Single Human Muscle Fibers Research Articles

Revisiting specific force loss in human permeabilised single skeletal muscle fibres obtained from older individuals.

Specific force (SF) has been shown to be reduced in some but not all studies of human ageing using chemically skinned single muscle fibres. This may be due in part to the health status/physical activity levels of different older cohorts, but also from methodological differences in studying skinned fibres. The aim of the present study was to compare SF in fibres from older hip fracture patients (HFP), healthy master cyclists (MC) and healthy non-trained young adults (YA) using two different activating solutions. Quadriceps muscle samples and 316 fibres were obtained from HFPs (74.6±4 years, n=5), MCs (74.8±1, n=5) and YA (25.5±2, n=6). Fibres were activated (pCa 4.5, 15oC) in solutions containing either 60 mM TES or 20 mM imidazole. SF was determined by normalising force to fibre CSA assuming either an elliptical or circular shape and to fibre myosin heavy chain content. Activation in TES resulted in significantly higher MHC-I SF in all groups and YA MHC-IIA fibres, irrespective of normalisation method. Whilst there were no differences in SF between the participant groups, the ratio of SF between the TES and imidazole solutions was lower in HFPs compared to YAs (MHC-I p<0.05; MHC-IIA p=0.055). Activating solution composition, as opposed to donor characteristics, had a more notable effect on single fibre SF. However, this two-solution approach revealed an age-related difference in sensitivity in HFPs which was not shown in MCs. This suggests further novel approaches may be required to probe age/activity-related differences in muscle contractile quality.

Sphingomyelinase activity promotes atrophy and attenuates force in human muscle fibres and is elevated in heart failure patients.

BackgroundActivation of sphingomyelinase (SMase) as a result of a general inflammatory response has been implicated as a mechanism underlying disease‐related loss of skeletal muscle mass and function in several clinical conditions including heart failure. Here, for the first time, we characterize the effects of SMase activity on human muscle fibre contractile function and assess skeletal muscle SMase activity in heart failure patients.MethodsThe effects of SMase on force production and intracellular Ca2+ handling were investigated in single intact human muscle fibres. Additional mechanistic studies were performed in single mouse toe muscle fibres. RNA sequencing was performed in human muscle bundles exposed to SMase. Intramuscular SMase activity was measured from heart failure patients (n = 61, age 69 ± 0.8 years, NYHA III‐IV, ejection fraction 25 ± 1.0%, peak VO2 14.4 ± 0.6 mL × kg × min) and healthy age‐matched control subjects (n = 10, age 71 ± 2.2 years, ejection fraction 60 ± 1.2%, peak VO2 25.8 ± 1.1 mL × kg × min). SMase activity was related to circulatory factors known to be associated with progression and disease severity in heart failure.ResultsSphingomyelinase reduced muscle fibre force production (−30%, P < 0.05) by impairing sarcoplasmic reticulum (SR) Ca2+ release (P < 0.05) and reducing myofibrillar Ca2+ sensitivity. In human muscle bundles exposed to SMase, RNA sequencing analysis revealed 180 and 291 genes as up‐regulated and down‐regulated, respectively, at a FDR of 1%. Gene‐set enrichment analysis identified ‘proteasome degradation’ as an up‐regulated pathway (average fold‐change 1.1, P = 0.008), while the pathway ‘cytoplasmic ribosomal proteins’ (average fold‐change 0.8, P < 0.0001) and factors involving proliferation of muscle cells (average fold‐change 0.8, P = 0.0002) where identified as down‐regulated. Intramuscular SMase activity was ~20% higher (P < 0.05) in human heart failure patients than in age‐matched healthy controls and was positively correlated with markers of disease severity and progression, and with several circulating inflammatory proteins, including TNF‐receptor 1 and 2. In a longitudinal cohort of heart failure patients (n = 6, mean follow‐up time 2.5 ± 0.2 years), SMase activity was demonstrated to increase by 30% (P < 0.05) with duration of disease.ConclusionsThe present findings implicate activation of skeletal muscle SMase as a mechanism underlying human heart failure‐related loss of muscle mass and function. Moreover, our findings strengthen the idea that SMase activation may underpin disease‐related loss of muscle mass and function in other clinical conditions, acting as a common patophysiological mechanism for the myopathy often reported in diseases associated with a systemic inflammatory response.

