Reinventing the wheel: comparison of two wheel cage styles for assessing mouse voluntary running activity.

Voluntary wheel cage assessment of mouse activity is commonly employed in exercise and behavioral research. Currently, no standardization for wheel cages exists resulting in an inability to compare results among data from different laboratories. The purpose of this study was to determine whether the distance run or average speed data differ depending on the use of two commonly used commercially available wheel cage systems. Two different wheel cages with structurally similar but functionally different wheels (electromechanical switch vs. magnetic switch) were compared side-by-side to measure wheel running data differences. Other variables, including enrichment and cage location, were also tested to assess potential impacts on the running wheel data. We found that cages with the electromechanical switch had greater inherent wheel resistance and consistently led to greater running distance per day and higher average running speed. Mice rapidly, within 1-2 days, adapted their running behavior to the type of experimental switch used, suggesting these running differences are more behavioral than due to intrinsic musculoskeletal, cardiovascular, or metabolic limits. The presence of enrichment or location of the cage had no detectable impact on voluntary wheel running. These results demonstrate that mice run differing amounts depending on the type of cage and switch mechanism used and thus investigators need to report wheel cage type/wheel resistance and use caution when interpreting distance/speed run across studies. NEW & NOTEWORTHY The results of this study highlight that mice will run different distances per day and average speed based on the inherent resistance present in the switch mechanism used to record data. Rapid changes in running behavior for the same mouse in the different cages demonstrate that a strong behavioral factor contributes to classic exercise outcomes in mice. Caution needs to be taken when interpreting mouse voluntary wheel running activity to include potential behavioral input and physiological parameters.

Automated image segmentation of haematoxylin and eosin stained skeletal muscle cross-sections.

The ability to accurately and efficiently quantify muscle morphology is essential to determine the physiological relevance of a variety of muscle conditions including growth, atrophy and repair. There is agreement across the muscle biology community that important morphological characteristics of muscle fibres, such as cross-sectional area, are critical factors that determine the health and function (e.g. quality) of the muscle. However, at this time, quantification of muscle characteristics, especially from haematoxylin and eosin stained slides, is still a manual or semi-automatic process. This procedure is labour-intensive and time-consuming. In this paper, we have developed and validated an automatic image segmentation algorithm that is not only efficient but also accurate. Our proposed automatic segmentation algorithm for haematoxylin and eosin stained skeletal muscle cross-sections consists of two major steps: (1) A learning-based seed detection method to find the geometric centres of the muscle fibres, and (2) a colour gradient repulsive balloon snake deformable model that adopts colour gradient in Luv colour space. Automatic quantification of muscle fibre cross-sectional areas using the proposed method is accurate and efficient, providing a powerful automatic quantification tool that can increase sensitivity, objectivity and efficiency in measuring the morphometric features of the haematoxylin and eosin stained muscle cross-sections.

Aging does not alter the mechanosensitivity of the p38, p70S6k, and JNK2 signaling pathways in skeletal muscle.

The capacity for skeletal muscle to recover its mass following periods of unloading (regrowth) has been reported to decline with age. Although the mechanisms responsible for the impaired regrowth are not known, it has been suggested that aged muscles have a diminished capacity to sense and subsequently respond to a given amount of mechanical stimuli (mechanosensitivity). To test this hypothesis, extensor digitorum longus muscles from young (2-3 mo) and old (26-27 mo) mice were subjected to intermittent 15% passive stretch (ex vivo) as a source of mechanical stimulation and analyzed for alterations in the phosphorylation of stress-activated protein kinase (p38), ribosomal S6 kinase (p70S6k), and the p54 jun N-terminal kinase (JNK2). The results indicated that the average magnitude of specific tension (mechanical stimuli) induced by 15% stretch was similar in muscles from young and old mice. Young and old muscles also revealed similar increases in the magnitude of mechanically induced p38, p70S6k (threonine/serine 421/424 and threonine 389), and JNK2 phosphorylation. In addition, coincubation experiments demonstrated that the release of locally acting growth factors was not sufficient for the induction of JNK2 phosphorylation, suggesting that JNK2 was activated by a mechanical rather than a mechanical/growth factor-dependent mechanism. Taken together, the results of this study demonstrate that aging does not alter the mechanosensitivity of the p38, p70S6k, and JNK2 signaling pathways in skeletal muscle.

Mechanotransduction and the regulation of protein synthesis in skeletal muscle.

Repeated bouts of resistance exercise produce an increase in skeletal muscle mass. The accumulation of protein associated with the growth process results from a net increase in protein synthesis relative to breakdown. While the effect of resistance exercise on muscle mass has long been recognized, the mechanisms underlying the link between high-resistance contractions and the regulation of protein synthesis and breakdown are, to date, poorly understood. In the present paper skeletal muscle will be viewed as a mechanosensitive cell type and the possible mechanisms through which mechanically-induced signalling events lead to changes in rates of protein synthesis will be examined.

