Fast Fiber Types Research Articles

Effect Of Slow-speed Vs. Traditional Resistance Training Regimens On Myonuclear Domain And Satellite Cell Content

PURPOSE: The purpose of this study was to investigate muscle fiber adaptations in myonuclear domain and satellite cell content in conjunction with varying degrees of fiber hypertrophy following slow-speed vs. "normal-speed“ resistance training protocols. METHODS: Thirty-four healthy adult females (21.1 ± 2.7 yr.) were randomly divided into four groups: slow-speed (SS), traditional strength (TS), traditional muscular endurance (TE), and non-exercising control (C). Workouts, consisting of 3 sets each of 3 exercises (leg press, squat, and knee extension), were performed 2d/wk for the first week and 3d/wk for the remaining 5 weeks. For each training session, the SS group performed 6-10RM (6-10 repetitions maximum) for each set with 10s con and 4s ecc contractions for each repetition. TS and TE performed 6-10RM and 20-30RM, respectively, at "normal“ speed (1-2s/con and ecc contraction). Both TE and SS trained at the same relative intensity (40-60% 1RM), whereas TS trained at 80-85% 1RM. Pre- and post-training vastus lateralis muscle biopsies were analyzed for fiber cross-sectional area (CSA), myonuclear domain and number, NCAM + (anti-neural cell adhesion molecule) satellite cells, and fiber type. RESULTS: 1) As previously reported, the CSA of all major fiber types (I, IIA, and IIX) increased post-training for TS, whereas only the fast fiber types increased in size for SS. No significant change in fiber size was found for either the TE or C groups. 2) Myonuclear domain was significantly larger post-training in TS for all major fiber types, whereas only Type IIA domain increased in SS. There were no changes in myonuclear domain for TE or C. 3) Myonuclear number did not differ significantly from pre- to post-training in any group. 4) Satellite cell content increased post-training for TS in all major fiber types, while only NCAM + nuclei associated with Type IIX and Type IIAX fibers increased in SS. No changes in the satellite cell population were observed for TE or C. CONCLUSIONS: Slow-speed resistance training resulted in a larger degree of fiber hypertrophy, increased myonuclear domain, and stimulus to muscle stem cells compared to training with a similar resistance at a normal speed. However, training at a higher intensity (80-85% 1RM) at normal speed resulted in the greatest overall fiber response for each of these variables.

Functional Characterization of Muscle Fibres from Patients with Chronic Fatigue Syndrome: Case-Control Study

Chronic fatigue syndrome (CFS) is a disabling condition characterized by unexplained chronic fatigue that impairs normal activities. Although immunological and psychological aspects are present, symptoms related to skeletal muscles, such as muscle soreness, fatigability and increased lactate accumulation, are prominent in CFS patients. In this case-control study, the phenotype of the same biopsy samples was analyzed by determining i) fibre-type proportion using myosin isoforms as fibre type molecular marker and gel electrophoresis as a tool to separate and quantify myosin isoforms, and ii) contractile properties of manually dissected, chemically made permeable and calcium-activated single muscle fibres. The results showed that fibre-type proportion was significantly altered in CSF samples, which showed a shift from the slow- to the fast-twitch phenotype. Cross sectional area, force, maximum shortening velocity and calcium sensitivity were not significantly changed in single muscle fibres from CSF samples. Thus, the contractile properties of muscle fibres were preserved but their proportion was changed, with an increase in the more fatigue-prone, energetically expensive fast fibre type. Taken together, these results support the view that muscle tissue is directly involved in the pathogenesis of CSF and it might contribute to the early onset of fatigue typical of the skeletal muscles of CFS patients.

The cell nuclei of skeletal muscle cells are transcriptionally active in hibernating edible dormice

