Temperature acclimation: improved sustained swimming performance in carp at low temperatures.

At low temperatures, the reduction in mechanical power output of the aerobic muscle forces cold-blooded animals, such as carp, to recruit their rapidly fatiguing anaerobic fibers at relatively slow swimming speeds. Previous experimental data have suggested that changes in the biochemistry and morphology of the aerobic muscle during cold acclimation might increase its output of mechanical power. The present experiments show that, because of these changes, carp can swim faster at low temperature using only their aerobic muscle, which results in an increase in their sustainable swimming speed. By modifying their musculature, cold-blooded animals can achieve some independence from the effects of seasonal changes in environmental temperature.

High-throughput ultra-high-performance liquid chromatography/tandem mass spectrometry quantitation of insulin-like growth factor-I and leucine-rich alpha-2-glycoprotein in serum as biomarkers of recombinant human growth hormone administration.

Insulin-like growth factor-I (IGF-I) is a known biomarker of recombinant human growth hormone (rhGH) abuse, and is also used clinically to confirm acromegaly. The protein leucine-rich alpha-2-glycoprotein (LRG) was recently identified as a putative biomarker of rhGH administration. The combination of an ACN depletion method and a 5-min ultra-high-performance liquid chromatography/tandem mass spectrometry (uHPLC/MS/MS)-based selected reaction monitoring (SRM) assay detected both IGF-I and LRG at endogenous concentrations. Four eight-point standard addition curves of IGF-I (16-2000 ng/mL) demonstrated good linearity (r(2) = 0.9991 and coefficients of variance (CVs) <13%). Serum samples from two rhGH administrations were extracted and their uHPLC/MS/MS-derived IGF-I concentrations correlated well against immunochemistry-derived values. Combining IGF-I and LRG data improved the separation of treated and placebo states compared with IGF-I alone, further strengthening the hypothesis that LRG is a biomarker of rhGH administration. Artificial neural networks (ANNs) analysis of the LRG and IGF-I data demonstrated an improved model over that developed using IGF-I alone, with a predictive accuracy of 97%, specificity of 96% and sensitivity of 100%. Receiver operator characteristic (ROC) analysis gave an AUC value of 0.98. This study demonstrates the first large scale and high throughput uHPLC/MS/MS-based quantitation of a medium abundance protein (IGF-I) in human serum. Furthermore, the data we have presented for the quantitative analysis of IGF-I suggest that, in this case, monitoring a single SRM transition to a trypsin peptide surrogate is a valid approach to protein quantitation by LC/MS/MS.

Therapeutic use of growth factors in the musculoskeletal system in sports-related injuries.

Growth factors (GFs) act as signalling agents for cells and become a more and more popular mean to influence the human body and its tissues. This review gives an overview of the current possibilities to use such agents in the field of sports related injuries and thus providing the athlete with a whole new potential to minimize recovery time. GFs and its application have been studied intensively for a long time starting with animal studies. For some of this GFs this research has been brought onto the next level to clinical phase trials. Agents such as insulin like growth factor 1 (IGF-1), mechano growth factor (MGF), basic fibroblast growth factor (B-FGF), platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor b (TGF-b), bone morphogenetic protein (BMP) and leukemia inhibitory factor (LIF) are being discussed in this review. These GFs not only have the potential to be used to cure injuries but also are being in the centre of interest for doping abusers and are a powerful yet not fully understood technique to gain performance.

Effects of eccentric cycling exercise on IGF-I splice variant expression in the muscles of young and elderly people.

Recovery from micro damage resulting from intensive exercise has been shown to take longer in older muscles. To investigate the factors that may contribute to muscle repair, we have studied the expression of two splice variants of the insulin-like growth factor-I (IGF-I) gene. IGF-IEa and mechano growth factor (MGF) were studied in response to 1 h of eccentric cycling exercise in young and old individuals. Subjects (nine young, aged 20-27 years and eight elderly, aged 67-75 years) completed an eccentric exercise protocol that consisted of 60 min of reverse pedal cycling. Workloads were chosen to give the same relative increases in oxygen uptake (VO2max) and heart rate in young and old subjects. Muscle biopsy samples were obtained from the quadriceps muscle before and 2 1/4 h after completion of the exercise bout and were analyzed for IGF-IEa and MGF mRNA levels using real-time quantitative PCR. No difference was observed between the baseline levels of the two splice variants between the two subject groups. Eccentric cycling exercise resulted in a significant increase in the mean MGF mRNA in both young and old subjects but did not alter IGF-IEa mRNA levels in either age group. As reported previously (Toft et al., 2002), the levels of serum creatine kinase and myoglobin, markers of muscle damage, were increased significantly from baseline and to 5 days after exercise in both young and old subjects. This supports previous research in suggesting that the MGF splice variant is sensitive to muscle damage-inducing exercise and is differentially regulated compared with IGF-IEa.

