Type 2A Fibers Research Articles

Skeletal muscle phenotypic switching in heart failure with preserved ejection fraction.

Skeletal muscle (SkM) phenotypic switching is associated with exercise intolerance in heart failure with preserved ejection fraction (HFpEF). Patients with HFpEF have decreased type-1 oxidative fibers and mitochondrial dysfunction, indicative of impaired oxidative capacity. The SAUNA (SAlty drinking water/Unilateral Nephrectomy/Aldosterone) mice are commonly used in HFpEF pre-clinical studies and demonstrate cardiac, lung, kidney, and white adipose tissue impairments. However, the SkM (specifically the oxidative-predominant, soleus muscle) has not been described in this preclinical HFpEF model. We sought to characterize the soleus skeletal muscle in the HFpEF SAUNA mice and investigate its translational potential. HFpEF was induced in mice by uninephrectomy, d-aldosterone or saline (Sham) infusion by osmotic pump implantation, and 1% NaCl drinking water was given for 4 weeks. Mice were euthanized, and the oxidative-predominant soleus muscle was collected. We examined fiber composition, fiber cross-sectional area, capillary density, and fibrosis. Molecular analyses were also performed. To investigate the clinical relevance of this model, the oxidative-predominant, vastus lateralis muscle from patients with HFpEF was biopsied and examined for molecular changes in mitochondrial oxidative phosphorylation, vasculature, fibrosis, and inflammation. Histological analyses demonstrated a reduction in the abundance of oxidative fibers, type-2A fiber atrophy, decreased capillary density, and increased fibrotic area in the soleus muscle of HFpEF mice compared to Sham. Expression of targets of interest such as a reduction in mitochondrial oxidative-phosphorylation genes, increased VEGF-α and an elevated inflammatory response was also seen. The histological and molecular changes in HFpEF mice are consistent and comparable with changes seen in the oxidative-predominant SkM of patients with HFpEF. The HFpEF SAUNA model recapitulates the SkM phenotypic switching seen in HFpEF patients. This model is suitable and relevant to study SkM phenotypic switching in HFpEF.

Dominantly inherited myosin IIa myopathy caused by aberrant splicing of MYH2

BackgroundMyosin heavy chain (MyHC) isoforms define the three major muscle fiber types in human extremity muscles. Slow beta/cardiac MyHC (MYH7) is expressed in type 1 muscle fibers. MyHC IIa (MYH2) and MyHC IIx (MYH1) are expressed in type 2A and 2B fibers, respectively. Whereas recessive MyHC IIa myopathy has been described in many cases, myopathy caused by dominant MYH2 variants is rare and has been described with clinical manifestations and muscle pathology in only one family and two sporadic cases.MethodsWe investigated three patients from one family with a dominantly inherited myopathy by clinical investigation, whole-genome sequencing, muscle biopsy, and magnetic resonance imaging (MRI).ResultsThree siblings, one woman and two men now 54, 56 and 66 years old, had experienced muscle weakness initially affecting the lower limbs from young adulthood. They have now generalized proximal muscle weakness affecting ambulation, but no ophthalmoplegia. Whole-genome sequencing identified a heterozygous MYH2 variant, segregating with the disease in the three affected individuals: c.5673 + 1G > C. Analysis of cDNA confirmed the predicted splicing defect with skipping of exon 39 and loss of residues 1860–1891 in the distal tail of the MyHC IIa, largely overlapping with the filament assembly region (aa1877–1905). Muscle biopsy in two of the affected individuals showed prominent type 1 muscle fiber predominance with only a few very small, scattered type 2A fibers and no type 2B fibers. The small type 2A fibers were frequently hybrid fibers with either slow MyHC or embryonic MyHC expression. The type 1 fibers showed variation in fiber size, internal nuclei and some structural alterations. There was fatty infiltration, which was also demonstrated by MRI.ConclusionDominantly inherited MyHC IIa myopathy due to a splice defect causing loss of amino acids 1860–1891 in the distal tail of the MyHC IIa protein including part of the assembly competence domain. The myopathy is manifesting with slowly progressive muscle weakness without overt ophthalmoplegia and markedly reduced number and size of type 2 fibers.

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.

Sexual and Regional Differences in Myosin Heavy Chain Expression in the Rat External Urethral Sphincter.

