Muscle Fiber Splitting Is a Physiological Response to Extreme Loading in Animals

Abstract

Skeletal muscle fiber branching and splitting typically is associated with damage and regeneration and is considered pathological when observed during loading-induced hypertrophy. We hypothesize that fiber splitting is a nonpathological component of extreme loading and hypertrophy, which is primarily supported by evidence in animals, and propose that the mechanisms and consequences of fiber splitting deserve further exploration.

Figure 1.. Skeletal muscle architectural adaptations in the mouse plantaris following synergist ablation overload, an extreme model for rodent hypertrophy.

The synergist ablation technique induces robust hypertrophy of the plantaris muscle, which can be evaluated at the muscle fiber level via histology. During eight weeks of overload, pennation angle in the mouse plantaris increases from ~14° to ~16° (21). If muscle fiber hypertrophy (lines) keeps pace with increased pennation angle, then more muscle fibers will not appear on cross section when the muscle is mounted upright and cut perpendicular to the long axis of the muscle (circles). However, if muscle fiber hypertrophy does not sync with increased pennation angle, more muscle fibers could theoretically appear on cross-section as a consequence of increased pennation angle. The plantaris is a bi-articular muscle (61) and may not adapt architecturally as expected during hypertrophy. It is conceivable that changes in muscle geometry during hypertrophy influences the number of fibers appearing on cross section, but we do not think this could entirely account for increased muscle fiber number during hypertrophy.

Figure 2.. The appearance of split muscle fibers following 14 days of modified synergist ablation overload of the mouse plantaris.

A-D illustrate fiber splitting over ~50 µm on serial cross sections of a frozen plantaris muscle. Representative images show laminin and dystrophin to identify muscle fiber borders, and myonuclei. Dark arrows point to central myonuclei that appear prior to the appearance of each new branch in the muscle fiber (light arrows). E shows a phase-contrast image of a trifurcated single muscle fiber from this same mouse, along with myonuclei. F illustrates the extent of fiber splitting morphology in this mouse, with minimal eMyHC expression (a marker of regeneration). Note that many fibers contain >1 mis-positioned myonucleus, which appears to signify the splitting process. Immunohistochemistry images were captured at 20x and 40x magnification, and single fiber image was captured at 40x; Scale bars = 50 µm. (Reprinted from (44). Copyright © 2017 BioMed Central. Used with permission.)

Figure 3.. Potential explanations for increased fiber number on cross-section during robust hypertrophy.

In scenario A-G, a muscle fiber response to an extreme hypertrophic stimulus but is damaged in the process. This leads to a degeneration-regeneration response in which the sarcolemma is damaged but the basal lamina initially remains intact. The muscle fiber is ultimately degraded while satellite cells are activated to initiate regeneration. During regeneration, aberrant fusion of satellite cells leads to the appearance of a branched (assymetrical, not depicted) or split (symmetrical) muscle fiber. In scenario A’-G’, there is focal damage to the sarcolemma in response to a robust hypertrophic stimulus. Satellite cells are activated and form a myocyte that “grafts” to the existing muscle fiber, ultimately resulting in a branching or splitting phenotype. In scenario A”-G”, an existing muscle fiber grows and reaches a critical point, upon which it splits. Whether a split fiber can become two separate muscle fibers, and whether satellite cells are required to fuse in to maintain a new muscle fiber is unknown.

Figure 4.. Muscle fiber size and number in the soleus muscle with progressive weighted wheel running (PoWeR) in adult mice.

PoWeR is a non-invasive minimally-injurious method for inducing muscle fiber hypertrophy via exercise in mice. A Relative to untrained age-matched control mice, muscle fiber cross sectional area (CSA) measured on entire histological cross-sections using dystrophin immunohistochemistry and automated software (62) is greater after 8 weeks and muscle fiber number is not different. After 12 weeks of PoWeR, muscle fiber size is lower relative to 8 week-trained mice, and muscle fiber number is greater. B The leftward shift in the muscle fiber size distribution curve suggests that the largest muscle fibers may have split sometime between 8 and 12 weeks of training. Data were normally distributed, significance was determined via one-way ANOVA with a Tukey’s post-hoc test and significance set at P<0.05, shared letters = NOT significantly different, ^#p=0.12 vs untrained, n = 5 female >4 month old C57BL6 mice per group.