BackgroundSkeletal muscle is able to react in a rapid, dynamic way to metabolic and mechanical stimuli. In particular, exposure to either prolonged starvation or disuse results in muscle atrophy. At variance, in hibernating animals muscle atrophy may be scarce or absent after bouts of hibernation i.e., periods of prolonged (months) inactivity and food deprivation, and muscle function is fully preserved at arousal. In this study, myocytes from the quadriceps muscle of euthermic and hibernating edible dormice were investigated by a combination of morphological, morphometrical and immunocytochemical analyses at the light and electron microscopy level. The focus was on cell nuclei and mitochondria, which are highly sensitive markers of changing metabolic rate.ResultsFindings presented herein demonstrate that: 1) the general histology of the muscle, inclusive of muscle fibre shape and size, and the ratio of fast and slow fibre types are not affected by hibernation; 2) the fine structure of cytoplasmic and nuclear constituents is similar in euthermia and hibernation but for lipid droplets, which accumulate during lethargy; 3) during hibernation, mitochondria are larger in size with longer cristae, and 4) myonuclei maintain the same amount and distribution of transcripts and transcription factors as in euthermia.ConclusionIn this study we demonstrate that skeletal muscle cells of the hibernating edible dormouse maintain their structural and functional integrity in full, even after months in the nest. A twofold explanation for that is envisaged: 1) the maintenance, during hibernation, of low-rate nuclear and mitochondrial activity counterbalancing myofibre wasting, 2) the intensive muscle stimulation (shivering) during periodic arousals in the nest, which would mimic physical exercise. These two factors would prevent muscle atrophy usually occurring in mammals after prolonged starvation and/or inactivity as a consequence of prevailing catabolism. Understanding the mechanisms responsible for skeletal muscle preservation in hibernators could pave the way to prevention and treatment of muscle wasting associated with pathological conditions or ageing as well as life in extreme environments, such as ocean deeps or spaceflights.

Post-Tetanic Calcium Transients In Adult Skeletal Muscle Fibers Are Frequency-Dependent And Fiber Type Specific

Isolated adult Flexor digitorum brevis fibers from 4-6 weeks-old mice, loaded with Fluo-3 were stimulated with trains of 270, 0.3 ms pulses at different frequencies. We observed a fast calcium tetanus (associated with contraction) and a second, slower signal, similar to those previously described in cultured myotubes. The slow signal (more than the fast one) was inhibited by 25 micro M nifedipine, suggesting a role for DHPR in its onset and by the IP3R inhibitor Xestospongin-C (5 micro M). The amplitude of post-tetanic calcium transients depended on both stimulus train frequency and duration; a bell shaped curve frequency was obtained with a maximum at 10-20 Hz. Likewise, signal amplitude was proportional to stimulus train duration. Fibers isolated from soleus muscle completely lack slow calcium transients. Using immunofluorescence, we have found that all three IP3R isoforms are present in adult muscle at different levels and that IP3R-1 is differentially expressed (with a mosaic pattern) in different types of muscle fibers, being higher in a subset of fast-type fibers. ERK 1/2 phosphorylation of adult muscle fibers after tetanic stimulation appears to relate slow calcium signals to transcription-related events. These results support the idea that different calcium kinetics for the slow signals mediated by IP3R may exist in different types of muscle fibers and participate in the activation of specific transcriptional programs of slow and fast phenotype.FONDAP 15010006, Bicentenario-PSD24, FONDECYT 1080120

Fiber-types of sarcomeric proteins expressed in cultured myogenic cells are modulated by the dose of myogenin activity

The myogenic basic helix–loop–helix regulatory factors (MRFs) maintain commitment of proliferating cells to the skeletal myogenic lineage, and contribute to activation of transcription of muscle-specific genes in myocytes and muscle fibers. A clear role for any or all of the MRFs in muscle fiber-type determination, however, has not emerged from expression or genetic studies. During fetal, neonatal and adult life, diversification of muscle fiber types, and the dynamics of slow or fast fiber type adaptation and growth, are controlled by exogenous factors, including innervation, work load, and hormonal signaling. In contrast, stereotypical development of muscle fibers preferentially expressing slow or fast isoforms of sarcomeric proteins in the embryo occurs in the absence of these factors, and appears to be mediated both by input from the surrounding interstitial milieu, as well by cell autonomous mechanisms. We report here that diversification of myogenic cells in culture towards the expression of fast or slow skeletal muscle fiber types can be determined by the activity and dose of at least one MRF, myogenin. The dose of myogenin is modulated by two parameters: the phosphorylation state of the transcriptional activation domains, and the level of expression. Low doses of myogenin promoted a fast phenotype, whereas higher doses promoted a slow phenotype, and further studies suggested that diversification is mediated by both transcriptional and post-transcriptional mechanisms. The potential for dose or numeration signaling by basic helix–loop–helix regulators has been revealed by studies in Drosophila melanogaster, while the present results support the notion that this mechanism may be more commonly employed to generate subdiversity among developing cell types.

