Type 2X Fibers Research Articles

Skeletal Muscle Fiber Type Composition and Citrate Synthase Activity in Fit and Unfit Warmbloods and Quarter Horses

Selective breeding and discipline specific training has led to equine breeds adept at various athletic disciplines. Breed-specific skeletal muscle adaptations have been studied in many breeds but not Warmbloods (WB). We evaluated gluteal muscle contractile muscle fiber types and citrate synthase activity (CS), a marker for mitochondrial volume density, in WB trained for dressage (second level-Grand Prix) contrasted with Quarter Horses (QH). Gluteus medius muscle biopsies from 14 unfit/18 fit dressage-trained WB and 20 unfit/16 fit reining/working cow QH were analyzed fluorometrically and fiber types determined by ATPase activity. Comparisons were made by one-way ANOVA. Unfit and fit WB had significantly higher % type 1 and lower % type 2X fibers than QH. Unfit WB had significantly higher CS than unfit QH but CS did not differ between fit WB and fit QH. CS was only significantly higher in fit versus unfit QH, not fit versus unfit WB. In conclusion, WB gluteal muscle has an inherently high % type 1/low % type 2X fibers and high mitochondrial content whether unfit or trained for dressage, contrasting QH with an inherently low % type 1/high % type 2X and low mitochondrial content, that was enhanced in fit QH. Similar CS activity in fit WB versus QH despite a two-fold difference in % type 2X fibers indicates that mitochondrial volume density cannot accurately be predicted from contractile fiber type composition.

Protein profile of fiber types in human skeletal muscle: a single-fiber proteomics study

BackgroundHuman skeletal muscle is composed of three major fiber types, referred to as type 1, 2A, and 2X fibers. This heterogeneous cellular composition complicates the interpretation of studies based on whole skeletal muscle lysate. A single-fiber proteomics approach is required to obtain a fiber-type resolved quantitative information on skeletal muscle pathophysiology.MethodsSingle fibers were dissected from vastus lateralis muscle biopsies of young adult males and processed for mass spectrometry-based single-fiber proteomics. We provide and analyze a resource dataset based on relatively pure fibers, containing at least 80% of either MYH7 (marker of slow type 1 fibers), MYH2 (marker of fast 2A fibers), or MYH1 (marker of fast 2X fibers).ResultsIn a dataset of more than 3800 proteins detected by single-fiber proteomics, we selected 404 proteins showing a statistically significant difference among fiber types. We identified numerous type 1 or 2X fiber type–specific protein markers, defined as proteins present at 3-fold or higher levels in these compared to other fiber types. In contrast, we could detect only two 2A-specific protein markers in addition to MYH2. We observed three other major patterns: proteins showing a differential distribution according to the sequence 1 > 2A > 2X or 2X > 2A > 1 and type 2–specific proteins expressed in 2A and 2X fibers at levels 3 times greater than in type 1 fibers. In addition to precisely quantifying known fiber type–specific protein patterns, our study revealed several novel features of fiber type specificity, including the selective enrichment of components of the dystrophin and integrin complexes, as well as microtubular proteins, in type 2X fibers. The fiber type–specific distribution of some selected proteins revealed by proteomics was validated by immunofluorescence analyses with specific antibodies.ConclusionWe here show that numerous muscle proteins, including proteins whose function is unknown, are selectively enriched in specific fiber types, pointing to potential implications in muscle pathophysiology. This reinforces the notion that single-fiber proteomics, together with recently developed approaches to single-cell proteomics, will be instrumental to explore and quantify muscle cell heterogeneity.