The external urethral sphincter is a unique striated muscle surrounding the urethra that plays a crucial role in urinary continence, and a comprehensive understanding of its morphology is needed to determine the pathophysiology underlying urinary incontinence and find suitable therapies. Differences between the sexes and among species regarding the fiber types present remain controversial. This study used triple immunofluorescence labeling to visualize one slow (Type 1) and two fast (Types 2A and 2B) myosin isoforms in rat external urethral sphincters from both sexes. Type 2A fibers predominated throughout the sphincter and Type 2B fibers were restricted to the proximal one-third of the external urethral sphincter in the female rats. Type 1 fibers were present adluminally and were concentrated in the proximal and distal segments of the sphincter. While most of the male external urethral sphincter comprised Type 2B fibers, Type 2A fibers intermingled among these fibers in the proximal one-third of the sphincter, and a few Type 1 fibers were present that were restricted to the adluminal region of the proximal segment. The fiber-type compositions and their areal densities changed in both sexes after gonadectomy. The areal density of the Type 1 fibers increased significantly in the ovariectomized females, especially in the distal segment. In the orchidectomized males, the areal densities of the Types 1 and 2A fibers increased significantly, but that of the Type 2B fibers decreased. These results indicate that myosin heavy chain expression in the rat external urethral sphincter is sexually dimorphic and shows regional differences. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:2058-2069, 2017. © 2017 Wiley Periodicals, Inc.

Two novel MYH7 proline substitutions cause Laing Distal Myopathy-like phenotypes with variable expressivity and neck extensor contracture.

BackgroundHuman skeletal muscles express three major myosin heavy chain (MyHC) isoforms: MyHCIIx (MYH1) in fast type 2B muscle fibers, MyHCIIa (MYH2) in fast type 2A fibers and MyHCI/β-cardiac MyHC (MYH7) in slow type I skeletal fibers and cardiac ventricles. In line with its expression pattern, MYH7 mutations have been reported in association with hypertrophic or dilated cardiomyopathy, skeletal myopathies or a combination of both. We analyzed the clinical and molecular phenotype of two unrelated families of Jewish Moroccan ancestry that presented with apparently autosomal dominant inheritance of progressive Laing-like distal myopathy with non-specific myopathic changes, but uncommon marked contractures and wasting of the neck extensors.MethodsClinical phenotyping, whole exome sequencing and restriction analysis, generation of mutants followed by cell culture transfection and imaging.ResultsUsing whole exome sequencing we identified in both families two novel heterozygous proline substitutions located in exon 31 of MYH7 within its rod domain: c.4309G>C (p.Ala1437Pro) and c.4301G>C (p.Arg1434Pro). Here we show that the phenotype caused by these mutations includes marked cervical muscle contracture, and report that the severity of the phenotype varies significantly, to the extent of non-penetrance in one of the families. Finally, we provide evidence that both proline substitutions impair myosin self-assembly in non-muscle cells transfected with β-myosin constructs carrying the mutations, but do not prevent incorporation of the mutant molecules into the sarcomere.ConclusionsThis study expands our clinical and molecular knowledge of MYH7 rod mutations causing skeletal myopathies, and underscores the importance of discussing disease penetrance during genetic counseling.

G.P.85: Truncated dystrophin with exon 45–55 deletion induced muscle atrophy and fiber type change through the hyper-nitrosylation of the ryanodine receptor type-1 and constant release of Ca2+to the cytosol

Duchenne muscular dystrophy (DMD), the most common form of muscular dystrophy, is caused by lack of dystrophin. Exon skipping has been developed as a possible treatment of DMD and exon 45–55 skipping strategy could be one of goals of this therapy, partly because patients with exon 45–55 in-frame deletion have shown very mild skeletal muscle symptoms. Moreover, this strategy could theoretically rescue up to 63% of DMD deletion patients, however function of exon 45–55 deleted dystrophin is not well understood. To clarify the functional role of truncated dystrophin, we generated the transgenic (Tg) mice which expressed the exon 45–55 lacking human dystrophin and two Tg lines were established. We, then, mated Tg male mice with <i>mdx</i> female mice to get Tg/<i>mdx</i> male mice and extensively analyzed the phenotypes. These Tg/<i>mdx</i> mice expressed only truncated dystrophin, accompanied by recovery of the dystrophin–glycoprotein complex at the sarcolemma. The Tg/<i>mdx</i> also showed improvement of the function, including serum creatine kinase level, muscle pathology and specific tetanic force, to a level of the wild type. These results suggest that truncated dystrophin with exon 45–55 deletion has a equivalent function to the full length dystrophin. On the other hand, atrophy of type2B fibers and increasing of type2A fibers were observed at 20weeks of Tg/<i>mdx</i> mice. Moreover, the localization of neuronal NOS (nNOS) in Tg/<i>mdx</i> was changed from sarcolemma to cytosol and nNOS itself was activated. This activation induced hyper-nitrosylation of the ryanodine receptor type-1 and Ca²⁺ was constantly released from sarcoplasmic reticulum to cytosol. This Ca²⁺ release might be related to the findings, up-reguletion of Atrogin-1 and MuRF1, which could induce muscle atrophy and PGC1<i>α</i>, which could alter the muscle fiber types. This Tg/<i>mdx</i> mouse could be also a very useful model for analyzing pathogenesis of BMD phenotypes.

