal increase in membrane permeability, plasma membrane depolarization, and Iso-evoked electrical activity in these cells. Additionally, Iso treatment promotes nitric oxide (NO) production and S-nitrosylation of Cx43 hemichannels in Dmdmdx heart. Importantly, inhibition of NO production prevents arrhythmias evoked by Iso. We found that NO directly activates Cx43 hemichannels by S-nitrosylation of cysteine at position 271. Our results demonstrate that opening of remodeled and S-nitrosylated Cx43 hemichannels plays a key role in the development of arrhythmias in DMD mice and that these channels may serve as therapeutic targets to prevent fatal arrhythmias in patients with DMD .

Authors

Mauricio A. Lillo, Eric Himelman, Natalia Shirokova, Lai-Hua Xie, Diego Fraidenraich, Jorge E. Contreras

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Figure 3

Iso increases S-nitrosylated levels of Cx43 at the lateral side of Dmdmdx cardiomyocytes.

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Iso increases S-nitrosylated levels of Cx43 at the lateral side of Dmdmd...
(A) Top and middle gels were loaded with S-nitrosylated proteins pulled down from heart samples using the biotin switch assay. Top gel was, then, blotted against Cx43, and the middle gel is the corresponding Ponceau staining. Lower blot was loaded using total cardiac proteins and blotted against Cx43. The bottom graph is the quantification for 5 independent blots using the ratio for S-nitrosylated Cx43/Ponceau. The number in parentheses indicates the n value. Comparisons between groups were made using 2-way ANOVA plus Tukey’s post hoc test. *P < 0.05 vs. WT control, and †P < 0.05 vs. WT Iso. (B) Analysis performed by PLA of the interaction between Cx43 and S-nitrosylation. Plasma membrane stained with wheat germ agglutinin (WGA) and S-nitrosylated Cx43 (Cx43-SNO) are shown in green and red, respectively. Representative images of n = 5 per group. (C) Assessment of Cx43 hemichannel activity in isolated Dmdmdx hearts perfused with buffer containing 5 μM ethidium bromide after or without treatment with Iso. Arrows show nuclei. The number in parentheses indicates the n value. Comparisons between groups were made using 2-way ANOVA test plus Tukey’s post hoc test. *P < 0.05 vs. vehicle WT; †P < 0.05 vs. vehicle Dmdmdx. (D) Top and middle gels were loaded with S-nitrosylated proteins pulled down from human heart samples using the biotin switch assay. Top gel was, then, blot against Cx43 and the middle gel is the corresponding ponceau staining. Lower blot was load using total cardiac proteins and blot against Cx43. (E) Analysis performed by PLA of the interaction between Cx43 and S-nitrosylation in human samples. Note that Cx43 is S-nitrosylated at the lateral side of DMD human samples compare to non-DMD.