Characterisation of flexor digitorum profundus, flexor digitorum superficialis and extensor digitorum communis by electrophoresis and immunohistochemical analysis of myosin heavy chain isoforms in older men

IntroductionFew data exist on the fiber type composition of the extrinsic finger muscles. The aim of the present study was to describe myosin heavy chain (MyHC) composition of flexor digitorum profundus (FDP), flexor digitorum superficialis (FDS) and extensor digitorum communis (EDC). MyHC composition is relevant for whole muscle contractile performance and several studies on single muscle fibers demonstrated that fibers expressing only slow MyHC-1 develop less specific force than fibers expressing fast MyHCs. Since contraction force of finger extensors is smaller than of finger flexors a hypothesis was posited that the content of MyHC-1 is higher in EDC than in extrinsic finger flexors. MethodsAutopsy samples of FDP, FDS, and EDC in 27 healthy older men were analyzed and compared with each other and with biceps brachii (BB). MyHC isoforms were quantified on silver-stained 6% SDS-PAGE. Muscle fibers were classified immunohistochemically according to the expression of adult MyHC isoforms and their morphometric parameters were determined. ResultsEDC stood out for its higher proportion of slow MyHC-1 (50%) compared to FDP (37%), FDS (38%) and BB (35%) (p<0.001 in all), and its lower proportion of fast MyHC-2x (13%) compared to FDP (26%, p=0.001), FDS (22%, p=0.028) and BB (29%, p<0.001) detected on SDS-PAGE. Immunohistochemically, EDC had a higher area proportion of pure slow type-1 fibers (63%) than FDP (47%, p=0.002), FDS (49%, p=0.007) and BB (47%, p=0.002), and lower area proportion of pure fast type-2x fibers (2%) than FDP (12%, p=0.014), FDS (8%, p=0.256) and BB (14%, p=0.002). All muscles contained a similar area proportion of pure type-2a fibers and hybrid type-2a/2x fibers. ConclusionsThe study results support the hypothesis that the content of MyHC-1 is higher in EDC than in extrinsic finger flexors, which is in agreement with the lower contraction force of finger extensors compared to finger flexors.

Changes in local capillarity of pure and hybrid MyHC muscle fiber types after nerve injury in rat extensor digitorum longus muscle (EDL).

Recently, we evaluated capillary indices without discrimination by fiber type in rat extensor digitorum longus muscle (EDL) 4weeks after nerve cut (NC), after double nerve crush (double NCR) and in two controls, from the start (CON-1) and the end (CON-2) of the experiment. In the present study, we determined the capillary indices related to specific myosin heavy chain (MyHC) fiber types. Fiber-type composition and local capillarity were assessed from a single, composite, multicolor image, where different MyHC-fiber types and capillaries were shown simultaneously. Applying local capillary indices [the number of capillaries around fiber (CAF) and the CAF scaled to fiber perimeter (CAF/FP)], to specific MyHC-fiber types, we found changes relevant to neuro-muscular studies. In the NC group, only type-2x fibers had a significantly lower CAF, and in the double NCR group, only type-2a fibers had a higher CAF in comparison with both controls. Both types of nerve injury elicited two responses: a coupled regulation of fiber size and capillarity in the oxidative, type 2a fibers and a capillarity independent regulation of fiber size in the glycolytic type-2b fibers. All subtypes of type-2 fibers had a better capillary supply (higher CAF/FP) in the NC and double NCR than in CON-2. The highest improvement was observed in type-2b fibers; this change was mirrored in an oxidative shift only in the double NCR group. Adopting fiber-type-specific capillary indices improves data analysis of rat EDL muscle samples.

Skeletal muscle fiber characteristics and oxidative capacity in hemiparetic stroke survivors.

Skeletal muscle is changed after stroke, but conflicting data exist concerning muscle morphology and oxidative enzyme capacity. In 36 chronic stroke patients bilateral rectus femoris muscle biopsies were analyzed, and fiber type proportions and cross-sectional areas were determined by ATPase histochemistry. Enzymatic concentrations of citrate synthase (CS) and 3-Hydroxyacyl-coenzymeA-dehydrogenase (HAD) were determined using freeze-dried muscle tissue. Findings were correlated with clinical outcomes. In the paretic muscles the mean fiber area was smaller (P = 0.0004), and a lower proportion of type 1 fibers (P = 0.0016) and a higher proportion of type 2X fibers (P = 0.0002) were observed. The paretic muscle had lower CS (P = 0.013) and HAD concentrations (P = 0.037). Mean fiber area correlated with muscle strength (r = 0.43; P = 0.041), and CS concentration correlated with aerobic capacity (r = 0.47; P = 0.01). In stroke survivors there is a phenotypic shift toward more fatigable muscle fibers with reduced oxidative enzymatic capacity that relates to clinical outcomes.

