Human Muscle Fibers Research Articles

Fukutin-Related Protein Resides in the Golgi Cisternae of Skeletal Muscle Fibres and Forms Disulfide-Linked Homodimers via an N-Terminal Interaction

Limb-Girdle Muscular Dystrophy type 2I (LGMD2I) is an inheritable autosomal, recessive disorder caused by mutations in the FuKutin-Related Protein (FKRP) gene (FKRP) located on chromosome 19 (19q13.3). Mutations in FKRP are also associated with Congenital Muscular Dystrophy (MDC1C), Walker-Warburg Syndrome (WWS) and Muscle Eye Brain disease (MEB). These four disorders share in common an incomplete/aberrant O-glycosylation of the membrane/extracellular matrix (ECM) protein α-dystroglycan. However, further knowledge on the FKRP structure and biological function is lacking, and its intracellular location is controversial. Based on immunogold electron microscopy of human skeletal muscle sections we demonstrate that FKRP co-localises with the middle-to-trans-Golgi marker MG160, between the myofibrils in human rectus femoris muscle fibres. Chemical cross-linking experiments followed by pairwise yeast 2-hybrid experiments, and co-immune precipitation, demonstrate that FKRP can exist as homodimers as well as in large multimeric protein complexes when expressed in cell culture. The FKRP homodimer is kept together by a disulfide bridge provided by the most N-terminal cysteine, Cys6. FKRP contains N-glycan of high mannose and/or hybrid type; however, FKRP N-glycosylation is not required for FKRP homodimer or multimer formation. We propose a model for FKRP which is consistent with that of a Golgi resident type II transmembrane protein.

Activation of skeletal muscle calpain-3 by eccentric exercise in humans does not result in its translocation to the nucleus or cytosol

The skeletal muscle-specific calpain-3 protease is likely involved in muscle repair, although the mechanism is not known. Physiological activation of calpain-3 occurs 24 h following eccentric exercise in humans. Functional consequences of calpain-3 activation are not known; however, calpain-3 has been suggested to be involved in nuclear signaling via NF-κB. To test this and help identify how/where calpain-3 acts, we investigated whether calpain-3 autolysis (hence, activation) following eccentric exercise results in translocation from its normal myofibrillar location to the nucleus or the cytosol. In resting human skeletal muscle, the majority (87%) of calpain-3 was present in myofibrillar fractions, with only a small proportion (<10%) in an autolyzed state. Enriched nuclear fractions contained ∼8% of the total calpain-3, which was present in a predominantly (>80%) autolyzed state. Using freshly dissected human muscle fibers to identify freely diffusible proteins, we showed that only ∼5% of the total calpain-3 pool was cytosolic. At 3 and 24 h following eccentric step exercise, there was an ∼70% increase in autolysis in whole muscle samples (n = 11, P < 0.05, by 1-way ANOVA with repeated measures and Newman-Keuls post hoc analysis). This exercise-induced autolysis was attributed to myofibrillar-bound calpain-3, since neither the amount of calpain-3 nor the proportion autolyzed was significantly changed in enriched nuclear or cytosolic fractions following the exercise intervention. We present a model for calpain-3 localization at rest and following activation in human skeletal muscle and suggest that the functional importance of calpain-3 remains predominantly tightly associated with its localization within the myofibrillar compartment.

Retraction: Exercise-induced metallothionein expression in human skeletal muscle fibres

Exercise induces free oxygen radicals that cause oxidative stress, and metallothioneins (MTs) are increased in states of oxidative stress and possess anti-apoptotic effects. We therefore studied expression of the antioxidant factors metallothionein I and II (MT-I + II) in muscle biopsies obtained in response to 3 h of bicycle exercise performed by healthy men and in resting controls. Both MT-I + II proteins and MT-II mRNA expression increased significantly in both type I and II muscle fibres after exercise. Moreover, 24 h after exercise the levels of MT-II mRNA and MT-I + II proteins were still highly increased and the MT-II mRNA expression reached a 15-fold increase. As expected, immunohistochemical detection of malondialdehyde (MDA) and nitrotyrosine (NITT) showed that formation of free radicals and oxidative stress were clearly increased in exercising muscle peaking shortly after the end of exercise in both type I and II muscle fibres. This is the first report demonstrating that MT-I + II are significantly induced in human skeletal muscle fibres following exercise. As MT-I + II are antioxidant factors that protect various tissues during pathological conditions, the MT-I + II increases post exercise may represent a mechanism whereby contracting muscle fibres are protected against cellular stress and injury.

