Proteomic Analysis Reveals New Cardiac-Specific Dystrophin-Associated Proteins

Abstract

Mutations affecting the expression of dystrophin result in progressive loss of skeletal muscle function and cardiomyopathy leading to early mortality. Interestingly, clinical studies revealed no correlation in disease severity or age of onset between cardiac and skeletal muscles, suggesting that dystrophin may play overlapping yet different roles in these two striated muscles. Since dystrophin serves as a structural and signaling scaffold, functional differences likely arise from tissue-specific protein interactions. To test this, we optimized a proteomics-based approach to purify, identify and compare the interactome of dystrophin between cardiac and skeletal muscles from as little as 50 mg of starting material. We found selective tissue-specific differences in the protein associations of cardiac and skeletal muscle full length dystrophin to syntrophins and dystrobrevins that couple dystrophin to signaling pathways. Importantly, we identified novel cardiac-specific interactions of dystrophin with proteins known to regulate cardiac contraction and to be involved in cardiac disease. Our approach overcomes a major challenge in the muscular dystrophy field of rapidly and consistently identifying bona fide dystrophin-interacting proteins in tissues. In addition, our findings support the existence of cardiac-specific functions of dystrophin and may guide studies into early triggers of cardiac disease in Duchenne and Becker muscular dystrophies.

Dystrophin–glycoprotein complex regulates muscle nitric oxide production through mechanoregulation of AMPK signaling

“…Immunofluorescence labeling revealed that nNOS was localized in the cytoplasm, in a striated pattern, and in the nucleus and was not specifically enriched at the sarcolemma of WT or mdx cardiomyocytes at rest (Fig. S1), in agreement with previous reports (33,34). Moreover, stretch did not lead to membrane recruitment of nNOS or otherwise discernibly alter nNOS localization in either WT or mdx cells (Fig.…”

“…Despite this apparent similarity of the effect of dystrophin deficiency on cardiac and skeletal muscle NOS signaling, another recent study has demonstrated that in contrast to skeletal muscle, nNOS is not physically associated with the DGC in cardiac muscle (34). These interesting observations suggest that dystrophin and the DGC's impact on NO signaling might not result from a purely scaffolding function of the DGC for nNOS in striated muscle.…”

Nicorandil, a Nitric Oxide Donor and ATP-Sensitive Potassium Channel Opener, Protects Against Dystrophin-Deficient Cardiomyopathy

“…28,29 Neuronal NOS (nNOS) interacts with dystrophin-associated glycoprotein complex at the sarcolemma in skeletal myocytes, and this association is lost in dystrophin deficiency. 28 However, in cardiomyocytes isolated from the mdx mouse, nNOS does not colocalize with dystrophin 30,31 but instead colocalizes with the ryanodine receptor 2 (RyR2) at the sarcoplasmic reticulum. 32 Endothelial NOS localizes to the Golgi complex and the sarcolemma caveolae.…”

A multi-model statistical approach for proteomic spectral count quantitation

“…Traditional maximum-likelihood techniques based on the negative binomial distribution have been used to model count data from transcriptomic experiments but it has been suggested that they can routinely be applied to proteomic spectral count data [57]. However, current approaches used to model RNA sequencing data, namely, edgeR [26], DESeq [27] and baySeq [28] have not been routinely applied to spectral count proteomics experiments, with few exceptions [58–61]. Each of these statistical approaches is uniquely valuable when determining differential expression and can be applied to small scale discovery proteomics experiments with as few as three biological replicates.…”