Absence of staircase following disuse in rat gastrocnemius muscle

Repetitive stimulation of mammalian fast-twitch skeletal muscles will normally result in a positive staircase response. This phenomenon was investigated in the rat gastrocnemius muscle following a 2-week period of tetrodotoxin-induced disuse. Muscle inactivity was imposed by superfusing tetrodotoxin in saline over the left sciatic nerve via an implanted osmotic pump. In situ isometric contractile responses to double pulse stimulation and repetitive stimulation at 10 Hz were determined the day after removal of the pump. Two weeks of disuse resulted in 40% muscle weight loss. A twitch contraction gave the same force when expressed per gram of wet muscle weight in control muscles, 317 +/- 24.6 (means +/- SE) g/g, as compared with tetrodotoxin-treated muscles, 328 +/- 24.2 g/g. Both contraction time and half-relaxation time were prolonged following treatment with tetrodotoxin. Repetitive stimulation at 10 Hz resulted in a positive staircase response in the control muscles, but not in muscles of the tetrodotoxin-treated rats. The observed changes in the time course of the twitch contraction with repetitive stimulation following tetrodotoxin-induced disuse are consistent with alterations in sarcoplasmic reticulum handling of calcium. It is not certain if there is a change following disuse in the mechanism normally associated with staircase or if this mechanism is merely opposed by an early fatigue.     

Terminal sprouting in rat sternocostalis muscle following partial denervation

Summary The sternocostalis muscle of the rat was examined at one to five days after partial denervation and levels of terminal sprouting were assessed.The removal of one intercostal nerve caused localised degeneration which did not extend more than a few muscle fibres deep into the field of distribution of the adjacent nerve. Terminal sprouting was clearly seen at 24 h after operation and did not appear to develop further up to five days.There was no difference in the sprouting responses to section of either intercostal nerve 2, 4 or 5. There was, however, a decrease in the response with increasing distance from the cut nerve. No sprouting response was observed in the contralateral muscle.Comparison of sprouting levels of B and C type end plates revealed a greater percentage of C type end plates with sprouts. However, the response of B type end plates, considered in relation to the levels of spontaneous sprouting, was greater than that of C type end plates.     

Mouse gastrocnemius muscle regeneration after mechanical or cardiotoxin injury

The goal of our study was to compare the skeletal muscle regeneration induced by two types of injury: either crushing, that causes muscle degeneration as a result of mechanical devastation of myofibers, or the injection of a cardiotoxin that is a myotoxic agent causing myolysis of myofibers leading to muscle degeneration. Regenerating muscles were analyzed at selected intervals, until the 14th day following the injury. We analyzed their weight and morphology. We also studied the expression of different myosin heavy chain isoforms as a molecular marker of the regeneration progress. Histological analysis revealed that inflammatory response and myotube formation in crushed muscles was delayed compared to cardiotoxin-injected ones. Moreover, the expression of myosin heavy chain isoforms was observed earlier in cardiotoxin-injured versus crushed muscles. We conclude that the dynamics of skeletal muscle regeneration depends on the method of injury.     

Mitochondrial AIF protein involved in skeletal muscle regeneration

The mitochondrial flavoprotein apoptosis-inducing factor (AIF) has proved to be either the main mediator of apoptosis or an anti-apoptotic factor via its putative oxidoreductase and peroxide scavenging activities. We report here that 100 muM hydrogen peroxide (H2O2) induced the proliferation of C2C12 myoblasts and over-expression of AIF simultaneously in vitro. Immunofluorescence showed that the over-expression of AIF was located in the cytoplasm. The immunopositive AIF was detected in nuclei 27 days after denervation of skeletal muscle, but in the cytoplasm it was detected 27 days after fiber-damaged skeletal muscle. AIF may be a factor involved in skeletal muscle regeneration.     

Influence of muscle shortening on the geometry of gastrocnemius medialis muscle of the rat

For static and dynamic conditions muscle geometry of the musculus gastrocnemius medialis of the rat was compared at different muscle lengths. The dynamic conditions differed with respect to isokinetic shortening velocity (25, 50 and 75 mm/s) of the muscle-tendon complex and in constancy of force (isotonic) and velocity (isokinetic) during shortening. Muscle geometry was characterized by fibre length and angle as well as aponeurosis length and angle. At high isokinetic shortening velocities (50 and 75 mm/s) small differences in geometry were found with respect to isometric conditions: aponeurosis lengths differed maximally by -2%, fibre length only showed a significant increase (+3.2%) at the highest shortening velocity. The isotonic condition only yielded significant differences of fibre angle (-4.5%) in comparison with isometric conditions. No significant differences of muscle geometry were found when comparing isotonic with isokinetic conditions of similar shortening velocity. The small differences of geometry between isometric and dynamic conditions are presumably due to the lower muscle force in the dynamic condition and the elastic behaviour of the aponeurosis. It is concluded that, unless very high velocities of shortening are used, the relationship between muscle geometry and muscle length in the isometric condition may be used to describe muscle geometry in the dynamic condition.     