S7.5 Effects of membrane polarisation on velocity recovery cycles of human muscle fibres in-vivo

S7.5 Effects of membrane polarisation on velocity recovery cycles of human muscle fibres in-vivo

Notice of Concern.

The editors of The FASEB Journal have been informed by Bente Klarlund Pedersen, MD, DMSc, Professor of Integrative Medicine, Director of Centre of Inflammation and Metabolism, Department of Infectious Diseases and CMRC Rigshospitalet at the University of Copenhagen Faculty of Health Sciences, Copenhagen, Denmark, of an ongoing investigation into the validity of the data contained in an article published in The FASEB Journal in 2003, which is part of a larger investigation involving other articles appearing in other journals. We await a final report from the investigating institution to learn the outcome of this investigation. The article is: Penkowa, M., Keller, C., Keller, P., Jauffred, S., and Pedersen, B. K. (2003) Immunohistochemical detection of interleukin-6 in human skeletal muscle fibers following exercise.

Multi-color Immunofluorescence Procedure for Rapid Identification of MHC Expression in Human Skeletal Muscle

We have previously outlined an immunofluorescence staining protocol for evaluating the fiber type composition of rat and mouse skeletal muscles. The main advantage of this protocol compared to typical myosin ATPase and immunoperoxidase procedures is that multiple myosin heavy chain (MHC) isoforms can be identified on a single cross-section, eliminating the need for multiple incubations. Additionally, detection of MHC expression directly allows for more accurate fiber type identification as differential pH-dependant inactivation of myosin ATPase has been noted across species. However, antibody cross-reactivity with human muscle must also be considered. PURPOSE: To examine the effectiveness of a rapid immunofluorescence fiber-typing procedure on human skeletal muscle. METHODS: Cross-sections of vastus lateralis muscle from eight subjects were incubated with antibodies against MHCI (BA-F8), IIa (SC-71 or 2F7), and IIx (6H1). An additional antibody, specific for all MHC isoforms except IIx (BF-35) was also used. Fiber-type specific oxidative and glycolytic capacity was measured by metabolic enzyme activity staining for succinate dehydrogenase (SDH) and glycerophosphate dehydrogenase (GPD), respectively. RESULTS: Fibers demonstrating strong reactivity with BA-F8 were classified as type I (48.9%). A considerable number of fibers stained intensely for SC-71 and 2F7 and were classified as type IIA (42.8%). A very small subset of fibers stained intermediate for both BA-F8 and SC-71 and were classified as type I/IIA (0.1%). A population of fibers displayed reactivity against 6H1; however, this population also showed intermediate staining for both MHCIIa antibodies. These fibers were identified either as pure type IIX based on their non-reactivity with BF-35 (5.6%), or as type IIAX hybrid fibers based on intermediate staining for both 6H1 and BF-35 (2.6%). Type I fibers were observed as being most oxidative and type IIX fibers as being most glycolytic, with hybrid fibers displaying enzyme activity between their respective pure counterparts. CONCLUSIONS: These results suggest that a multi-color approach using three commercially available antibodies simultaneously is effective for identifying both pure and hybrid fibers in human muscle.

Novel Application of Western Blotting for Human Skeletal Muscle Fiber Type Specific Protein Content Assessment