The MEF2 site is necessary for induction of the myosin light chain 2 slow promoter in overloaded regenerating plantaris muscle.

Functional overload (OV) of the rat plantaris muscle results in a fast to slow change in muscle phenotype with induction of the slow contractile protein genes including myosin light chain 2 slow (MLC2s). To identify potential cis-acting DNA sites regulating MLC2s following ablation, plasmid constructs were transfected in vivo into regenerating overloaded plantaris muscles. Activity of the 270bp promoter (-270MLC2s) was increased in OV muscles at 28 days. Mutation of the MEF2 site (-270MEF2) knocked out the overload-induced activity of the promoter. Mutation of the Ebox (-270Ebox) resulted in an earlier induction with OV and mutation of the CACC site (-270CACC) resulted in increased activity in the CON PLN with OV induction detected by 21 days. These results demonstrate that the -270MLC2s promoter contains the elements necessary for expression of MLC2s in regenerating OV PLN. More importantly, mutation analysis of -270MLC2s promoter demonstrates that mechanical loading induced expression shares some common molecular mechanisms with slow nerve dependent model regulation. In these two models of physiological induction of MLC2s, the CACC site acts as a repressor region (on/off switch) and the MEF2 site acts to modulate quantitative expression.

Regulation of translation factors during hindlimb unloading and denervation of skeletal muscle in rats.

In the rat, denervation and hindlimb unloading are two commonly employed models used to study skeletal muscle atrophy. In these models, muscle atrophy is generally produced by a decrease in protein synthesis and an increase in protein degradation. The decrease in protein synthesis has been suggested to occur by an inhibition at the level of protein translation. To better characterize the regulation of protein translation, we investigated the changes that occur in various translation initiation and elongation factors. We demonstrated that both hindlimb unloading and denervation produce alterations in the phosphorylation and/or total amount of the 70-kDa ribosomal S6 kinase, eukaryotic initiation factor 2 alpha-subunit, and eukaryotic elongation factor 2. Our findings indicate that the regulation of these protein translation factors differs between the models of atrophy studied and between the muscles evaluated (e.g., soleus vs. extensor digitorum longus).

Intracellular signaling specificity in skeletal muscle in response to different modes of exercise.

The aim of this study was to understand better the specific signaling events resulting from different modes of exercise. Three different exercise protocols were employed based on their well-characterized, long-term training effects on either muscle hypertrophy or endurance phenotypes. Rats were subjected to a single bout of either a high-frequency electrical stimulation, a low-frequency electrical stimulation, or a running exercise protocol. Postexercise intracellular signaling was analyzed in the tibialis anterior and soleus muscles at 0, 3, and 6 h. A prolonged increase in p70(S6k) and a transient increase in protein kinase B phosphorylation were only observed in response to a growth-inducing stimulus (e.g., tibialis anterior in high-frequency electrical stimulation). In contrast, extracellular regulated kinase and 38-kDa stress-activated protein kinase were activated in response to all forms of exercise at 0 h, but only extracellular regulated kinase phosphorylation was found significantly elevated at 6 h after running exercise. These results demonstrate that different exercise protocols resulted in the selective activation of specific intracellular signaling pathways, which may determine the specific adaptations induced by different forms of exercise.

The calcineurin-NFAT pathway and muscle fiber-type gene expression.

To test for a role of the calcineurin-NFAT (nuclear factor of activated T cells) pathway in the regulation of fiber type-specific gene expression, slow and fast muscle-specific promoters were examined in C2C12 myotubes and in slow and fast muscle in the presence of calcineurin or NFAT2 expression plasmids. Overexpression of active calcineurin in myotubes induced both fast and slow muscle-specific promoters but not non-muscle-specific reporters. Overexpression of NFAT2 in myotubes did not activate muscle-specific promoters, although it strongly activated an NFAT reporter. Thus overexpression of active calcineurin activates transcription of muscle-specific promoters in vitro but likely not via the NFAT2 transcription factor. Slow myosin light chain 2 (MLC2) and fast sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) reporter genes injected into rat soleus (slow) and extensor digitorum longus (EDL) (fast) muscles were not activated by coinjection of activated calcineurin or NFAT2 expression plasmids. However, an NFAT reporter was strongly activated by overexpression of NFAT2 in both muscle types. Calcineurin and NFAT protein expression and binding activity to NFAT oligonucleotides were different in slow vs. fast muscle. Taken together, these results indicate that neither calcineurin nor NFAT appear to have dominant roles in the induction and/or maintenance of slow or fast fiber type in adult skeletal muscle. Furthermore, different pathways may be involved in muscle-specific gene expression in vitro vs. in vivo.

