Abstract 20426: Study of S-Nitrosylation in Duchenne Muscular Dystrophy
Introduction: Severe cardiac myopathy occurs with Duchenne muscular dystrophy (DMD), a fatal disease characterized by absence of functional dystrophin protein. The mechanisms of the disease in cardiac muscle and whether it mimics some of the pathophysiological mechanisms present in skeletal muscle are not clear. The loss of dystrophin was reported to disrupt neuronal nitric oxide synthase mu, presumably altering redox-signaling. Nitric oxide-induced S-nitrosylation (SNO) on cysteine residues is known as a redox-sensor, which responds to cellular dynamic redox-environment. Here we identified and quantified the SNO-modified sites of cardiac tissue in dystrophy model using a novel proteomics approach.
Methods: SNO-proteins from cardiac tissues of 10 months-old mdx/utrn+/- (n=3) and C57BL/6 (n=3) mice were detected with our improved TMT (tandem mass tag)-switch technique coupled with liquid chromatography/tandem mass spectrometry. The identified SNO of each site was quantified across DMD and WT samples and the fold change of SNO on each site in DMD compared to WT was displayed as ‘SNO-index (I)’, calculated by SNODMD/SNOWT.
Results: Proteomics analysis identified total 866 SNO-modified peptides on 287 SNO-proteins in the dystrophy cardiac tissue. The magnitudes of SNO-fold change were various across peptides (I=0.1 least to 1702.7 most). 32% of these, 278 peptides were more than two-fold oxidized in DMD than WT. Interestingly, a termed ‘Dilated cardiomyopathy’ protein group by KEGG-pathway analysis has extremely oxidized sites than other groups, which are more than 10-folds S-nitrosylated in DMD. Myosin-6 at Cys949 and Cys1750 (I=11.4, 117.3), Cys432 of myosin-binding protein C (I=135.0) and Cys191 of myosin light chain 3 (I=81.2) displayed a considerable SNO-increase in dystrophy sample and Cys35 of troponin C was also more modified (I=2.9), while other sites such as Cys397 of creatine kinase S-type was less oxidized (I=0.26) in DMD.
Conclusions: Our novel TMT-switch assay allowed quantification of the very labile SNO-modification as well as site-identification. More than 500 cysteine residues sensed the oxidative stimuli and each had a specific susceptibility to SNO. The SNO-based redox switches were shifted in DMD compared with WT.
Author Disclosures: H.S. Chung: None. P.P. Rainer: None. D.A. Kass: None. J.E. Van Eyk: Ownership Interest; Modest; ImmunArray. Consultant/Advisory Board; Modest; ImmunArray Inc.
- © 2014 by American Heart Association, Inc.