Unexpected dependence of RyR1 splice variant expression in human lower limb muscles on fiber-type composition.

The skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1), essential for excitation-contraction (EC) coupling, demonstrates a known developmentally regulated alternative splicing in the ASI region. We now find unexpectedly that the expression of the splice variants is closely related to fiber type in adult human lower limb muscles. We examined the distribution of myosin heavy chain isoforms and ASI splice variants in gluteus minimus, gluteus medius and vastus medialis from patients aged 45 to 85 years. There was a strong positive correlation between ASI(+)RyR1 and the percentage of type 2 fibers in the muscles (r = 0.725), and a correspondingly strong negative correlation between the percentages of ASI(+)RyR1 and percentage of type 1 fibers. When the type 2 fiber data were separated into type 2X and type 2A, the correlation with ASI(+)RyR1 was stronger in type 2X fibers (r = 0.781) than in type 2A fibers (r = 0.461). There was no significant correlation between age and either fiber-type composition or ASI(+)RyR1/ASI(-)RyR1 ratio. The results suggest that the reduced expression of ASI(-)RyR1 during development may reflect a reduction in type 1 fibers during development. Preferential expression of ASI(-) RyR1, having a higher gain of in Ca(2+) release during EC coupling than ASI(+)RyR1, may compensate for the reduced terminal cisternae volume, fewer junctional contacts and reduced charge movement in type 1 fibers.

Altered Fiber Types in Regenerated Muscle of Fktn‐KO Muscular Dystrophy Mice

Secondary dystroglycanopathies are a class of muscular dystrophy caused by mutations in genes, including fukutin (FKTN), that are important for the glycosylation of alpha‐dystroglycan (aDG). Loss of glycosylation disrupts aDG binding to laminin, causing defects in muscle and nervous tissues. To assess the role of aDG in fiber type specification of regenerating muscle, we compared muscle fiber types 14 days after toxin injury in skeletal muscle‐specific (Myf5, developmental) and whole animal, post‐development‐induced (TAM) models of Fktn deficiency (KO) at equivalent times post‐gene deletion. Embryonic myosin heavy chain‐expressing fibers, an early regeneration marker, were increased in TAM‐Fktn KO mice 14 days after injury, suggesting that aDG disruption may delay muscle differentiation in this model. Oxidative fibers (type 1 and type 2A) were increased after injury in the TAM‐Fktn KO compared to Myf5‐Fktn KO mice. Type 2X fibers were reduced in toxin regenerated muscle compared to vehicle‐treated muscle for all study groups except the Myf5‐Fktn KO mice. Instead, Myf5‐Fktn KO showed a trend towards fewer type 2X fibers on the uninjured side. These data suggest that aDG contributes to skeletal muscle maturation and fiber type specification in a manner that may be timing and/or location dependent.

Effect of Diet on Haflinger Horses With GYS1 Mutation (Polysaccharide Storage Myopathy Type 1)

Describing the effects of a carbohydrate-rich diet (CRD) on Haflingers with R309 H glycogensynthase 1 (GYS1) mutation. Haflinger mares heterozygous positive (polysaccharide storage myopathy [PSSM] type 1, n = 7) and negative (control, n = 7) for GYS1 mutation received 6 weeks of CRD and hay only (hay), with a wash out period of two. Each diet was followed by oral glucose tolerance test (oGTT), submaximal treadmill exercise test (STET), and biopsies of semitendinosus (STm) and gluteus medius (GMm) muscles. Insulin sensitivity was evaluated by oGTT. Plasma creatine kinase, aspartate aminotransferase, lactate dehydrogenase, and lactate were measured before and after STET. Semitendinosus and GMm biopsies were evaluated for distribution of fiber types (1, 2A, and 2X), chronic myopathic changes, and complex Periodic Acid Schiff (cPAS) positive amylase resistant inclusions. One Haflinger of control group had cPAS inclusions indicating PSSM type 2; therefore, it was excluded. In PSSM1 horses, cPAS were found in every STm sample, and in 10 of 14 GMm samples, furthermore, STm samples showed a greater total myopathy score and significantly more type 2A and fewer type 2X fibers compared with controls. There were no significant differences between groups regarding enzyme levels and for oGTT. Diet had no effect on muscle enzyme levels after STET and on cPAS inclusions in PSSM1 group. Glycogensynthase 1 mutation in Haflingers was not accompanied by clinical signs, even after STET. Carbohydrate-rich diet for 6 weeks did not induce clinical signs or increase cPAS inclusions. As PSSM type 2 is a possibility in Haflingers, STm muscle biopsy and cPAS evaluation should be performed if GYS1 gene test is negative.