Summation of motor unit forces in rat medial gastrocnemius muscle

The summation of contractile forces of motor units (MUs) was analyzed by comparing the recorded force during parallel stimulation of two and four individual MUs or four groups of MUs to the algebraic sum of their individual forces. Contractions of functionally-isolated single MUs of the medial gastrocnemius muscle were evoked by electrical stimulation of thin filaments of the split L5 or L4 ventral roots of spinal nerves. Additionally, contractions of large groups of MUs were evoked by stimuli delivered to four parts of the divided L5 ventral root. Single twitches, 40 Hz unfused tetani, and 150 Hz fused maximum tetani were recorded. In these experimental situations the summation was more effective for unfused tetani than for twitches or maximum tetani. The results obtained for pairs of MUs were highly variable (more- or less-than-linear summation), but coactivation of more units led to progressively weaker effects of summation, which were usually less-than-linear in comparison to the algebraic sums of the individual forces. The variability of the results highlights the importance of the structure of the muscle and the architecture of its MUs. Moreover, the simultaneous activity of fast and slow MUs was considerably more effective than that of two fast units.     

Effects of training on fibre composition in rat gastrocnemius muscle

Forty-eight adult Wistar rats were divided into four groups for experimentation. The group I was used as a control and groups II, III and IV underwent progressive treadmill training. Samples of the red and mixed portions of m. gastrocnemius (lateral head) were stained with the histochemical technique of m-ATPase to determine the percentage of type I, IIA and IIB fibres, and with NADH-TR, in order to quantify variations in "low-oxidative" fibre percentages. The results showed that progressive training did not lead to statistical variations in the percentage of type I fibres. However, the proportion of type IIA fibres rose, while that of IIB fibres fell, in both cases significantly. Variations were more marked in mixed than in red Gastrocnemius muscle. A clear decrease was noted in "low-oxidative" fibres, which were virtually absent from red portion. This decrease was more marked, and occurred more rapidly, than in type IIB fibres.     

Agent-based model provides insight into the mechanisms behind failed regeneration following volumetric muscle loss injury

Skeletal muscle possesses a remarkable capacity for repair and regeneration following a variety of injuries. When successful, this highly orchestrated regenerative process requires the contribution of several muscle resident cell populations including satellite stem cells (SSCs), fibroblasts, macrophages and vascular cells. However, volumetric muscle loss injuries (VML) involve simultaneous destruction of multiple tissue components (e.g., as a result of battlefield injuries or vehicular accidents) and are so extensive that they exceed the intrinsic capability for scarless wound healing and result in permanent cosmetic and functional deficits. In this scenario, the regenerative process fails and is dominated by an unproductive inflammatory response and accompanying fibrosis. The failure of current regenerative therapeutics to completely restore functional muscle tissue is not surprising considering the incomplete understanding of the cellular mechanisms that drive the regeneration response in the setting of VML injury. To begin to address this profound knowledge gap, we developed an agent-based model to predict the tissue remodeling response following surgical creation of a VML injury. Once the model was able to recapitulate key aspects of the tissue remodeling response in the absence of repair, we validated the model by simulating the tissue remodeling response to VML injury following implantation of either a decellularized extracellular matrix scaffold or a minced muscle graft. The model suggested that the SSC microenvironment and absence of pro-differentiation SSC signals were the most important aspects of failed muscle regeneration in VML injuries. The major implication of this work is that agent-based models may provide a much-needed predictive tool to optimize the design of new therapies, and thereby, accelerate the clinical translation of regenerative therapeutics for VML injuries.     

Changes in geometry of activily shortening unipennate rat gastrocnemius muscle

Muscle geometry of the unipennate medial gastrocnemius (GM) muscle of the rat was examined with photographic techniques during isometric contractions at different muscle lengths. It was found that the length of fibers in different regions of GM differs significantly, and proximal aponeurosis length varies significantly from distal aponeurosis length; the angle of the aponeurosis with the muscular action differs significantly among regions at short muscle lengths (full contraction). These data support the idea that the unipennate GM cannot be represented by a parallelogram in a two-dimensional analysis. As the muscle shortens, the area of the mid-longitudinal plane of the GM decreases by 24%, a decrease that may be explained by assuming fiber diameter to increase in all directions. The angle between fiber and aponeurosis is determined by more than fiber length. Hence, such important assumptions as a parallelogram with constant area and fiber angle γ changes determined by fiber length changes, freqently used in the theoretical analysis of the morphological mechanism of unipennate muscle contraction, do not hold for the unipennate GM of the rat. Length of the sarcomere within the mid-longitudinal plane of GM varies from 1.92 to 2.14 μm among the different muscle regions at muscle optimum length (length at which force production is highest), whereas shortening to 6 mm less than optimum length produces a range of sarcomere lengths from 0.89 to 1.52 μm. These data suggest that fibers located in different regions of the GM reach their optimum and slack lengths at various muscle lengths. © 1993 Wiley-Liss, Inc.     

