Single Muscle Fibers Research Articles

A Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics.

Skeletal muscle is a major regulatory tissue of whole-body metabolism and is composed of a diverse mixture of cell (fiber) types. Aging and several diseases differentially affect the various fiber types, and therefore, investigating the changes in the proteome in a fiber-type specific manner is essential. Recent breakthroughs in isolated single muscle fiber proteomics have started to reveal heterogeneity among fibers. However, existing procedures are slow and laborious, requiring 2 h of mass spectrometry time per single muscle fiber; 50 fibers would take approximately 4 days to analyze. Thus, to capture the high variability in fibers both within and between individuals requires advancements in high throughput single muscle fiber proteomics. Here we use a single cell proteomics method to enable quantification of single muscle fiber proteomes in 15 min total instrument time. As proof of concept, we present data from 53 isolated skeletal muscle fibers obtained from two healthy individuals analyzed in 13.25 h. Adapting single cell data analysis techniques to integrate the data, we can reliably separate type 1 and 2A fibers. Ninety-four proteins were statistically different between clusters indicating alteration of proteins involved in fatty acid oxidation, oxidative phosphorylation, and muscle structure and contractile function. Our results indicate that this method is significantly faster than prior single fiber methods in both data collection and sample preparation while maintaining sufficient proteome depth. We anticipate this assay will enable future studies of single muscle fibers across hundreds of individuals, which has not been possible previously due to limitations in throughput.

Visualization, modeling and analysis of salmon muscle structure: Based on micro-CT

Seafood analogs using culture cells or plant-based materials are rising for future food supply, but their bionic design is still an issue on finely simulating the multiscale structure and function of fishes. For salmon, we compared the back, belly and tail tissues via micro computed tomography (micro-CT) scanning with optimized tissue contrast and spatial resolution. The optimum staining conditions of salmon muscle tissue for micro-CT was 3.75 % IKI (iodine-potassium iodide) solution for 7 days, and the muscle could be separated well from fat and connective tissue. The results of visualization analysis were correlated with the texture profile analysis. A relatively high magnification (3 µm/pixel) was used with fiber tracing procedure to analyze the connection of muscle fiber bundles surrounded by soft tissue. Single muscle fiber (e.g., diameter 117 µm) was successfully and artificially separated from whole visualized tissue through manual division. Overall, this study showed a novel ultra-high-resolution method based on micro-CT to conduct digital analysis of muscle fascicle architecture, and constructed printable model of bionic animal/plant tissue for food design.

Single fibre cytoarchitecture in ventilator-induced diaphragm dysfunction (VIDD) assessed by quantitative morphometry second harmonic generation imaging: Positive effects of BGP-15 chaperone co-inducer and VBP-15 dissociative corticosteroid treatment.

Ventilator-induced diaphragm dysfunction (VIDD) is a common sequela of intensive care unit (ICU) treatment requiring mechanical ventilation (MV) and neuromuscular blockade (NMBA). It is characterised by diaphragm weakness, prolonged respirator weaning and adverse outcomes. Dissociative glucocorticoids (e.g., vamorolone, VBP-15) and chaperone co-inducers (e.g., BGP-15) previously showed positive effects in an ICU-rat model. In limb muscle critical illness myopathy, preferential myosin loss prevails, while myofibrillar protein post-translational modifications are more dominant in VIDD. It is not known whether the marked decline in specific force (force normalised to cross-sectional area) is a pure consequence of altered contractility signaling or whether diaphragm weakness also has a structural correlate through sterical remodeling of myofibrillar cytoarchitecture, how quickly it develops, and to which extent VBP-15 or BGP-15 may specifically recover myofibrillar geometry. To address these questions, we performed label-free multiphoton Second Harmonic Generation (SHG) imaging followed by quantitative morphometry in single diaphragm muscle fibres from healthy rats subjected to five or 10days of MV + NMBA to simulate ICU treatment without underlying confounding pathology (like sepsis). Rats received daily treatment of either Prednisolone, VBP-15, BGP-15 or none. Myosin-II SHG signal intensities, fibre diameters (FD) as well as the parameters of myofibrillar angular parallelism (cosine angle sum, CAS) and in-register of adjacent myofibrils (Vernier density, VD) were computed from SHG images. ICU treatment caused a decline in FD at day 10 as well as a significant decline in CAS and VD from day 5. Vamorolone effectively recovered FD at day 10, while BGP-15 was more effective at day 5. BGP-15 was more effective than VBP-15 in recovering CAS at day 10 although not to control levels. In-register VD levels were restored at day 10 by both compounds. Our study is the first to provide quantitative insights into VIDD-related myofibrillar remodeling unravelled by SHG imaging, suggesting that both VBP-15 and BGP-15 can effectively ameliorate the structure-related dysfunction in VIDD.

