Calcineurin-Aα alpha activation enhances the structure and function of regenerating muscles after myotoxic injury

Calcineurin signaling is essential for successful muscle regeneration. Although calcineurin inhibition compromises muscle repair, it is not known whether calcineurin activation can enhance muscle repair after injury. Tibialis anterior (TA) muscles from adult wild-type (WT) and transgenic mice overexpressing the constitutively active calcineurin-Aα transgene under the control of the mitochondrial creatine kinase promoter (MCK-CnAα*) were injected with the myotoxic snake venom Notexin to destroy all muscle fibers. The TA muscle of the contralateral limb served as the uninjured control. Muscle structure was assessed at 5 and 9 days postinjury, and muscle function was tested in situ at 9 days postinjury. Calcineurin stimulation enhanced muscle regeneration and altered levels of myoregulatory factors (MRFs). Recovery of myofiber size and force-producing capacity was hastened in injured muscles of MCK-CnAα* mice compared with control. Myogenin levels were greater 5 days postinjury and myocyte enhancer factor 2a (MEF2a) expression was greater 9 days postinjury in muscles of MCK-CnAα* mice compared with WT mice. Higher MEF2a expression in regenerating muscles of MCK-CnAα* mice 9 days postinjury may be related to an increase of slow fiber genes. Calcineurin activation in uninjured and injured TA muscles slowed muscle contractile properties, reduced fatigability, and enhanced force recovery after 4 min of intermittent maximal stimulation. Therefore, calcineurin activation can confer structural and functional benefits to regenerating skeletal muscles, which may be mediated in part by differential expression of MRFs.

Activated calcineurin ameliorates contraction-induced injury to skeletal muscles of mdx dystrophic mice

Utrophin expression is regulated by calcineurin and up-regulating utrophin can decrease the susceptibility of dystrophic skeletal muscle to contraction-induced injury. We overexpressed the constitutively active calcineurin-A α in skeletal muscle of mdx dystrophic mice (mdx CnA*) and examined the tibialis anterior muscle to determine whether the presence of activated calcineurin promotes resistance to muscle damage after lengthening contractions. Two stretches (10 s apart) of 40% strain relative to muscle fibre length were initiated from the plateau of a maximal isometric tetanic contraction. Muscle damage was assessed 1, 5 and 15 min later by the deficit in maximum isometric force and by quantifying the proportion of muscle fibres staining positive for intracytoplasmic albumin. The force deficit at all time points after the lengthening contractions was approximately 80% in mdx muscles and 30% in mdxCnA* muscles. The proportion of albumin-positive fibres was significantly less in control and injured muscles from mdxCnA* mice than from mdx mice. Compared with mdx mice, mean fibre cross-sectional area was 50% less in muscles from mdxCnA* mice. Furthermore, muscles frommdxCnA* mice exhibited a higher proportion of fibres expressing the slow(er) myosin heavy chain (MyHC) I and IIa isoforms, prolonged contraction and relaxation times, lower absolute and normalized maximum forces, and a clear leftward shift of the frequency–force relationship with greater force production at lower stimulation frequencies. These are structural and functional markers of a slower muscle phenotype. Taken together, our findings show that muscles from mdxCnA* mice have a smaller mean fibre cross-sectional area, a greater sarcolemmal to cytoplasmic volume ratio, and an increase in utrophin expression, promoting an attenuated susceptibility to contraction-induced injury. We conclude that increased calcineurin activity may confer functional benefits to dystrophic skeletal muscles.