Effect of the activation of macrophages on the course of regeneration of rat liver following partial hepatectomy

Stimulation of the Kupffer cells with E. coli endotoxin (the purified lipopolysaccharide) or with prodigiosan (a polysaccharide from Serratia marcescens) 24 h before partial hepatectomy (resection of 65-70% of the liver) stimulated and intensified the onset of liver regenerative activity (evaluated from changes in liver DNA synthesis, the H5 labelling index and the mitotic activity of the hepatocytes). Liver DNA synthesis increased together with the dose of endotoxin (i.v., from 25 to 1000 micrograms/kg body weight). If E. coli endotoxin was injected during or 3 h after partial hepatectomy, partial inhibition of liver DNA synthesis was observed. In mice stimulated with zymosan (a polysaccharide isolated from yeast), administered 5 days before performing partial hepatectomy, proliferation of the hepatocytes (evaluated from changes in the 3H labelling index and in the mitotic activity of the hepatocytes) was evaluated. The results confirm that proliferation is correlated to the state of reactivity of the Kupffer cells.     

Increase in the amount of adenylate cyclase in rat gastrocnemius muscle after denervation

After section of the sciatic nerve, the basal adenylate cyclase (AC) activity in rat gastrocnemius muscle increased 6-7 times per membrane protein and about 2 times per whole muscle in the following 30 or 40 days. The AC activity in the muscle 30 days after denervation was increased about 4 times by forskolin. Calcitonin gene-related peptide (CGRP) also increased the adenylate cyclase activity in the denervated muscle. The binding of [3H]-forskolin (10nM) to cells isolated from gastrocnemius muscle was examined to determine the amount of AC molecules. Inhibition of [3H]-forskolin binding by increasing amounts of unlabeled forskolin gave a sigmoid curve with a IC50 value of 3 x 10(-7) M. Results showed that the number of [3H]-forskolin binding sites per cell was higher on the denervated side than on the control side, like the basal AC activity. The IC50 values for inhibition by unlabeled forskolin of binding of [3H]-forskolin were similar to muscles on the control and denervated sides. These results suggest that an increase in the AC activity induced by denervation was due to an increase in the numbers of AC molecules in the muscle.     

Unilateral immobilization affects contralateral rat gastrocnemius muscle architecture.

In order to study the effects of unilateral short length immobilization on the contralateral gastrocnemius muscle (GM), length measurements were conducted on photographs taken in the active condition (tetanic plateau). Comparison of geometry of experimental and control muscles was made at optimum muscle length. The results show that a process occurred in the muscle which can be ascribed predominantly in terms of atrophy. This atrophy did not reach a maximum after 4 weeks but gradually increased in time. The altered conditions imposed on the muscle changed its architecture. It was shown that variables of the contralateral GM muscle are not representative of those of normally used muscles and should therefore not be used as control muscles for the determination of immobilization effects.     

Morphometric properties and innervation of muscle compartments in rat medial gastrocnemius

The rat medial gastrocnemius (MG) muscle is composed of the proximal and distal compartments. In this study, morphometric properties of the compartments and their muscle fibres at five levels of the muscle length and the innervation pattern of these compartments from lumbar segments were investigated. The size and number of muscle fibres in the compartments were different. The proximal compartment at the largest cross section (25% of the muscle length) had 34% smaller cross-sectional area but contained a slightly higher number of muscle fibres (max. 5521 vs. 5360) in comparison to data for the distal compartment which had the largest cross-sectional area at 40% of the muscle length. The muscle fibre diameters revealed a clear tendency within both compartments to increase along the muscle (from the knee to the Achilles tendon) up to 46.9?μm in the proximal compartment and 58.4?μm in the distal one. The maximal tetanic and single twitch force evoked by stimulation of L4, L5, and L6 ventral roots in whole muscle and compartments were measured. The MG was innervated from L4 and L5, only L5, or L5 and L6 segments. The proximal compartment was innervated by axons from L5 or L5 and L4, and the distal one from L5, L5 and L6, or L5 and L4 segments. The forces produced by the compartments summed non-linearly. The tetanic forces of the proximal and distal compartments amounted to 2.24 and 4.86?N, respectively, and their algebraic sums were 11% higher than the whole muscle force (6.37?N).