Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.     

Fiber architecture of the intrinsic muscles of the shoulder and arm in semiterrestrial and arboreal guenons

The internal organization of myofibers and connective tissues has important physiologic implications for muscle function and for naturalistic behavior. In this study of forelimb muscle morphology and primate locomotion, fiber architecture is examined in the intrinsic muscles of the shoulder (musculi deltoideus, infraspinatus, supraspinatus, subscapularis, teres major, and t. minor) and arm (m. coracobrachialis, biceps brachii, brachialis, and triceps brachii) in the semiterrestrial vervets (Chlorocebus aethiops) and arboreal red-tailed guenons (Cercopithecus ascanius). Wet weights and lengths of whole muscles, lengths of fasciculi and their associated proximal and distal tendons, and angles of pinnation were measured to estimate morphologic correlates of physiologic properties of individual muscles: force, velocity/excursion, energy expense, and relative isometric or isotonic contraction. Neither mean total-shoulder:total-arm ratios for muscle mass nor total reduced physiological cross-sectional area exhibited significant (P < 0.05) interspecific differences, thus emphasizing the importance of fine-tuning musculoskeletal analyses by the data collected here. The results generally support those previously published for quadriceps femoris and triceps surae of the hind limb in these species (Anapol and Barry [1996] Am. J. Phys. Anthropol. 99:429-447). The fiber architecture of the semiterrestrial vervets is largely suited for higher velocity while running on the ground. By contrast, the architectural configuration of red-tailed monkeys implies relatively isometric muscle contraction and passive storage of elastic strain energy for exploitation of the compliant canopy, where substrate components are situated beneath the sagittal plane of the animal. With respect to relative distribution of maximum potential force output among muscles of either shoulder or arm groups in these otherwise hind limb-dominated quadrupedal primates, statistically significant interspecific differences are best interpreted in light of braking, climbing, and, for vervets, the transition between ground and canopy.The interspecific differences shown here for the intrinsic muscles of the shoulder and arm underscore the significance of intramuscular morphology in reconciling structure and function with regard to locomotor behavior. Its analysis and interpretation lend support to consideration of "semiterrestrial" as a bona fide locomotor category uniquely different from what is practiced by dedicated arboreal and terrestrial quadrupeds that occasionally visit the habitat of one another. Data from a more committed terrestrial species would clarify this enigma.     

Age-related changes in muscle fiber type frequencies and cross-sectional areas in straightbred and crossbred rabbits

This study was designed to investigate the effects of the interaction among genetic group, sex and age on the frequencies and cross-sectional areas of myofiber types in rabbits. A total of 48 straightbred and crossbred Botucatu rabbits, males and females, were involved in a split plot design with a 2 × 2 (genetic groups × genders) factorial arrangement. Young rabbits were weaned at 35 days of age and sequentially slaughtered, four per genetic group × sex combination, at 42, 63 and 84 days of age. The flexor carpi radialis muscle was dissected, histological sections (10 μm) were obtained and the frequencies and cross-sectional areas of myofiber types: I, IIA and IIB/X were determined. An effect of the genetic group × sex × slaughter age interaction was found on the frequency distribution of myofiber types. A transition from type IIA to type IIB/X fibers was observed (P < 0.01) with advancing age, except in crossbred females, but the frequency of IIA fibers was already lower (57.3%) and of IIB/X fibers numerically higher (33.7%) in this group at 42 days. The proportions of IIA fibers in straightbred males, crossbred males and straightbred females decreased from 80.1%, 89.4% and 68.8% at 42 days to 43.9%, 52.3% and 40.1% at 63 days, respectively, whereas the proportions of type IIB/X fibers, in the same groups, increased from 10.3%, 1.6% and 22.3% at 42 days to 42.2%, 37.0% and 49.8% at 63 days, respectively. In all three age points, type IIA fibers showed the largest cross-sectional areas, followed by type I and IIB/X fibers. The cross-sectional areas of IIB/X fibers were larger in crossbreds, but no differences were found between genetic groups concerning fiber types IIA and I. All three types of fibers showed positive linear association with age, but relative to the initial area type IIB/X fibers presented a higher degree of hypertrophy (144% up to 84 days) than type IIA and I fibers (86% and 85%, respectively). The flexor carpi radialis muscle was, on average, heavier in crossbred than in straightbred females, but no difference was observed between crossbred and straightbred males. Differences in the weight of flexor carpi radialis muscle were attributed to the hypertrophy of type IIB/X fibers in the crossbreds.     

