Generating fast-twitch myotubes in vitro with an optogenetic-based, quantitative contractility assay.

The composition of fiber types within skeletal muscle impacts the tissue's physiological characteristics and susceptibility to disease and ageing. In vitro systems should therefore account for fiber-type composition when modelling muscle conditions. To induce fiber specification in vitro, we designed a quantitative contractility assay based on optogenetics and particle image velocimetry. We submitted cultured myotubes to long-term intermittent light-stimulation patterns and characterized their structural and functional adaptations. After several days of in vitro exercise, myotubes contract faster and are more resistant to fatigue. The enhanced contractile functionality was accompanied by advanced maturation such as increased width and up-regulation of neuron receptor genes. We observed an up-regulation in the expression of fast myosin heavy-chain isoforms, which induced a shift towards a fast-twitch phenotype. This long-term in vitro exercise strategy can be used to study fiber specification and refine muscle disease modelling.

Quercetin regulates skeletal muscle fiber type switching via adiponectin signaling.

This study aimed to investigate the role and underlying molecular mechanism of quercetin in regulating skeletal muscle fiber type transition. We found that dietary quercetin supplementation in mice significantly increased oxidative fiber-related gene expression, slow-twitch fiber percentage and succinic dehydrogenase (SDH) activity. By contrast, quercetin decreased lactate dehydrogenase (LDH) activity, fast MyHC protein expression, fast-twitch fiber percentage, and MyHC IIb mRNA expression. Furthermore, quercetin significantly increased serum adiponectin (AdipoQ) concentration, and the expression levels of AdipoQ and AdipoR1. However, inhibition of adiponectin signaling by AdipoR1 siRNA significantly attenuated the effects of quercetin on muscle fiber type-related gene expression, the percentages of slow MyHC-positive and fast MyHC-positive fibers, and metabolic enzyme activity in C2C12 myotubes. Together, our data indicated that quercetin could promote skeletal fiber switching from glycolytic type II to oxidative type I through AdipoQ signaling.

Patients with MELAS with negative myopathology for characteristic ragged-red fibers.

BACKGROUND: Muscle pathology usually contributes to mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episode (MELAS), even in patients without prominent muscle symptoms. We report a series of patients with MELAS without significant myopathic changes. METHODS: Twelve patients without ragged-red fibers (RRFs) on muscle pathology (RRF-negative group) and 99 patients with MELAS and RRFs and/or cytochrome c oxidase (COX)-deficient fibers (control RRF-positive group) were recruited. We analyzed clinical features, neuroimaging and pathological findings, gene mutation data, immunofluorescence assay of key respiratory chain subunits of complexes I and IV and mitochondrial DNA (mtDNA) mutation load in biopsied muscle samples. RESULTS: None of the RRF-negative patients had RRF or COX-negative fibers, but four patients had strongly succinate dehydrogenase-stained vessels (SSVs). There was a lower proportion of m.3243A>G and higher proportion of mitochondria-encoded ND gene mutations in RRF-negative than RRF-positive patients. The proportion of aphasia was relatively higher, while complex I and IV subunit abundance in muscle and mutation load were lower in RRF-negative than in RRF-positive patients. CONCLUSION: RRF-negative patients had a similar disease course, clinical symptoms, and neuroimaging results to RRF-positive patients with MELAS. SSV is a valuable diagnostic indicator for MELAS. For highly suspected MELAS yet without positive myopathological findings, combined immunofluorescence and genetic studies should be used to achieve final diagnosis.

Comparative transcriptome analysis of fast twitch muscle and slow twitch muscle in Takifugu rubripes.

Fast twitch muscle and slow twitch muscle are two important organs of Takifugu rubripes. Both tissues are of ectodermic origin, and the differences between the two muscle fibers reflect the differences in their myofibril protein composition and molecular structure. In order to identify and characterize the gene expression profile in the two muscle fibers of T. rubripes, we generated 54 million and 44 million clean reads from the fast twitch muscle and slow twitch muscle, respectively, using RNA-Seq and identified a total of 580 fast-muscle-specific genes, 1533 slow-muscle-specific genes and 11,806 genes expressed by both muscles. Comparative transcriptome analysis of fast and slow twitch muscles allowed the identification of 1508 differentially expressed genes, of which 34 myosin and 30 ubiquitin family genes were determined. These differentially expressed genes (DEGs) were also analyzed by Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. In addition, alternative splicing analysis was also performed. The generation of larger-scale transcriptomic data presented in this work would enrich the genetic resources of Takifugu rubripes, which could be valuable to comparative studies of muscles.