Little is known about protein profiles in slow-twitch (MHC I) and fast-twitch (MHC IIa and MHC IIx) muscle fiber types from human skeletal muscle. PURPOSE:To develop a method of assessing fiber type specific protein content with low amounts of protein via Western blotting following single fiber SDS-PAGE MHC identification. METHODS:Two hundred and sixty four individual muscle fibers from a muscle biopsy of the vastus lateralis were isolated. Approximately ¼ of each single fiber was clipped and fiber-typed based on MHC composition using SDS-PAGE combined with silver staining. The remaining ¾ was stored in individually labeled wells of an RNALater filled 96-well plate. Following MHC identification, the preserved portions were combined into pools of 20 like fibers. Homogenized pools of known fiber types were loaded onto Western blots and probed for GAPDH, Actin, and total p38 to establish reliable protein detection using this methodology. RESULTS:At low amounts of protein, good reliability of this method was shown as MHC I and MHC IIa pools loaded in quintuplicate displayed 11% and 4% variance in GAPDH expression, respectively. When the amount of sample loaded on the blot doubled, GAPDH expression doubled in both MHC I and MHC IIa samples, implying consistent transferring and equal protein loading. Actin content did not differ between fiber types and was therefore found to be an appropriate loading control. GAPDH content was 67% (MHC IIa) and 84% (MHC IIx) more abundant when compared to MHC I pools. Similarly, total p38 expression was 40% greater in MHC IIa fibers when compared to MHC I fibers. CONCLUSIONS:These data establish a novel application of Western blotting for fiber type specific protein quantification in human skeletal muscle. These initial results show content of specific proteins are 40-84% higher in fast-twitch muscle fibers as compared to slow-twitch muscle fibers (MHC IIx>MHC IIa>MHC I). Application of these methods can enhance our knowledge on fiber type specific differences in relation to skeletal muscle health profiles among physically active and clinically based populations.

Pharmacologic Specificity of Nicotinic Receptor Mediated Relaxation of Muscarinic Receptor Pre-Contracted Human Gastric Sling and Clasp Muscle Fibers

Pharmacologic Specificity of Nicotinic Receptor Mediated Relaxation of Muscarinic Receptor Pre-Contracted Human Gastric Sling and Clasp Muscle Fibers

Myosin‐actin cross‐bridge kinetics explain variation in single skeletal muscle fiber function in humans

Our objective was to determine if myosin-actin cross-bridge kinetics, measured in their intact myofilament lattice, could account for variation in skeletal muscle fiber type specific performance among healthy and diseased humans. We applied small amplitude sinusoidal analysis for the first time to human skeletal muscle fibers obtained from 9 heart failure patients and 6 controls of similar age and activity level. In both groups, myosin heavy chain (MHC) isoform contractile velocity (I<IIA<IIX) was inversely related to myosin-actin attachment time (ton). The rate of myosin transition between weakly- and strongly-bound states (2πb) increased with MHC velocity, suggesting the myosin-actin detachment time (toff) decreases with faster contractions. These results suggest that total myosin-actin cycle time (ton + toff) decreases with contractile velocity due to reductions in both ton and toff in humans. Heart failure patients had an increased ton, which compensated for their reduced cross-bridge number due to MHC content loss, resulting in similar isometric tensions compared to healthy controls. Collectively, these results indicate the usefulness of cross-bridge kinetics and highlight their potential use for examining the effects of disease, muscle activity and aging on single fiber function. Support: NIH HL-077418 and AG-031303

Relationship between force and size in human single muscle fibres

When the contractile properties of single muscle fibres are studied, force is typically normalized by fibre cross-sectional area and expressed as specific force. We studied a set of 2725 chemically skinned human single muscle fibres from 119 healthy adults to determine whether specific force is the optimal way to express the relationship between single-fibre force and size. A linear mixed effects model was used to estimate the slope and slope variability among individuals of log-log plots of force and diameter. For type I fibres, the slope estimate was 0.99 (95% confidence interval 0.36-1.62), and for type IIa fibres it was 0.94 (95% confidence interval 0.77-1.11), indicating that force is proportional to fibre diameter, rather than to cross-sectional area. If force were proportional to cross-sectional area, the slope estimate would be 2.0. In future studies using the chemically skinned single fibre preparation, force may be normalized to fibre diameter rather than cross-sectional area. We propose that a new term, 'normalized force', be used for this variable, with units of newtons per metre. We demonstrate using our data set that when populations of single fibres are compared with one another, the determination of whether the size and force relationship is the same or different is dependent upon the method used to account for fibre size (i.e. specific force versus 'normalized force').