Use of DNA injection for identification of slow nerve-dependent regions of the MLC2s gene.

It has been well established that expression of slow contractile protein genes in skeletal muscle is regulated, in part, by activity from slow motoneurons. However, very little is understood about the mechanism by which neural activity regulates transcription of slow isoform genes. The purpose of this investigation was first to more fully define the in vivo DNA injection technique for use in both fast-twitch and slow-twitch muscles and second to use the injection technique for the identification of slow nerve-dependent regions of the myosin light chain 2 slow (MLC2s) gene. Initial experiments determined that the same amount of plasmid DNA was taken up by both the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles and that injection of from 0.5 to 10 micrograms DNA/muscle is ideal for analysis of promoter activity during regeneration. This technique was subsequently used to identify that the region from -800 to +12 base pairs of MLC2s gene directed approximately 100 times higher activity in the innervated soleus than in innervated EDL, denervated soleus, or denervated EDL muscles. Placing the introns upstream of either the MLC2s or SV40 promoter increased expression 5- and 2.7-fold, respectively, in innervated soleus but not in innervated EDL, denervated soleus, or denervated EDL muscles. These results demonstrate that 1) in vivo DNA injection is a sensitive assay for promoter analysis in both fast-twitch and slow-twitch skeletal muscles and 2) both 5' flanking and intronic regions of the MLC2s gene can independently and synergistically direct slow nerve-dependent transcription in vivo.

Changes in contractile protein mRNA accumulation in response to spaceflight.

Ten rats were exposed to 9 days of zero gravity aboard the National Aeronautics and Space Administration SLS-1 space mission (June 1991). Levels of fast and slow isoform mRNAs from six contractile protein gene families were quantified in the flight soleus and extensor digitorum longus (EDL) muscles. The gene families studied were myosin light chain-1 (MLC-1), myosin light chain-2 (MLC-2), troponin (Tn) T, TnI, TnC, and tropomyosin. In the EDL muscle there was no change in slow mRNA levels with a general increase in fast mRNA levels from 23 to 232%. Changes in slow mRNA levels were seen in the flight soleus muscle with TnCslow and TnTslow levels increasing slightly, and MLC-1slow a, MLC-1slow b, TnIslow, alpha-Tmslow, and MLC-2slow levels decreasing. All fast mRNA levels increased in the flight soleus muscle from 170 to 1,100%. We can conclude that exposure to zero gravity results in 1) a general increase in fast mRNA levels in both fast and slow muscles and 2) differing directional changes in slow mRNA accumulation in the soleus muscle.

Mechanical load affects growth and maturation of skeletal muscle grafts.

The purpose of our study was to determine whether the early patterns of growth and maturation of regenerating soleus muscle grafts are sensitive to alterations in mechanical load. We hypothesized that decreased and increased mechanical loading of grafts would reduce and accelerate, respectively, the rate and magnitude of growth and impair and enhance, respectively, the pattern of maturation. On day 0, soleus muscles were grafted and rats were assigned to one of three groups: cage sedentary (normal load), hindlimb suspension (decreased load), or ablation of synergist muscle (increased load). From days 7 to 35, graft mass in cage-sedentary rats increased at a rate of 1.85 mg mass/day. Rates were less for grafts of suspended rats and greater in grafts of ablated rats (-1.06 and 3.89 mg mass/day, respectively; P < 0.01). Neonatal myosin heavy chain (MHC) in grafts reached 10 +/- 1.6% of total MHC at day 7 for cage-sedentary rats, whereas in the suspended animals it reached 11 +/- 2.4% of total MHC at day 14. At days 21 and 35, grafts from the suspended animals had a lower proportion of slow MHC (45 +/- 2.4%) than did grafts from the control and ablated groups (95 +/- 1.5%; P < 0.05). Decreased mechanical load impaired the rate and degree of growth and maturation during regeneration, whereas increased mechanical load enhanced growth characteristics but not maturation.

Identification of a program of contractile protein gene expression initiated upon skeletal muscle differentiation.

The functional diversity of skeletal muscle is largely determined by the combinations of contractile protein isoforms that are expressed in different fibers. Just how the developmental expression of this large array of genes is regulated to give functional phenotypes is thus of great interest. In the present study, we performed a comprehensive analysis of contractile protein isoform mRNA profiles in skeletal muscle systems representing each generation of fiber formed: primary, secondary, and regenerating fibers. We find that in each system examined there is a common pattern of isoform gene expression during early differentiation for 5 of the 6 gene families we have investigated: myosin light chain (MLC)1, MLC2, tropomyosin, troponin (Tn)C, and TnI. We suggest that the common isoform patterns observed together represent a genetic program of skeletal muscle differentiation that is independent of the mature fiber phenotype and is found in all newly formed myotubes. Within each of these contractile protein gene families the program is independent of the isoforms of myosin heavy chain (MHC) expressed. The maintenance of such a program may reflect a specific requirement of the initial differentiation process.