Fatty acid binding protein 3 is associated with skeletal muscle strength in polymyositis and dermatomyositis.

OBJECTIVE: To determine serum levels and histopathological characteristics of fatty acid binding protein 3 (FABP3) in patients with polymyositis (PM) and dermatomyositis (DM), and to evaluate the correlation between the FABP3 and muscle strength. METHODS: The study population included 24 subjects with PM/DM, 12 subjects with asymptomatic or minimally symptomatic hyper-creatine kinase-emia (AMSH), and 10 healthy control subjects. Muscle strength was measured by using the Manual Muscle Test (MMT8). Serum CK, myoglobin and FABP3 levels were tested. Correlations between variables were studied by using Spearman's rank and partial correlation analysis methods. Immunohistochemical and double immunofluorescent stainings of FABP3 were performed to investigate its distribution in skeletal muscle. RESULTS: PM/DM patients had significantly higher (P < 0.05) serum FABP3 levels (35.46 ± 38.45 ng/mL) than did AMSH patients (3.77 ± 1.21 ng/mL) and healthy control subjects (4.30 ± 3.18 ng/mL). MMT8 scores correlated negatively with CK, myoglobin and FABP3 levels. Partial correlation analysis was performed and showed that the correlation coefficients between MMT8 score and serum CK, myoglobin and FABP3 levels were -0.276 (P > 0.05), -0.228 (P > 0.05) and -0.927 (P < 0.001), respectively. Immunohistochemical and double immunofluorescent staining showed that FABP3 expression increased in the skeletal muscle of PM/DM patients and was mainly distributed in slow twitch muscle fibers. CONCLUSION: Serum levels of FABP3 in PM/DM patients were significantly increased and were mainly distributed in slow twitch muscle fibers, displaying a closer association with muscle weakness than did serum levels of CK and myoglobin. FABP3 is likely to be a useful serum biomarker in PM/DM patients.

Effects of strength training on muscle cellular outcomes in prostate cancer patients on androgen deprivation therapy.

Androgen deprivation therapy (ADT) improves life expectancy in prostate cancer (PCa) patients, but is associated with adverse effects on muscle mass. Here, we investigated the effects of strength training during ADT on muscle fiber cross-sectional area (CSA) and regulators of muscle mass. PCa patients on ADT were randomized to 16 weeks of strength training (STG) (n = 12) or a control group (CG; n = 11). Muscle biopsies were obtained from m. vastus lateralis and analyzed by immunohistochemistry and western blot. Muscle fiber CSA increased with strength training (898 µm(2) , P = 0.04), with the only significant increase observed in type II fibers (1076 µm(2) , P = 0.03). There was a trend toward a difference in mean change between groups myonuclei number (0.33 nuclei/fiber, P = 0.06), with the only significant increase observed in type I fibers, which decreased the myonuclear domain size of type I fibers (P = 0.05). Satellite cell numbers and the content of androgen receptor and myostatin remained unchanged. Sixteen weeks of strength training during ADT increased type II fiber CSA and reduced myonuclear domain in type I fibers in PCa patients. The increased number of satellite cells normally seen following strength training was not observed.

Characterization of α-actin isoforms in white and red skeletal muscle types of Leporinus macrocephalus (Characiformes, Anostomidae).

Two α-actin genes of the fish Leporinus macrocephalus, referring to white and red muscle tissues, were isolated. Actin isoforms, that mainly differed by a Ser/Ala155 substitution, can have a functional significance related to actin-ATP interaction. An Ala155 residue, as observed in the α-skeletal actin from red muscle, results in a decrease in actin's affinity for ATP, which may also be associated to the slow contractile performance of this tissue. Furthermore, a Phe/Ile262 substitution at the red muscle actin leads to a hydrophobicity variation at the D-plug of the protein, which could alter its stability. Data on qRT-PCR evidenced a significant higher actin mRNA level in white muscle when compared to red muscle (T=105 Mann Whitney; p<0.001). This finding could be related to the energetic demands of the white muscle tissue, with fast contraction fibers and glycolytic metabolism for energy supply. Available data on muscle actins lead to the proposal that white and red α-skeletal actins are genetically and functionally distinguishable in fish species, a feature that is not found in other vertebrate groups.