Comparison of the contractile properties, oxidative capacities and fibre type profiles of the voluntary sphincters of continence in the rat

The external urethral sphincter (EUS) and external anal sphincter (EAS) are the principal voluntary striated muscles that sustain continence of urine and faeces. In light of their common embryological origin, shared tonic sphincteric action and synchronized electrical activity in vivo, it was expected that they would exhibit similar physiological and structural properties. However, the findings of this study using paired observations of both sphincters isolated from the rat show clearly that this is not the case. The anal sphincter is much more fatigable than the urethral sphincter. On completion of a fatigue protocol, the amplitude of the last twitch of the EAS had declined to 42 +/- 3% of the first twitch, whereas the last twitch of the EUS was almost identical to that of the first (95 +/- 3%). Immunocytochemical detection of myosin heavy-chain isoforms showed that this difference was not due to the presence of more slow-twitch oxidative type 1 fibres in the EUS compared with the EAS (areal densities 4 +/- 1% and 5 +/- 1%, respectively; P = 0.35). In addition, the fatigue difference was not explained by a greater contribution to force production by fast oxidative type 2A fibres in the urethral sphincter. In fact, the anal sphincter contained a higher areal density of type 2A fibres (56 +/- 5% vs. 37 +/- 4% in the EUS, P = 0.017). The higher oxidative capacity of the EUS, measured histochemically, explained its fatigue resistance. These results were surprising because the fatigue-resistant urethral muscle exhibited faster single-twitch contraction times compared with the anal sphincter (56 +/- 0.87 ms vs. 72.5 +/- 1.16 ms, P < 0.001). Neither sphincter expressed the type 2X myosin isoform but the fast-twitch isoform type 2B was found exclusively in the EUS (areal density 16 +/- 2%). The type 2B fibres of the EUS were small (diameter 19.5 +/- 0.4 mum) in comparison to typical type 2B fibres of other muscles. As a whole the EUS is a more oxidative than glycolytic muscle. In conclusion, analysis of the twitch mechanics and fatigue of two sphincters showed that the EUS contained more fatigue-resistant muscle fibres compared with the EAS.

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.

Spastic wrist flexors are more severely affected than wrist extensors in children with cerebral palsy

Morphological properties of skeletal muscle were compared between wrist flexors and extensors within the same children (n=8, six females, two males; age range 4 to 9y, median age 7y)with wrist muscle imbalance secondary to spastic cerebral palsy (CP). Five patients had hemiplegic CP, two diplegic CP, and one patient had tetraplegic CP. Muscle biopsies were taken during either tendon transfer or tendon lengthening procedures. Analyses included distribution of muscle fibre types, fibre sizes, and expression of developmental myosins. Extensor fibre area was significantly greater than flexor fibre area for type 2A fibres and type 2B fibres but not for type 1 fibres. Coefficient of variation (CV) of fibre size for all three fibre types was greater for flexors compared with extensors. The greatest CV was observed for the type 2A fibres in flexors (39.5 [3.6%]). A wide variation was observed for expression of developmental myosin with the magnitude of the expression being greater, but not statistically significant, in flexors compared with extensors (5.4/mm2 vs 0.53/mm2). These data demonstrate that significant secondary myopathy of wrist flexor muscles results from CP.