Enzyme‐ and immunohistochemical aspects of skeletal muscle fibers in brown bear (Ursus arctos)

To further elucidate the pattern of MHC isoform expression in skeletal muscles of large mammals, in this study the skeletal muscles of brown bear, one of the largest mammalian predators with an extraordinary locomotor capacity, were analyzed. Fiber types in longissimus dorsi, triceps brachii caput longum, and rectus femoris muscles were determined according to the myofibrillar ATPase (mATPase) histochemistry and MHC isoform expression, revealed by a set of antibodies specific to MHC isoforms. The oxidative (SDH) and glycolytic enzyme (alpha-GPDH) capacity of fibers was demonstrated as well. By mATPase histochemistry five fiber types, i.e., I, IIC, IIA, IIAX, IIX were distinguished. Analyzing the MHC isoform expression, we assume that MHC-I, -IIa, and -IIx are expressed in the muscles of adolescent bears. MHC-I isoform was expressed in Type-I fibers and coexpressed with presumably -IIa isoform, in Type-IIC fibers. Surprisingly, two antibodies specific to rat MHC-IIa stained those fast fibers, that were histochemically and immunohistochemically classified as Type IIX. This assumption was additionally confirmed by complete absence of fiber staining with antibody specific to rat MHC-IIb and all fast fiber staining with antibody that according to our experience recognizes MHC-IIa and -IIx of rat. Furthermore, quite high-oxidative capacity of all fast fiber types and their weak glycolytic capacity also imply for MHC-IIa and -IIx isoform expression in fast fibers of bear. However, in adult, full-grown animal, only MHC-I and MHC-IIa isoforms were expressed. The expression of only two fast isoforms in bear, like in many other large mammals (humans, cat, dog, goat, cattle, and horse) obviously meets the weight-bearing and locomotor demands of these mammals.

20-Hydroxyecdysone increases fiber size in a muscle-specific fashion in rat

20-Hydroxyecdysone (20E) is an ecdysteroid hormone that regulates moulting in insects. Interestingly, 20E is also found most abundantly in plant species and has anabolic effects in vertebrates, i.e. increasing muscle size without androgen influence. The effect of 20E on slow and fast fiber types of skeletal muscle has not been reported yet. Here we present that 20E affects the size (cross-sectional area, CSA) of the different fiber types in a muscle-specific manner. The effect on fiber size was modified by the distance from the site of the treatment and the presence of a regenerating soleus muscle in the animal. Besides the fiber size, 20E also increased the myonuclear number in the fibers of normal and regenerating muscles, suggesting the activation of satellite cells. According to our results 20E may provide an alternative for substitution of anabolic-androgenic steroids in therapeutic treatments against muscle atrophy.

Changes in exercise tolerance and body composition in myostatin overexpressing transgenic mice

Myostatin (Mst) regulates skeletal muscle mass. Mst‐null (KO) mice have double muscle size, while Mst overexpression leads to skeletal muscle wasting. We aimed at identifying the role of Mst in adult‐onset atrophy, hypertrophy, physical performance and the involved molecular cascades. Fiber type composition of Mst overexpressing transgenic (Tg), Mst KO and WT male 1 year old mice (n=6 in each group) was assessed by immunohistochemistry, MHC isoforms by gel electrophoresis and gene expression levels by real time RT‐PCR from muscle tissues. Exercise tolerance was measured by incremental treadmill tests performed to exhaustion 3 times a week for 8 weeks. The initial speed was set to 8 m/min at no inclination. The speed and inclination were subsequently incremented by 4 m/min and 5% every 5 min. Exercise tolerance of KO animals was significantly lower than Tg animals (1.5±1.6 mJ/g vs. 7.3±4.1 mJ/g, P<0.05). In Tg animals the abdominal fat increased by 150% while the muscle mass decreased by 25%. Compared to wild type animals, the fast type fibers decreased by 22% in Tg animals while it increased by 25% in KO. The amount of MHC IIb protein was 2.2 fold higher in KO mice and MHC I was 1.8 fold higher in Tg animals. The Tg mice had a 37% increase in Mst mRNA expression and Akt2 expression was down‐regulated. We conclude that Mst plays a crucial role in regulating body composition, muscle structure and physical performance.

