Muscle injuries and repair: the role of prostaglandins and inflammation

Skeletal muscle injuries are a common problem in trauma and orthopaedic surgery. Muscle injuries undergo the healing phases of degeneration, inflammation, regeneration, and fibrosis. Current and experimental therapies to improve muscle regeneration and limit muscle fibrosis include conservative and surgical principles with the adjuvant use of non-steroidal anti-inflammatory drugs (NSAIDs) and growth factor manipulation. NSAIDs appear to have a paradoxical effect on the healing of muscle injuries with early signs of improvement and subsequent late impairment in functional capacity and histology. In vitro and in vivo studies have explored the role of the cyclooxygenases and prostaglandins in the biological processes of healing muscle, including precursor cell activation, myoblast proliferation, myoblast fusion, and muscle protein synthesis. Through use of more specific cyclooxygenase inhibitors, we may be able to better understand the role of inflammation in muscle healing.     

Capillary density and capillary-to-fibre ratio in vastus lateralis muscle of untrained and trained men

Muscle fibre profile area (Af), volume density (Vv), capillary-to-fibre ratio (CF) and number of capillaries per fibre square millimetre (CD) were determined from needle biopsies of vastus lateralis of twenty-four male volunteers (mean +/- SD: age 25.4+/-5.8 years, height 178.6+/-5.5 cm, body mass 72.1+/-7.7 kg) of different training background. Seven subjects were untrained students (group A), nine were national and sub-national level endurance athletes (group B) with the background of 7.8+/-2.9 years of specialised training, and eight subjects were sprint-power athletes (group C) with 12.8+/-8.7 years of specialised training. Muscle biopsies of vastus lateralis were analysed histochemically for mATPase. Capillaries were visualized and counted using CD31 antibodies against endothelial cells. There were significant differences in the Vv of type I and type II muscle fibres in both trained groups, B (51.8%; 25.6%) and C (50.5%; 26.4%). However, in untrained group A that was treated as a reference group, the difference between Vv of type I and type II fibres was less prominent, nevertheless statistically significant (42.1%; 35.1%). There was also a significant difference in CF: 1.9 in group A and 2.1 in groups B and C. The number of capillaries per mm2 (CD) was 245 (group A), 308 (group B) and 325 (group C). Significant differences (P<0.05) in CF and CD, were found only between group A (1.9; 245) and both groups of trained men, B and C (2.1; 308 and 325). However, endurance athletes (group B), such as long-distance runners, cyclists and cross country skiers, did not differ from the athletes representing short term, high power output sports (group C) such as ice hockey, karate, ski-jumping, volleyball, soccer and modern dance.     

Endurance training does not alter the level of neuronal nitric oxide synthase in human skeletal muscle.

The effect of endurance training on neuronal nitric oxide synthase (nNOS) content and distribution in muscle was investigated. Seven male subjects performed 6 wk of one-legged knee-extensor endurance training (protocol A). Muscle biopsies, obtained from vastus lateralis muscle in the untrained and the trained leg, were analyzed for nNOS protein and activity as well as immunohistochemical distribution of nNOS and endothelial nitric oxide synthase (eNOS). Muscle biopsies were also obtained from another seven male subjects before and after 6 wk of training by endurance running (protocol B) and analyzed for nNOS protein. No difference was found in the amount of nNOS protein in the untrained and the trained muscle either with protocol A or protocol B (P > 0.05). In protocol A, the activity of nNOS was 4.76 +/- 0.56 pmol. mg protein(-1). min(-1) in the control leg, and the level was not different in the trained leg (P > 0.05). nNOS was present in the sarcolemma and cytosol of type I and type II muscle fibers, and the qualitative distribution was similar in untrained and trained muscle. The number of eNOS immunoreactive structures and the number of capillaries per muscle fiber were higher (P < 0.05) after training than before. The present findings demonstrate that, in contrast to findings on animals, nNOS levels remain unaltered with endurance training in humans. Evidence is also provided that endurance training may increase the amount of eNOS, in parallel with an increase in capillaries in human muscle.     