Single skeletal muscle fiber mechanical properties: a muscle quality biomarker of human aging.

Skeletal muscle strength, mass, and function should be carefully monitored for signs of decline with advanced adult age. An understanding of the pathophysiology and severity of sarcopenia can be improved with the exploration of changes in muscle fiber properties. Furthermore, although functional decline with increase age is a well-known phenomenon, the mechanisms underlying this decline, and the features that characterize it, are complex and variable. The age-related decline of muscle function is a result of not only a decrease of muscle mass but also a decline in the intrinsic properties of muscle fibers that are independent of size. We believe it is important to understand changes in muscle quality (force adjusted for size), and not to focus solely on muscle mass, because muscle quality is closely related to measurements of function and could potentially predict clinical outcomes such as morbidity, disability, and mortality. Neurological and metabolic mechanisms contribute to muscle quality, but the intrinsic properties of muscle cells are central to the maintenance of force-generating capacity. Muscle quality can be evaluated with the assessment of morphological, physiological, and mechanical properties in single permeabilized or skinned fibers. This approach excludes the influence of the nervous system, tendons, and the extracellular matrix. In this review, we summarized the changes in active and passive mechanical properties at the single muscle cell level in older skeletal muscles. We argue that intrinsic mechanical changes in human single muscle fibers are useful biomarkers and indicators of muscle quality.

In Vivo Fatigue Reduces In Vitro Performance In Human Skeletal Muscle Fibers

Muscle fatigue, the acute reduction in muscle force-generating capacity associated withrepeated or prolonged activation, derives from the accumulation of intracellular metabolites.However, reduced contractile performance (low-frequency force depression) persists long afterrecovery of metabolic homeostasis. Few studies have investigated in vivo fatigue on in vitromyofilament function in humans and the potential role of post-translational proteinmodification in this phenomenon. Therefore, the PURPOSE of this study was to examine theeffect of in vivo fatigue on the phosphorylation status of sarcomeric proteins and contractileperformance of human single muscle fibers. METHODS: In two separate studies, seven healthyadults performed repeated knee extensions until task failure with their dominant leg.Immediately following, bilateral, percutaneous biopsy of the vastus lateralis muscle wasperformed on the fatigued and non-fatigued (control) limbs. Tissue was divided and portionswere blotted, snap frozen, and stored at -80 °C until analysis by high resolution massspectrometry or prepared for single fiber mechanical analysis. Mass spec data were assessedusing edgeR while mechanical outcomes were assed via 2-way ANOVA using SPSS. RESULTS: Mass spectrometry confirmed differential phosphorylation of multiple residues in sarcomericproteins, including regulatory light chain and troponin I. We also observed phospho-enrichmentof myosin binding protein C (MyBP-C) and tropomyosin (β-Tm), and a reduction in titinphosphorylation. Mechanical analysis showed that fatigued fibers produced significantly lesspower (8.0 ± 6.2 mN/mm2) than controls (5.9 ± 3.9 mN/mm2; p = 0.05). Differences in tension(p = 0.11) or velocity (p = 0.11) relative to non-fatigued fibers were not significant. No MHCisoform by fatigue interactions were observed. CONCLUSION: These data suggest fatiguingcontractions alter phosphorylation of select sarcomeric proteins and alter contractileperformance in isolated single muscle fibers regardless of intracellular environment, supportingthe notion that altered contractile protein function potentiates in vivo fatigue and maycontribute to longer-lasting contractile deficits.