Expression of Ankrd2 in fast and slow muscles and its response to stretch are consistent with a role in slow muscle function.

In striated muscle, the structural genes associated with muscle fiber phenotype determination as well as muscle mass accretion are regulated largely by mechanical stimuli. Passive stretch of skeletal muscle stimulates muscle growth/hypertrophy and an increased expression of slow muscle genes. We previously identified Ankyrin repeat-domain protein (Ankrd2) as a novel transcript expressed in fast tibialis anterior muscles after 7 days of passive stretch immobilization in vivo. Here, we test the hypothesis that the expression of Ankrd2 in stretched fast muscle is associated with the stretch-induced expression of slow muscle phenotype rather than the hypertrophic response. Our results show that, in 4- and 7-day stretched tibialis anterior muscle, the expression of Ankrd2 mRNA and protein was significantly upregulated (P > 0.001). However, in fast muscles of kyphoscoliotic mutant mice, which lack the hypertrophic response to overload but have a slower muscle phenotype than wild-type, Ankrd2 expression was significantly upregulated. The distribution pattern of Ankrd2 in fast and slow muscle is also in accord with their slow fiber composition. Furthermore, it was markedly downregulated in denervated rat soleus muscle, which produces a pronounced shift toward the fast muscle phenotype. Using a sensitive proteomics approach (Ciphergen Technology), we observed that Ankrd2 protein was undetectable in soleus after 4 wk of denervation. We suggest that Ankrd2, which is also a titin binding protein, is a stretch-response gene associated with slow muscle function and that it is part of a separate mechanotransduction system to the one that regulates muscle mass.

Variable adaptation of molecular mechanisms in relation to the use of autologous striated muscle to augment myocardial function.

Significant recent advances in molecular biology and protein biochemistry have allowed the examination of muscle function at gene and protein level. This article reviews the variable adaptation of molecular mechanisms in striated muscle. The adaptation and transformation of fast skeletal muscle in response to chronic electrical stimulation may have considerable importance in relation to the use of autologous skeletal muscle to augment myocardial function.

Age and sex influence expression of plasmid DNA directly injected into mouse skeletal muscle.

Direct injection of plasmid DNA into the skeletal muscle has been proposed as a means of effecting somatic gene therapy. We examined the influence of age and sex on the level of expression of an SV40-CAT construct injected into mouse muscle. Age markedly affected expression, with peak values in the 4-6 week age class which were significantly higher than in animals older than 10 weeks. Sex also altered expression, with higher levels of CAT activity seen in males compared to females. We conclude that the rate of growth is important in determining levels of expression of directly injected DNA.

Age-related loss of skeletal muscle function and the inability to express the autocrine form of insulin-like growth factor-1 (MGF) in response to mechanical overload.

The response of insulin-like growth factor-1 (IGF-1) signalling and the capacity of skeletal muscle to adapt to mechanical overload was studied using synergistic muscle ablation. Overload of the plantaris and soleus resulted in marked hypertrophy and activation of satellite cells (as indicated by MyoD expression), particularly in young rats. Two muscle IGF-1 splice variants were measured and found to be differentially regulated at the RNA level. The significant changes associated with the inability of the older muscles to respond to mechanical overload included the considerably lower expression of the local splice variant mechano growth factor, and the failure to up-regulate IGF-1 receptor and MyoD mRNA.

Strong expression of foreign genes following direct injection into fish muscle.

We report here for the first time direct injection of genes into fish muscle in vivo. Plasmids used contain either SV40 early promoter, rabbit beta-cardiac myosin heavy chain promoter, human MxA promoter or an artificial promoter, fused to a chloramphenicol acetyltransferase (CAT) or beta-galactosidase reporter gene. CAT assays revealed that most gene constructs were highly expressed. Histochemical analysis showed that beta-galactosidase was strongly expressed at the site of injection within muscle fibres. This method provides an excellent system for testing expression of gene constructs, including those of mammalian origin, in fish muscle in vivo and has the potential for fish vaccination.

Age-related changes in collagen gene expression in the muscles of mdx dystrophic and normal mice.