Immunohistochemical analysis of orbicularis oris muscle fiber distribution at the philtrum in healthy infants.

OBJECTIVE: To characterize the fiber-type distribution of the orbicularis oris muscle at the philtrum in healthy infants by immunohistochemistry and examine the relationship between orbicularis oris and philtrum structure. METHODS: Samples of the upper lip were obtained from two infant cadavers. Serial sagittal sections were obtained at the midline of the philtral dimple, unilateral philtral ridge, and the lateral side. Three sections from each site were prepared for immunohistochemical staining using myosin heavy chain fast fiber (MHCf) and myosin heavy chain slow fiber (MHCs) antibodies to determine the ratio of fast to slow skeletal muscle fibers. RESULTS: The ratio of fast to slow muscle fibers differed significantly among the superficial orbicularis oris muscle (98.30%:1.13%), deep pars peripheralis (95.30%:3.14%), and deep pars marginalis (91.31%:5.74%), with a significantly higher percentage of slow fibers in the pars marginalis compared to pars peripheralis (P=0.002) and fast fibers in the superficial muscle compared to pars marginalis and peripheralis (both P=0.000). Similarly, the fast:slow fiber ratio differed among the superficial philtral dimple (95.88%:2.41%), superficial philtral ridge (98.52%:1.11%), and superficial midlateral philtral ridge (99.07%:0.66%), with a higher percentage of fast fibers higher on the lateral side of the superficial philtral ridge than at the philtral ridge (P=0.030) and higher at the philtral ridge than the philtral dimple (P=0.001). The fast:slow fiber ratio did not differ within the pars peripheralis at the philtral dimple (93.94%:4.19%), philtral ridge (94.49%:3.84%), and lateral philtral ridge (95.79%:2.70%) (all P>0.05). CONCLUSIONS: Philtum structure is likely determined in part by the distribution of muscle fiber types among philtral dimple, ridge, and lateral side. These differences should be considered in cleft lip repair.

Chronic sleep deprivation alters the myosin heavy chain isoforms in the masseter muscle in rats.

To investigate the changes in myosin heavy chain (MyHC) isoforms of rat masseter muscle fibres caused by chronic sleep deprivation and a possible link with the pathogenesis of disorders of the temporomandibular joint (TMJ). A total of 180 male rats were randomly divided into three groups (n=60 in each): cage controls, large platform controls, and chronic sleep deprivation group. Each group was further divided into three subgroups with different observation periods (7, 14, and 21 days). We investigated he expression of MyHC isoforms in masseter muscle fibres by real-time quantitative polymerase chain reaction (PCR), Western blotting, and immunohistochemical staining. In rats with chronic sleep deprivation there was increased MyHC-I expression in layers of both shallow and deep muscles at 7 and 21 days compared with the control groups, whereas sleep deprivation was associated with significantly decreased MyHC-II expression. At 21 days, there were no differences in MyHC-I or MyHC-II expression between the groups and there were no differences between the two control groups at any time point. These findings suggest that chronic sleep deprivation alters the expression of MyHC isoforms, which may contribute to the pathogenesis of disorders of the TMJ.

The effects of short-term exercise training on peak-torque are time- and fiber-type dependent.