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.

Rapid restitution of contractile dysfunction by synthetic copolymers in dystrophin-deficient single live skeletal muscle fibers

Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin, a cytoskeletal protein essential for the preservation of the structural integrity of the muscle cell membrane. DMD patients develop severe skeletal muscle weakness, degeneration, and early death. We tested here amphiphilic synthetic membrane stabilizers in mdx skeletal muscle fibers (flexor digitorum brevis; FDB) to determine their effectiveness in restoring contractile function in dystrophin-deficient live skeletal muscle fibers. After isolating FDB fibers via enzymatic digestion and trituration from thirty-three adult male mice (9 C57BL10, 24 mdx), these were plated on a laminin-coated coverslip and treated with poloxamer 188 (P188; PEO75-PPO30-PEO75; 8400 g/mol), architecturally inverted triblock (PPO15-PEO200-PPO15, 10,700 g/mol), and diblock (PEO75-PPO16-C4, 4200 g/mol) copolymers. We assessed the twitch kinetics of sarcomere length (SL) and intracellular Ca2+ transient by Fura-2AM by field stimulation (25 V, 0.2 Hz, 25 °C). Twitch contraction peak SL shortening of mdx FDB fibers was markedly depressed to 30% of the dystrophin-replete control FDB fibers from C57BL10 (P < 0.001). Compared to vehicle-treated mdx FDB fibers, copolymer treatment robustly and rapidly restored the twitch peak SL shortening (all P < 0.05) by P188 (15 μM = + 110%, 150 μM = + 220%), diblock (15 μM = + 50%, 150 μM = + 50%), and inverted triblock copolymer (15 μM = + 180%, 150 μM = + 90%). Twitch peak Ca2+ transient from mdx FDB fibers was also depressed compared to C57BL10 FDB fibers (P < 0.001). P188 and inverted triblock copolymer treatment of mdx FDB fibers increased the twitch peak Ca2+ transient (P < 0.001). This study shows synthetic block copolymers with varied architectures can rapidly and highly effectively enhance contractile function in live dystrophin-deficient skeletal muscle fibers.

Modeling Human Muscular Dystrophies in Zebrafish: Mutant Lines, Transgenic Fluorescent Biosensors, and Phenotyping Assays.

Muscular dystrophies (MDs) are a heterogeneous group of myopathies characterized by progressive muscle weakness leading to death from heart or respiratory failure. MDs are caused by mutations in genes involved in both the development and organization of muscle fibers. Several animal models harboring mutations in MD-associated genes have been developed so far. Together with rodents, the zebrafish is one of the most popular animal models used to reproduce MDs because of the high level of sequence homology with the human genome and its genetic manipulability. This review describes the most important zebrafish mutant models of MD and the most advanced tools used to generate and characterize all these valuable transgenic lines. Zebrafish models of MDs have been generated by introducing mutations to muscle-specific genes with different genetic techniques, such as (i) N-ethyl-N-nitrosourea (ENU) treatment, (ii) the injection of specific morpholino, (iii) tol2-based transgenesis, (iv) TALEN, (v) and CRISPR/Cas9 technology. All these models are extensively used either to study muscle development and function or understand the pathogenetic mechanisms of MDs. Several tools have also been developed to characterize these zebrafish models by checking (i) motor behavior, (ii) muscle fiber structure, (iii) oxidative stress, and (iv) mitochondrial function and dynamics. Further, living biosensor models, based on the expression of fluorescent reporter proteins under the control of muscle-specific promoters or responsive elements, have been revealed to be powerful tools to follow molecular dynamics at the level of a single muscle fiber. Thus, zebrafish models of MDs can also be a powerful tool to search for new drugs or gene therapies able to block or slow down disease progression.

High sensitivity top-down proteomics captures single muscle cell heterogeneity in large proteoforms.

