The study of the interactome of dystrophin has been hampered by the large size of dystrophin and by the complexity of its direct and indirect interactions with extracellular, transmembrane, and intracellular proteins. Yet the identification of dystrophin-associated proteins is fundamental to our knowledge of the functions of dystrophin in healthy striated muscles and, by correlate, to our understanding of the molecular events that trigger cardiac and skeletal muscle disease in dystrophinopathies.
This study describes a rapid and simple protocol that allows for the high confidence and robust identification of proteins that interact either directly or indirectly with dystrophin within muscle tissues. Compared to previous studies 
, this approach offers the advantage of specifically purifying dystrophin-containing complexes with the MANDYS1 antibody rather than using lectins that bind to multiple glycosylated proteins. We are also directly interrogating the entire protein eluate with minimal losses in sensitivity associated with gel separation of proteins. This resulted in impressive protein coverage, high confidence scores and reliable detection of all core DAPC members. This high level of sensitivity likely played a key role in our ability to identify with high confidence Cypher, Ahnak1, Cavin-1, and CRYAB as new cardiac-specific dystrophin-associated proteins. Indeed, our Western blot results indicate that these proteins are not abundant in dystrophin immunoprecipitations, possibly explaining why they have not been previously detected. Finally, we downsized the amount of starting material required to 50 mg of tissue. The ability to study the interactome of dystrophin from small amounts of tissue is not of trivial importance. It enables the study of DAPC composition by proteomics in the mouse, an animal model for many human muscle disorders 
. In combination with the spectral count analysis described here, this opens the door to future comparative studies in mouse models of muscular dystrophies aimed at understanding how disease-causing mutations may affect the composition of the DAPC. In addition, 50 mg is within the size range of a human tissue biopsy, suggesting that our protocol may be adapted in the future to directly study the DAPC in human muscles. Analysis of human cardiac samples may be particularly informative since our results with α-dystrobrevin suggest a higher complexity of the human cardiac DAPC compared to the mouse.
The successful comparison of affinity purified dystrophin protein complexes between cardiac and skeletal muscles required a tissue-specific background strategy and was further made possible by a multi-factor quantitative bio-informatics approach applied to spectral counts. From a biological standpoint, our approach provided a global yet detailed view of the DAPC in cardiac and skeletal muscle that clearly revealed differences specifically affecting the syntrophin-dystrobrevin sub-complex and its interaction with nNOS. The finding that nNOS is not part of the cardiac DAPC suggests that dystrophin is not involved in nNOS membrane localization in cardiomyocytes as it is in skeletal muscle fibers 
. This agrees with the reported interaction of nNOS with the ryanodine receptor in the heart and its spatial confinement to the sarcoplasmic reticulum 
where dystrophin is absent. A link between cardiac dystrophin and nNOS has been postulated based on decreased nNOS activity in mdx
and on improved cardiac histopathology upon nNOS over-expression 
. Our data favors a model where perturbations in cardiac nNOS activity are secondary to the loss of dystrophin, while in skeletal muscle deregulated nNOS function is a direct consequence of lack of dystrophin at the myofiber membrane 
Our findings also raise the question of the identity of the proteins that interact with the cardiac syntrophin/dystrobrevin complex and the nature of the intracellular functions they may mediate. Given a general paucity of information on binding partners for syntrophins and dystrobrevins, we cannot at this time address the functional significance of the observed preferential inclusion of β2-syntrophin and α3-dystrobrevin in the cardiac DAPC. Interestingly, mice genetically engineered to lack α1-, α2- and α3-dystrobrevins show cardiomyocyte degeneration, inflammation and fibrosis indicating that α-dystrobrevins are important for cardiac integrity 
. In addition, in
studies have indicated that all three syntrophins can interact with and may regulate the activity of the cardiac sodium channel Nav1.5 which is disrupted in the dystrophin-deficient heart 
Finally, our findings open the door to studies into the functional significance of the novel cardiac-specific association of dystrophin with Cavin-1, Ahnak1, Cypher and CRYAB. These novel associations suggest a direct link between dystrophin and two major cardiac systems that are disrupted in the dystrophin-deficient heart: the regulation of ion channels that initiate and pace contraction, and the sarcomeric apparatus that ultimately mediates cardiac contraction. Indeed, the majority of dystrophinopathy patients have arrhythmias, long QT syndrome and contractile dysfunction 
, and therefore overlap in phenotype with patients with mutations in cypher, CRYAB, Ahnak1, or Cavin-1 
. Therefore, our proteomics findings suggest a novel molecular link between these different cardiac diseases that warrants further investigation.