Passive mechanical properties of rat abdominal wall muscles suggest an important role of the extracellular connective tissue matrix

Abstract

Abdominal wall muscles have a unique morphology suggesting a complex role in generating and transferring force to the spinal column. Studying passive mechanical properties of these muscles may provide insights into their ability to transfer force among structures. Biopsies from rectus abdominis (RA), external oblique (EO), internal oblique (IO), and transverse abdominis (TrA) were harvested from male Sprague-Dawley rats, and single muscle fibers and fiber bundles (4-8 fibers ensheathed in their connective tissue matrix) were isolated and mechanically stretched in a passive state. Slack sarcomere lengths were measured and elastic moduli were calculated from stress-strain data. Titin molecular mass was also measured from single muscle fibers. No significant differences were found among the four abdominal wall muscles in terms of slack sarcomere length or elastic modulus. Interestingly, across all four muscles, slack sarcomere lengths were quite long in individual muscle fibers (>2.4 µm), and demonstrated a significantly longer slack length in comparison to fiber bundles (p < 0.0001). Also, the extracellular connective tissue matrix provided a stiffening effect and enhanced the resistance to lengthening at long muscle lengths. Titin molecular mass was significantly less in TrA compared to each of the other three muscles (p < 0.0009), but this difference did not correspond to hypothesized differences in stiffness.

Figure 1

Mean elastic modulus of single fibers and fiber bundles for each abdominal wall muscle. Moduli of single fibers were calculated as the slope of the linear region of the stress-strain relationship. Moduli of fiber bundles were calculated by fitting 2^nd-order polynomials to the stress-strain data of each sample, differentiating and solving for modulus at a sarcomere length of 3.2 µm. No significant differences were found among any of the muscles. Error bars represent standard error of the mean.

Figure 2

Representative stress-strain raw data points and curve fits for 3 individual RA muscle fibers and 3 RA fiber bundles. Individual fibers were linearly fit after exceeding an initial toe region; fiber bundles were fit with 2^nd-order polynomials over the stress-strain relationship.

Figure 3

A. 2^nd-order polynomial fits of fiber bundle stress-sarcomere length relationship across all animals for each muscle. Data were fit to the stress-strain relationship for each tested fiber bundle, and coefficients were averaged across all samples for each muscle. Strain was then converted to sarcomere length based on average slack sarcomere lengths for fiber bundles for each muscle. Stress SEM, calculated from the polynomial fits at sarcomere length 4.24 µm, were: RA 14.1 kPa; EO 31.0 kPa; IO 23.6 kPa; and TrA 24.5 kPa. B. Piece-wise linear fits of individual fiber stress-sarcomere length relationship across all animals for each muscle. Data were fit to the stress-strain relationship for each tested fiber, and coefficients were averaged across all samples for each muscle. The steeper linear region for each muscle represents the modulus values reported in Figure 1.

Figure 4

Mean slack sarcomere length of single fibers and fiber bundles for each muscle. A significant difference was observed between single fibers and fiber bundles (p < 0.0001). Error bars represent standard error of the mean.

Figure 5

A. Mean titin molecular mass for single fibers from each muscle. Star indicates TrA titin was significantly smaller compared to each of the other three muscles (p < 0.0009). Error bars represent standard error of the mean. B. Example SDS-VAGE gel lanes. Molecular masses were calculated by the migration of titin relative to standards of known mass (human soleus and rat cardiac). STD represents the standard lane containing titin from human soleus (sample) and rat cardiac muscle. RA represents the lane containing titin from RA (sample) and rat cardiac muscle. TrA represents the lane containing titin from TrA (sample) and rat cardiac muscle. T2 degradation bands are degradation forms of the intact titin molecule.