Rate of force development is Ca2+-dependent and influenced by Ca2+-sensitivity in human single muscle fibres from older adults

Natural adult aging is associated with declines in skeletal muscle performance, including impaired Ca2+ sensitivity and a slowing of rapid force production (rate of force redevelopment; ktr). The purpose of this study was to investigate the relationship between impaired Ca2+ sensitivity and ktr of single muscle fibres from young and older adults. Participants included 8 young (22-35yrs) and 8 older (60-81yrs) males who were living independently. A percutaneous muscle biopsy of the vastus lateralis of each participant was performed. Single muscle fibre mechanical tests included maximal Ca2+-activated force (Po), force-pCa curves, and ktr. We showed a decrease in pCa50 in old type II fibres compared to young, indicating impaired Ca2+ sensitivity in older adults. The ktr behaved in a Ca2+-dependent manner such that with increasing [Ca2+], ktr increases, to a plateau. Interestingly, ktr was not different between young and old muscle fibres. Furthermore, we found strong associations between pCa50 and ktr in both old type I and type II fibres, such that those fibres with lower Ca2+ sensitivity had a slowed ktr. This Ca2+ association, combined with impaired Ca2+ handling in older adults suggests a potential Ca2+-dependent mechanism affecting the transition from weakly- to strongly-bound cross-bridge states, leading to a decline in skeletal muscle performance. Future research is needed to explore the role alterations to Ca2+ sensitivity/handling could be playing in age-related whole muscle performance declines.

Spatial Proteomic Approach to Characterize Skeletal Muscle Myofibers.

Skeletal muscle myofibers have differential protein expression resulting in functionally distinct slow- and fast-twitch types. While certain protein classes are well-characterized, the depth of all proteins involved in this process is unknown. We utilized the Human Protein Atlas (HPA) and the HPASubC tool to classify mosaic expression patterns of staining across 49,600 unique tissue microarray (TMA) images using a visual proteomic approach. We identified 2164 proteins with potential mosaic expression, of which 1605 were categorized as "likely" or "real." This list included both well-known fiber-type-specific and novel proteins. A comparison of the 1605 mosaic proteins with a mass spectrometry (MS)-derived proteomic dataset of single human muscle fibers led to the assignment of 111 proteins to fiber types. We additionally used a multiplexed immunohistochemistry approach, a multiplexed RNA-ISH approach, and STRING v11 to further assign or suggest fiber types of newly characterized mosaic proteins. This visual proteomic analysis of mature skeletal muscle myofibers greatly expands the known repertoire of twitch-type-specific proteins.

Novel Method To Visualize AMPK Protein Localization In Single Human Muscle Fibers Via Confocal Microscopy

PURPOSE: AMP-activated protein kinase (AMPK) is the energy regulator of skeletal muscle cells. Current methods can identify the magnitude of AMPK expression in skeletal muscle cells via Western blotting and Capillary Nano-Immunoassay (CNIA); however, these methods lack the ability to visually identify AMPK localization within single muscle fibers. Identifying AMPK in human muscle is important because it is involved in various exercise training adaptations such as mitochondrial biogenesis and glucose transport. Therefore, we aimed to develop a novel confocal microscopy method to identify AMPK protein expression (relative intensity) and localization within human single muscle fibers. METHODS: A vastus lateralis muscle biopsy was obtained from a healthy male and immediately fixed (4% PFA). Twenty fibers were isolated, placed on microscope slides, incubated in 0.1% Triton (15min), then incubated in 5% normal goat serum (blocking solution; 4h). This was followed by exposure to a 1 antibody (Ab) (anti-AMPKα2) in 5% bovine serum albumin (14h at 4°C). Fibers were then exposed to a 2 Ab (anti-rabbit IgG conjugated w/AlexaFluor 488) and phalloidin (AlexaFluor 568) to label actin (2h). Finally, fibers were mounted under coverslips with AntiFade Gold w/DAPI for myonuclei detection. Confocal microscopy imaging was conducted using a Zeiss LSM 710 with 63x plan apochromatic objective (oil emersion). Images were processed via ImageJ. RESULTS: Muscle fiber contractile proteins (actin; red), myonuclei (blue), and AMPK proteins (green) were successfully visually identified at rest (AMPK fluorescence intensity = 1199.64 + 630 AU). To ensure that no auto-fluorescence or non-specific binding was observed, images were compared to control slides: 1) DAPI only, 2) 1 Ab only, 3) 2 Ab only, and 4) no staining. CONCLUSION: These methods allow for the successful visualization (relative intensity) and localization of AMPK proteins within single human muscle fibers. This method could be used in future research to investigate the response and myonuclear co-localization of AMPK following exercise in human skeletal muscle to elucidate how they may play a role in these physiological processes.