Single-cell proteomics has emerged as a powerful method to characterize cellular phenotypic heterogeneity and the cell-specific functional networks underlying biological processes. However, significant challenges remain in single-cell proteomics for the analysis of proteoforms arising from genetic mutations, alternative splicing, and post-translational modifications. Herein, we have developed a highly sensitive functionally integrated top-down proteomics method for the comprehensive analysis of proteoforms from single cells. We applied this method to single muscle fibers (SMFs) to resolve their heterogeneous functional and proteomic properties at the single-cell level. Notably, we have detected single-cell heterogeneity in large proteoforms (>200kDa) from the SMFs. Using SMFs obtained from three functionally distinct muscles, we found fiber-to-fiber heterogeneity among the sarcomeric proteoforms which can be related to the functional heterogeneity. Importantly, we detected multiple isoforms of myosin heavy chain (~223kDa), a motor protein that drives muscle contraction, with high reproducibility to enable the classification of individual fiber types. This study reveals single muscle cell heterogeneity in large proteoforms and establishes a direct relationship between sarcomeric proteoforms and muscle fiber types, highlighting the potential of top-down proteomics for uncovering the molecular underpinnings of cell-to-cell variation in complex systems.

Influence of 2,3-Butanedione Monoxime on the Actin–Myosin Interaction in Modeling Healthy Function and Congenital Myopathy in a Single Muscle Fiber

Influence of 2,3-Butanedione Monoxime on the Actin–Myosin Interaction in Modeling Healthy Function and Congenital Myopathy in a Single Muscle Fiber

ECR Spotlight – Luke Flewwelling

ECR Spotlight is a series of interviews with early-career authors from a selection of papers published in Journal of Experimental Biology and aims to promote not only the diversity of early-career researchers (ECRs) working in experimental biology during our centenary year but also the huge variety of animals and physiological systems that are essential for the ‘comparative’ approach. Luke Flewwelling is an author on ‘ Thermoregulatory trade-offs underlie the effects of warming summer temperatures on deer mice’, published in JEB. Luke conducted the research described in this article while a graduate student (MSc) in Dr Graham Scott's lab at McMaster University, Canada. He is now a PhD student in the lab of Dr Arthur Cheng at York University, Canada, investigating muscle physiology and the mechanisms by which exercise can create physiological change in individuals. Currently he is studying how various types of exercise training impact muscle fatigue tolerance and calcium signalling in single muscle fibres.

Titin activates myosin filaments in skeletal muscle by switching from an extensible spring to a mechanical rectifier

Titin is a molecular spring in parallel with myosin motors in each muscle half-sarcomere, responsible for passive force development at sarcomere length (SL) above the physiological range (>2.7 μm). The role of titin at physiological SL is unclear and is investigated here in single intact muscle cells of the frog (Rana esculenta), by combining half-sarcomere mechanics and synchrotron X-ray diffraction in the presence of 20 μM para-nitro-blebbistatin, which abolishes the activity of myosin motors and maintains them in the resting state even during activation of the cell by electrical stimulation. We show that, during cell activation at physiological SL, titin in the I-band switches from an SL-dependent extensible spring (OFF-state) to an SL-independent rectifier (ON-state) that allows free shortening while resisting stretch with an effective stiffness of ~3 pN nm-1 per half-thick filament. In this way, I-band titin efficiently transmits any load increase to the myosin filament in the A-band. Small-angle X-ray diffraction signals reveal that, with I-band titin ON, the periodic interactions of A-band titin with myosin motors alter their resting disposition in a load-dependent manner, biasing the azimuthal orientation of the motors toward actin. This work sets the stage for future investigations on scaffold and mechanosensing-based signaling functions of titin in health and disease.

Six weeks of N-acetylcysteine antioxidant in drinking water decreases pathological fiber branching in MDX mouse dystrophic fast-twitch skeletal muscle.