Capillary supply, fibre types and fibre morphometry in rat tibialis anterior and diaphragm muscles after intermittent exposure to hypobaric hypoxia

Three groups of sedentary male rats were exposed to intermittent hypobaric hypoxia (IHH) for 22 days (4 h/day, 5 days/week) in a hypobaric chamber at a simulated altitude of 5,000 m. Tibialis anterior (TA) and diaphragm (DG) were removed at the end of the programme (H group), and 20 or 40 days later (P20 and P40 groups). A control group (C) was maintained at sea-level pressure and their TA and DG were compared to those of the experimental rats at the end of the IHH programme, and also 20 and 40 days later. We measured the fibre morphometry and capillaries of each muscle. Our results demonstrate that IHH does not change the fibre type composition (with reference to either their contractile or oxidative properties) for most muscle regions of the muscles analysed analysed. We found few significant differences in muscle capillarity and fibre morphometry for TA after IHH. However, IHH did induce some statistically significant changes in DG: capillary density of the H rats (736 capillaries/mm2) increased compared to C animals (610 capillaries/mm2). Although IHH did not change the fibre capillarization or morphometric parameters of fast fibre types, we observed reductions ranging from 7 to 13% in fibre area, perimeter and diffusion distances between C and H for slow fibres. Moreover, these morphometric changes accounted for increases of 10-20% in capillarization, fibre unit area and fibre unit perimeter. This indicates that SO fibres are more sensitive to IHH than both fast fibre types.

Expression of Myosin Heavy-chain mRNA in Cultured Myoblasts Induced by Centrifugal Force

Ballistic muscle training leads to hypertrophy of fast type fibers and training for endurance induces that of slow type fibers. Numerous studies have been conducted on electrical, extending and magnetic stimulation of cells, but the effect of centrifugal force on cells remains to be investigated. In this study, we investigated the effect of stimulating cultured myoblasts with centrifugal force at different speeds on cell proliferation and myosin heavy-chain (MyHC) mRNA expression in muscle fiber. Stimulation of myoblasts was carried out at 2 different speeds for 20 min using the Himac CT6D, a desk centrifuge, and cells were observed at 1, 3 and 5 days later. Number of cells 1 and 5 days after centrifugal stimulation was significantly larger in the 62.5 x g and 4,170 x g stimulation groups than in the control group. Expression of MyHC-2b mRNA 1 day after centrifugal stimulation was significantly higher in the 2 stimulation groups than in the control group. Almost no expression of MyHC-2a was observed in any group at 1 and 3 days after centrifugal stimulation. However, 5 days after stimulation, MyHC-2a was strongly expressed in the 2 stimulation groups in comparison to the control group. Three days after centrifugal stimulation, expression of MyHC-1 was significantly higher in the 2 stimulation groups than in the control group. The results of this study clarified the effect of different centrifugal stimulation speeds on muscle fiber characteristics, and suggest that centrifugal stimulation of myoblasts enhances cell proliferation.

Is Fiber-Type Composition Related to Daily Jaw Muscle Activity during Postnatal Development?

Aim: Muscles containing large numbers of slow-contracting fibers are generally more active than muscles largely composed of fast fibers. This relationship between muscle activity and phenotype suggests that (1) changes in fiber-type composition during postnatal development are accompanied by changes in daily activity and (2) individual variations in fiber-type composition are related to similar variations in daily muscle activity. Methods: The masseter and digastric muscles of 23 New Zealand White rabbits (young, juvenile and adult) were examined for their phenotype (myosin heavy chain content) and their daily activity (total daily number of activity bursts). Results: During development, the masseter showed a strong increase in the number of fast-type fibers compared to the number of slow-type fibers. During development, also the number of powerful bursts in the masseter increased. The digastric showed no significant changes in fiber types or burst numbers. Within each muscle, across individual animals, no significant correlations (R < 0.70) were found between any of the fiber types and daily burst numbers in any of the age groups. Conclusions: The results suggest that activity-related influences are of relatively minor importance during development and that other factors are dominant in determining fiber-type composition.