We examined the susceptibility of fast and slow twitch muscle fibers in the quadriceps muscle to eccentric exercise-induced muscle damage. Nine healthy men (age: 22.5 ± 1.6 years) performed maximal eccentric quadriceps contractions at 120°·s-1 over a 120° of knee joint range of motion for 6 consecutive days. Biopsies were taken from the vastus lateralis muscle before repeated bouts of eccentric exercise on the third and seventh day. Immunohistochemical procedures were used to determine fiber composition and fibronectin activity. Creatine kinase (CK) and lactate dehydrogenase (LDH) were determined in serum. Average torque was calculated in each day for each subject. Relative to baseline, average torque decreased 37.4% till day 3 and increased 43.0% from the day 3 to day 6 (p < 0.001). Creatine kinase and LDH were 70.6 and 1.5 times higher on day 3 and 75.5 and 1.4 times higher on day 6. Fibronectin was found in fast fibers in subjects with high CK level on day 3 and 7 after exercise, but on day 7, fibronectin seemed in both slow and fast fibers except in muscles of 2 subjects with high fast fiber percentage. Peak torque and muscle fiber-type composition measured at baseline showed a strong positive association on day 3 (r = 0.76, p < 0.02) and strong negative association during recovery between day 3 and day 6 (r = -0.76, p < 0.02), and day 1 and day 6 (r = 0.84, p < 0.001). We conclude that the damage of fast fibers preceded the damage of slow fibers, and muscles with slow fiber dominance were more susceptible to repeated bouts of eccentric exercise than fast fiber dominance muscles. The data suggest that the responses to repeated bouts of eccentric exercise are fiber-type-dependent in the quadriceps muscle, which can be the basis for the design of individualized strength training protocols.

Fiber typing in aging muscle.

It is accepted widely that fast-twitch muscle fibers are preferentially impacted in aging muscle, yet we hypothesize that this is not valid when aging muscle atrophy becomes severe. In this review, we summarize the evidence of fiber type-specific effect in aging muscle and the potential confounding roles of fibers coexpressing multiple myosin heavy-chain isoforms and their histochemical identification.

Age-related changes in rat genioglossus, geniohyoid and masseter muscles.

OBJECTIVES: The aim of this study was to elucidate age-related changes from adult to middle age in the contractile properties of the masseter, genioglossus and geniohyoid muscles of the rat. MATERIALS AND METHODS: We analysed the expressions of myosin heavy chain (MyHC) mRNAs and proteins as indicators of the contractile properties in these muscles obtained from rats at 6, 12, 18 and 24 months of age using real-time PCR and SDS-PAGE. RESULTS: We found no marked age-related changes in the expressions of MyHC mRNAs and proteins in rat masseter and geniohyoid muscles, suggesting that the biological ageing process does not affect contractile properties in these muscles. However, we found a decrease in the expression of MyHC IIb mRNA with ageing in the rat genioglossus muscle, suggesting that biological ageing process induces at least some fast-to-slow myofibre phenotype transition. CONCLUSION: The biological ageing process from adult to middle age appears to differentially affect different types of craniofacial muscles.

Morphometric analyses of normal pediatric brachial biceps and quadriceps muscle tissue.

Pediatric normal brachial biceps (14 specimens) and quadriceps muscles (14 specimens) were studied by immunohistochemistry to quantify fiber-type, diameter and distribution, capillary density, presence of inflammatory cells (CD3, CD20, CD68) and expression of neonatal myosin and MHC class 1 proteins. Brachial biceps showed more fast-twitch fibers and lower capillary/fiber ratio than quadriceps. The mean diameter of both fiber types was smaller in biceps than quadriceps. Fast-fibers were smaller than slow-fibers, and capillary/fiber ratio was < 1.0 in both muscles. Fiber size and capillary / fiber ratio increased with age. Normal limits for infiltrating haematopoietic cells were <4 T lymphocytes, or CD68+ cells, very few B cells, < 6 neonatal myosin positive fibers, and no fibers MHC class 1 positive in one x20 field, for both muscles. The present comparison of quantitative findings between brachial biceps and quadriceps may allow standardization of the assessment of pathological changes in both pediatric muscles.

[Changes in titin and myosin heavy chain isoform composition in skeletal muscles of Mongolian gerbil (Meriones unguiculatus) after 12-day spaceflight].

Changes of titin and myosin heavy chain isoform composition in skeletal muscles (m. soleus, m. gastrocnemius, m. tibialis anterior, m. psoas major) in Mongolian Gerbil (Meriones unguiculatus ) were investigated after 12-day spaceflight on board of Russian space vehicle "Foton-M3". In m. psoas and m. soleus in the gerbils from "Flight" group the expected increase in the content of fast myosin heavy chain isoforms (IIxd and IIa, respectively) were observed. No significant differences were found in the content of IIxd and IIa isoforms of myosin heavy chain in m. tibialis anterior in the gerbils from control group as compared to that in "Flight" group. An unexpected increase in the content of slow myosin heavy chain I isoform and a decrease in the content of fast IIx/d isoform in m. gastrocnemius of the gerbils from "Flight" group were observed. In skeletal muscles of the gerbils from "Flight" group the relative content of titin N2A-isoform was reduced (by 1,2-1,7 times), although the content of its NT-isoform, which was revealed in striated muscles of mammals in our experiments earlier, remained the same. When the content of titin N2A-isoform was decreased, no predictable abnormalities in sarcomeric structure and contractile ability of skeletal muscles in the gerbils from "Flight" group were found. An assumption on the leading role of titin NT-isoform in maintenance of structural and functional properties of striated muscles of mammals was made.