Introduction: It has been proposed that an increased susceptivity to oxidative stress caused by the absence of the protein dystrophin from the inner surface of the sarcolemma is a trigger of skeletal muscle necrosis in the destructive dystrophin deficient muscular dystrophies. Here we use the mdx mouse model of human Duchenne Muscular Dystrophy to test the hypothesis that adding the antioxidant NAC at 2% to drinking water for six weeks will treat the inflammatory phase of the dystrophic process and reduce pathological muscle fiber branching and splitting resulting in a reduction of mass in mdx fast-twitch EDL muscles. Methods: Animal weight and water intake was recorded during the six weeks when 2% NAC was added to the drinking water. Post NAC treatment animals were euthanised and the EDL muscles dissected out and placed in an organ bath where the muscle was attached to a force transducer to measure contractile properties and susceptibility to force loss from eccentric contractions. After the contractile measurements had been made the EDL muscle was blotted and weighed. In order to assess the degree of pathological fiber branching mdx EDL muscles were treated with collagenase to release single fibers. For counting and morphological analysis single EDL mdx skeletal muscle fibers were viewed under high magnification on an inverted microscope. Results: During the six-week treatment phase NAC reduced body weight gain in three- to nine-week-old mdx and littermate control mice without effecting fluid intake. NAC treatment also significantly reduced the mdx EDL muscle mass and abnormal fiber branching and splitting. Discussion: We propose chronic NAC treatment reduces the inflammatory response and degenerative cycles in the mdx dystrophic EDL muscles resulting in a reduction in the number of complexed branched fibers reported to be responsible for the dystrophic EDL muscle hypertrophy.

Thin filament regulation of striated muscle power output: implications for targets to improve failing human hearts.

The heart's pumping capacity is determined by myofilament power generation. Power is work done per unit time and measured as the product of force and velocity. At a sarcomere level, these contractile properties are linked to the number of attached cross-bridges and their cycling rate; and many signaling pathways modulate one or both of these factors. We previously showed that rodent permeabilized cardiac myocyte power is increased following PKA-mediated phosphorylation of myofibrillar proteins (Herron, Korte, McDonald, Circ. Res. 2001). This increase in power demonstrates a capacity for improved myofilament function and this therapeutic potential adds significance for identification of the molecular specificity underlying enhanced power output. To address molecular specificity at the thin filament level, myofilament mechanical properties were measured before and after exchange of endogenous troponin with recombinant human Tn complex, which contained cardiac (c)TnT, cTnC and either WT cTnI or pseudo-phosphorylated cTnI at sites Ser23/24Asp, Y26E, or the combinatorial Ser23/24Asp and Y26E in single permeabilized rat slow-twitch skeletal muscle fibers. We found that cTnI Ser23/24Asp, Y26E, and combinatorial Ser23/24Asp and Y26E were sufficient for a ∼20% increase in power. We then tested the power reserve capacity in myofilaments from failing human hearts. We found that PKA increased power by ∼35% in permeabilized cardiac myocyte preparations from human failing hearts. Next, we determined if pseudo-phosphorylated cTnI at Ser23/24 was sufficient to increase power in cardiac myocytes from human failing hearts. Following cTn exchange that included cTnI Ser23/24Asp, power output increased ∼20% in permeabilized cardiac myocyte preparations from the left ventricle of human failing hearts. These results implicate cTnI N-terminal phosphorylation is a molecular regulator of myocyte power and could serve as a target to augment myocyte power reserve capacity in failing human hearts.

Myonuclear alterations associated with exercise are independent of age in humans.

Age-related decline in skeletal muscle structure and function can be mitigated by regular exercise. However, the precise mechanisms that govern this are not fully understood. The nucleus plays an active role in translating forces into biochemical signals (mechanotransduction), with the nuclear lamina protein lamin A regulating nuclear shape, nuclear mechanics and ultimately gene expression. Defective lamin A expression causes muscle pathologies and premature ageing syndromes, but the roles of nuclear structure and function in physiological ageing and in exercise adaptations remain obscure. Here, we isolated single muscle fibres and carried out detailed morphological and functional analyses on myonuclei from young and older exercise-trained individuals. Strikingly, myonuclei from trained individuals were more spherical, less deformable, and contained a thicker nuclear lamina than those from untrained individuals. Complementary to this, exercise resulted in increased levels of lamin A and increased myonuclear stiffness in mice. We conclude that exercise is associated with myonuclear remodelling, independently of age, which may contribute to the preservative effects of exercise on muscle function throughout the lifespan. KEY POINTS: The nucleus plays an active role in translating forces into biochemical signals. Myonuclear aberrations in a group of muscular dystrophies called laminopathies suggest that the shape and mechanical properties of myonuclei are important for maintaining muscle function. Here, striking differences are presented in myonuclear shape and mechanics associated with exercise, in both young and old humans. Myonuclei from trained individuals were more spherical, less deformable and contained a thicker nuclear lamina than untrained individuals. It is concluded that exercise is associated with age-independent myonuclear remodelling, which may help to maintain muscle function throughout the lifespan.

