Spatial and age-related changes in the microstructure of dystrophic and healthy diaphragms

PLOS ONE, Nov 2019

Duchenne muscular dystrophy (DMD) is a progressive degenerative disease that results in fibrosis and atrophy of muscles. The main cause of death associated with DMD is failure of the diaphragm. The diaphragm is a dome-shaped muscle with a fiber microstructure that differs across regions of the muscle. However, no studies to our knowledge have examined spatial variations of muscle fibers in dystrophic diaphragm or how aging affects those variations in DMD. In this study, diaphragms were obtained from mdx and healthy mice at ages three, seven, and ten months in the dorsal, midcostal, and ventral regions. Through immunostaining and confocal imaging, we quantified sarcomere length, interstitial space between fibers, fiber branching, fiber cross sectional area (CSA), and fiber regeneration measured by centrally located nuclei. Because DMD is associated with chronic inflammation, we also investigated the number of macrophages in diaphragm muscle cross-sections. We saw regional differences in the number of regenerating fibers and macrophages during the progression of DMD in the mdx diaphragm. Additionally, the number of regenerating fibers increased with age, while CSA and the number of branching fibers decreased. Dystrophic diaphragms had shorter sarcomere lengths than age-matched controls. Our results suggest that the dystrophic diaphragm in the mdx mouse is structurally heterogeneous and remodels non-uniformly over time. Understanding regional changes in dystrophic diaphragms over time will facilitate the development of targeted therapies to prevent or minimize respiratory failure in DMD patients.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0183853&type=printable

Spatial and age-related changes in the microstructure of dystrophic and healthy diaphragms

September Spatial and age-related changes in the microstructure of dystrophic and healthy diaphragms Catherine C. Henry 1 2 Kyle S. Martin 1 2 Bridget B. Ward 1 2 Geoffrey G. Handsfield 1 2 Shayn M. Peirce 0 1 2 Silvia S. Blemker 1 2 3 0 Department of Ophthalmology, University of Virginia , Charlottesville, Virginia , United States of America 1 Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia, United States of America, 2 Auckland Bioengineering Institute, University of Auckland , Auckland , New Zealand 2 Editor: Gerhard Wiche, Universitat Wien , AUSTRIA 3 Department of Mechanical and Aerospace Engineering, University of Virginia , Charlottesville, Virginia , United States of America Duchenne muscular dystrophy (DMD) is a progressive degenerative disease that results in fibrosis and atrophy of muscles. The main cause of death associated with DMD is failure of the diaphragm. The diaphragm is a dome-shaped muscle with a fiber microstructure that differs across regions of the muscle. However, no studies to our knowledge have examined spatial variations of muscle fibers in dystrophic diaphragm or how aging affects those variations in DMD. In this study, diaphragms were obtained from mdx and healthy mice at ages three, seven, and ten months in the dorsal, midcostal, and ventral regions. Through immunostaining and confocal imaging, we quantified sarcomere length, interstitial space between fibers, fiber branching, fiber cross sectional area (CSA), and fiber regeneration measured by centrally located nuclei. Because DMD is associated with chronic inflammation, we also investigated the number of macrophages in diaphragm muscle cross-sections. We saw regional differences in the number of regenerating fibers and macrophages during the progression of DMD in the mdx diaphragm. Additionally, the number of regenerating fibers increased with age, while CSA and the number of branching fibers decreased. Dystrophic diaphragms had shorter sarcomere lengths than age-matched controls. Our results suggest that the dystrophic diaphragm in the mdx mouse is structurally heterogeneous and remodels non-uniformly over time. Understanding regional changes in dystrophic diaphragms over time will facilitate the development of targeted therapies to prevent or minimize respiratory failure in DMD patients. - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: C.C.H. was supported by the Beckman Scholars Program (http://www.beckmanfoundation.org) and the Harrison Undergraduate Research Award at the University of Virginia (http:// www.virginia.edu/cue/harrison.html). Research funding was provided by NSF Grant #1235244 (https://www.nsf.gov) and NIH Grant #U01AR069393 awarded to S.S.B. and S.M.P. The funders had no role in study design, data collection Introduction Duchenne muscular dystrophy (DMD) affects 1 in 3,500 live male births and results from an x-linked gene mutation that causes a deficiency of the protein dystrophin [1±3]. Loss of dystrophin results in progressive damage of muscle, as evidenced by increased fibrosis, branched and analysis, decision to publish, or preparation of the manuscript. muscle fibers, chronic inflammation, muscle fiber atrophy, and fatty infiltration [4±8]. Ultimately, respiratory failure is the leading cause of death in DMD patients, which is largely a result of degeneration and associated weakening of the diaphragm muscle [ 3,9 ]. In order to understand the age-related progression of diaphragm degeneration, it is important to develop a greater understanding of diaphragm microstructure in DMD and how microstructure changes with age. It is known that diaphragm microstructure changes as a result of the aging process in both healthy and dystrophic diaphragms. Healthy, aging diaphragms undergo muscle weakening and atrophy and exhibit decreased cross sectional area (CSA) of type IIx/IIb fibers [ 10 ]. In contrast to these healthy age-related changes, diaphragms of the mdx mouse, a widely used mouse model of DMD [ 8,11,12 ], display varied muscle fiber size, necrosis, and an increase in connective tissue after only 6 months [ 8 ]. Connective tissue continues to increase and fibrosis develops by 270 days (~9 months) [ 13 ]. By this time, the mdx diaphragm also displays significant fiber CSA decreases compared to controls. In mdx diaphragms, slow myosin is doubled relative to controls at 16 months, a phenomenon also documented in DMD [ 8,14 ]. By 16±22 months of age, the optimal fiber length in mdx diaphragms is shorter than age-matched controls and, at 24 months, mdx diaphragms have seven times the collagen density of controls [8]. Due to the dramatic microstructural changes in mdx diaphragms over time, age is an important factor to consider when studying DMD [ 8,13 ]. In addition to temporal changes, the microstructure of healthy diaphragms also varies spatially. Muscle fiber area, perimeter, (...truncated)


This is a preview of a remote PDF: https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0183853&type=printable

Catherine C. Henry, Kyle S. Martin, Bridget B. Ward, Geoffrey G. Handsfield, Shayn M. Peirce, Silvia S. Blemker. Spatial and age-related changes in the microstructure of dystrophic and healthy diaphragms, PLOS ONE, 2017, Volume 12, Issue 9, DOI: 10.1371/journal.pone.0183853