Duchenne muscular dystrophy (DMD) is an incurable, rapidly-worsening neuromuscular degenerative disease that affects nearly one in every 3,600 male infants (MedlinePlus-NIH). Although most research has focused on skeletal muscle, the development of cardiomyopathy, seen in 100% of patients over 18 years of age, is an important cause of death of dystrophic patients. While DMD is a recessive X-linked form of muscular dystrophy, dystrophic cardiomyopathy also affects female carriers (dystrophin +/-), found to harbor hearts mosaic for dystrophin expression. As cardiac fibers do not form a syncytium, cardiomyocytes (CMs) are compartmentalized in dystrophin positive and dystrophin negative fibers in DMD carriers. However, previous findings have shown that 20% wild type (WT) incorporation in mdx (dystrophin -/-) mice relieved myopathic symptoms, suggesting that in the mosaic heart the mechanism by which WT CMs prevent DMD pathology does not involve the structural role of dystrophin, but may involve dystrophin-dependent intercellular signaling. In this project, modulation of the nitric oxide (NO) pathway, specifically related to neuronal nitric oxide synthase (nNOS), will be examined in DMD carrier models to understand both the cell type requirement of functional pathway members and also if induced overexpression of a critical upstream factor can lead to correction.
Specific Aim #1 will focus on the use of chimeric mouse models to most accurately represent and examine the mosaicism seen in DMD carrier patient hearts. Through the injection of WT embryonic stem (ES) cells into mdx:nNOS knock out (KO) blastocysts, and the injection of nNOS KO ES cells into mdx blastocysts, mosaic models with compartment specific nNOS ablation will be generated. By knocking out nNOS in separate compartments, the determination of which cell type is required for proper function and rescue can be discerned through the use of immunofluorescence, western blotting, and experiments viewing pathway activity. In addition, by assessing degrees of chimerism it is possible to understand relative levels of incorporation required for correction.
In Specific Aim #2, the L-Arginine transporter CAT-2a, a critical upstream factor in the NO pathway, will be overexpressed in CMs obtained from WT, mdx, and nNOS KO mice, by use of adenoviral- CAT-2a in vitro infection. Through measurements of NO pathway members downstream of CAT-2a, normalization can be directly viewed. In addition, siRNA targeted to CAT-2a will be used in similar conditions to view the degree of dysregulation of the NO pathway and associated downstream activity and function. The modeling of specific mosaicism seen in DMD carrier patients, through the use of chimeric mice, allows for direct clinical implications from this projet. By examining distinct populations of cells, as well as associated morphological alterations, functional and phenotypic changes can be assessed. In understanding the most critical components of the NO pathway in regards to DMD associated cardiomyopathy, results gained through CAT-2a modulation will serve as a basis for further therapeutic research for DMD patients.

Public Health Relevance

Duchenne Muscular Dystrophy carrier patients represent a distinct population wherein those who are symptomatic experience severe cardiomyopathy, while those who are asymptomatic utilize novel uncharacterized rescue mechanisms. The use of mosaic mice in this research project most accurately models human carrier patients, allowing for the precise determination of canonical nitric oxide pathway factors affected and employed in correction of disease. Most importantly, by direct modulation of these compensatory mechanisms, namely the overexpression of the nitric oxide pathway critical L-Arginine transporter CAT in cardiomyocytes, this project will serve as a basis for therapeutic research to benefit all DMD patients.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1-F10A-S (20))
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Wang, Wayne C
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Rutgers University
Anatomy/Cell Biology
Schools of Medicine
United States
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