De‐phosphorylation of MyoD is linking nerve‐evoked activity to fast myosin heavy chain expression in rodent adult skeletal muscle

Elucidating the molecular pathways linking electrical activity to gene expression is necessary for understanding the effects of exercise on muscle. Fast muscles express higher levels of MyoD and lower levels of myogenin than slow muscles, and we have previously linked myogenin to expression of oxidative enzymes. We here report that in slow muscles, compared with fast, 6 times as much of the MyoD is in an inactive form phosphorylated at T115. In fast muscles, 10 h of slow electrical stimulation had no effect on the total MyoD protein level, but the fraction of phosphorylated MyoD was increased 4-fold. Longer stimulation also decreased the total level of MyoD mRNA and protein, while the level of myogenin protein was increased. Fast patterned stimulation did not have any of these effects. Overexpression of wild type MyoD had variable effects in active slow muscles, but increased expression of fast myosin heavy chain in denervated muscles. In normally active soleus muscles, MyoD mutated at T115 (but not at S200) increased the number of fibres containing fast myosin from 50% to 85% in mice and from 13% to 62% in rats. These data establish de-phosphorylated active MyoD as a link between the pattern of electrical activity and fast fibre type in adult muscles.

A mathematical model of fatigue in skeletal muscle force contraction

The ability for muscle to repeatedly generate force is limited by fatigue. The cellular mechanisms behind muscle fatigue are complex and potentially include breakdown at many points along the excitation-contraction pathway. In this paper we construct a mathematical model of the skeletal muscle excitation-contraction pathway based on the cellular biochemical events that link excitation to contraction. The model includes descriptions of membrane voltage, calcium cycling and crossbridge dynamics and was parameterised and validated using the response characteristics of mouse skeletal muscle to a range of electrical stimuli. This model was used to uncover the complexities of skeletal muscle fatigue. We also parameterised our model to describe force kinetics in fast and slow twitch fibre types, which have a number of biochemical and biophysical differences. How these differences interact to generate different force/fatigue responses in fast- and slow- twitch fibres is not well understood and we used our modelling approach to bring new insights to this relationship.

The p38α/β Mitogen-activated Protein Kinases Mediate Recruitment of CREB-binding Protein to Preserve Fast Myosin Heavy Chain IId/x Gene Activity in Myotubes

In skeletal muscle, the transformation of fast into slow fiber type is accompanied by shifts in fiber type-specific gene expression that includes down-regulation of the adult fast fiber myosin heavy chain IId/x (MyHCIId/x) gene. Here, we report that the mitogen-activated protein kinases (MAPKs) p38alpha/beta regulate MyHCIId/x gene expression. Electrical stimulation of rabbit skeletal muscle cells with a slow fiber type activity pattern and treatment of C2C12 myotubes with Ca(2+)-ionophore inhibited p38alpha/beta MAPKs and reduced fast fiber type MyHC protein expression and promoter activity. Pharmacological inhibition of p38alpha/beta also down-regulated MyHCII gene expression. In controls, binding of the myocyte enhancer factor-2 (MEF-2) isoforms C and D as a heterodimer to a proximal consensus site within the MyHCIId/x promoter and recruitment of a transcriptional coactivator, the CREB-binding protein CBP, were observed. Overexpression of wild type MEF-2C but not of a MEF-2C mutant that cannot be phosphorylated by p38 induced promoter activity. Mutation of the MEF-2-binding site decreased the inducing effect of overexpressed CBP. Inhibition of p38alpha/beta MAPKs abolished CBP binding, whereas enforced induction of p38 by activated MAPK kinase 6 (MKK6EE) enhanced binding of CBP and increased promoter activity. Furthermore, knockdown of endogenous CBP by RNA interference eliminated promoter activation by MEF-2C or MKK6EE. In electrical stimulated and Ca(2+)-ionophore-treated myotubes, CBP was absent in complex formation at that site. Taken together, the data indicate that p38alpha/beta MAPKs-mediated coactivator recruitment at a proximal MEF-2 site is important for MyHCIId/x gene regulation in skeletal muscle.