Fiber Type and Size as Sources of Variation in Human Single Muscle Fiber Passive Elasticity.

Studies on single muscle fiber passive material properties often report relatively large variation in elastic modulus (or normalized stiffness), and it is not clear where this variation arises. This study was designed to determine if the stiffness, normalized to both fiber cross-sectional area and length, is inherently different between types 1 and 2 muscle fibers. Vastus lateralis fibers (n = 93), from ten young men, were mechanically tested using a cumulative stretch-relaxation protocol. SDS-PAGE classified fibers as types 1 or 2. While there was a difference in normalized stiffness between fiber types (p = 0.0019), an unexpected inverse relationship was found between fiber diameter and normalized stiffness (r = -0.64; p < 0.001). As fiber type and diameter are not independent, a one-way analysis of covariance (ANCOVA) including fiber diameter as a covariate was run; this eliminated the effect of fiber type on normalized stiffness (p = 0.1935). To further explore the relationship between fiber size and elastic properties, we tested whether stiffness was linearly related to fiber cross-sectional area, as would be expected for a homogenous material. Passive stiffness was not linearly related to fiber area (p < 0.001), which can occur if single muscle fibers are better represented as composite materials. The rule of mixtures for composite materials was used to explore whether the presence of a stiff perimeter-based fiber component could explain the observed results. The model (R2 = 0.38) predicted a perimeter-based normalized stiffness of 8800 ± 2600 kPa/μm, which is within the range of basement membrane moduli reported in the literature.

Single-cell transcriptional profiles in human skeletal muscle

Skeletal muscle is a heterogeneous tissue comprised of muscle fiber and mononuclear cell types that, in addition to movement, influences immunity, metabolism and cognition. We investigated the gene expression patterns of skeletal muscle cells using RNA-seq of subtype-pooled single human muscle fibers and single cell RNA-seq of mononuclear cells from human vastus lateralis, mouse quadriceps, and mouse diaphragm. We identified 11 human skeletal muscle mononuclear cell types, including two fibro-adipogenic progenitor (FAP) cell subtypes. The human FBN1+ FAP cell subtype is novel and a corresponding FBN1+ FAP cell type was also found in single cell RNA-seq analysis in mouse. Transcriptome exercise studies using bulk tissue analysis do not resolve changes in individual cell-type proportion or gene expression. The cell-type gene signatures provide the means to use computational methods to identify cell-type level changes in bulk studies. As an example, we analyzed public transcriptome data from an exercise training study and revealed significant changes in specific mononuclear cell-type proportions related to age, sex, acute exercise and training. Our single-cell expression map of skeletal muscle cell types will further the understanding of the diverse effects of exercise and the pathophysiology of muscle disease.

Fibre and extracellular matrix contributions to passive forces in human skeletal muscles: An experimental based constitutive law for numerical modelling of the passive element in the classical Hill-type three element model.

The forces that allow body movement can be divided into active (generated by sarcomeric contractile proteins) and passive (sustained by intra-sarcomeric proteins, fibre cytoskeleton and extracellular matrix (ECM)). These are needed to transmit the active forces to the tendon and the skeleton. However, the relative contribution of the intra- and extra- sarcomeric components in transmitting the passive forces is still under debate. There is limited data in the literature about human muscle and so it is difficult to make predictions using multiscale models, imposing a purely phenomenological description for passive forces. In this paper, we apply a method for the experimental characterization of the passive properties of fibres and ECM to human biopsy and propose their clear separation in a Finite Element Model. Experimental data were collected on human single muscle fibres and bundles, taken from vastus lateralis muscle of elderly subjects. Both were progressively elongated to obtain two stress-strain curves which were fitted to exponential equations. The mechanical properties of the extracellular passive components in a bundle of fibres were deduced by the subtraction of the passive tension observed in single fibres from the passive tension observed in the bundle itself. Our results showed that modulus and tensile load bearing capability of ECM are higher than those of fibres and defined their quantitative characterization that can be used in macroscopic models to study their role in the transmission of forces in physiological and pathophysiological conditions.