Spastic wrist flexors are more severely affected than wrist extensors in children with cerebral palsy

Morphological properties of skeletal muscle were compared between wrist flexors and extensors within the same children (n = 8, six females, two males; age range 4 to 9y, median age 7 y) with wrist muscle imbalance secondary to spastic cerebral palsy (CP). Five patients had hemiplegic CP, two diplegic CP, and one patient had tetraplegic CP. Muscle biopsies were taken during either tendon transfer or tendon lengthening procedures. Analyses included distribution of muscle fibre types, fibre sizes, and expression of developmental myosins. Extensor fibre area was significantly greater than flexor fibre area for type 2A fibres and type 2B fibres but not for type 1 fibres. Coefficient of variation (CV) of fibre size for all three fibre types was greater for flexors compared with extensors. The greatest CV was observed for the type 2A fibres in flexors (39.5 [3.6%]). A wide variation was observed for expression of developmental myosin with the magnitude of the expression being greater, but not statistically significant, in flexors compared with extensors (5.4/mm2 vs 0.53/mm2). These data demonstrate that significant secondary myopathy of wrist flexor muscles results from CP.

Myostatin, insulin-like growth factor-1, and leukemia inhibitory factor mRNAs are upregulated in chronic human disuse muscle atrophy.

Human disuse muscle atrophy frequently accompanies orthopedic injury, arthritis, or bed rest, and recovery is often incomplete despite current rehabilitation programs. We have studied the vastus lateralis muscle in 12 patients with chronic disuse atrophy associated with chronic osteoarthritis of the hip both preoperatively and after total hip arthroplasty. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated an increase in the level of expression of myostatin, insulin-like growth factor-1 (IGF-1) and leukemia inhibitory factor (LIF) mRNAs compared to healthy control muscle. In all patients there was a significant correlation preoperatively between increasing myostatin mRNA expression and reduction in type 2A and 2B fiber area. In the 8 female patients there was a significant correlation between increased myostatin mRNA expression and the atrophy factor calculated for 2A and 2B fiber types preoperatively. We hypothesize that a complex interaction occurs between muscle growth regulating factors in the genesis of muscle wasting. Our results indicate that myostatin is a muscle-wasting factor contributing to type 2B and 2A atrophy. Other muscle growth factors, such as IGF-1 and LIF, may be upregulated in a counterregulatory fashion or may be involved in the fiber type switching seen in disuse muscle wasting.

Autosomal dominant myopathy: missense mutation (Glu-706 --> Lys) in the myosin heavy chain IIa gene.

We here report on a human myopathy associated with a mutation in a fast myosin heavy chain (MyHC) gene, and also the genetic defect in a hereditary inclusion body myopathy. The disorder has previously been described in a family with an "autosomal dominant myopathy, with joint contractures, ophthalmoplegia, and rimmed vacuoles." Linkage analysis and radiation hybrid mapping showed that the gene locus (Human Genome Map locus name: IBM3) is situated in a 2-Mb region of chromosome 17p13, where also a cluster of MyHC genes is located. These include the genes encoding embryonic, IIa, IIx/d, IIb, perinatal, and extraocular MyHCs. Morphological analysis of muscle biopsies from patients from the family indicated to us that the type 2A fibers frequently were abnormal, whereas other fiber types appeared normal. This observation prompted us to investigate the MyHC-IIa gene, since MyHC-IIa is the major isoform in type 2A fibers. The complete genomic sequence for this gene was deduced by using an "in silico" strategy. The gene, found to consist of 38 exons, was subjected to a complete mutation scan in patients and controls. We identified a missense mutation, Glu-706 --> Lys, which is located in a highly conserved region of the motor domain, the so-called SH1 helix region. By conformational changes this region communicates activity at the nucleotide-binding site to the neck region, resulting in the lever arm swing. The mutation in this region is likely to result in a dysfunctional myosin, compatible with the disorder in the family.

Muscle biopsy as a tool for assessing muscular adaptation to training in horses

Abstract Objective To describe an applied method for quantitative estimation of training condition in horses. Animals 17 sedentary adult (5 to 14 years old) stallions of several breeds endurance trained for 3 months. Procedure Muscle biopsy specimens from 2 depths (20 and 60 mm) of the gluteus medius muscle were obtained before and after training and were analyzed for fiber type distribution, mean cross-sectional area, relative fiber area, and mean number of capillaries in contact with each fiber type relative to their mean area. Fiber types were designated as types 1, 2A, and 2B (high, low, and moderate myosin ATPase activities at pH 4.5, respectively). Data were subjected to discriminant and principal component analyses. Results 77% of the observations were correctly discriminated according to training condition of horses by use of discriminant analyses when histochemical and morphometric data from the 2 muscle biopsy specimens were used. Principal component analysis indicated that the most efficient variables describing muscular adaptation to training are fiber area and number of capillaries in contact with type-1 and type-2A fibers, particularly from the deepest sampling site of the muscle. Conclusion It is possible to satisfactorily classify an individual horse in a given training category on the basis of a physiologic muscle index. Clinical Relevance These methods could become a useful aid for evaluation of the individual horse's response to training. (Am J Vet Res 1996;57:1412-1416)

Skeletal muscle changes after short term vibration.

