S-nitrosylation of connexin43 hemichannels elicits cardiac stress–induced arrhythmias in Duchenne muscular dystrophy mice
Mauricio A. Lillo, Eric Himelman, Natalia Shirokova, Lai-Hua Xie, Diego Fraidenraich, Jorge E. Contreras
Mauricio A. Lillo, Eric Himelman, Natalia Shirokova, Lai-Hua Xie, Diego Fraidenraich, Jorge E. Contreras
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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 7

Position C271, but not C260 and C298, was S-nitrosylated and mediated NO-induced hemichannel currents.

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Position C271, but not C260 and C298, was S-nitrosylated and mediated NO...
(A) Representative current traces for oocytes expressing Cx43 with a deleted CT and Cx43 mutants C260S, C271S, and C298S. Black and red traces correspond to voltage step–evoked currents in the absence or presence of 10 μM DEENO, respectively. Oocytes were clamped to −80 mV, and square pulses from −80 mV to +90 mV (in 10-mV steps) were then applied for 2 seconds. At the end of each pulse, the membrane potential was returned to −80 mV. Graph shows normalized fold increase of DEENO current after treatment at different voltages. 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. Cx43ΔCT; †P < 0.05 vs. Cx43C271. (B) DEENO decreased the resting membrane potential in oocytes expressing Cx43 mutants C260S and C298S but not in those expressing the Cx43ΔCT or Cx43 mutant C271S. The number in parentheses indicates the n value. Comparisons between groups were made using 2-way ANOVA test; *P < 0.05 vs. control. (C) Top gel was loaded with S-nitrosylated proteins pulled down using the biotin switch assay and blotted against Cx43. Bottom Western blot was loaded with total proteins of oocytes expressing Cx43 against Cx43. The number in parentheses indicates the n value. Comparisons between groups were made using Student’s t test; *P < 0.05 vs. control.