Differential calcineurin signalling activity and regeneration efficacy in diaphragm and limb muscles of dystrophic mdx mice

Calcineurin activity is essential for successful skeletal muscle regeneration in young mdx mice and in wild type mice following myotoxic injury and cryodamage. In mature myofibres of adult mdx mice, calcineurin stimulation can ameliorate the dystrophic pathology. The aim of this study was to test the hypothesis that the more severe dystrophic pathology of the diaphragm compared with hindlimb muscles of mdx mice could be attributed to aberrant calcineurin signalling and that due to ongoing regeneration calcineurin activity would be greater in muscles of adult mdx than wild type mice. Differences in markers of regeneration between tibialis anterior and diaphragm muscles were also characterised, to determine whether there was an association between regeneration efficacy and calcineurin activity in dystrophic muscles. In diaphragm muscles of adult mdx mice, the proportion of centrally nucleated fibres and developmental myosin heavy chain protein expression was lower and myogenin protein expression was higher than in tibialis anterior muscles. Calcineurin and activated NFATc1 protein content and calcineurin phosphatase activity were higher in muscles from mdx than wild type mice and calcineurin activation was greater in diaphragm than tibialis anterior muscles of mdx mice. Thus, despite greater calcineurin activity in diaphragm compared to hindlimb muscles, regeneration events downstream of myoblast differentiation and mediated by the injured myofibre were severely compromised.

Examination of EMG normalisation methods for the study of the posterior and posterolateral neck muscles in healthy controls

The purpose of this study was to examine the reliability of normalisation methods used in the study of the posterior and posterolateral neck muscles in a group of healthy controls. Six asymptomatic male subjects performed a total of 12 maximum voluntary isometric contractions (MVIC) and 60%-submaximal isometric contractions (60%-MVIC) against the torque arm of an isokinetic dynamometer whilst surface and intramuscular electromyography (EMG) was recorded unilaterally from representative posterior and posterolateral locations. Reliability was calculated using intra-class correlation coefficient (ICC), relative standard error of measurement (%SEM) and relative coefficient of variation (%CV). Maximal torque output was found to be highly reliable in the directions of extension and right lateral bending when the first of three MVIC contractions was excluded. When averaged across contraction direction, high reliability was found for both surface (MVIC: ICC = 0.986, %SEM = 7.5, %CV = 9.2; 60%-MVIC: ICC = 0.975, %SEM = 10, %CV = 13.7) and intramuscular (MVIC: ICC = 0.910, %SEM = 20, %CV = 19.1; 60%-MVIC: ICC = 0.952, %SEM = 16.5, %CV = 13.5) electrodes. Intramuscular electrodes displayed the least reliability in right lateral bending. The use of visual feedback markedly increased the reliability of 60%-MVIC contractions.

Reliability of normalisation methods for EMG analysis of neck muscles

Acceptable reliability of normalisation contractions in electromyography (EMG) is paramount for testing conducted over a number of days or if normal laboratory strength testing equipment is unavailable. This study examined the reliability of maximal voluntary isometric contractions (MVIC) and sub-maximal (60%) isometric contractions for use in neck muscle EMG studies. Surface EMG was recorded bilaterally from eight sites around the neck at C4/5 level from five healthy male subjects. Subjects performed MVIC and sub-maximal normalisation contractions using an isokinetic dynamometer (ID) and a portable cable dynamometer with attached strain gauge (PCD) in addition to a MVIC against a manual resistance (MR). Subjects were tested in flexion, extension, left and right lateral bending and were retested by the same tester within a two-week period. Intra class correlation co-efficients (ICC) were calculated for each testing method and contraction direction and a mean ICC was calculated across all contraction directions. All normalisation methods produced excellent within-day reliability (mean ICC >0.80) but only the MVICs using the ID and PCD had acceptable reliability when assessed between-days. This study confirmed the validity of using MVICs elicited using the ID and PCD as reliable reference contractions for the normalisation of neck EMG.

Endurance training adaptations modulate the redox–force relationship of rat isolated slow-twitch skeletal muscles

1. Studies have shown that, in isolated skeletal muscles, maximum isometric force production (Po) is dependent on muscle redox state. Endurance training increases the antioxidant capacity of skeletal muscles, a factor that could impact on the force-producing capacity following exogenous exposure to an oxidant. We tested the hypothesis that 12 weeks treadmill training would increase anti-oxidant capacity in rat skeletal muscles and alter their response to exogenous oxidant exposure.

2. At the conclusion of the 12 week endurance-training programme, soleus (slow-twitch) muscles from trained rats had greater citrate synthase (CS) and catalase (CAT) activity compared with soleus muscles from untrained rats (P < 0.05).
In contrast, CAT activity of extensor digitorum longus (EDL; fast-twitch) muscles from trained rats was not different to EDL muscles of untrained rats. The CS activity was lower in EDL muscles from trained compared with untrained rats (P < 0.05).