Sex- and fiber-type-related contractile properties in human single muscle fiber.

This study aimed to examine the distribution and contractile properties of single muscle fiber sex/myosin heavy chain (MHC) type-related differences and to evaluate the correlation of cross-sectional area (CSA) and specific force (SF) in a single muscle fiber. Six young men and six young women were participated in this study. Muscle sample was obtained from vastus lateralis muscle. To examine potential gender differences within each fiber contractile properties (CSA, maximal isometric force, SF, maximal shortening velocity) and relationship between CSA and SF of single fiber using Pearson correlation. After mechanical measurements, single muscle fiber determined MHC isoforms using silver stain. MHC isoform composition did not differ by sex (chi-square=6.978, P=0.073). There were sex-related differences in CSA and maximal isometric force (P<0.05), but no fiber type-related differences (P>0.05). Related to SF and maximal shortening velocity, there were no sex-related differences only fiber type-related differences (P<0.05). However, there were differences in SF between single fiber types in men but not in women. A negative correlation was found between CSA and SF in both men and women (P<0.05). It is suggested that there might be different mechanical properties of cross-bridges according to sex.

Residual force enhancement and force depression in human single muscle fibres

Residual force depression (rFD) and residual force enhancement (rFE) are intrinsic contractile properties of muscle. rFD is characterized as a decrease in steady-state isometric force following active shortening compared with a purely isometric contraction at the same muscle length and level of activation. By contrast, isometric force is increased following active lengthening compared to a reference isometric contraction at the same muscle length and level of activation; this is termed rFE. To date, there have been no investigations of rFD and rFE in human muscle fibres, therefore the purpose of this study was to determine whether rFD and rFE occur at the single muscle fibre level in humans. rFD and rFE were investigated in maximally activated single muscle fibres biopsied from the vastus lateralis of healthy adults. To induce rFD, fibres were activated and shortened from an average sarcomere length (SL) of 3.2–2.6 μm. Reference isometric contractions were performed at an average SL of 2.6 μm. To induce rFE, fibres were actively lengthened from an average SL of 2.6–3.2 μm and a reference isometric contraction was performed at an average SL of 3.2 μm. Isometric steady-state force was lower following active shortening (p < 0.05), and higher following active lengthening (p < 0.05), as compared to the reference isometric contractions. We demonstrated rFD and rFE in human single fibres which is consistent with previous animal models. The non-responder phenomenon often reported in rFE studies involving voluntary contractions at the whole human level was not observed at the single fibre level.

PL - 039 Heat Shock Proteins in human single skeletal muscle fibres resist age associated alterations and differentially respond to high-intensity exercise training