Age effects on myosin subunit and biochemical alterations with skeletal muscle hypertrophy.

The purpose of this study was to determine whether skeletal muscle mass, myofibrillar adenosinetriphosphatase activity, and the expression of myosin heavy (MHC) and light chain subunits are differentially affected in juvenile (4 wk) and young adult (12 wk) rats by a hypertrophic growth stimulus. Hypertrophy of the plantaris or soleus was studied 4 wk after ablation of either two [gastrocnemius (GTN) and soleus or plantaris] or one (GTN) synergistic muscle(s). There was no difference in the relative magnitude of hypertrophy because of age. Plantaris myofibrillar adenosinetriphosphatase activity was decreased 21 and 12% in juvenile and adult rats, respectively, as a result of ablation of both the GTN and soleus. Slow myosin light chain isoforms (1s and 2s) were expressed to a greater extent in hypertrophied plantaris muscles of both ages, but the increase in 1s was greater in juvenile rats. The relative expression of slow beta-MHC in hypertrophied plantaris muscles increased by 470 and 350%, whereas MHC IIb decreased by 70 and 33% in juvenile and adult rats, respectively. The relative expression of MHC IIa increased (56%) in the plantaris after ablation in juvenile rats only. These shifts in myosin subunit expression and the increases in mass were generally about one-half the magnitude when only the GTN was removed. There were no detectable myosin shifts in hypertrophied soleus muscles. Although the extent of muscle hypertrophy is similar, the shifts in myosin subunits were greater in juvenile than in young adult rats.

Prior running reduces hypertrophic growth of skeletal muscle grafts.

Run training can increase the mass of soleus muscle grafts, yet values remain lower than nongrafted muscle even with continued training. Thus we tested the hypothesis that nerve-implant soleus grafts of rats previously run trained would be refractory to the hypertrophic stimulus of ablation of synergistic muscle. We also compared the magnitude of growth of the nerve-implant soleus graft after ablation with that reported by others for the nerve-intact soleus graft. We studied eight groups that differed relative to the combination and order of treatments (running and ablation of synergistic muscle) and the graft age at the time of the ablation operation and study. Graft mass, protein concentration, and histochemical fiber composition were measured. Compared with grafts from cage-sedentary rats, the mass and protein content of the nerve-implant soleus grafts were higher (16-63%) at all times after ablation. When the ablation operation was performed at 56 days postgrafting, there was a 33% increase in protein content of the soleus graft by 84 days for cage-sedentary animals. This increase was twofold greater (P less than or equal to 0.02) than the 15% increase that followed ablation for the grafts from the animals that had been run trained before the ablation operation. Four weeks of run training before the ablation operation impaired the adaptive response of muscle grafts to the ablation of synergistic muscles, which may reflect alterations in motor unit recruitment and/or satellite cell activity. Ablation of synergistic muscles resulted in an absolute growth of the nerve-implant soleus grafts that was comparable with that reported for nerve-intact soleus grafts.

Satellite cell and growth factor involvement in skeletal muscle growth.

The activity of the satellite cell, discovered by Alexander Mauro, is of fundamental importance in postnatal skeletal muscle development, muscle adaptation to certain activity stimuli, and to muscle fiber regeneration following injury and transplantation operations. There are numerous mitogens and growth factors that influence satellite cell proliferation and differentiation in vitro and likely in vivo. The best understood purified growth factors are fibroblast growth factor (FGF), the insulin-like growth factors (IGF-I and -II), and transforming growth factor-beta (TGF-beta). Soluble extracts from injured muscle and chronically stretched muscle are also known to be mitogenic and are yet to be purified. Skeletal muscle development, hypertrophy, and regeneration can be viewed as points on a continuum with respect to the regulatory mechanisms of myogenic cell growth. The occurrence of fiber hyperplasia differs amongst some models of activity-induced growth and may reflect differences in the magnitude of the stimulus relative to the capacity of fibers to adapt. The relationships between the mechanical and environmental events coincident with an activity or injury stimulus and the role of specific muscle fiber satellite cell populations and growth factors are fertile areas for investigation. Insights from these experiments will yield a comprehensive understanding of the muscle growth process at the molecular, cellular, and tissue levels, and have implications for development and aging, health, disease, and adaptation.