Slow to fast muscle transformation following heterochronous isotransplantation is influenced by host thyroid hormone status.

We studied the effect of regeneration, altered innervation and thyroid hormone (TH) levels on fiber type transitions in slow soleus (SOL) muscles grafted (GRAFT) into host extensor digitorum longus (EDLh) muscles of euthyroid (EU), hyperthyroid (HT) and hypothyroid (HY) Lewis strain rats. SOL muscles were excised from 3-week to 4-week-old inbred Lewis rats and intramuscularly transplanted into EDLh muscles of 2-month-old female rats of the same strain. The proportions of type 1, 2A, 2X and 2B fibers of GRAFT were determined by immunohistochemistry and compared with those of EDLh muscle and EDL and SOL muscles of the unoperated contralateral hind limb. After an average regeneration period of 6-7 months and after being reinnervated by the "fast" peroneal nerve of EDLh muscle, GRAFT was transformed into a fast muscle. However, the extent of GRAFT transformation varied with different TH states. In the EU rats, GRAFT contained about 95 % of fast fibers, among which type 2X and 2B fibers predominated (about 75 %). The transition toward fast muscle phenotype was more pronounced in HT status, where the fastest type 2B fibers predominated. On the contrary, in HY status, the slow to fast transformation was less pronounced, as GRAFT contained less type 2B and 2X but more type 2A and 1 fibers. We conclude that the type of innervation is the crucial factor for the slow to fast fiber type transitions in GRAFT, but the extent of muscle transformation is further modulated by altered TH status.

Improved Glucose Tolerance after High-Load Strength Training in Patients Undergoing Dialysis

Background/Aims: The aim of this controlled study was to investigate the effect of high-load strength training on glucose tolerance in patients undergoing dialysis. Methods: 23 patients treated by dialysis underwent a 16-week control period followed by 16 weeks of strength training three times a week. Muscle fiber size, composition and capillary density were analyzed in biopsies obtained in the vastus lateralis muscle. Glucose tolerance and the insulin response were measured by a 2-hour oral glucose tolerance test. Results: All outcome measures remained unchanged during the control period. After strength training the relative area of type 2X fibers was decreased. Muscle fiber size and capillary density remained unchanged. After the strength training, insulin concentrations were significantly lower in patients with impaired glucose tolerance or type 2 diabetes (n = 14) (fasting insulin from 68 ± 12 (46-96) to 54 ± 10 (37-77) pmol/l, p < 0.05, 2-hour insulin from 533 ± 104 (356-776) to 344 ± 68 (226-510) pmol/l, p < 0.05, total insulin area under the curve from 1,868 ± 334 (1,268-2,536) to 1,465 ± 222 (1,094-1,913), p < 0.05). Insulin concentrations were unchanged in patients with normal glucose tolerance (n = 9). Conclusion: The conducted strength training was associated with a significant improvement in glucose tolerance in patients with impaired glucose tolerance or type 2 diabetes undergoing dialysis. The effect was apparently not associated with muscle hypertrophy, whereas the muscle fiber type composition was changed.