A new method for non-invasive estimation of human muscle fiber type composition

Background

It has been established that excellence in sports with short and long exercise duration requires a high proportion of fast-twitch (FT) or type-II fibers and slow-twitch (ST) or type-I fibers, respectively. Until today, the muscle biopsy method is still accepted as gold standard to measure muscle fiber type composition. Because of its invasive nature and high sampling variance, it would be useful to develop a non-invasive alternative.

Methodology

Eighty-three control subjects, 15 talented young track-and-field athletes, 51 elite athletes and 14 ex-athletes volunteered to participate in the current study. The carnosine content of all 163 subjects was measured in the gastrocnemius muscle by proton magnetic resonance spectroscopy (¹H-MRS). Muscle biopsies for fiber typing were taken from 12 untrained males.

Principal Findings

A significant positive correlation was found between muscle carnosine, measured by ¹H-MRS, and percentage area occupied by type II fibers. Explosive athletes had ∼30% higher carnosine levels compared to a reference population, whereas it was ∼20% lower than normal in typical endurance athletes. Similar results were found in young talents and ex-athletes. When active elite runners were ranked according to their best running distance, a negative sigmoidal curve was found between logarithm of running distance and muscle carnosine.

Conclusions

Muscle carnosine content shows a good reflection of the disciplines of elite track-and-field athletes and is able to distinguish between individual track running distances. The differences between endurance and sprint muscle types is also observed in young talents and former athletes, suggesting this characteristic is genetically determined and can be applied in early talent identification. This quick method provides a valid alternative for the muscle biopsy method. In addition, this technique may also contribute to the diagnosis and monitoring of many conditions and diseases that are characterized by an altered muscle fiber type composition.     

Skeletal muscle enzymes and fiber composition in male and female track athletes.

Muscle samples were obtained from the gastrocnemius of 17 female and 23 male track athletes, 10 untrained women, and 11 untrained men. Portions of the specimen were analyzed for total phosphorylase, lactic dehydrogenase (LDH), and succinate dehydrogenase (SDH) activities. Sections of the muscle were stained for myosin adenosine triphosphatase, NADH2 tetrazolium reductase, and alpha-glycerophosphate dehydrogenase. Maximal oxygen uptake (VO2max) was measured on a treadmill for 23 of the volunteers (6 female athletes, 11 male athletes, 10 untrained women, and 6 untrained men). These measurements confirm earlier reports which suggest that the athlete's preference for strength, speed, and/or endurance events is in part a matter of genetic endowment. Aside from differences in fiber composition and enzymes among middle-distance runners, the only distinction between the sexes was the larger fiber areas of the male athletes. SDH activity was found to correlate 0.79 with VO2max, while muscle LDH appeared to be a function of muscle fiber composition. While sprint- and endurance-trained athletes are characterized by distinct fiber compositions and enzyme activities, participants in strength events (e.g., shot-put) have relatively low muscle enzyme activities and a variety of fiber compositions.     

Circulating plasma VEGF response to exercise in sedentary and endurance-trained men.

The skeletal muscle capillary supply is an important determinant of maximum exercise capacity, and it is well known that endurance exercise training increases the muscle capillary supply. The muscle capillary supply and exercise-induced angiogenesis are regulated in part by vascular endothelial growth factor (VEGF). VEGF is produced by skeletal muscle cells and can be secreted into the circulation. We investigated whether there are differences in circulating plasma VEGF between sedentary individuals (Sed) and well-trained endurance athletes (ET) at rest or in response to acute exercise. Eight ET men (maximal oxygen consumption: 63.8 +/- 2.3 ml x kg(-1) x min(-1); maximum power output: 409.4 +/- 13.3 W) and eight Sed men (maximal oxygen consumption: 36.3 +/- 2.1 ml x kg(-1) x min(-1); maximum power output: 234.4 +/- 13.3 W) exercised for 1 h at 50% of maximum power output. Antecubital vein plasma was collected at rest and at 0, 2, and 4 h postexercise. Plasma VEGF was measured by ELISA analysis. Acute exercise significantly increased VEGF at 0 and 2 h postexercise in ET subjects but did not increase VEGF at any time point in Sed individuals. There was no difference in VEGF between ET and Sed subjects at any time point. When individual peak postexercise VEGF was analyzed, exercise did increase VEGF independent of training status. In conclusion, exercise can increase plasma VEGF in both ET athletes and Sed men; however, there is considerable variation in the individual time of the peak VEGF response.     