Six female Wistar rats were anaesthetised and attached to a vibrating table, (80 Hz/32 m/s2) five hours daily for two days. Histological, enzyme and immunohistochemical, and morphometric analyses of the directly exposed and opposite control plantar muscles were made. Neither fibre necrosis nor regenerative activity were seen. The mean (SD) area (micron2) of vibration-exposed muscle fibres was significantly increased compared with controls (682 (274) and 642 (230), p < 0.05). Both type 1 and 2C fibres were significantly larger after vibration (773 (293) and 650 (223) compared with 683 (209) and 579 (149), p < 0.05) while type 2A and 2AB fibres were not significantly enlarged. The percentage of centrally located nuclei was significantly increased after vibration. This study shows that short term vibration can induce changes in size in muscle fibres. We postulate that this is the first indication of a vibration-induced muscle injury that may develop into chronic impairment of muscle function if the exposure continues for an extended period of time.

Histochemical properties of intrinsic laryngeal muscles in cats

As one means in elucidating the contractile properties of the intrinsic laryngeal muscles, we examined the histochemical properties of the posterior cricoarytenoid (PCA), thyroarytenoid (TA), lateral cricoarytenoid (LCA), arytenoid (A) cricothyroid (CT) muscles in cats using the reaction for myosin ATPase following acid and alkali preincubation. The muscle fiber compositions of the intrinsic laryngeal muscles differed not only across the muscles but also across the muscle fascicles within a single muscle. The relative frequency of type-1 fibers was the smallest (9%) in the TA and the largest (45%) in the CT, and that of type-2A fibers was 39–48% in each muscle. The relative frequency of type-2B fibers was the smallest (9%) in the CT and the largest (42–45%) in the TA, LCA and A, and that of type-2C fibers was less than 1.0% in each muscle. Across the muscle fascicles, the TA was the most heterogeneous in muscle fiber distribution. The mediocaudal part of the TA was mainly composed of the muscle fascicles with type-1 fibers, while the laterocaudal and rostral parts of the TA were mostly composed of the muscle fascicles without type-1 fibers. The PCA was the most homogeneous in muscle fiber distribution. Our results demonstrate that the contractile properties of the intrinsic laryngeal muscles differ across the muscles and across the muscle fascicles within a single muscle and suggest that the delicate laryngeal movements are established by the coordinated activities of the intrinsic laryngeal muscle fibers with different contractile properties.

Histochemical and morphometric study of fiber types in ten skeletal muscles of healthy young adult cats

SUMMARY A histochemical and morphometric study of fiber types in a variety of skeletal muscles of healthy young adult cats was undertaken to provide normative data not available previously. Using a standardized system of nomenclature, fiber types 1, 2A, 2B, and 2C were identified in most cat muscles on the basis of myosin ATPase staining at pH 4.45. Type-2M fibers were present in temporalis (tem) and masseter (mas) muscles. Type-1 fibers predominated in medial head of triceps (mht) and soleus muscles. Type-2B fibers were dominant in biceps femoris, lateral head of gastrocnemius, cranial tibial, long head of triceps, and superficial digital flexor muscles; type-2A fibers were dominant in buccinator muscle samples; and type-2M fibers were dominant in tem and mas muscles. Numbers of type-2C fibers did not exceed 2 to 3% of the myofiber population in any muscle. In CT and LHT muscles, a gradient of fiber type distribution was observed, with significant (P &lt; 0.05) increase in numbers of type-1 and type-2A fibers in deeper regions of the muscles. The distribution of fiber types was compartmentalized in mht and mas specimens. Diameter of type-2B fibers was significantly (P &lt; 0.05) larger than that of type-1 and type-2A fibers in biceps femoris, lateral head of gastrocnemius, cranial tibial, long head of triceps, and superficial mht muscles. Diameter of type-2M fibers was significantly (P &lt; 0.05) larger than that of type-1 fibers in tem and mas muscles. The soleus type-1 muscle fibers were the largest fibers encountered in any muscle. In mht muscle, fiber diameter of type-1 and type-2B fibers varied significantly (P &lt; 0.05) in oxidative and glycolytic compartments. Variability coefficients were less than 200 in all muscles. In every muscle specimen, the number of fibers with internal nuclei was less than or equal to 2%.