Objective Heat shock proteins (HSPs) are ubiquitously expressed proteins that help preserve cellular homeostasis. Within mammalian skeletal muscle three of the better characterised HSPs are HSP72, HSP27 and αB-crystallin. Among other roles, these three HSPs are involved in regulation of muscle mass and function and may be of importance in ageing. HSP’s are fibre-type dependent in rat skeletal muscle and thus examining these proteins in humans should be completed on the single fibre level, particularly in ageing where maladaptations primarily occur in Type II fibres. High-Intensity Training (HIT) is a commonly used method to improve muscle health and function in the elderly, but HSP adaptability to training has not yet been investigated. &#x0D; Methods This study examined isolated single muscle fibre segments collected from freeze-dried vastus lateralis muscle samples from young (25 /- 3 year old) and older (70 /- 4 year old) healthy individuals. A further sample was collected from the older individuals following 12 weeks of HIT, where they performed 4 x 4 min @ ~90-95% of peak heart rate (HR), with 4 min active recovery at 50-60% peak HR&#x0D; Results Basal expression of HSP’s in skeletal muscle: HSP70 tended to be higher in Type I fibres compared to Type II in young adults (p=0.08) and was higher in Type I compared to Type II fibres of older adults (p=0.03). HSP27 abundance was higher in Type I fibres compared to Type II in young adults (p=0.01) and tended to be higher in Type I compared to Type II fibres in older adults (p=0.07). The abundance of αβ-crystallin was more abundant in Type I fibres compared to Type II in both young and older adults (p&lt;0.05). Preliminary data revealed that the abundance of pABCser59 and pHSP2782 displayed no fibre-type specific abundances in either young or older adults.&#x0D; Age effects on HSP’s: There was no difference in the abundance of HSP70, HSP27, ABC or pHSP2782 between young and older adults in either Type I or Type II fibres. There was an increase in the abundance of pABCser59 in Type I fibres in older adults compared to Type I fibres of young adults (p=0.03), with no difference in Type II fibres.&#x0D; Effects of HIT on HSP’s: HIT in the older individuals increased the abundance of HSP70 in Type I fibres (p&lt;0.01) but not Type II. HIT tended to decrease the abundance of HSP27 in Type I fibres (0.92±0.66, p=0.06) and tended to increase the abundance of αβ-crystallin in Type I fibres (1.03±1.51 p=0.07).&#x0D; Conclusions These results revealed that in healthy, older individuals, the basal levels of HSP27, ABC or pHSP2782 are not different to those in young adults in either Type I or Type II fibres. This could indicate that the muscle from the older individuals was not compromised. Interestingly, in response to HIT there were varying changes between these HSP’s, and of note these occurred only in Type I fibres. Given that during HIT Type II fibres would be activated to a greater extent, it appears that the recovery phases of the HIT were most responsive to HSPs.

From single muscle fiber to whole muscle mechanics: a finite element model of a muscle bundle with fast and slow fibers.

Muscles exhibit highly complex, multi-scale architecture with thousands of muscle fibers, each with different properties, interacting with each other and surrounding connective structures. Consequently, the results of single-fiber experiments are scarcely linked to the macroscopic or whole muscle behavior. This is especially true for human muscles where it would be important to understand of how skeletal muscles disorders affect patients' life. In this work, we developed a mathematical model to study how fast and slow muscle fibers, well characterized in single-fiber experiments, work and generate together force and displacement in muscle bundles. We characterized the parameters of a Hill-type model, using experimental data on fast and slow single human muscle fibers, and comparing experimental data with numerical simulations obtained from finite element (FE) models of single fibers. Then, we developed a FE model of a bundle of 19 fibers, based on an immunohistochemically stained cross section of human diaphragm and including the corresponding properties of each slow or fast fiber. Simulations of isotonic contractions of the bundle model allowed the generation of its apparent force-velocity relationship. Although close to the average of the force-velocity curves of fast and slow fibers, the bundle curve deviates substantially toward the fast fibers at low loads. We believe that the present model and the characterization of the force-velocity curve of a fiber bundle represents the starting point to link the single-fiber properties to those of whole muscle with FE application in phenomenological models of human muscles.

Repeated high-intensity exercise modulates Ca(2+) sensitivity of human skeletal muscle fibers.

The effects of short-term high-intensity exercise on single fiber contractile function in humans are unknown. Therefore, the purposes of this study were: (a) to access the acute effects of repeated high-intensity exercise on human single muscle fiber contractile function; and (b) to examine whether contractile function was affected by alterations in the redox balance. Eleven elite cross-country skiers performed four maximal bouts of 1300 m treadmill skiing with 45 min recovery. Contractile function of chemically skinned single fibers from triceps brachii was examined before the first and following the fourth sprint with respect to Ca(2+) sensitivity and maximal Ca(2+) -activated force. To investigate the oxidative effects of exercise on single fiber contractile function, a subset of fibers was incubated with dithiothreitol (DTT) before analysis. Ca(2+) sensitivity was enhanced by exercise in both MHC I (17%, P < 0.05) and MHC II (15%, P < 0.05) fibers. This potentiation was not present after incubation of fibers with DTT. Specific force of both MHC I and MHC II fibers was unaffected by exercise. In conclusion, repeated high-intensity exercise increased Ca(2+) sensitivity in both MHC I and MHC II fibers. This effect was not observed in a reducing environment indicative of an exercise-induced oxidation of the human contractile apparatus.