Satellite cell proliferation and differentiation during postnatal growth of porcine skeletal muscle

Age-related changes in satellitecell proliferation and differentiation during rapid growth of porcineskeletal muscle were examined. Satellite cells were isolated fromhindlimb muscles of pigs at 1, 7, 14, and 21 wk of age (4 animals/agegroup). Satellite cells were separated from cellular debris by usingPercoll gradient centrifugation and were adsorbed to glass coverslipsfor fluorescent immunostaining. Positive staining for neural celladhesion molecule (NCAM) distinguished satellite cells from nonmyogeniccells. The proportion of NCAM-positive cells (satellite cells) inisolates decreased from 1 to 7 wk of age. Greater than 77% ofNCAM-positive cells were proliferating cell nuclear antigen positive atall ages studied. Myogenin-positive satellite cells decreased from 30%at 1 wk to 14% at 7 wk of age and remained at constant levels thereafter. These data indicate that a high percentage of satellite cells remain proliferative during rapid postnatal muscle growth. Thereduced proportion of myogenin-positive cells during growth may reflecta decrease in the proportion of differentiating satellite cells oraccelerated incorporation of myogenin-positive cells into myofibers.

     

Carnosine content of the middle gluteal muscle in thoroughbred horses with relation to age, sex and training

1. Muscle biopsies were collected from 85 thoroughbred horses and analysed for carnosine content by an automated HPLC method. 2. No significant sex difference was found between colts, geldings and fillies. 3. There was a trend towards lower muscle carnosine contents with age, which was only significant between 1-year-old untrained horses and 4+ year-old horses (P less than 0.002).     

Regulation and activity-dependence of N-cadherin, NCAM isoforms, and polysialic acid on chick myotubes during development

Muscle development in vivo involves a complex sequence of cell-cell interactions in which secondary myotubes first form in association with primary myotubes and subsequently separate from them. We show here that during this process N-cadherin and the different structural forms of NCAM are regulated in a pattern that involves both temporal changes in expression and localization to particular regions of the muscle cell surface. In particular, levels of N-cadherin on maturing myotubes are decreased, and the form of NCAM synthesized by the muscle changes from a transmembrane non-polysialylated to a lipid-linked polysialylated membrane protein. Moreover, while NCAM was distributed on all myotube surfaces, the polysialyated form of NCAM was restricted to regions of the myotube surface that had recently separated from neighboring cells. We previously found that blockade of nerve-induced activity by d- Tubocurarine perturbed muscle cell interactions, resulting in a failure of myotubes to separate. We now show that this activity blockade also alters adhesion molecule expression. First, N-cadherin was no longer down-regulated in maturing myotubes, and its persistence on the surfaces of mature myotubes may partly explain their failure to separate. Secondly, the developmental switch from transmembrane to lipid-linked NCAM did not occur, and polysialylated NCAM was no longer formed. As the unusual physical properties of PSA have been proposed to impede cell-cell interactions, this alteration would also be expected to compromise cell separation. Together, these results suggest that the regulated expression of both N-cadherin and NCAM isoforms including their polysialylation, is an essential mechanism for the normal separation of secondary myotubes from primary myotubes.     

