Analysis of muscle fiber clustering in the diaphragm muscle of sarcopenic mice

Abstract

Introduction: Sarcopenia likely comprises muscle fiber denervation and re-innervation, resulting in clustering of muscle fibers of the same type (classified by myosin heavy chain isoform composition). Development of methodology to quantitatively evaluate clustering of muscle fibers according to fiber type is necessary.

Methods: Fiber type specific immunofluorescence histology was used to quantify fiber clustering in murine diaphragm muscle (n = 15) at ages 6 and 24 months.

Results: With age, fiber type clustering is evidenced by fiber type specific changes in distances between fibers, specifically a 14% decrease to the closest fiber for type I and 24% increase for type IIx and/or IIb fibers (P < 0.001). Additionally, a 34% increase to the 3 closest type IIx and/or IIb fibers was found (P < 0.001).

Conclusions: This novel method of analyzing fiber type clustering may be useful in examining pathophysiological conditions of motor unit loss in neuromuscular disorders, myopathies, dystrophies, injuries, or amyotrophic lateral sclerosis.

Keywords: aging; cross-sectional area; fiber type; motor unit; myosin heavy chain; sarcopenia.

Figure 1

Representative histologic images as used for analysis of fiber type clustering. A) The color combined image of the myosin heavy chain (MyHC) isoforms, blue denotes type I fibers, purple type IIa fibers, and the black type IIx and/or IIb fibers (scale bars are 50 µm). Note that type IIa fibers are weakly immunoreactive to the anti-MyHC_Slow antibody used for triple labeling giving a purple hue to type IIa fibers (see Methods). B) Using MetaMorph the threshold was set resulting in a black and white image. The black outline is indicative of the sarcolemma of the diaphragm (DIAm) section. The individual fibers types are separated for analysis; C) type I fibers, D) type IIa fibers, and E) type IIx and/or IIb. The coordinate plots for these figures show the interfiber distance to for all fibers (gray lines) and for the 3 closest fibers (red lines) for a randomly selected fiber. Representative plots are shown for; F) all fibers, G) type I fibers, H) type IIa fibers, and I) type IIx and/or IIb.

Figure 2

Diaphragm muscle fiber cross-sectional area from young and old mice, presented as the distribution of fiber cross-sectional areas independent of fiber type. There was a significant left shit in the old fiber cross-sectional areas in comparison to young; distribution was analyzed by chi-squared analysis (P<0.001).

Figure 3

Diaphragm muscle fiber type proportions from young and old mice. Data were analyzed was analyzed by two-way ANOVA (age×fiber type); there was an interaction of age and fiber type (P<0.001). Interaction post hoc results are as follows: *significantly different than old age group of same fiber type; ^†significantly different than type I fibers; ^‡significantly different than type IIa fibers.

Figure 4

The type dependent distance to fibers was determined for A) the closest individual fiber and B) the 3 closest fibers across ages. C) The distance to the 3 closest fibers dependent on fiber type was also normalized by the type specific average interfiber distance to account for the change in fiber type proportions with age. Data was analyzed with a mixed linear model with an individual animal as a random effect (age×fiber type×animal); there was a main effect of age and fiber type P<0.001.*significantly different than old age group of same fiber type; ^†significantly different than type I fibers; ^‡significantly different than type IIa fibers.