3. Equilibration with exogenous hydrogen peroxide (H2O2, 5 mmol/L) increased the Po of soleus muscles from untrained rats for the duration of treatment (30 min), whereas the Po of EDL muscles was affected biphasically, with a small increase initially (after 5 min), followed by a more marked decrease in Po (after 30 min). The H2O2-induced increase in Po of soleus muscles from trained rats was less than that in untrained rats (P < 0.05), but no differences were observed in the Po of EDL muscles following training.

4. The results indicate that 12 weeks endurance running training conferred adaptations in soleus but not EDL muscles. These adaptations were associated with an attenuation of the oxidant-induced increase in Po of soleus muscles from trained compared with untrained rats. We conclude that endurance training-adapted soleus muscles have a slightly altered redox - force relationship.

Oxygen dependence of metabolism and cellular adaptation in vertebrate muscles: a review

The key roles the cardiovascular system play in the complex distribution of blood, and consequently oxygen, have been extensively studied in vertebrates. Numerous studies have also revealed the complex and varied ways in which tissues cope with compromised oxygen supply. The links between these two processes are the subject of much current research. This article aims to review how blood supply influences tissue oxygenation and affects metabolism, and how this might have played a role in the evolution of the complex muscle arrangements which characterise vertebrates. Muscle tissue is the greatest proportion of body mass in most vertebrates and undergoes dramatic alterations in metabolism and associated oxygen flux. Special attention is given to the myotome of fishes, in which the partitioning of the fibre types contrasts with the mosaic arrangement of tetrapods. This gives us the opportunity to study pure whole vascularised muscle blocks, rather than single fibres, and further explore the interrelationship between oxygen supply and tissue energetics.

Oxygen-dependence of metabolic rate in the muscles of craniates

We present evidence that oxygen consumption (V_O2 ) is oxygen partial pressure (P_O2) dependent in striated muscles and P_O2 -independent in the vasculature in representatives of three craniate taxa: two teleost fish, a hagfish and a rat. Blood vessel V_O2 displayed varying degrees of independence in a P_O2 range of 15–95 mmHg, while V_O2 by striated muscle tissue slices from all species related linearly to P_O2 between 0 and 125 mmHg, despite V_O2 rates varying greatly between species and muscle type. In salmon red muscle, lactate concentrations fell in slices incubated at a P_O2 of either 30 or 100 mmHg, suggesting aerobic rather than anaerobic metabolism. Consistent with this finding, potential energy, a proxy of ATP turnover, was P_O2 -dependent. Our data suggest that the reduction in V_O2 with falling P_O2 results in a decrease in ATP demand, suggesting that the hypoxic signal is sensed and cellular changes effected. Viability and diffusion limitation of the preparations were investigated using salmon cardiac and skeletal muscles. Following the initial P_O2 depletion, reoxygenation of the Ringer bathing salmon cardiac muscle resulted in V_O2^s that was unchanged from the first run. V_O2 increased in all muscles uncoupled with p-trifluoromethoxylphenyl-hydrazone (FCCP) and 2,4-dinitrophenol (DNP). Mitochondrial succinate dehydrogenase activity, quantified by reduction of 3-(4,5-dimethylthiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan, was constant over the course of the experiment. These three findings indicate that the tissues remained viable over time and ruled out diffusion-limitation as a constraint on V_O2.

Artificial Muscles Based on Polypyrrole/Carbon Nanotube Laminates

A novel method for the periodical assembly of laminates of forest-drawn carbon nanotube (CNT) sheets and polypyrrole (PPy) is described. The method produces composite films in which the volume fraction and orientation of CNTs can be controlled. Actuator stroke and strength is increased and work capacity per cycle doubled when nanotube orientation is perpendicular to the actuation direction. Most importantly, these PPy/CNT laminates have dramatically decreased creep during actuation, which has been a major barrier for the application of PPy actuators.