Metabolic reprogramming underlies cavefish muscular endurance despite loss of muscle mass and contractility

Physical inactivity is a scourge to human health, promoting metabolic disease and muscle wasting. Interestingly, multiple ecological niches have relaxed investment into physical activity, providing an evolutionary perspective into the effect of adaptive physical inactivity on tissue homeostasis. One such example, the Mexican cavefish Astyanax mexicanus, has lost moderate-to-vigorous activity following cave colonization, reaching basal swim speeds ~3.7-fold slower than their river-dwelling counterpart. This change in behavior is accompanied by a marked shift in body composition, decreasing total muscle mass and increasing fat mass. This shift persisted at the single muscle fiber level via increased lipid and sugar accumulation at the expense of myofibrillar volume. Transcriptomic analysis of laboratory-reared and wild-caught cavefish indicated that this shift is driven by increased expression of pparγ-the master regulator of adipogenesis-with a simultaneous decrease in fast myosin heavy chain expression. Exvivo and invivo analysis confirmed that these investment strategies come with a functional trade-off, decreasing cavefish muscle fiber shortening velocity, time to maximal force, and ultimately maximal swimming speed. Despite this, cavefish displayed a striking degree of muscular endurance, reaching maximal swim speeds ~3.5-fold faster than their basal swim speeds. Multi-omic analysis suggested metabolic reprogramming, specifically phosphorylation of Pgm1-Threonine 19, as a key component enhancing cavefish glycogen metabolism and sustained muscle contraction. Collectively, we reveal broad skeletal muscle changes following cave colonization, displaying an adaptive skeletal muscle phenotype reminiscent to mammalian disuse and high-fat models while simultaneously maintaining a unique capacity for sustained muscle contraction via enhanced glycogen metabolism.

Faster triceps surae muscle cyclic contractions alter muscle activity and whole body metabolic rate.

Hundred years ago, Fenn demonstrated that when a muscle shortens faster, its energy liberation increases. Fenn's results were the first of many that led to the general understanding that isometric muscle contractions are energetically cheaper than concentric contractions. However, this evidence is still primarily based on single fiber or isolated (ex vivo) muscle studies and it remains unknown whether this translates to whole body metabolic rate. In this study, we specifically changed the contraction velocity of the ankle plantar flexors and quantified the effects on triceps surae muscle activity and whole body metabolic rate during cyclic plantar flexion (PF) contractions. Fifteen participants performed submaximal ankle plantar flexions (∼1/3 s activation and ∼2/3 s relaxation) on a dynamometer at three different ankle angular velocities: isometric (10° PF), isokinetic at 30°/s (5-15° PF), and isokinetic at 60°/s (0-20° PF) while target torque (25% MVC) and cycle frequency were kept constant. In addition, to directly determine the effect of ankle angular velocity on muscle kinematics we collected gastrocnemius medialis muscle fascicle ultrasound data. As expected, increasing ankle angular velocity increased gastrocnemius medialis muscle fascicle contraction velocity and positive mechanical work (P < 0.01), increased mean and peak triceps surae muscle activity (P < 0.01), and considerably increased net whole body metabolic rate (P < 0.01). Interestingly, the increase in triceps surae muscle activity with fast ankle angular velocities was most pronounced in the gastrocnemius lateralis (P < 0.05). Overall, our results support the original findings from Fenn in 1923 and we demonstrated that greater triceps surae muscle contraction velocities translate to increased whole body metabolic rate.NEW & NOTEWORTHY Single muscle fiber studies or research on isolated (ex vivo) muscles demonstrated that faster concentric muscle contractions yield increased energy consumption. Here we translated this knowledge to muscle activation and whole body metabolic rate. Increasing ankle angular velocity increased triceps surae contraction velocity and mechanical work, increasing triceps surae muscle activity and substantially elevating whole body metabolic rate. Additionally, we demonstrated that triceps surae muscle activation strategy depends on the mechanical demands of the task.