Three‐dimensional reconstruction of the human skeletal muscle mitochondrial network as a tool to assess mitochondrial content and structural organization

Mitochondria undergo continuous changes in shape as result of complex fusion and fission processes. The physiological relevance of mitochondrial dynamics is still unclear. In the field of mitochondria bioenergetics, there is a need of tools to assess cell mitochondrial content. To develop a method to visualize mitochondrial networks in high resolution and assess mitochondrial volume. Confocal fluorescence microscopy imaging of mitochondrial network stains in human vastus lateralis single muscle fibres and focused ion beam/ scanning electron microscopy (FIB/SEM) imaging, combined with 3D reconstruction was used as a tool to analyse mitochondrial morphology and measure mitochondrial fractional volume. Most type I and type II muscle fibres have tubular highly interconnected profusion mitochondria, which are thicker and more structured in type I muscle fibres (Fig. 1). In some muscle fibres, profission-isolated ellipsoid-shaped mitochondria were observed. Mitochondrial volume was significantly higher in type I muscle fibres and showed no correlation with any of the investigated molecular and biochemical mitochondrial measurements (Fig. 2). Three-dimensional reconstruction of FIB/SEM data sets shows that some subsarcolemmal mitochondria are physically interconnected with some intermyofibrillar mitochondria (Fig. 3). Two microscopy methods to visualize skeletal muscle mitochondrial networks in 3D are described and can be used as tools to investigate mitochondrial dynamics in response to life-style interventions and/or in certain pathologies. Our results question the classification of mitochondria into subsarcolemmal and intermyofibrillar pools, as they are physically interconnected.

Transient impairments in single muscle fibre contractile function after prolonged cycling in elite endurance athletes

Prolonged muscle activity impairs whole-muscle performance and function. However, little is known about the effects of prolonged muscle activity on the contractile function of human single muscle fibres. The purpose of this study was to investigate the effects of prolonged exercise and subsequent recovery on the contractile function of single muscle fibres obtained from elite athletes. Nine male triathletes (26±1years, 68±1mL O2 min(-1) kg(-1) , training volume 16±1hweek(-1) ) performed 4h of cycling exercise (at 73% of HRmax ) followed by 24h of recovery. Biopsies from vastus lateralis were obtained before and following 4h exercise and following 24h recovery. Measurements comprised maximal Ca(2+) -activated specific force and Ca(2+) sensitivity of slow type I and fast type II single muscle fibres, as well as cycling peak power output. Following cycling exercise, specific force was reduced to a similar extent in slow and fast fibres (-15 and -18%, respectively), while Ca(2+) sensitivity decreased in fast fibres only. Single fibre-specific force was fully restored in both fibre types after 24h recovery. Cycling peak power output was reduced by 4-9% following cycling exercise and fully restored following recovery. This is the first study to demonstrate that prolonged cycling exercise transiently impairs specific force in type I and II fibres and decreases Ca(2+) sensitivity in type II fibres only, specifically in elite endurance athletes. Further, the changes in single fibre-specific force induced by exercise and recovery coincided temporally with changes in cycling peak power output.

Role of alpha-actinin-3 in contractile properties of human single muscle fibers: a case series study in paraplegics.

