Non-linear Scaling of Passive Mechanical Properties in Fibers, Bundles, Fascicles and Whole Rabbit Muscles

Abstract

Defining variations in skeletal muscle passive mechanical properties at different size scales ranging from single muscle fibers to whole muscles is required in order to understand passive muscle function. It is also of interest from a muscle structural point-of-view to identify the source(s) of passive tension that function at each scale. Thus, we measured passive mechanical properties of single fibers, fiber bundles, fascicles, and whole muscles in three architecturally diverse muscles from New Zealand White rabbits (n = 6) subjected to linear deformation. Passive modulus was quantified at sarcomere lengths across the muscle's anatomical range. Titin molecular mass and collagen content were also quantified at each size scale, and whole muscle architectural properties were measured. Passive modulus increased non-linearly from fiber to whole muscle for all three muscles emphasizing extracellular sources of passive tension (p < 0.001), and was different among muscles (p < 0.001), with significant muscle by size-scale interaction, indicating quantitatively different scaling for each muscle (p < 0.001). These findings provide insight into the structural basis of passive tension and suggest that the extracellular matrix (ECM) is the dominant contributor to whole muscle and fascicle passive tension. They also demonstrate that caution should be used when inferring whole muscle properties from reduced muscle size preparations such as muscle biopsies.

Keywords: collagen; muscle architecture; passive tension; scaling; titin.

FIGURE 1

Photomicrograph of samples at the single fiber, fiber bundle (approximately 20 fibers), and fascicle (∼300 fibers, defined by natural ECM divisions) scale. Each sample (1.5–3.0 mm in length) was secured using suture to a force transducer on one end and a titanium wire rigidly attached to a rotational bearing on the other end. Each stretch was held for 3 min to permit stress-relaxation before measuring passive tension. See text for details.

FIGURE 2

Passive stress-strain curves for (A) TA, (B) EDL, and (C) ED2 with each curve representing a different size scale: fibers (open circles), bundles (filled circles), fascicles (open squares), and whole muscle (filled squares). As size scale increased, samples increased in stiffness and became highly non-linear, presumably due to changes in ECM. Data are presented as mean ± SEM.

FIGURE 3

Passive modulus-strain curves for (A) TA, (B) EDL, and (C) ED2 with each curve representing a different size scale: fibers (dotted lines), bundles (squares), fascicles (triangles), and whole muscle (inverted triangles). As size scale increased, modulus increases. Data are presented as mean ± SEM.

FIGURE 4

Scaling of passive tension modulus across four size scales and three muscles. (A) Modulus calculated at 30% strain, (B) Modulus calculated at 20% strain, (C) Modulus calculated at 10% strain. In all cases, two-way ANOVA revealed a significant effect of scale and muscle with significant scale × muscle interaction (p < 0.0001).

FIGURE 5

Measurement of muscle biochemical content to provide insights into the sources of passive tension. (A) Average collagen content was determined for fibers, bundles, fascicles, and whole muscle homogenate using a hydroxyproline assay. Collagen content increased with size scale (p < 0.0001) and was significantly different across muscles (p < 0.0001) with significant scale × muscle interaction (p < 0.0001). Collagen content is expressed as a percentage of wet weight. (B) Average titin molecular weight is shown for fibers, bundles, and fascicles and a whole muscle homogenate from TA, EDL, and ED2. Titin molecular mass was not significantly different across size scales for any of the muscles tested (p = 0.24). However, titin isoform size for TA was significantly smaller compared to EDL and ED2 (p < 0.0001). Data are presented as mean ± SEM.