It has been shown that there is a marked accumulation of collagen (notably type III) in the muscles of Duchenne muscular dystrophy patients. Although not as marked, there is also an accumulation of collagen in normal skeletal muscle with age. To attempt to determine whether dystrophy-related and/or age-related collagen accumulation are the result of increased collagen gene expression, sections from muscles of mdx and control mice at different ages were subjected to in situ hybridization with cRNA probes for procollagen I and III to estimate changes in specific mRNA levels. Greater amounts of collagen I and III mRNA were noted in skeletal muscles and the diaphragm of the mdx mice than in the same muscles of age matched controls. The over-expression of these collagen genes was particularly noticeable in the mdx diaphragm and may possibly be explained by the greater activity of this muscle as compared to the limb muscles of these dystrophic mice. There was, however, an age-related decline of collagen expression in both normal and dystrophic muscle and this strongly suggests that the increased fibrosis of skeletal muscle with age is not the result of increased collagen gene expression but is most likely due to an impairment of degradation. In contrast, the excess accumulation of collagen in dystrophic muscle compared to normal muscle is related to increased gene expression, probably triggered by an injured tissue response induced by muscle fibre damage due to the lack of dystrophin.

Age, diet and injury affect the survival of facial motoneurons.

Using the model of facial nerve avulsion, we have compared the effects of injury, age and diet on motoneuronal survival. One to four weeks after nerve avulsion, 50-75% motoneuron loss was quantified in ad libitum-fed rats aged 7 days (neonate), 6 months (adult) and 24 months (aging) at the time of injury. Evidence of apoptosis was found for neonatal rats at 3 days post-injury, but not for neonates examined 7 days or adult or aging rats examined 1 month after injury. Non-operated, ad libitum-fed rats showed no significant loss of facial motoneurons by 24 months. Surprisingly, non-operated rats whose food intake was restricted to 15 g standard rat chow per day from the age of 6 months lost 50% of their motoneurons by 24 months. Facial nerve avulsion of 24-month-old rats raised on this restricted diet did not result in any additional loss of motoneurons one month after injury. These results challenge the common view that aging results in neuronal loss and that dietary restriction is universally beneficial.

Using skeletal muscle as an artificial endocrine tissue.

Gene transfer into muscle tissue is currently being developed as a method for the production, secretion and delivery of therapeutic proteins. This methodology has been used to produce a variety of physiologically active proteins and may ultimately be applied to the treatment of several diseases. In this review, we consider several applications of this methodology and discuss approaches for modulating therapeutic protein production and secretion from muscle, using growth hormone as an example. In addition, factors limiting the effectiveness of muscle gene transfer are also discussed, as these shall determine the efficacy of muscle gene transfer when applied to humans.

Optimisation of growth hormone production by muscle cells using plasmid DNA.

The production of peptide hormones by skeletal muscle tissue is a promising area of gene therapy. Skeletal muscle myogenesis can be induced in vitro, resulting in the fusion of mononucleate myoblasts to form multinucleate myotubes, and delivery vectors are first tested in vitro. C2C12 myoblasts transfected with pcDNA3-GH, which used the human cytomegalovirus (CMV) promoter, secreted immunoreactive GH with comparable biological activity to pituitary GH. Mouse myeloid leukaemia cells, which express the mouse GH receptor were used for the bioassay, and activation of these cells by GH was measured by a colorimetric microculture tetrazolium assay. Cells were incubated with a tetrazolium salt (MTS) and an intermediate electron acceptor (phenazine methosulphate, PMS), and formazan production was measured as optical density (O.D.) at 490 nm. The efficiencies of several plasmid expression vectors were compared in differentiated and non-differentiated muscle cells, as a function of bioactive GH secreted by the transfected cells. Ten-day differentiated C2C12 myotubes transfected with pcDNA3E-GH, which used the CMV promoter and a rat myosin light chain enhancer element, secreted significantly more biologically active GH than myotubes transfected with pcDNA3-GH (0.82 O.D. units+/-0.06 vs 0.57+/-0.05 respectively, P<0.001). This was consistent with reduced CMV promoter activity in myotubes. Myoblasts transfected with pcDNA3-GH secreted more bioactive GH than 10-day transfected myotubes (1.1+/-0. 1 vs 0.77+/-0.07 respectively). However, the responses were indistinguishable (both 1.0+/-0.09) if both the myotubes and myoblasts had been transfected with pcDNA3E-GH. Substitution of the vector pMHLC-GH, which used a muscle-specific truncated rabbit myosin heavy chain promoter, and the myosin enhancer resulted in a marked decrease in the responses to the conditioned medium from fused myotubes compared with the vectors pcDNA3-GH and pcDNA3E-GH (0. 24+/-0.02 vs 0.57+/-0.05 vs 0.82+/-0.06 respectively). We concluded that the combination of CMV promoter and myosin light chain enhancer in pcDNA3E-GH had the greatest expression efficiency of the several plasmid vectors which we investigated.