Signaling mechanisms involved in disuse muscle atrophy

Prolonged periods of skeletal muscle inactivity due to bed rest, denervation, hindlimb unloading, immobilization, or microgravity can result in significant muscle atrophy. The muscle atrophy is characterized as decreased muscle fiber cross-sectional area and protein content, reduced force, increased insulin resistance as well as a slow to fast fiber type transition. The decreases in protein synthesis and increases in protein degradation rates account for the majority of the rapid loss of muscle protein due to disuse. However, we are just beginning to pay more attention on the identification of genes involved in triggering initial responses to physical inactivity/microgravity. Our review mainly focuses on the signaling pathways involved in protein loss during disuse atrophy, including two recently identified ubiquitin ligases: muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx). Recent reports suggest that inhibition of the IGF-1/PI3K/Akt pathway in muscle may be involved in the progression of disuse atrophy. NF-kappaB seems to be a key intracellular signal transducer in disuse atrophy. Factors such as myostatin, p38 and calcineurin can induce muscle protein loss under specified conditions, but further experiments are needed to determine whether they are necessary components of disuse atrophy. Where possible, the molecular mechanisms underlying the slow to fast fiber type transition and increased insulin resistance in atrophic muscles are discussed as well. Collectively, the disuse-induced muscle atrophy is a highly ordered process that is controlled by interactions between intracellular signaling pathways rather than isolated pathways.

Changes in Soleus Muscle Function and Fiber Morphology with One Week of Locomotor Training in Spinal Cord Contusion Injured Rats

The purpose of this study is two-fold: (1) to examine skeletal muscle function in a rat model of midthoracic contusion spinal cord injury (SCI) and (2) to evaluate the therapeutic influence of a short bout (1 week) of treadmill locomotor training on soleus muscle function (peak force, fatigability, contractile properties, fiber types), size (fiber area), and motor deficit and recovery (BBB scores) after SCI. The rats were injured with a moderate T8 spinal cord contusion and were assigned to either receive treadmill locomotor training (TM), starting 1 week after SCI for 5 consecutive days (20 min/trial, 2 trials/day) or not to receive any exercise intervention (no TM). Locomotor training resulted in a significant improvement in overall locomotor function (32% improvement in BBB scores) when compared to no TM. Also, the injured animals that trained for 1 week had 38% greater peak soleus tetanic forces (p < 0.05), a 9% decrease in muscle fatigue (p < 0.05), 23% larger muscle fiber CSA (p < 0.05), and decreased immunoexpression of fast heavy chain fiber types than did rats receiving no TM. In addition, there was a good correlation (0.704) between the BBB scores of injured animals and peak soleus muscle force regardless of group assignment. No significant differences were seen in twitch or time to peak tension values across groups. Collectively, these results indicate that 1 week of treadmill locomotor training, initiated early after SCI, can significantly improve motor recovery following SCI. The magnitude of these changes is remarkable considering the relatively short training interval and clearly illustrates the potential that initiating treadmill locomotor training shortly after injury may have on countering some of the functional deficits resulting from SCI.

Heterogeneous postnatal transitions in myosin heavy chain isoforms within the rabbit temporalis muscle

Postnatal changes in the fiber type composition and fiber cross-sectional area were investigated in the superficial (TEM1) and deep (TEM23) temporalis of male rabbits. It was hypothesized that, due to the transition from suckling to chewing during early postnatal development, the proportion of fast fiber types would decrease, while the proportion of fibers positive for myosin heavy chain (MyHC) cardiac alpha would increase, and that, due to the influence of testosterone during late postnatal development, the proportion of these alpha fibers would decrease again. Classification of the fibers types was performed by immunohistochemistry according to their MyHC content. The proportion of alpha fiber types significantly increased in both muscle portions from 2% and 8% for TEM1 and TEM23 at week 1 to 29% and 54% at week 8, respectively,. While in TEM1 the proportion of this fiber type did not change thereafter, it decreased again to 27% in TEM23 at week 20. The change for the fast fiber types was opposite to that of the alpha fiber types. Significantly more MyHC IIX fibers were found in TEM1 than in TEM23 in adult rabbits. In the first 8 weeks, the cross-sectional areas of all fibers increased. After this period, only MyHC cardiac alpha + I fibers continued to increase significantly. It was concluded that there are developmental differences in the myosin heavy chain transitions of the two portions of the temporalis muscle.

