Intrinsic stiffness of extracellular matrix increases with age in skeletal muscles of mice

Abstract

Advanced age is associated with increases in muscle passive stiffness, but the contributors to the changes remain unclear. Our purpose was to determine the relative contributions of muscle fibers and extracellular matrix (ECM) to muscle passive stiffness in both adult and old animals. Passive mechanical properties were determined for isolated individual muscle fibers and bundles of muscle fibers that included their associated ECM, obtained from tibialis anterior muscles of adult (8-12 mo old) and old (28-30 mo old) mice. Maximum tangent moduli of individual muscle fibers from adult and old muscles were not different at any sarcomere length tested. In contrast, the moduli of bundles of fibers from old mice was more than twofold greater than that of fiber bundles from adult muscles at sarcomere lengths >2.5 μm. Because ECM mechanical behavior is determined by the composition and arrangement of its molecular constituents, we also examined the effect of aging on ECM collagen characteristics. With aging, muscle ECM hydroxyproline content increased twofold and advanced glycation end-product protein adducts increased threefold, whereas collagen fibril orientation and total ECM area were not different between muscles from adult and old mice. Taken together, these findings indicate that the ECM of tibialis anterior muscles from old mice has a higher modulus than the ECM of adult muscles, likely driven by an accumulation of densely packed extensively crosslinked collagen.

Keywords: age crosslinking; collagen; muscle mechanics; passive tension; tangent modulus.

Fig. 1.

Representative passive tension responses of mouse tibialis anterior fiber to step length increases. Five stretches were applied at a velocity of 1 fiber length (L_f/s), all from a resting sarcomere length (SL) of 2.1 μm and increasing in 0.2 μm/sarcomere increments, resulting in a mean sarcomere length of 3.1 μm after the final stretch. Detailed records for the final 2 stretches are shown. Tension was measured 2 min after each stretch as indicated by the arrows. Fibers were forced to slacken for 50 ms immediately after each tension measurement to indicate the location of zero on the tension record. Fibers were then returned to a sarcomere length of 2.1 μm and held there for 1.3 s before the next stretch.

Fig. 2.

Stress-strain responses (A) and tangent moduli (B) for single fibers and fiber bundles from adult and old TA muscles. At long sarcomere lengths, old muscle bundles exhibited larger stresses than old single fibers, indicating increased bundle modulus at sarcomere lengths at 2.7, 2.9, and 3.1 μm. Moduli of adult fibers and bundles were similar, and single fiber modulus was unchanged with age. *Significantly different from all other groups at a given strain (P < 0.05). Data presented as means ± SD.

Fig. 3.

Representative adult (A) and old (B) tibialis anterior muscle cross sections immunostained with wheat germ agglutinin and DAPI for visualization of extracellular matrix (ECM) and cell nuclei, respectively. Total ECM area remained unchanged with age (P = 0.79). Scale bars = 75 μm.

Fig. 4.

Representative cross sections of tibialis anterior (TA) muscles of adult (A) and old (B) mice stained with picrosirius red and viewed under polarized light. Red staining indicates collagen fibrils that are oriented at a large angle with respect to the muscle fiber, and yellow and green staining indicates collagen fibrils that are more parallel to the fibers. Scale bars = 200 μm. Total number of colored pixels increased with aging, suggesting an age-associated increase in total collagen content in the muscle cross sections (C). When analyzed as a percent of all colored pixels, the distribution of red, yellow, and green pixels remained constant with aging, indicating no age-associated change in collagen orientation (D). *Significantly different from adult group (P < 0.05). Data presented as means ± SD.

Fig. 5.

Hydroxyproline (A) and advanced glycation end product (AGE) protein adduct (B) concentration in whole TA muscles. Old muscles had increased hydroxyproline content and AGE adduct concentration compared with muscles from adult animals. *Significantly different from adult group (P < 0.05). Data presented as means ± SD.