Protection of human muscle acetylcholinesterase from soman by pyridostigmine bromide

Pretreatment with pyridostigmine bromide (PB) of human intercostal muscle fibers exposed to the irreversible acetylcholinesterase (AChE) inhibitor soman was investigated. Muscles were pretreated with 3 × 10(-6) M PB or saline for 20 minutes, then exposed to 10(-7) M soman for 10 minutes. AChE of muscles treated with soman alone was inhibited >95%. In contrast, PB pretreatment of soman-exposed bundles protected 20% of AChE activity. AChE of bundles exposed to PB alone recovered after 4 hours, but bundles exposed to both PB and soman did not. Soman-induced reduction of resting membrane potentials and increment of amplitudes and decay times of miniature endplate potentials (MEPPs) were partially corrected by PB pretreatment. In vitro pretreatment of human muscles with PB protected up to 20% of muscle AChE and ameliorated some deleterious effects on endplate physiology induced by soman.

Estimating the duration of intracellular action potentials in muscle fibres from single-fibre extracellular potentials

In situ recording of the intracellular action potential (IAP) of human muscle fibres is not yet possible, and consequently, knowledge concerning certain IAP characteristics is still limited. According to the core-conductor theory, close to a fibre, a single fibre action potential (SFAP) can be assumed to be proportional to the IAP second derivative. Thus, we might expect to be able to derive some characteristics of the IAP, such as the duration of its spike, from the SFAP waveform. However, SFAP properties not only depend on the IAP shape but also on the fibre-to-electrode (radial) distance and other physiological properties of the fibre. In this paper we, first, propose an SFAP parameter (the negative phase duration, NPD) appropriate for estimating the IAP spike duration and, second, show that this parameter is largely independent of changes in radial distance and muscle fibre propagation velocity. Estimation of the IAP spike duration from a direct measurement taken from the SFAP waveform provides a possible way to enhance the accuracy of SFAP models. Because IAP spike duration is known to be sensitive to the effects of fatigue and calcium accumulation, the proposed SFAP parameter, the NPD, has potential value in electrodiagnosis and as an indicator of IAP profile changes due to peripheral fatigue.

Force-Generating Capacity of Human Myosin Isoforms Extracted from Single Muscle Fiber Segments

Muscle, motor unit and muscle fibre type-specific differences in force-generating capacity have been investigated for many years, but there is still no consensus regarding specific differences between slow- and fast- twitch muscles, which may relate to a number of different confounding factors disguising the myosin function, i.e. the molecular motor protein. The aim of this study is to evaluate the force-generating capacity of specific myosin isoforms extracted from human muscle fiber segments in a modified single fiber in vitro motility assay, in which an internal load (α-actinin, actin-binding protein) was added in different concentrations to inhibit force generation of the myosin. After the negative linear relationship between the fraction of moving filaments and the α-actinin concentration was plotted, both the slope and x-axis intercept were used as force index to evaluate force production. The force-generating capacity of the β/slow myosin isoform (type I) was weaker (p<0.05) than the fast myosin isoform (type II), but the force-generating capacity of the different fast myosin isoforms types IIa, IIx or IIax were inseparable. In conclusion, (i) a significant difference in force-generating capacity was observed between human slow and fast myosin isoforms; (ii) the modified single fiber in vitro motility assay presents a unique possibility to measure the force-generating capacity of specific myosin isoforms, and also provides a platform for studies on myosin function in the growing disease entity called “myosinopathies” as well as in the different post-translational modifications.

When less is more: a simple Western blotting amendment allowing data acquisition on human single fibers

the subdivision of skeletal muscle into distinct fiber types based on myosin heavy chain (MHC) isoform expression [I, IIa, and IIx in humans; I, IIa, IIx, and IIb in rodents ([9][1])], has proven to be a practical and lasting working concept not only in studies of contractile properties but in the

Force-generating capacity of human myosin isoforms extracted from single muscle fibre segments