Visualizing MyoD Oscillations in Muscle Stem Cells.

The bHLH transcription factor MyoD is a master regulator of myogenic differentiation, and its sustained expression in fibroblasts suffices to differentiate them into muscle cells. MyoD expression oscillates in activated muscle stem cells of developing, postnatal and adult muscle under various conditions: when the stem cells are dispersed in culture, when they remain associated with single muscle fibers, or when they reside in muscle biopsies. The oscillatory period is around3h and thus much shorter than the cell cycle or circadian rhythm. Unstable MyoD oscillations and long periods of sustained MyoD expression are observed when stem cells undergo myogenic differentiation. The oscillatory expression of MyoD is driven by the oscillatory expression of the bHLH transcription factor Hes1 that periodically represses MyoD. Ablation of the Hes1 oscillator interferes with stable MyoD oscillations and leads to prolonged periods of sustained MyoD expression. This interferes with the maintenance of activated muscle stem cells and impairs muscle growth and repair. Thus, oscillations of MyoD and Hes1 control the balance between the proliferation and differentiation of muscle stem cells. Here, we describe time-lapse imaging methods using luciferase reporters, which can monitor dynamic MyoD gene expression in myogenic cells.

THRIFTY-A High-throughput Single Muscle Fiber Typing Method Based on Immunofluorescence Detection.

Skeletal muscle consists of a mixture of fiber types with different functional and metabolic characteristics. The relative composition of these muscle fiber types has implications for muscle performance, whole-body metabolism, and health. However, analyses of muscle samples in a fiber type-dependent manner are very time consuming. Therefore, these are often neglected in favor of more time-efficient analyses on mixed muscle samples. Methods such as western blot and myosin heavy chain separation by SDS-PAGE have previously been utilized to fiber type-isolated muscle fibers. More recently, the introduction of the dot blot method significantly increased the speed of fiber typing. However, despite recent advancements, none of the current methodologies are feasible for large-scale investigations because of their time requirements. Here, we present the protocol for a new method, which we have named THRIFTY (high-THRoughput Immunofluorescence Fiber TYping), that enables rapid fiber type identification using antibodies towards the different myosin heavy chain (MyHC) isoforms of fast and slow twitch muscle fibers. First, a short segment (<1 mm) is cut off from isolated muscle fibers and mounted on a customized gridded microscope slide holding up to 200 fiber segments. Second, the fiber segments attached to the microscope slide are stained with MyHC-specific antibodies and then visualized using a fluorescence microscope. Lastly, the remaining pieces of the fibers can either be collected individually or pooled together with fibers of the same type for subsequent analyses. The THRIFTY protocol is approximately three times as fast as the dot blot method, which enables not only time-sensitive assays to be performed but also increases the feasibility to conduct large-scale investigations into fiber type specific physiology. Graphical Overview Graphical overview of the THRIFTY workflow. Cut off a small segment (0.5 mm) of an individually dissected muscle fiber and mount it onto the customized microscope slide containing a printed grid system. Using a Hamilton syringe, fixate the fiber segment by applying a small droplet of distilled water on the segment and let it fully dry (1A). The remaining large segment of the fiber should be placed in the corresponding square on a black A4 paper (1B). Once the microscope slide has been fully mounted with fiber segments, submerge the slide in a polypropylene slide mailer (illustrated as a Coplin jar in the figure) containing acetone to permeabilize the fiber segments. Thereafter, incubate the slide with primary antibodies targeting MyHC-I and MyHC-II. Following washes in PBS solution, incubate the slides with fluorescently labeled secondary antibodies, wash again, and mount with a cover glass and antifade reagent (2). Identification of fiber type can be performed using a digital fluorescence microscope (3), whereafter the remaining pieces of the fiber segments (large) are pooled together according to their fiber type or individually collected for experiments on single fibers (4). Image modified from Horwath et al. (2022).

Preservation of shortening velocity and power output in single muscle fibres from patients with idiopathic inflammatory myopathies.

