Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.     

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.²⁶ that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 μM in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.     

Citrulline enhances myofibrillar constituents expression of skeletal muscle and induces a switch in muscle energy metabolism in malnourished aged rats

Citrulline (Cit) actions on muscle metabolism remain unclear. Those latter were investigated using a proteomic approach on Tibialis muscles from male Sprague‐Dawley rats. At 23 months of age, rats were either fed ad libitum (AL group) or subjected to dietary restriction for 12 weeks. At the end of the restriction period, one group of rats was euthanized (R group) and two groups were refed for one week with a standard diet supplemented with nonessential amino acids group or Cit (CIT group). Results of the proteomic approach were validated using targeted Western blot analysis and assessment of gene expression of the related genes. Maximal activities of the key enzymes involved in mitochondrial functioning were also determined. Cit supplementation results in a significant increase in the protein expression of the main myofibrillar constituents and of a few enzymes involved in glycogenolysis and glycolysis (CIT vs. AL and R, p < 0.05). Conversely, the expression of oxidative enzymes from Krebs cycle and mitochondrial respiratory chain was significantly decreased (CIT vs. AL, p < 0.05). However, maximal activities of key enzymes of mitochondrial metabolism were not significantly affected, except for complex 1 which presented an increased activity (CIT vs. AL and R, p < 0.05). In conclusion, Cit supplementation increases expression of the main myofibrillar proteins and seems to induce a switch in muscle energy metabolism, from aerobia toward anaerobia.     

Effect of resistance training to muscle failure vs non-failure on strength,hypertrophy and muscle architecture in trained individuals

The aim of this study was to compare the effects of resistance training to muscle failure (RT-F) and non-failure (RT-NF) on muscle mass, strength and activation of trained individuals. We also compared the effects of these protocols on muscle architecture parameters. A within-subjects design was used in which 14 participants had one leg randomly assigned to RT-F and the other to RT-NF. Each leg was trained 2 days per week for 10 weeks. Vastus lateralis (VL) muscle cross-sectional area (CSA), pennation angle (PA), fascicle length (FL) and 1-repetition maximum (1-RM) were assessed at baseline (Pre) and after 20 sessions (Post). The electromyographic signal (EMG) was assessed after the training period. RT-F and RT-NF protocols showed significant and similar increases in CSA (RT-F: 13.5% and RT-NF: 18.1%; P < 0.0001), PA (RT-F: 13.7% and RT-NF: 14.4%; P < 0.0001) and FL (RT-F: 11.8% and RT-NF: 8.6%; P < 0.0001). All protocols showed significant and similar increases in leg press (RT-F: 22.3% and RT-NF: 26.7%; P < 0.0001) and leg extension (RT-F: 33.3%, P < 0.0001 and RT-NF: 33.7%; P < 0.0001) 1-RM loads. No significant differences in EMG amplitude were detected between protocols (P > 0.05). In conclusion, RT-F and RT-NF are similarly effective in promoting increases in muscle mass, PA, FL, strength and activation.     

Diurnal Variation in Wingate-Test Performance and Associated Electromyographic Parameters

The present study was designed to evaluate time-of-day effects on electromyographic (EMG) activity changes during a short-term intense cycling exercise. In a randomized order, 22 male subjects were asked to perform a 30-s Wingate test against a constant braking load of 0.087?kg·kg^?1 body mass during two experimental sessions, which were set up either at 07:00 or 17:00?h. During the test, peak power (P_peak), mean power (P_mean), fatigue index (FI; % of decrease in power output throughout the 30 s), and evolution of power output (5-s span) throughout the exercise were analyzed. Surface EMG activity was recorded in both the vastus lateralis and vastus medialis muscles throughout the test and analyzed over a 5-s span. The root mean square (RMS) and mean power frequency (MPF) of EMG were calculated. Neuromuscular efficiency (NME) was estimated from the ratio of power to RMS. Resting core temperature, P_peak, P_mean, and FI were significantly higher (p?<?.05) in the evening than morning test (e.g., P_peak: 11.6?±?0.8 vs. 11.9?±?1 W·kg^?1). The results showed that power output decreased following two phases. During the first phase (first 20s), power output decreased rapidly and values were higher (p?<?.05) in the evening than in the morning. During the second phase (last 10s), power decreased slightly and appeared independent of the time of day of testing. This power output decrease was paralleled by evolution of the MPF and NME. During the first phase, NME and MPF were higher (p <?.05) in the evening. During the second phase, NME and MPF were independent of time of day. In addition, no significant differences were noticed between 7:00 and 17:00?h for EMG RMS during the whole 30 s. Taken together, these results suggest that peripheral mechanisms (i.e., muscle power and fatigue) are more likely the cause of the diurnal variation of the Wingate-test performance rather than central mechanisms. (Author correspondence: n_souissi@yahoo.fr)     

