Diaphragm muscle sarcopenia in aging mice

Abstract

Sarcopenia, defined as muscle weakness and fiber atrophy, of respiratory muscles such as the diaphragm (DIAm) has not been well characterized. The DIAm is the main inspiratory muscle and knowledge of DIAm sarcopenia is important for establishing the effects of aging on respiratory function. We hypothesized that aging is associated with a loss of DIAm force and reduced fiber cross-sectional area (CSA), and that these changes vary across fiber types. DIAm sarcopenia was assessed in young (5 month; n = 11) and old (23 month; n = 12) wild-type mice reflecting ~100 and 75% survival, respectively. In addition, DIAm sarcopenia was evaluated in BubR1(H/H) mice (n = 4) that display accelerated aging (~60% survival at 5 months) as a result of expression of a hypomorphic allele (H) of the mitotic checkpoint protein BubR1. Maximum specific force (normalized for CSA) of the DIAm was 34% less in old mice and 57% lower in BubR1(H/H) mice compared to young mice. Mean CSA of type IIx and/or IIb DIAm fibers was 27% smaller in old wild-type mice and 47% smaller in BubR1(H/H) mice compared to young mice. Mean CSA of type I or IIa fibers was not different between groups. Collectively these results demonstrate sarcopenia of the DIAm in aging wild-type mice and in BubR1(H/H) mice displaying accelerated aging. Sarcopenia may limit the ability of the DIAm to accomplish expulsive, non-ventilatory behaviors essential for airway clearance. As a result, these changes in the DIAm may contribute to respiratory complications with aging.

Keywords: Atrophy; Contractile properties; Cross-sectional area; Fiber type; Myosin heavy chain; Respiratory muscles.

Figure 1

Contractility of the DIAm from natural and accelerated aging mice (mean±SE). Specific forces are corrected for physiological cross-sectional area. Both naturally-aged mice at 75% survival (old; n=10) and the BubR1^H/H accelerated-aging model (n=4) at ~60% survival displayed less force generating capacity than young mice (n=7). Data analyzed by one-way ANOVA. *Significantly different than young mice.

Figure 2

Representative serial cross-sections of DIAm fibers from natural and accelerated aging mice. A) Hematoxylin and eosin stained cross-sections, indicating a normal and healthy appearance for all age groups. B) Fiber types were classified according to myosin heavy chain (MyHC) expression based on immunoreactivity to isoforms MyHC_slow (blue) and MyHC_2A (purple), the absence of staging (black) was classified as MyHC IIx and/or IIb. Anit-laminin (green) was used to define the fiber sarcolemma. Scale bar represents 50 μm.

Figure 3

Fiber cross-sectional area (CSA) of DIAm muscles from natural and accelerated aging mice (mean±SE). A) The distribution of fiber CSAs of the DIAm with significantly shifted toward the left in both old (n= 5) and BubR1^H/H (n= 4) mice compared to young (n= 9) mice, distribution analyzed by chi-squared analysis. B) Fiber CSA of type identified DIAm fibers; there was a significant decrease in the CSA of type IIx and/or IIb fibers of old and BubR1^H/H mice compared to young mice. Data analyzed by one-way ANOVA. *Significantly different than young mice. n=4-9 per group.

Figure 4

The proportion of DIAm fibers by fiber type and age. Data were analyzed by two-way ANOVA (group × fiber type). Interaction post hoc results are as follows: ^‡significantly different than type I fibers of the same group; ^§significantly different than type IIa fibers of the same group;*significantly different than young mice within the same fiber type; ^†significantly different than old mice within the same fiber type. n=4-9 per group.