Effects of fibre type and kefir, wine lemon, and pineapple marinades on texture and sensory properties of wild boar and deer longissimus muscle.

Fibre type percentage and changes in textural parameters, sensory properties as well as mean fibre cross sectional area (CSA), fibre shape, endomysium and perimysium thickness of wild boar and deer longissimus (L) muscle subjected to ageing with kefir, dry red wine, lemon and pineapple juice marinades for 4 days were studied. Among the non-marinated and non-aged samples of muscles it was found that wild boar meat with its higher percentage of red fibres, higher CSA, thicker connective tissue as compared with deer meat, was harder, more springy and stringy. Muscles ageing, regardless of methods, resulted in a decrease in both the CSA and thickness of the connective tissue, and improve in fibre shape. As a consequence ageing caused a reduction in hardness, cohesiveness, springiness, and stringiness as well as in augmentation of tenderness, juiciness and general attractiveness of the muscles studied. As demonstrated by obtained data, regardless of ageing methods, deer L muscle contained more white fibres compared to wild boar muscle, were more susceptible to tenderization. The highest structural and textural changes, but the worst general attractiveness was found in muscles marinated with pineapple juice addition. Insignificantly lower changes in both quality traits were found in muscles aged with kefir marinade which at the same time were characterized by the high tenderness, the highest juiciness and general attractiveness.

Comparison of new ELISA method with established SDS-PAGE method for determination of muscle myosin heavy chain isoforms.

We developed a new method for the quantitative determination of myosin heavy chain (MyHC) isoforms taking advantage of immunochemical differences and based on the ELISA principle. In the present paper we compare analysis of MyHC isoforms using the SDS-PAGE and the ELISA methods in the same samples of adult female inbred Lewis strain euthyroid, hyperthyroid and hypothyroid rats. In all thyroid states, the same composition and corresponding changes of MyHC isoforms were determined using both methodological approaches in the slow soleus and the fast extensor digitorum longus muscles. Our results showed that ELISA can be used for a "semi-quantitative" or "comparative" measurement of MyHC isoforms in multiple muscle samples, but that it is neither more exact nor faster compared to SDS-PAGE.

Differential effect of calsequestrin ablation on structure and function of fast and slow skeletal muscle fibers.

We compared structure and function of EDL and Soleus muscles in adult (4-6 m) mice lacking both Calsequestrin (CASQ) isoforms, the main SR Ca²âº-binding proteins. Lack of CASQ induced ultrastructural alterations in ~30% of Soleus fibers, but not in EDL. Twitch time parameters were prolonged in both muscles, although tension was not reduced. However, when stimulated for 2 sec at 100 hz, Soleus was able to sustain contraction, while in EDL active tension declined by 70-80%. The results presented in this paper unmask a differential effect of CASQ1&2 ablation in fast versus slow fibers. CASQ is essential in EDL to provide large amount of Ca²âº released from the SR during tetanic stimulation. In contrast, Soleus deals much better with lack of CASQ because slow fibers require lower Ca²âº amounts and slower cycling to function properly. Nevertheless, Soleus suffers more severe structural damage, possibly because SR Ca²âº leak is more pronounced.

