Alterations of Extracellular Matrix Mechanical Properties Contribute to Age-Related Functional Impairment of Human Skeletal Muscles

Abstract

Aging of human skeletal muscles is associated with increased passive stiffness, but it is still debated whether muscle fibers or extracellular matrix (ECM) are the determinants of such change. To answer this question, we compared the passive stress generated by elongation of fibers alone and arranged in small bundles in young healthy (Y: 21 years) and elderly (E: 67 years) subjects. The physiological range of sarcomere length (SL) 2.5-3.3 μm was explored. The area of ECM between muscle fibers was determined on transversal sections with picrosirius red, a staining specific for collagen fibers. The passive tension of fiber bundles was significantly higher in E compared to Y at all SL. However, the resistance to elongation of fibers alone was not different between the two groups, while the ECM contribution was significantly increased in E compared to Y. The proportion of muscle area occupied by ECM increased from 3.3% in Y to 8.2% in E. When the contribution of ECM to bundle tension was normalized to the fraction of area occupied by ECM, the difference disappeared. We conclude that, in human skeletal muscles, the age-related reduced compliance is due to an increased stiffness of ECM, mainly caused by collagen accumulation.

Keywords: aging; collagen; extracellular matrix; resting tension; single muscle fiber; skeletal muscle.

Figure 1

Left: passive tension vs. sarcomere length for fibers (open squares) and bundles (open circles) in elderly subjects. Fibers N = 11, cross-sectional area (CSA) 7718 ± 1499 μm², bundles N = 11 CSA 18,360 ± 2648 μm², (mean ± SEM), average 5 fibers in each bundle. Right: passive tension vs. sarcomere length for fibers (filled squares) and bundles (filled circles) in young subjects. Fibers N = 12, fiber CSA 7777 ± 886 μm², bundles N = 13 CSA 25,115 ± 2095 μm², (mean ± SEM), average 5 fibers in each bundle. Data are shown as mean value +/- SEM (standard error of the mean), after interpolation with a cubic function as described in Experimental Procedures section.

Figure 2

Histogram of the passive tension in the fibers (pale color) and in the bundles (dark color) at different sarcomere lengths for the young (blue) and elderly (red) subjects. Post-hoc comparisons of ANOVA are shown. * p < 0.05, statistical difference between fibers and bundles in the same age group. # p < 0.05, statistical difference of bundles between age groups. Data are shown as mean value +/- SEM. Note that the difference between fibers of young and elderly subjects does not reach statistical significance at any sarcomere length.

Figure 3

Increase of the relative area occupied by collagen and extracellular matrix (ECM) with age. Typical examples of transverse sections of biopsy samples stained with picrosirius red from subjects of different ages: (A), young, 18 years; (B), young adult, 32 years; and (C), elderly, 74 years.

Figure 4

ECM area fraction vs. age of subjects. Solid line is the regression, while dashed lines identify the confidence interval at p = 0.05. Males (N = 13) are indicated with filled circles, while females (N = 3) are indicated with empty circles.

Figure 5

Normalized extra-tension in young and elderly subjects. Left: the same fraction (α=3.3%) of ECM in CSA is considered in the two groups. Right: fraction of ECM (parameter α) is imposed at 3.3% of fibers CSA in young subjects and 8.2% in elderly subjects. Data are shown as mean value +/- SEM. The differences between elderly and young subjects at each sarcomere length level of the left histogram are statistically significant (p < 0.05), while the same differences in the right histogram are not.