NSAIDs Modulate Clonal Evolution in Barrett's Esophagus

Cancer is considered an outcome of decades-long clonal evolution fueled by acquisition of somatic genomic abnormalities (SGAs). Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to reduce cancer risk, including risk of progression from Barrett''s esophagus (BE) to esophageal adenocarcinoma (EA). However, the cancer chemopreventive mechanisms of NSAIDs are not fully understood. We hypothesized that NSAIDs modulate clonal evolution by reducing SGA acquisition rate. We evaluated thirteen individuals with BE. Eleven had not used NSAIDs for 6.2±3.5 (mean±standard deviation) years and then began using NSAIDs for 5.6±2.7 years, whereas two had used NSAIDs for 3.3±1.4 years and then discontinued use for 7.9±0.7 years. 161 BE biopsies, collected at 5–8 time points over 6.4–19 years, were analyzed using 1Million-SNP arrays to detect SGAs. Even in the earliest biopsies there were many SGAs (284±246 in 10/13 and 1442±560 in 3/13 individuals) and in most individuals the number of SGAs changed little over time, with both increases and decreases in SGAs detected. The estimated SGA rate was 7.8 per genome per year (95% support interval [SI], 7.1–8.6) off-NSAIDs and 0.6 (95% SI 0.3–1.5) on-NSAIDs. Twelve individuals did not progress to EA. In ten we detected 279±86 SGAs affecting 53±30 Mb of the genome per biopsy per time point and in two we detected 1,463±375 SGAs affecting 180±100 Mb. In one individual who progressed to EA we detected a clone having 2,291±78 SGAs affecting 588±18 Mb of the genome at three time points in the last three of 11.4 years of follow-up. NSAIDs were associated with reduced rate of acquisition of SGAs in eleven of thirteen individuals. Barrett''s cells maintained relative equilibrium level of SGAs over time with occasional punctuations by expansion of clones having massive amount of SGAs.     

Exercise adaptation attenuates VEGF gene expression in human skeletal muscle

Angiogenesis is a component of the multifactoral adaptation to exercise training, and vascular endothelial growth factor (VEGF) is involved in extracellular matrix changes and endothelial cell proliferation. However, there is limited evidence supporting the role of VEGF in the exercise training response. Thus we studied mRNA levels of VEGF, using quantitative Northern analysis, in untrained and trained human skeletal muscle at rest and after a single bout of exercise. Single leg knee-extension provided the acute exercise stimulus and the training modality. Four biopsies were collected from the vastus lateralis muscle at rest in the untrained and trained conditions before and after exercise. Training resulted in a 35% increase in muscle oxygen consumption and an 18% increase in number of capillaries per muscle fiber. At rest, VEGF/18S mRNA levels were similar before (0.38 +/- 0.04) and after (1.2 +/- 0.4) training. When muscle was untrained, acute exercise greatly elevated VEGF/18S mRNA levels (16.9 +/- 6.7). The VEGF/18S mRNA response to acute exercise in the trained state was markedly attenuated (5.4 +/- 1.3). These data support the concept that VEGF is involved in exercise-induced skeletal muscle angiogenesis and appears to be subject to a negative feedback mechanism as exercise adaptations occur.     

Skeletal muscle properties and performance in elite female track athletes

Selected biochemical and physiological properties of skeletal muscle were studied in light of performance capabilities in 24 elite female track athletes. The feasibility of quantifying end point histochemistry and relating oxidative staining density (reduced nicotinomide adenine dinucleotide diaphorase: NADH-D) to whole body maximal oxygen consumption (VO2 max) was also investigated, while muscle fiber types, classified according to alkaline APTase stains, were studied and related to muscle oxidative capacity (succinate dehydrogenase: SDH), VO2 max and "in vivo" torque-velocity properties. Muscle biopsies were taken from the vastus lateralis of each subject and maximal knee extensor torques were recorded at 30 degrees from full extension at four selected velocities. While results confirm earlier reports on skeletal muscle properties and performance it was concluded that end point histochemistry could be reliably quantified and that an "oxidative" stain such as NADH-D correlates extremely well with VO2 max (r = 0.86, p less than 0.001) whereas correlations between % slow twitch fibres (Alkaline ATPase stain) and VO2 max were lower (r = 0.44, p less than 0.05). Additionally, as knee extension velocity increased from 0-1.7 rad x s-1 angle specific extensor torque production did not decline as observed in vitro and pentathletes displayed significantly larger torques at all velocities when compared to the other athletes. These data confirm that while myofibrillar ATPase staining correlates with force-velocity properties of muscle, VO2 max is better correlated with quantified oxidative staining.     

