In Duchene muscular dystrophy (DMD), muscle stiffness increases due to intramuscular fibrosis, characterized by excessive accumulation of extracellular matrix (ECM) components such as collagen. Although many studies focus on the impact of amount of collagen in DMD, only some report a positive correlation between increased amount of collagen and increased stiffness. Collagen has a complex organization that may also impact stiffness and our goal was to determine if, and the extent to which, collagen organization changes with DMD progression. We used scanning electron microscopy (SEM) to determine collagen fiber and fibril organization in the epimysial layer of the ECM of diaphragm muscle in mdx mice.
Samples were collected from 3 (n=3), 6 (n=5), and 9-month (n=4) mdx mice and collagen structure was isolated by digesting muscle fibers. SEM images were taken of collagen fibers (1kX) and fibrils (50kX). An image processing algorithm was developed to measure collagen orientation in local windows for each image. Mean orientation, quantified in degrees relative to muscle fiber orientation, was used to measure collagen fiber or fibril preferred direction. Strength of alignment, quantified as the length of the mean resultant vector (0<R<1, 1=isotropic, 0=anisotropic), was used to measure the circular spread in local orientations per image. Significance was measured with t test and Bonferroni correction.
Changes in collagen organization were observed with aging and disease progression. Mean preferred direction was significantly different between collagen fibers at 6 (55.0±17.8°) and 9-months (77.4±14.9°) and between collagen fibrils at 6 (63.4±9.9°) and 9-months (82.0±13.1°). At 6-months, strength of alignment was significantly different between fibers (0.94±0.02) and fibrils (0.88±0.05). Our findings suggest that both collagen fiber and fibril organization are affected with DMD progression. We will further investigate their role on diaphragm function in dystrophic muscle.