Aortic counterpulsation for up to 28 days with autologous latissimus dorsi in sheep.

This article reports the development and assessment of an entirely autologous extraaortic counterpulsation system using skeletal muscle (latissimus dorsi). The technique has been performed and assessed in 16 sheep to quantify the effectiveness of counterpulsation over periods up to 28 days and to optimize the stimulation regimens for muscle contraction and fiber-type transformation. Hemodynamic changes have been quantified by calculation of the endocardial viability ratio. This has shown an increase of between 12% and 89% for 28 days. The wide variety of increase observed has been related to the development of an optimum flap configuration. The technique of surface impedance monitoring of flap blood flow has allowed the start of electrical stimulation after 48 hours with the introduction of hemodynamic benefit (1:4 mode) during the process of fiber-type transformation (in situ training). Extraaortic counterpulsation with autologous latissimus dorsi has been shown to be effective and safe for as long as 28 days. It has not been associated with any thromboembolic or infective complications, which we attribute to the exclusion of any foreign material in the design.

Effect of inactivity and passive stretch on protein turnover in phasic and postural rat muscles.

A state of hypokinesia and hypodynamia has been induced in the hindlimb muscles of the rat (100 g) using a suspension model. The ensuing muscle atrophy was assessed by reference to muscles in fully mobile control animals, which were either fed ad libitum or fed the same lower food intake of the suspended animals. Over a total of 7 days of suspension the slow-twitch postural soleus muscle underwent a much greater atrophy than the fast-twitch phasic extensor digitorum longus. Changes with respect to the position of the suspended foot, and hence muscle length, necessitate caution in comparing the extent of the atrophy between different muscle types. After 3 days of inactivity the atrophy of the soleus muscle was explained by a 21% decrease in the fractional rate of synthesis (measured in vivo) and a 100% increase in the rate of protein breakdown. The reduction in the synthetic rate was associated with a net loss (23%) of RNA and hence muscle ribosomes. In contrast when this inactive soleus muscle was permanently stretched the RNA content (44%) and protein synthetic rate increased (59%) markedly above control values. Although protein breakdown remained elevated in this stretched muscle, the extent of the atrophy in response to hypokinesia and hypodynamia was greatly reduced.

Biochemical and structural adaptation of autologous skeletal muscle used for counterpulsation.

We have studied in a normal animal model (sheep), the biochemical and morphological adaptation of electrically stimulated skeletal muscle used for extra aortic counterpulsation. Immunocytochemical analysis of latissimus dorsi, using monoclonal antibodies to slow and fast myosin heavy chains, indicated an increase in the population of mixed fibres after stimulation for one week. By one month, up to 70% of fibres expressed both slow and fast myosin heavy chains in addition to the 15% of fibres expressing only slow myosin heavy chains. After 4 months, the population of mixed fibres was further transformed towards purely slow fibres to give values of 40 and 67% of fibres expressing only slow myosin heavy chain at 4 and 6 months, respectively. Increased staining, both in intensity and area, for NADH tetrazolium reductase activity (an enzyme of the oxidative metabolic pathway) was detected by 28 days. An increase in mitochondrial number was observed also by 28 days, further indicating a shift towards an oxidative metabolism. The molecular adaptation of latissimus dorsi was achieved by stimulation every fourth cardiac cycle at 35 Hz, 3 V, initiated 48 hours after the operation; this being a marked reduction in the delay from operation prior to stimulation. Evaluation of other regimes indicated that more frequent modes, or an increase in voltage or frequency, caused damage to the muscle during the early phase of molecular adaptation. A thorough understanding of the time sequence of the different adaptive processes is required to determine the ideal regime of stimulation initiated promptly after mobilisation of the muscle; aimed at harvesting the maximum amount of energy from the autologous muscle.

Postprandial resynthesis of myofibrillar proteins is translationally rather than transcriptionally regulated in human skeletal muscle.