Automated cytometry of fibre size and spatial distribution in the superficial masseter muscle of the rat at three ages.

An image cytometry program was applied to sections of the superficial masseter muscle of female and male 21-, 42- and 105-day-old rats. Lesser diameter and spatial distribution of more than 6000 muscle fibres were automatically measured in digital images from muscle sections stained for myofibrillar ATPase activity. In this muscle, only type 2A, 2B and 2C fibres were observed. At the three ages and in both sexes, 2A fibres were found to have the largest diameter and were the most frequent (> 54%). In the 21-day-old animals, females presented larger diameters than did males; in contrast, in the 105-day-old animals, the three fibre types were larger in males than in females. At all ages and in both sexes, type 2A occupied 32 to 80% more cross-sectional area than type 2B. Most images (98%) showed a random spatial distribution of their fibre types. All fibre types grew in diameter with age. The coefficient of variation of the diameter was age-independent and remained under 23%. The finding of an age-independent variable could have a practical application: an increase of the coefficient of variation (> 25%) can be considered as pathological, even without a perfect age-matched control.

Changes in cross-sectional area and capillary supply of the muscle fiber population in equine gluteus medius muscle as a function of sampling depth

SUMMARY The right and left gluteus medius muscles of 4 mature Andalusian stallions were examined by repeated needle biopsy over a specific area and depth, as well as at different depths, to determine whether the cross-sectional area and capillary supply of the various fiber types are homogeneous throughout the muscle. The muscle biopsy specimens were histochemically analyzed for fiber types (myofibrillar adenosine triphosphatase) and capillaries (amylase-periodic acid-Schiff method). Differences between contralateral sites were not identifiable for any of the analyzed variables. Differences between sampling depths were larger than those between sample sites. The percentage of type-1 fibers increased, and that of type-2B fibers decreased as a function of increased sampling depth, but the relative frequency of type- 2A fibers did not change with depth. The mean crosssectional area of type-1 and type-2A fibers increased significantly, and that of type-2B fibers decreased between the most superficial and the deepest sampling sites. The capillary density and the mean number of capillaries per fiber increased as a function of sampling depth, but significant changes were not recorded between sampling depths for overall mean fiber area. The mean number of capillaries in contact with fibers of each histochemical type increased significantly with increasing sampling depth only for type- 1 fibers. However, the number of capillaries in contact with fibers changed significantly, relative to the fiber area, as a function of sampling depth; for type- 28 fibers this variable increased, whereas for type-2A and type-1 fibers, it decreased. These results indicate that fiber size and capillary supply of the various fiber types in the gluteus medius muscle of horses vary as a function of depth. These patterns of variation most likely indicate the functional difference between various portions of the muscle, and support the general opinion that the functional demands placed on the fiber population are an important factor in the development of muscle fiber properties.

Mammalian skeletal muscle fibers distinguished by contents of phosphocreatine, ATP, and Pi.

We tested the proposition that muscle cell types have different contents of phosphocreatine (PCr), ATP, and Pi by 31P NMR spectroscopy and HPLC analyses of adult rat and mouse muscles containing various volume fractions of different fiber types. There was a 2-fold difference in the PCr content between muscles with a high volume fraction of fiber types 1 and 2x versus those with fast-twitch (types 2a and 2b) fiber types. Pi content was low, and PCr and ATP contents were high in muscles with large contents of type 2b and 2a fibers; the reverse was true in muscles with a large volume fraction of type 1 and 2x fibers. There is a large range in the Pi/PCr ratios in normal resting muscles, from less than 0.05 in type 2 to 0.51 in type 1 fibers, depending upon the distribution of their component fiber types. In all muscles, the peak area resulting from the beta phosphate of ATP constituted approximately 13% of the sum of all peak areas observable in the 31P spectrum. Fiber types 2a and 2b were not distinguishable, and the content of type 2x fibers was similar to type 1 fibers. From the profile of these metabolites, we could distinguish only two classes of fibers. For type 2a and 2b fibers, the intracellular concentrations were 8 mM ATP, 39 mM total creatine, 32 mM PCr, 0.8 mM Pi, and 8 microM ADP. For type 1 and 2x fibers, these quantities were 5 mM ATP, 23 mM total creatine, 16 mM PCr, 6 mM Pi, and 11 microM ADP. Thus our results establish an additional criterion upon which to distinguish skeletal muscle cells, one based on the resting content of bioenergetically important metabolites. These results also provide the basis for estimating skeletal muscle fiber-type composition from noninvasive NMR spectroscopic data.