Neuromuscular dysfunction with the experimental arm acting as its own reference following eccentric and isometric exercise

Eccentric exercise has been extensively used as a model to study muscle damage-induced neuromuscular impairment, adopting mainly a bilateral matching task between the reference (unexercised) arm and the indicator (exercised) arm. However, little attention has been given to the muscle proprioceptive function when the exercised arm acts as its own reference. This study investigated muscle proprioception and motor control, with the arm acting both as reference and indicator, following eccentric exercise and compared them with those observed after isometric exercise. Fourteen young male volunteers were equally divided into two groups and performed an eccentric or isometric exercise protocol with the elbow flexors of the non-dominant arm on an isokinetic dynamometer. Both exercise protocols induced significant changes in indicators of muscle damage, that is, muscle soreness, range of motion and maximal isometric force post-exercise (p < 0.05–0.001), and neuromuscular function was similarly affected following both protocols. Perception of force was impaired over the 4-day post-exercise period (p < 0.001), with the applied force being systematically overestimated. Perception of joint position was significantly disturbed (i.e., target angle was underestimated) only at one elbow angle on day 4 post-exercise (p < 0.05). The misjudgements and disturbed motor output observed when the exercised arm acted as its own reference concur with the view that they could be a result of a mismatch between the central motor command and an impaired motor control after muscle damage.     

Facilitated Hyperpolarization Signaling in Vascular Smooth Muscle-overexpressing TRIC-A Channels

The TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation-specific channels and likely mediate counterion movements to support efficient Ca²⁺ release from the sarco/endoplasmic reticulum. Vascular smooth muscle cells (VSMCs) contain both TRIC subtypes and two Ca²⁺ release mechanisms; incidental opening of ryanodine receptors (RyRs) generates local Ca²⁺ sparks to induce hyperpolarization and relaxation, whereas agonist-induced activation of inositol trisphosphate receptors produces global Ca²⁺ transients causing contraction. Tric-a knock-out mice develop hypertension due to insufficient RyR-mediated Ca²⁺ sparks in VSMCs. Here we describe transgenic mice overexpressing TRIC-A channels under the control of a smooth muscle cell-specific promoter. The transgenic mice developed congenital hypotension. In Tric-a-overexpressing VSMCs from the transgenic mice, the resting membrane potential decreased because RyR-mediated Ca²⁺ sparks were facilitated and cell surface Ca²⁺-dependent K⁺ channels were hyperactivated. Under such hyperpolarized conditions, L-type Ca²⁺ channels were inactivated, and thus, the resting intracellular Ca²⁺ levels were reduced in Tric-a-overexpressing VSMCs. Moreover, Tric-a overexpression impaired inositol trisphosphate-sensitive stores to diminish agonist-induced Ca²⁺ signaling in VSMCs. These altered features likely reduced vascular tonus leading to the hypotensive phenotype. Our Tric-a-transgenic mice together with Tric-a knock-out mice indicate that TRIC-A channel density in VSMCs is responsible for controlling basal blood pressure at the whole-animal level.     

The Superfast Human Extraocular Myosin Is Kinetically Distinct from the Fast Skeletal IIa,IIb, and IId Isoforms

Humans express five distinct myosin isoforms in the sarcomeres of adult striated muscle (fast IIa, IId, the slow/cardiac isoform I/β, the cardiac specific isoform α, and the specialized extraocular muscle isoform). An additional isoform, IIb, is present in the genome but is not normally expressed in healthy human muscles. Muscle fibers expressing each isoform have distinct characteristics including shortening velocity. Defining the properties of the isoforms in detail has been limited by the availability of pure samples of the individual proteins. Here we study purified recombinant human myosin motor domains expressed in mouse C₂C₁₂ muscle cells. The results of kinetic analysis show that among the closely related adult skeletal isoforms, the affinity of ADP for actin·myosin (K_AD) is the characteristic that most readily distinguishes the isoforms. The three fast muscle myosins have K_AD values of 118, 80, and 55 μm for IId, IIa, and IIb, respectively, which follows the speed in motility assays from fastest to slowest. Extraocular muscle is unusually fast with a far weaker K_AD = 352 μm. Sequence comparisons and homology modeling of the structures identify a few key areas of sequence that may define the differences between the isoforms, including a region of the upper 50-kDa domain important in signaling between the nucleotide pocket and the actin-binding site.