Muscle, motor unit and muscle fibre type-specific differences in force-generating capacity have been investigated for many years, but there is still no consensus regarding specific differences between slow- and fast-twitch muscles, motor units or muscle fibres. This is probably related to a number of different confounding factors disguising the function of the molecular motor protein myosin. We have therefore studied the force-generating capacity of specific myosin isoforms or combination of isoforms extracted from short single human muscle fibre segments in a modified single fibre myosin in vitro motility assay, in which an internal load (actin-binding protein) was added in different concentrations to evaluate the force-generating capacity. The force indices were the x-axis intercept and the slope of the relationship between the fraction of moving filaments and the α-actinin concentration. The force-generating capacity of the β/slow myosin isoform (type I) was weaker (P < 0.05) than the fast myosin isoform (type II), but the force-generating capacity of the different human fast myosin isoforms types IIa and IIx or a combination of both (IIax) were indistinguishable. A single fibre in vitro motility assay for both speed and force of specific myosin isoforms is described and used to measure the difference in force-generating capacity between fast and slow human myosin isoforms. The assay is proposed as a useful tool for clinical studies on the effects on muscle function of specific mutations or post-translational modifications of myosin.

Enhanced technique to measure proteins in single segments of human skeletal muscle fibers: fiber-type dependence of AMPK-α1 and -β1

Human physiological studies typically use skeletal muscle biopsies from the heterogeneous vastus lateralis muscle comprised of both fast-twitch and slow-twitch fiber types. It is likely that potential changes of physiological importance are overlooked because fiber-type specific responses may not be apparent in the whole muscle preparation. A technological advance in Western blotting is presented where proteins are analyzed in just one small segment (<2 mm) of individual fibers dissected from freeze-dried muscle samples using standard laboratory equipment. A significant advance is being able to classify every fiber at the level of both contractile (myosin heavy chain and tropomyosin) and sarcoplasmic reticulum [sarco(endo)plasmic reticulum Ca(2+)-ATPase type 1] properties and then being able to measure specific proteins in the very same segments. This removes the need to fiber type segments before further analyses and, as such, dramatically reduces the time required for sample collection. Compared with slow-twitch fibers, there was less AMP-activated protein kinase (AMPK)-α(1) (∼25%) and AMPK-β(1) (∼60%) in fast-twitch fibers from human skeletal muscle biopsies.

Chronic heart failure decreases cross-bridge kinetics in single skeletal muscle fibres from humans

Skeletal muscle function is impaired in heart failure patients due, in part, to loss of myofibrillar protein content, in particular myosin. In the present study, we utilized small-amplitude sinusoidal analysis for the first time in single human skeletal muscle fibres to measure muscle mechanics, including cross-bridge kinetics, to determine if heart failure further impairs contractile performance by altering myofibrillar protein function. Patients with chronic heart failure (n = 9) and controls (n = 6) were recruited of similar age and physical activity to diminish the potentially confounding effects of ageing and muscle disuse. Patients showed decreased cross-bridge kinetics in myosin heavy chain (MHC) I and IIA fibres, partially due to increased myosin attachment time (t(on)). The increased t(on) compensated for myosin protein loss previously found in heart failure patients by increasing the fraction of the total cycle time myosin is bound to actin, resulting in a similar number of strongly bound cross-bridges in patients and controls. Accordingly, isometric tension did not differ between patients and controls in MHC I or IIA fibres. Patients also had decreased calcium sensitivity in MHC IIA fibres and alterations in the viscoelastic properties of the lattice structure of MHC I and IIA fibres. Collectively, these results show that heart failure alters skeletal muscle contraction at the level of the myosin-actin cross-bridge, leading to changes in muscle mechanics which could contribute to impaired muscle function. Additionally, we uncovered a unique kinetic property of MHC I fibres, a potential indication of two distinct populations of cross-bridges, which may have important physiological consequences.

Prolonged space flight-induced alterations in the structure and function of human skeletal muscle fibres