Cyclooxygenase-dependent signalling: molecular events and consequences

Non-steroidal anti-inflammatory drugs (NSAIDs) currently attract large interest. Next to pain relief, NSAIDs have important anti-thrombotic and anti-oncogenic effects. NSAIDs exert their action by inhibition of cyclooxygenase, the enzyme responsible for the production of prostanoids. Prostanoid signal transduction is still poorly understood, but it has become clear that these inflammatory lipids influence cellular physiology at three different levels: (1) activation of a 7 x transmembrane receptor coupled to heterotrimeric G proteins, (2) the inhibition of inflammation by activating corticosteroid-like receptors, (3) participation in receptor protein tyrosine kinase signal transduction. In this review prostanoid signalling at these three different levels will be reviewed and the relevance in (patho)physiological processes will be evaluated.     

NSAIDs counteract H. pylori VacA toxin-induced cell vacuolation in MKN 28 gastric mucosal cells

The relationship between nonsteroidal anti-inflammatory drugs (NSAIDs) and Helicobacter pylori-induced gastric mucosal injury is still under debate. VacA toxin is an important H. pylori virulence factor that causes cytoplasmic vacuolation in cultured cells. Whether and how NSAIDs affect VacA-induced cytotoxicity is unclear. This study was designed to evaluate the effect of NSAIDs on H. pylori VacA toxin-induced cell vacuolation in human gastric mucosal cells in culture (MKN 28 cell line). Our data show that 1) NSAIDs (indomethacin, aspirin, and NS-398) inhibit VacA-induced cell vacuolation independently of inhibition of cell proliferation and prostaglandin synthesis; 2) NSAIDs impair vacuole development/maintenance without affecting cell binding and internalization of VacA; and 3) NSAIDs, as well as the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid, also inhibit cell vacuolation induced by ammonia. We thus hypothesize that NSAIDs might protect MKN 28 cells against VacA-induced cytotoxicity by inhibiting VacA channel activity required for vacuole genesis.     

Skeletal muscle nNOS mu protein content is increased by exercise training in humans

The major isoform of nitric oxide synthase (NOS) in skeletal muscle is the splice variant of neuronal NOS, termed nNOS mu. Exercise training increases nNOS mu protein levels in rat skeletal muscle, but data in humans are conflicting. We performed two studies to determine 1) whether resting nNOS mu protein expression is greater in skeletal muscle of 10 endurance-trained athletes compared with 11 sedentary individuals (study 1) and 2) whether intense short-term (10 days) exercise training increases resting nNOS mu protein (within whole muscle and also within types I, IIa, and IIx fibers) in eight sedentary individuals (study 2). In study 1, nNOS mu protein was approximately 60% higher (P < 0.05) in endurance-trained athletes compared with the sedentary participants. In study 2, nNOS mu protein expression was similar in types I, IIa, and IIx fibers before training. Ten days of intense exercise training significantly (P < 0.05) increased nNOS mu protein levels in types I, IIa, and IIx fibers, a finding that was validated by using whole muscle samples. Endothelial NOS and inducible NOS protein were barely detectable in the skeletal muscle samples. In conclusion, nNOS mu protein expression is greater in endurance-trained individuals when compared with sedentary individuals. Ten days of intense exercise is also sufficient to increase nNOS mu expression in untrained individuals, due to uniform increases of nNOS mu within types I, IIa, and IIx fibers.     

Muscle satellite cells

Skeletal muscle satellite cells are quiescent mononucleated myogenic cells, located between the sarcolemma and basement membrane of terminally-differentiated muscle fibres. These are normally quiescent in adult muscle, but act as a reserve population of cells, able to proliferate in response to injury and give rise to regenerated muscle and to more satellite cells. The recent discovery of a number of markers expressed by satellite cells has provided evidence that satellite cells, which had long been presumed to be a homogeneous population of muscle stem cells, may not be equivalent. It is possible that a sub-population of satellite cells may be derived from a more primitive stem cell. Satellite cell-derived muscle precursor cells may be used to repair and regenerate damaged or myopathic skeletal muscle, or to act as vectors for gene therapy. CELL FACTS: (1) Number of cells in body: 2 x 10(7) to 3 x 10(7) myonuclei/g, 20-25 kg muscle in average man; 2 x 10(5) to 10 x 10(5) satellite cells/g, i.e. approximately 1 x 10(10) to 2 x 10(10) satellite cells per person. (2) Main functions: repair and maintenance of skeletal muscle. (3) Turnover rate: close to zero in non-traumatic conditions-high in disease or severe trauma.