Feeding stimulates protein synthesis in skeletal muscles, although the regulatory mechanisms are incompletely understood. The aim of this study was to determine whether this could be detected at the gene transcription level for postprandial stimulation of the synthesis of muscle proteins. Healthy male volunteers were investigated after an overnight fast. Open muscle biopsies were performed in the starved state and 3 h after meal intake, consisting of 0.15 gN/kg, 12 kcal/kg. Blood samples were drawn every 15 to 30 min for 5 h. Myosin mRNA and insulin growth factor-I (IGF-I) mRNA were measured by solution hybridization assay in homogenized muscle specimens. After food intake, plasma glucose concentrations increased from 5.0 +/- 0.1 to 7.3 +/- 0.3 (P < or = 0.001), and insulin concentration rose from 3.8 +/- 0.5 mU/L before to 75.3 +/- 11.4 15 min after the meal (P < or = 0.001). Plasma concentration of free fatty acids declined after food intake (P < or = 0.001). Plasma concentrations of amino acids increased from basal values (2864 +/- 128 microM) to 4419 +/- 262 microM (P < or = 0.05) 90 min after meal ingestion. Myosin mRNA concentration in the biopsied muscle tissue was higher during starvation and was reduced by 20% after food intake: 10.8 +/- 1.3 amol mRNA/microgram DNA in the starved state and 8.5 +/- 1.3 amol mRNA/microgram DNA after food intake (P < or = 0.05). Feeding did not alter IGF-I mRNA concentrations in muscle: 0.51 +/- 0.05 and 0.55 +/- 0.06 amol/microgram DNA in the starved and fed state, respectively (P < or = 0.48). Improved protein balance by stimulation of protein synthesis has been related to increased plasma amino acids. Interestingly, in the short term, this was not related to increases in gene transcription of either myofibrillar proteins (myosin) or muscle IGF-I. Thus, postprandial stimulation of protein synthesis appears not to be regulated by increased gene transcription but by increased translation using the increased concentrations of amino acids. In contrast, as far as the 2X myosin mRNA level is concerned, this is enhanced during starvation, which facilitates rapid recovery once the availability of substrate is resumed.

Age-related loss of skeletal muscle function; impairment of gene expression.

Mechano Growth Factor (MGF) is derived from the insulin-like growth factor (IGF-I) but its sequence differs from the systemic IGF-I produced by the liver. MGF is expressed by mechanically overloaded muscle and is involved in tissue repair and adaptation. It is expressed as a pulse following muscle damage and involved in the activation of muscle satellite (stem) cells. These donate nuclei to the muscle fibers that are required for repair and for the hypertrophy processes which may have similar regulatory mechanisms. Muscles in the elderly are unable to upregulate MGF in response to exercise. This is also true in certain diseases and this helps to explain muscle loss in those conditions. There is evidence that MGF is a local tissue repair factor as well as a growth factor and that it has an important role in damage limitation and inducing repair in other post-mitotic tissues. As there is no cell replacement in these tissues there has to be an effective local cellular repair mechanism. With advancing years this seems to become deficient and there is an increased chance that the damaged cells will undergo cell death leading to progressive loss of tissue function.

Selective gene expression during adaptation of muscle in response to different physiological demands.

Muscle is a very adaptable tissue in which gene expression is to a large extent influenced by physical signals. Adaptation to a different work regime is brought about by changes in fibre type and fibre cross-sectional area. We have shown both mass and phenotype are markedly altered by stretch and force production within a period as short as 4 days. This is associated with quantitative as well as qualitative changes in gene expression. The latter involves the expression of myosin heavy chain isogenes which encode different types of molecular motors. Some species of fish have exploited this and they are able to rebuild their myofibrillar systems for warm and cold temperature swimming by selective myosin gene expression. To understand how the different myosin isoform confer different contractile properties methods have been developed for cloning, sequencing and visualizing the structure of the ATPase site to explain how the molecular motors are designed. With regard to the chemical link between the physical signal and the upregulation of certain muscle genes we have cloned a new growth factor that is only expressed in muscles subjected to stretch and/or exercise and which is designed for autocrine/paracrine action. Experiments indicate that the expression of a local growth factor which induces repair, remodelling and hypertrophy is one of the ways cells respond to mechanical strain.

Characterisation of red and white muscle myosin heavy chain gene coding sequences from antarctic and tropical fish.

To understand molecular adaptation for locomotion at different environmental temperatures, we have studied the myosin heavy chain genes as these encode the molecular motors involved. For this purpose, cDNA libraries from white (fast) and red (slow) myotomal muscle of an Antarctic and a tropical fish were constructed and from these different myosin heavy chain cDNAs were isolated. Northern and in situ hybridisation confirmed in which type of muscle these isoform genes are expressed. The cDNAs were sequenced and the structure of the ATPase sites compared. There was a marked similarity between the tropical fast myosin and the Antarctic slow myosin in the loop 1 region, which has similar amino acid side chains, charge distribution and conformation. These findings help to explain why the myofibrils isolated from white muscle of tropical fish show a lower specific ATPase activity than the white muscle of Antarctic fish but a similar activity to the Antarctic red (slow) muscle. It also provides insight into the way molecular motors in Antarctic fish have evolved to produce more power and thus ensure effective swimming at near zero temperatures by the substitution or addition of a few residues in strategic regions, which include the ATPase site.