Diaphragm weakness in pulmonary arterial hypertension: role of sarcomeric dysfunction

We previously demonstrated that diaphragm muscle weakness is present in experimental pulmonary arterial hypertension (PH). However, the nature of this diaphragm weakness is still unknown. Therefore, the aim of this study was to investigate whether changes at the sarcomeric level contribute to diaphragm weakness in PH. For this purpose, in control rats and rats with monocrotaline-induced PH, contractile performance and myosin heavy chain content of demembranated single diaphragm fibers were determined. We observed a reduced maximal tension of 20% (P < 0.05), whereas tension cost was preserved in type 2X and 2B diaphragm fibers in PH compared with control. The reduced maximal tension was associated with a reduction of force generated per half-sarcomeric myosin heavy chain content. Additionally, reduced Ca(2+) sensitivity of force generation was found in type 2X fibers compared with control, which could exacerbate diaphragm muscle weakness at submaximal activation. No changes in maximal tension and Ca(2+) sensitivity of force generation were observed in fibers from the nonrespiratory extensor digitorum longus muscle. Together, these findings indicate that diaphragm weakness in PH is at least partly caused by sarcomeric dysfunction, which appears to be specific for the diaphragm.

Muscle Fiber Type-Dependent Differences in the Regulation of Protein Synthesis

This study examined fiber type-dependent differences in the regulation of protein synthesis in individual muscle fibers found within the same whole muscle. Specifically, the in vivo SUrface SEnsing of Translation (SUnSET) methodology was used to measure protein synthesis in type 1, 2A, 2X and 2B fibers of the mouse plantaris muscle, in response to food deprivation (FD), and mechanical overload induced by synergist ablation (SA). The results show that 48 h of FD induced a greater decrease in protein synthesis in type 2X and 2B fibers compared to type 1 and 2A fibers. Type 2X and 2B fibers also had the largest FD-induced decrease in total S6 protein and Ser240/244 S6 phosphorylation, respectively. Moreover, only type 2X and 2B fibers displayed a FD-induced decrease in cross-sectional area (CSA). Ten days of SA also induced fiber type-dependent responses, with type 2B fibers having the smallest SA-induced increases in protein synthesis, CSA and Ser240/244 S6 phosphorylation, but the largest increase in total S6 protein. Embryonic myosin heavy chain (MHCEmb) positive fibers were also found in SA muscles and the protein synthesis rates, levels of S6 Ser240/244 phosphorylation, and total S6 protein content, were 3.6-, 6.1- and 2.9-fold greater than that found in fibers from control muscles, respectively. Overall, these results reveal differential responses in the regulation of protein synthesis and fiber size between fiber types found within the same whole muscle. Moreover, these findings demonstrate that changes found at the whole muscle level do not necessarily reflect changes in individual fiber types.

Myosin and actin content of human skeletal muscle fibers following 35 days bed rest

Biopsy samples were taken from the vastus lateralis muscle of seven male subjects pre- and post-35 days bed rest (BR). The myosin heavy chain (MHC) isoform distribution of the samples was determined by densitometry of MHC bands separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Individual muscle fibers were dissected from biopsy samples pre-BR (n=143) and post-BR (n=144). They were studied as regards cross-sectional area (CSA), myosin content by quantitative electrophoresis and myosin actin (M/A) ratio by densitometry of myosin and actin bands of individual muscle fibers. All fibers were typed according to their MHC isoform content determined by SDS-PAGE. A decrease in MHC-1 relative content and an increase in MHC-2X content of whole muscle samples were found, suggesting a slow to fast shift in muscle phenotype. Consistently, fiber type distribution was shifted toward type 2X and 2AX fibers. Muscle fiber atrophy occurred at variable extent among fiber types. Myosin concentration was significantly lower in type 1 and type 2A muscle fibers post-BR than pre-BR, whereas M/A ratio did not vary. The latter findings indicate a disproportionate loss of myosin compared with fiber CSA and a proportional loss of myosin and actin.