Histochemistry and morphometric analysis of muscle fibers from patients with duchenne muscular dystrophy (DMD) / Histoquímica y análisis morfométrico de las fibras musculares de p0acientes con distrofia muscular de duchenne (DMD)

The aim of the study was to analyze the muscle fibers by histochemistry and morphometric methods from patients with Duchenne muscular dystrophy (DMD). Muscle biopsies were taken from the vastus lateralis muscle of five boys between 13 and 15-years of age, with clinical diagnosis of DMD. The histochemistry was performed using myofibrillar ATPases (9.6, 4.6 and 4.3). To morphometrical analysis a computerized semiautomatic system and software Image-Lab was used. ATPase staining showed atrophy of muscle fibers. Fibrosis and adipose deposition occurred in variable degree depending of muscular involvement. The morphometrical analysis showed an increase of size (percentage) to type I fiber than other types in all patients. Furthermore, the type I fiber had a larger cross-sectional area and mean diameter than type IIa and IIb fibers. Both histochemistry and morphometric analysis could be important tools for qualitative and quantitative diagnostics of muscle fibers attacked in this type of disease.

El objetivo del estudio fue analizar las fibras musculares mediante histoquímica y métodos morfométricos en pacientes con distrofia muscular de Duchenne (DMD). Se tomaron biopsias musculares del músculo vasto lateral de cinco niños entre 13 y 15 años de edad, con diagnóstico clínico de DMD. La histoquímica se realizó mediante ATPasa miofibrilar (9.6, 4.6 y 4.3). Para el análisis morfométrico se utilizó un sistema semiautomático computarizado y software de imagen de laboratorio. La tinción de ATPasa mostró una atrofia de las fibras musculares. La fibrosis y depósito adiposo se observó en grado variable dependiendo del compromiso muscular. El análisis morfométrico mostró un aumento de tamaño (porcentaje) de fibras tipo I en todos los pacientes. Además, la fibra tipo I tuvo un área de sección transversal y diámetro medio mayor que las fibras tipos IIa y IIb. Tanto la histoquímica y el análisis morfométrico pueden ser herramientas importantes para el diagnóstico cualitativo y cuantitativo de las fibras musculares comprometidas en este tipo de enfermedad.

Microgenomic analysis in skeletal muscle: expression signatures of individual fast and slow myofibers.

BACKGROUND: Skeletal muscle is a complex, versatile tissue composed of a variety of functionally diverse fiber types. Although the biochemical, structural and functional properties of myofibers have been the subject of intense investigation for the last decades, understanding molecular processes regulating fiber type diversity is still complicated by the heterogeneity of cell types present in the whole muscle organ. METHODOLOGY/PRINCIPAL FINDINGS: We have produced a first catalogue of genes expressed in mouse slow-oxidative (type 1) and fast-glycolytic (type 2B) fibers through transcriptome analysis at the single fiber level (microgenomics). Individual fibers were obtained from murine soleus and EDL muscles and initially classified by myosin heavy chain isoform content. Gene expression profiling on high density DNA oligonucleotide microarrays showed that both qualitative and quantitative improvements were achieved, compared to results with standard muscle homogenate. First, myofiber profiles were virtually free from non-muscle transcriptional activity. Second, thousands of muscle-specific genes were identified, leading to a better definition of gene signatures in the two fiber types as well as the detection of metabolic and signaling pathways that are differentially activated in specific fiber types. Several regulatory proteins showed preferential expression in slow myofibers. Discriminant analysis revealed novel genes that could be useful for fiber type functional classification. CONCLUSIONS/SIGNIFICANCE: As gene expression analyses at the single fiber level significantly increased the resolution power, this innovative approach would allow a better understanding of the adaptive transcriptomic transitions occurring in myofibers under physiological and pathological conditions.

Posterior cricoarytenoid bellies: relationship between their function and histology.

OBJECTIVES/HYPOTHESIS: Complete physiological information about human posterior cricoarytenoid muscle (PCA) is essential and is not only of basic science interest but also could lead directly to understanding phonation and many clinical issues in neurolaryngology. The purpose of the study was to investigate and compare the histochemical and morphological properties to know contractile muscle fiber characteristics of two bellies of the PCA. STUDY DESIGN: Cross-sectional experimental study. METHODS: The PCAs were harvested from the total laryngectomy simples. Serial transverse sections of the two PCA bellies were performed and studied by immunohistochemical analysis. RESULTS: Two separate muscle bellies were always identified within 15 PCA. The following muscle fiber types were observed: I, I-IIA, and IIA. Comparisons of the vertical and horizontal bellies of the PCA reveled differences in the fiber-type composition. CONCLUSION: In our experience, the PCA should be considered as a combination of two functional subunits, which significantly differ in their muscle fiber-type composition.