SIRT1 regulates nuclear number and domain size in skeletal muscle fibers

Skeletal muscle fibers are giant multinucleated cells wherein individual nuclei govern the protein synthesis in a finite volume of cytoplasm; this is termed the myonuclear domain (MND). The factors that control MND size remain to be defined. In the present study, we studied the contribution of the NAD⁺‐dependent deacetylase, sirtuin 1 (SIRT1), to the regulation of nuclear number and MND size. For this, we isolated myofibers from mice with tissue‐specific inactivation (mKO) or inducible overexpression (imOX) of SIRT1 and analyzed the 3D organisation of myonuclei. In imOX mice, the number of nuclei was increased whilst the average MND size was decreased as compared to littermate controls. Our findings were the opposite in mKO mice. Muscle stem cell (satellite cell) numbers were reduced in mKO muscles, a possible explanation for the lower density of myonuclei in these mice; however, no change was observed in imOX mice, suggesting that other factors might also be involved, such as the functional regulation of stem cells/muscle precursors. Interestingly, however, the changes in the MND volume did not impact the force‐generating capacity of muscle fibers. Taken together, our results demonstrate that SIRT1 is a key regulator of MND sizes, although the underlying molecular mechanisms and the cause‐effect relationship between MND and muscle function remain to be fully defined.     

Estrogen modulates 7/4 antigen distribution within eccentrically contracted injured skeletal muscle

Abstract

Eccentric contractions are skeletal muscle stretches with concurrent active force production; these contractions commonly occur during dynamic sports activities and can cause acute muscle injury. Recovery from this injury depends in part on pro-inflammatory processes, such as neutrophil infiltration at the injured site, which is affected by estrogen. This estrogen effect has been examined broadly, but without distinguishing between major compartments within muscle in which neutrophil infiltration can occur. Therefore, we compared neutrophil antigen expression in two compartments of eccentrically contracted muscle of ovariectomized mice with or without estrogen. To quantify neutrophil antigen expression, serial cross sections of muscle were immunolabeled with antibodies that recognize 7/4 or Ly6C/G, then quantified using computer-assisted image analysis. At 48 h post injury, estrogen-positive (E+) mice had more 7/4-positive and Ly6C/G-positive myofibers, increased 7/4 area percentage, and more 7/4-positive cells in the connective tissue. In addition, E+ mice showed more 7/4-positive myofibers that were Ly6C/G-negative and more Ly6C/G-positive myofibers that were 7/4-negative. These data suggest that in injured muscle, estrogen increases 7/4 antigen in connective tissue and myofibers and is associated with more Ly6C/G-positive myofibers when the 7/4 antigen is absent from these myofibers.     

Endocytosis in skeletal muscle fibers

Defining the organization of endocytic pathway in multinucleated skeletal myofibers is crucial to understand the routing of membrane proteins, such as receptors and glucose transporters, through this system. Here we analyzed the organization of the endocytic trafficking pathways in isolated rat myofibers. We found that sarcolemmal-coated pits and transferrin receptors were concentrated in the I band areas. Fluid phase markers were taken up into vesicles in the same areas along the whole length of the fibers and were then delivered into structures around and between the nuclei. These markers also accumulated beneath the neuromuscular and myotendinous junctions. The recycling compartment, labeled with transferrin, appeared as perinuclear and interfibrillar dots that partially colocalized with the GLUT4 compartment. Low-density lipoprotein, a marker of the lysosome-directed pathway, was transported into sparsely distributed perinuclear and interfibrillar dots that contacted microtubules. A majority of these dots did not colocalize with internalized transferrin, indicating that the recycling and the lysosome-directed pathways were distinct. In conclusion, the I band areas were active in endocytosis along the whole length of the multinucleated myofibers. The sorting endosomes distributed in a cross-striated fashion while the recycling and late endosomal compartments showed perinuclear and interfibrillar localizations and followed the course of microtubules.     

Transcriptomic Analysis of Single Isolated Myofibers Identifies miR-27a-3p and miR-142-3p as Regulators of Metabolism in Skeletal Muscle

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