Demonstration of myosin heavy chain isoforms in rat and humans: the specificity of seven available monoclonal antibodies used in immunohistochemical and immunoblotting methods

The aim of this paper was to present our experience with seven monoclonal antibodies, six of them were applied in immunohistochemistry and immunoblotting of MyHC isoforms in rats and humans, one of them, 6H1 (Lucas et al., 2000), was tested in human muscle sections only. The four antibodies specific to rat MyHC isoforms, BA-D5,SC-71, BF-35,BF-F3 (Schiaffino et al.,1989) reacted as declared both on muscle sections and immunoblots of rat except SC-71 antibody, which stained MyHC-2a and -2x bands in blots. One of the two commercially available antibodies, A4.74 antibody,reliably marked type 2a fibres of rat,but in blots it weakly stained MyHC-2a and -2x isoforms when used undiluted. The other one, F113.15F4, stained type 2a and 2x fibres and corresponding MyHC bands in blots. Therefore, using this antibody rat MyHC-2x can be additionally confirmed, which can be otherwise demonstrated only on the principle of exclusion with BF-35.Using the same set of antibodies human fast MyHC isoforms can be revealed less clearly. Namely, SC-71 and A4.74 antibodies intensively stained histochemical type 2a,predominantly expressing 2a MyHC transcripts and moderately type 2x fibres, expressing mostly 2x MyHC transcripts, in blots the antibodies recognized both fast isoforms. The 6H1antibody was the only one that selectively labelled type 2x fibres, whereas BF-35 left unstained only a variable proportion of histochemical type 2x fibres and MyHC-2x in blots. F113.15F4 did not distinguish between human fast fibre types and corresponding MyHC isoforms in blots. The negativeresults obtained with BF-F3 in muscle sections and in blots are in agreement with the absence of MyHC-2b in human skeletal muscles. Our results imply that the reactivity of antibodies specific to distinct MyHC isoforms should be carefully evaluated not only among various species but with the two different techniques used as well.

Long-term resistance training improves force and unloaded shortening velocity of single muscle fibres of elderly women

Four elderly women (78 +/- 4.3 years) were resistance trained (RT) for one year and needle biopsies of the vastus lateralis muscle were taken pre- and post-RT. A training intensity of 60% one-repetition maximum (1RM) was adopted for the entire duration of the study. Following RT, significant gain in isometric force of the quadriceps muscles was observed and MHC isoform distribution of vastus lateralis muscle showed a very significant shift from MHC-1 and MHC-2X towards MHC-2A, i.e. a bi-directional shift. A large population (n = 310) of individual skinned muscle fibres were dissected from biopsy samples and used for determination of cross-sectional area (CSA), specific force (Po/CSA) and unloaded shortening velocity (Vo) at 12 degrees C and maximum activation. Fibres were classified on the basis of their MHC isoform content determined by SDS-PAGE in three pure fibre types (1, 2A and 2X) and two hybrid fibre types (1-2A and 2AX). Whereas the CSA of individual muscle fibres did not change, Po/CSA was significantly higher in both type 1 and type 2A fibres post training. Vo of type 1 fibres was significantly higher post-training, whereas no change in Vo was observed in type 2A fibres. The number of pure type 2X fibres was very low especially post-training and did not enable a statistically significant comparison. The data suggest that in elderly women moderate and prolonged RT: (1) can determine similar adaptations in MHC distribution of skeletal muscle to those expected in young subjects; (2) can improve function of muscle fibres.

Adaptation of muscle fibre types and capillary network to acute denervation and shortlasting reinnervation