Postnatal transitions in myosin heavy chain isoforms of the rabbit superficial masseter and digastric muscle

We investigated the early (< 8 weeks) and late (> 8 weeks) postnatal development of the fibre type composition and fibre cross-sectional area in the superficial masseter and digastric muscle of male rabbits. It was hypothesized, first, that due to the transition between suckling and chewing, during early postnatal development the increase in the proportion of slow fibre types and in fibre cross-sectional areas would be larger in the masseter than in the digastric; and second, that due to the supposed influence of testosterone during late postnatal development, the proportion of slow fibre types in both muscles would decrease. Fibre types were classified by immunostaining according to their myosin heavy chain (MyHC) content. The proportion of slow fibre types significantly increased in the masseter, from 7% at week 1 to 47% at week 8, and then decreased to 21% at week 20, while in the digastric it increased from 5% in week 1 to 19% at week 8 and remained the same thereafter. The changes in the proportion of fast fibre types were the opposite. The remarkable increase and decrease in the proportion of slow fibre types in the masseter was attributed predominantly to MyHC-cardiac alpha fibres. During early development, the cross-sectional area of all fibres in both muscles increased. However, only the fast fibre types in the masseter continued to grow further after week 8. Before weaning, the fast fibre types in the digastric were larger than those in the masseter, but after week 8, they became larger in the masseter than in the digastric. In adult animals, masseter and digastric had the same percentage of fast fibre types, but these fibres were almost twice as large in masseter as in digastric.

Effect of calcineurin inhibitors on myotoxic activity of crotoxin and Bothrops asper phospholipase A 2 myotoxins in vivo and in vitro

Previous studies have shown that calcineurin activity plays a critical role in the myotoxic activity induced by crotoxin (CTX), a group II phospholipase A 2 (PLA 2) with neurotoxic and myotoxic actions. In order to address whether calcineurin is also important for the activity of non-neurotoxic group II PLA 2 myotoxins we have compared the effects of calcineurin inhibition on the myotoxic capacity of CTX and the non-neurotoxic PLA 2s, myotoxin II (Mt II) and myotoxin III (Mt III) from Bothrops asper venom. Rats were treated with cyclosporin A (CsA) or FK506, calcineurin inhibitors, and received an intramuscular injection of either CTX, Mt II or Mt III into the tibialis anterior. Animals were killed 24 h after injection of toxins. Tibialis anterior was removed and stored in liquid nitrogen. Myofibers in culture were also treated with CsA or FK506 and exposed to CTX, Mt II and Mt III. It was observed that, in contrast to CTX, CsA and FK506 do not attenuate myotoxic effects induced by both Mt II and Mt III in vivo and in vitro. The results of the present study suggest that calcineurin is not essential for the myotoxic activity of Mt II and Mt III, indicating that distinct intracellular pathways might be involved in myonecrosis induced by neurotoxic CTX and non-neurotoxic Bothrops sp. PLA 2 myotoxins. Alternatively, calcineurin dependent fast fiber type shift might render the muscle resistant to the action of CTX, without affecting its susceptibility to Bothrops sp. myotoxins.

An Alternative Domain Near the ATP Binding Pocket of Drosophila Myosin Affects Muscle Fiber Kinetics

We examined the importance of alternative versions of a region near the ATP binding site of Drosophila myosin heavy chain for muscle mechanical properties. Previously, we exchanged two versions of this region (encoded by alternative exon 7s) between the indirect flight muscle myosin isoform (IFI) and an embryonic myosin isoform (EMB) and found, surprisingly, that in vitro solution actin-activated ATPase rates were increased (higher V max) by both exon exchanges. Here we examined the effect of increased ATPase rate on indirect flight muscle (IFM) fiber mechanics and Drosophila locomotion. IFM expressing EMB with the exon 7a domain replaced by the IFM specific exon 7d domain (EMB-7d) exhibited 3.2-fold greater maximum oscillatory power ( P max) and 1.5-fold greater optimal frequency of power generation ( f max) versus fibers expressing EMB. In contrast, IFM expressing IFI with the exon 7d region replaced by the EMB exon 7a region (IFI-7a), showed no change in P max, f max, step response, or isometric muscle properties compared to native IFI fibers. A slight decrement in IFI-7a flight ability was observed, suggesting a negative influence of the increased ATPase rate on Drosophila locomotion, perhaps due to energy supply constraints. Our results show that exon 7 plays a substantial role in establishing fiber speed and flight performance, and that the limiting step that sets ATPase rate in Drosophila myosin has little to no direct influence in setting f max for fast muscle fiber types.