The primary goal of this study was to determine the effects of prolonged space flight (180 days) on the structure and function of slow and fast fibres in human skeletal muscle. Biopsies were obtained from the gastrocnemius and soleus muscles of nine International Space Station crew members 45 days pre- and on landing day (R+0) post-flight. The main findings were that prolonged weightlessness produced substantial loss of fibre mass, force and power with the hierarchy of the effects being soleus type I > soleus type II > gastrocnemius type I > gastrocnemius type II. Structurally, the quantitatively most important adaptation was fibre atrophy, which averaged 20% in the soleus type I fibres (98 to 79 μm diameter). Atrophy was the main contributor to the loss of peak force (P(0)), which for the soleus type I fibre declined 35% from 0.86 to 0.56 mN. The percentage decrease in fibre diameter was correlated with the initial pre-flight fibre size (r = 0.87), inversely with the amount of treadmill running (r = 0.68), and was associated with an increase in thin filament density (r = 0.92). The latter correlated with reduced maximal velocity (V(0)) (r = 0.51), and is likely to have contributed to the 21 and 18% decline in V(0) in the soleus and gastrocnemius type I fibres. Peak power was depressed in all fibre types with the greatest loss (55%) in the soleus. An obvious conclusion is that the exercise countermeasures employed were incapable of providing the high intensity needed to adequately protect fibre and muscle mass, and that the crew's ability to perform strenuous exercise might be seriously compromised. Our results highlight the need to study new exercise programmes on the ISS that employ high resistance and contractions over a wide range of motion to mimic the range occurring in Earth's 1 g environment.

Calcium-activated force of human muscle fibers following a standardized eccentric contraction

Peak Ca(2+)-activated specific force (force/fiber cross-sectional area) of human chemically skinned vastus lateralis muscle fiber segments was determined before and after a fixed-end contraction or an eccentric contraction of standardized magnitude (+0.25 optimal fiber length) and velocity (0.50 unloaded shortening velocity). Fiber myosin heavy chain (MHC) isoform content was assayed by SDS-PAGE. Posteccentric force deficit, a marker of damage, was similar for type I and IIa fibers but threefold greater for type IIa/IIx hybrid fibers. A fixed-end contraction had no significant effect on force. Multiple linear regression revealed that posteccentric force was explained by a model consisting of a fiber type-independent and a fiber type-specific component (r(2) = 0.91). Preeccentric specific force was directly associated with a greater posteccentric force deficit. When preeccentric force was held constant, type I and IIa fibers showed identical susceptibility to damage, while type IIa/IIx fibers showed a significantly greater force loss. This heightened sensitivity to damage was directly related to the amount of type IIx MHC in the hybrid fiber. Our model reveals a fiber-type sensitivity of the myofilament lattice or cytoskeleton to mechanical strain that can be described as follows: type IIa/IIx > type IIa = type I. If these properties extend to fibers in vivo, then alterations in the number of type IIa/IIx fibers may modify a muscle's susceptibility to eccentric damage.

Impaired Autophagy in Sporadic Inclusion-Body Myositis and in Endoplasmic Reticulum Stress-Provoked Cultured Human Muscle Fibers

The hallmark pathologies of sporadic inclusion-body myositis (s-IBM) muscle fibers are autophagic vacuoles and accumulation of ubiquitin-positive multiprotein aggregates that contain amyloid-beta or phosphorylated tau in a beta-pleated sheet amyloid configuration. Endoplasmic reticulum stress (ERS) and 26S proteasome inhibition, also associated with s-IBM, putatively aggrandize the accumulation of misfolded proteins. However, autophagosomal-lysosomal pathway formation and function, indicated by autophagosome maturation, have not been previously analyzed in this system. Here we studied the autophagosomal-lysosomal pathway using 14 s-IBM and 30 disease control and normal control muscle biopsy samples and our cultured human muscle fibers in a microenvironment modified to resemble aspects of s-IBM pathology. We report for the first time that in s-IBM, lysosomal enzyme activities of cathepsin D and B were decreased 60% (P < 0.01) and 40% (P < 0.05), respectively. We also detected two indicators of increased autophagosome maturation, the presence of LC3-II and decreased mammalian target of rapamycin-mediated phosphorylation of p70S6 kinase. Moreover, in cultured human muscle fibers, ERS induction significantly decreased activities of cathepsins D and B, increased levels of LC3-II, decreased phosphorylation of p70S6 kinase, and decreased expression of VMA21, a chaperone for assembly of lysosomal V-ATPase. We conclude that in s-IBM muscle, decreased lysosomal proteolytic activity might enhance accumulation of misfolded proteins, despite increased maturation of autophagosomes, and that ERS is a possible cause of s-IBM-impaired lysosomal function. Thus, unblocking protein degradation in s-IBM muscle fibers may be a desirable therapeutic strategy.