We postulated that, in rat extensor digitorum longus muscle (EDL), the length of capillaries per fibre surface area (Lcap/Sfib) and per fibre volume (Lcap/Vfib) could reflect fibre-type transformations accompanied by changes in oxidative metabolic profile and selective fibre-type atrophy. We excised rat EDL muscle 2 weeks after the sciatic nerve was cut (acute denervation; DEDL) and 4 weeks after the nerve was crushed (early reinnervation; REDL) and characterised muscle fibre-type transformation by the expression of myosin heavy-chain isoforms and by succinate dehydrogenase (SDH) and nicotinoamide adenine dinucleotide-tetrazolium reductase (NADH-TR) reactions. The numerical percentage (N/N) and area percentage (A/A) of pure and hybrid fibres and their diameter were determined, as was the A/A of SDH- and NADH-TR-positive fibres. The length of capillaries per fibre length (Lcap/Lfib), Lcap/Sfib and Lcap/Vfib were estimated in REDL and Lcap/Vfib in DEDL. In DEDL, the type 2x and 2b fibres evidently atrophied, with the N/N of type 2x fibres being lower and that of hybrid fibres higher. In REDL, the N/N of hybrid fibres was even higher, consequent to a lower N/N of type 2b fibres; however, fibre diameters approached values of the control EDL. Compared with control EDL, denervated and reinnervated muscles exhibited a higher A/A of oxidative fibres. This is probably the result of fibre-type transformation and selective fibre atrophy. We conclude that capillary length does not change during acute denervation and early reinnervation. The obtained higher values of Lcap/Sfib and Lcap/Vfib are related to changes in muscle fibre cross-sectional area.

Mitochondrial Respiration Is Decreased in Skeletal Muscle of Patients With Type 2 Diabetes

We tested the hypothesis of a lower respiratory capacity per mitochondrion in skeletal muscle of type 2 diabetic patients compared with obese subjects. Muscle biopsies obtained from 10 obese type 2 diabetic and 8 obese nondiabetic male subjects were used for assessment of 3-hydroxy-Acyl-CoA-dehydrogenase (HAD) and citrate synthase activity, uncoupling protein (UCP)3 content, oxidative stress measured as 4-hydroxy-2-nonenal (HNE), fiber type distribution, and respiration in isolated mitochondria. Respiration was normalized to citrate synthase activity (mitochondrial content) in isolated mitochondria. Maximal ADP-stimulated respiration (state 3) with pyruvate plus malate and respiration through the electron transport chain (ETC) were reduced in type 2 diabetic patients, and the proportion of type 2X fibers were higher in type 2 diabetic patients compared with obese subjects (all P < 0.05). There were no differences in respiration with palmitoyl-l-carnitine plus malate, citrate synthase activity, HAD activity, UCP3 content, or oxidative stress measured as HNE between the groups. In the whole group, state 3 respiration with pyruvate plus malate and respiration through ETC were negatively associated with A1C, and the proportion of type 2X fibers correlated with markers of insulin resistance (P < 0.05). In conclusion, we provide evidence for a functional impairment in mitochondrial respiration and increased amount of type 2X fibers in muscle of type 2 diabetic patients. These alterations may contribute to the development of type 2 diabetes in humans with obesity.

Myosin heavy-chain isoform distribution, fibre-type composition and fibre size in skeletal muscle of patients on haemodialysis

Chronic uraemia is associated with abnormalities in skeletal muscles, which can affect their working capacity. It is also well known that the fibre-type composition of skeletal muscles influences endurance, muscle strength and power. In this study we therefore determined the size and distribution of muscle fibres and the myosin heavy-chain (MHC) isoform composition in patients on haemodialysis (HD) in order to establish any differences with values for untrained control subjects. Muscle biopsies were obtained from the vastus lateralis muscle of 14 non-diabetic patients on HD. The size and distribution of muscle fibres were evaluated using adenosine triphosphate synthase (ATPase) histochemistry, whilst MHC isoform composition was determined in muscle homogenates using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Values were compared to those for a group of age-, gender- and BMI-matched untrained control subjects. The aerobic work capacity of the patients was also determined. The MHC composition for I, IIA and IIX isoforms was found to be 35.3% +/- 18.2%, 35.9% +/- 7.1% and 28.9% +/- 15.6%, respectively, findings supported by the ATPase histochemically determined fibre-type composition of the vastus lateralis muscle. The mean fibre area of type 1 and 2 fibres was 3283 +/- 873 and 3594 +/- 1483 MICROm2, respectively. The MHC composition and the size of the type 1 fibres of the patients on HD were significantly different from those of the control subjects. The data demonstrate relatively fewer type 1 and consequently more type 2x fibres, with a corresponding change in MHC isoforms (MHC I and MHC IIX) in the skeletal muscle of patients on HD. Several patients on HD were found to have <15% type 1 (or relative percentage of MHC I) fibres. Such a low percentage of type 1 fibres is very rarely observed in normal untrained subjects. Chronic uraemia more severely affects the composition than the size of fibres.