A common nonsense polymorphism in the ACTN3 gene results in the absence of α-actinin-3 in XX individuals. The wild type allele has been associated with power athlete status and an increased force output in numeral studies, though the mechanisms by which these effects occur are unclear. Recent findings in the Actn3−/− (KO) mouse suggest a shift towards ‘slow’ metabolic and contractile characteristics of fast muscle fibers lacking α-actinin-3. Skinned single fibers from the quadriceps muscle of three men with spinal cord injury (SCI) were tested regarding peak force, unloaded shortening velocity, force-velocity relationship, passive tension and calcium sensitivity. The SCI condition induces an ‘equal environment condition’ what makes these subjects ideal to study the role of α-actinin-3 on fiber type expression and single muscle fiber contractile properties. Genotyping for ACTN3 revealed that the three subjects were XX, RX and RR carriers, respectively. The XX carrier’s biopsy was the only one that presented type I fibers with a complete lack of type IIx fibers. Properties of hybrid type IIa/IIx fibers were compared between the three subjects. Absence of α-actinin-3 resulted in less stiff type IIa/IIx fibers. The heterozygote (RX) exhibited the highest fiber diameter (0.121±0.005 mm) and CSA (0.012±0.001 mm2) and, as a consequence, the highest peak force (2.11±0.14 mN). Normalized peak force was similar in all three subjects (P = 0.75). Unloaded shortening velocity was highest in R-allele carriers (P<0.001). No difference was found in calcium sensitivity. The preservation of type I fibers and the absence of type IIx fibers in the XX individual indicate a restricted transformation of the muscle fiber composition to type II fibers in response to long-term muscle disuse. Lack of α-actinin-3 may decrease unloaded shortening velocity and increase fiber elasticity.

A myopathy-related actin mutation increases contractile function

Nemaline myopathy (NM) is the most common congenital myopathy and is caused by mutations in various genes including NEB (nebulin), TPM2 (beta-tropomyosin), TPM3 (gamma-tropomyosin), and ACTA1 (skeletal alpha-actin). 20-25% of NM cases carry ACTA1 defects and these particular mutations usually induce substitutions of single residues in the actin protein. Despite increasing clinical and scientific interest, the contractile consequences of these subtle amino acid substitutions remain obscure. To decipher them, in the present study, we originally recorded and analysed the mechanics as well as the X-ray diffraction patterns of human membrane-permeabilized single muscle fibres with a particular peptide substitution in actin, i.e. p.Phe352Ser. Results unravelled an unexpected cascade of molecular and cellular events. During contraction, p.Phe352Ser greatly enhances the strain of individual cross-bridges. Paradoxically, p.Phe352Ser also slightly lowers the number of cross-bridges by altering the rate of myosin head attachment to actin monomers. Overall, at the cell level, these divergent mechanisms conduct to an improved steady-state force production. Such results provide new surprising scientific insights and crucial information for future therapeutic strategies.

Effects of ageing on single muscle fibre contractile function following short‐term immobilisation

Very little attention has been given to the combined effects of healthy ageing and short-term disuse on the contractile function of human single muscle fibres. Therefore, the present study investigated the effects of 2 weeks of lower limb cast immobilisation (i.e. disuse) on selected contractile properties of single muscle fibres (n = 378) from vastus lateralis of nine young (24 ± 1 years) and eight old (67 ± 2 years) healthy men with comparable levels of physical activity. Prior to immobilisation, MHC IIa fibres produced higher maximum Ca(2+)-activated force (approx. 32%) and specific force (approx. 33%) and had lower Ca(2+) sensitivity than MHC I fibres (P < 0.05), with no differences between young and old. After immobilisation, the decline in single fibre force (MHC I: young 21% and old 22%; MHC IIa: young 22% and old 30%; P < 0.05) as well as specific force (MHC I: young 14% and old 13%; MHC IIa: young 18% and old 25%; P < 0.05) was more pronounced in MHC IIa fibres compared to MHC I fibres (P < 0.05), with no differences between young and old. Notably, there was a selective decrease in Ca(2+) sensitivity in MHC IIa fibres of young (P < 0.05) and in MHC I fibres of old individuals (P < 0.05), respectively. In conclusion, 2 weeks of lower limb immobilisation caused greater impairments in single muscle fibre force and specific force in MHC IIa than MHC I fibres independently of age. In contrast, immobilisation-induced changes in Ca(2+) sensitivity that were dependent on age and MHC isoform.