Sheep genotype, age and muscle type affect the expression of metabolic enzyme markers

The objective of this study was to determine whether genotype, age (4, 8, 14 and 22 months), sex (ewe and wether) and muscle type influence ovine (n = 587) muscle metabolic characteristics. The genotypes represented were Poll Dorsetgrowth × Border Leicester Merino, Poll Dorsetgrowth × Merino, Poll Dorsetmuscling × Merino, Merino × Merino and Border Leicester × Merino. Between 4 and 22 months of age, myoglobin concentration within all muscles and all genotypes doubled, with the bulk of this response occurring between 4 and 8 months of age. Levels in the longissimus thoracis et lumborum (LT) and semimembranosus muscles were double those seen in the semitendinosus (ST) muscle, and Merinos had the lowest myoglobin concentrations of all genotypes. The other aerobic indicator, isocitrate dehydrogenase, had lower activity in the ST compared with the LT, was lower in 22-month-old sheep compared with all other ages, and decreased as selection for leanness increased. Both phosphofructokinase and lactate dehydrogenase activity tended to increase with age, were lower in the ST compared with the LT, and had higher activity in the Border Leicester × Merino sheep. The correlation between the percentage of total myofibre area comprising type 2X myofibres and metabolic markers was far higher for the oxidative indicators isocitrate dehydrogenase and myoglobin, which both decreased as relative area of type 2X fibres increased. However, the strongest correlations were with the relative area of type 2A myofibres, which were consistently high for both oxidative and glycolytic metabolic markers implying positive coregulation with both energy producing pathways.

Skeletal muscle hypertrophy and structure and function of skeletal muscle fibres in male body builders

Needle biopsy samples were taken from vastus lateralis muscle (VL) of five male body builders (BB, age 27.4+/-0.93 years; mean+/-s.e.m.), who had being performing hypertrophic heavy resistance exercise (HHRE) for at least 2 years, and from five male active, but untrained control subjects (CTRL, age 29.9+/-2.01 years). The following determinations were performed: anatomical cross-sectional area and volume of the quadriceps and VL muscles in vivo by magnetic resonance imaging (MRI); myosin heavy chain isoform (MHC) distribution of the whole biopsy samples by SDS-PAGE; cross-sectional area (CSA), force (Po), specific force (Po/CSA) and maximum shortening velocity (Vo) of a large population (n=524) of single skinned muscle fibres classified on the basis of MHC isoform composition by SDS-PAGE; actin sliding velocity (Vf) on pure myosin isoforms by in vitro motility assays. In BB a preferential hypertrophy of fast and especially type 2X fibres was observed. The very large hypertrophy of VL in vivo could not be fully accounted for by single muscle fibre hypertrophy. CSA of VL in vivo was, in fact, 54% larger in BB than in CTRL, whereas mean fibre area was only 14% larger in BB than in CTRL. MHC isoform distribution was shifted towards 2X fibres in BB. Po/CSA was significantly lower in type 1 fibres from BB than in type 1 fibres from CTRL whereas both type 2A and type 2X fibres were significantly stronger in BB than in CTRL. Vo of type 1 fibres and Vf of myosin 1 were significantly lower in BB than in CTRL, whereas no difference was observed among fast fibres and myosin 2A. The findings indicate that skeletal muscle of BB was markedly adapted to HHRE through extreme hypertrophy, a shift towards the stronger and more powerful fibre types and an increase in specific force of muscle fibres. Such adaptations could not be fully accounted for by well known mechanisms of muscle plasticity, i.e. by the hypertrophy of single muscle fibre (quantitative mechanism) and by a regulation of contractile properties of muscle fibres based on MHC isoform content (qualitative mechanism). Two BB subjects took anabolic steroids and three BB subjects did not. The former BB differed from the latter BB mostly for the size of their muscles and muscle fibres.