Idiopathic inflammatory myopathies (IIMs) are autoimmune disorders of skeletal muscle causing weakness and disability. Utilizing single fibre contractility studies, we have previously shown that contractility is affected in muscle fibres from individuals with IIMs. For the current study, we hypothesized that a compensatory increase in shortening velocity occurs in muscle fibres from individuals with IIMs in an effort to maintain power output. We performed in vitro single fibre contractility studies to assess force-velocity relationships and maximum shortening velocity (Vmax) of muscle fibres from individuals with IIMs (25 type I and 58 type IIA) and healthy controls (66 type I and 27 type IIA) and calculated maximum power output (Wmax) for each fibre. We found significantly higher Vmax (mean ± SEM) of fibres from individuals with IIMs, for both type I (1.40 ± 0.31 fibre lengths/s, n = vs. 0.63 ± 0.13 fibre lengths/s; p = 0.0019) and type IIA fibres (2.00 ± 0.17 fibre lengths/s vs 0.77 ± 0.10 fibre lengths/s; p < 0.0001). Furthermore, Wmax (mean ± SEM) was maintained compared to fibres from healthy controls, again for both type I and type IIA fibres (4.10 ± 1.00 kN/m2·fibre lengths/s vs. 2.00 ± 0.16 kN/m2·fibre lengths/s; p = ns and 9.00 ± 0.64 kN/m2·fibre lengths/s vs. 6.00 ± 0.67 kN/m2·fibre lengths/s; p = ns respectively). In addition, type I muscle fibres from individuals with IIMs was able to develop maximum power output at lower relative force. The findings of this study suggest that compensatory responses to maintain power output, including increased maximum shortening velocity and improved efficiency, may occur in muscle of individuals with IIMs. The mechanism underlying this response is unclear, and different hypotheses are discussed.

The relationship between single muscle fibre and voluntary rate of force development in young and old males.

It is suggested that the early phase (< 50ms) of force development during a muscle contraction is associated with intrinsic contractile properties, while the late phase (> 50ms) is associated with maximal force. There are no direct investigations of single muscle fibre rate of force development (RFD) as related to joint-level RFD METHODS: Sixteen healthy, young (n = 8; 26.4 ± 1.5 yrs) and old (n = 8; 70.1 ± 2.8 yrs) males performed maximal voluntary isometric contractions (MVC) and electrically evoked twitches of the knee extensors to assess RFD. Then, percutaneous muscle biopsies were taken from the vastus lateralis and chemically permeabilized, to assess single fibre function. At the joint level, older males were ~ 30% weaker and had ~ 43% and ~ 40% lower voluntary RFD values at 0-100 and 0-200ms, respectively, than the younger ones (p ≤ 0.05). MVC torque was related to every voluntary RFD epoch in the young (p ≤ 0.001), but only the 0-200ms epoch in the old (p ≤ 0.005). Twitch RFD was ~ 32% lower in the old compared to young (p < 0.05). There was a strong positive relationship between twitch RFD and voluntary RFD during the earliest time epochs in the young (≤ 100ms; p ≤ 0.01). While single fibre RFD was unrelated to joint-level RFD in the young, older adults trended (p = 0.052-0.055) towards significant relationships between joint-level RTD and Type I single fibre RFD at the 0-30ms (r2 = 0.48) and 0-50ms (r2 = 0.49) time epochs. Electrically evoked twitches are good predictors of early voluntary RFD in young, but not older adults. Only the older adults showed a potential relationship between single fibre (Type I) and joint-level rate of force development.

Redox Balance Differentially Affects Biomechanics in Permeabilized Single Muscle Fibres—Active and Passive Force Assessments with the Myorobot

An oxidizing redox state imposes unique effects on the contractile properties of muscle. Permeabilized fibres show reduced active force generation in the presence of H2O2. However, our knowledge about the muscle fibre's elasticity or flexibility is limited due to shortcomings in assessing the passive stress-strain properties, mostly due to technically limited experimental setups. The MyoRobot is an automated biomechatronics platform that is well-capable of not only investigating calcium responsiveness of active contraction but also features precise stretch actuation to examine the passive stress-strain behaviour. Both were carried out in a consecutive recording sequence on the same fibre for 10 single fibres in total. We denote a significantly diminished maximum calcium-saturated force for fibres exposed to ≥500 µM H2O2, with no marked alteration of the pCa50 value. In contrast to active contraction (e.g., maximum isometric force activation), passive restoration stress (force per area) significantly increases for fibres exposed to an oxidizing environment, as they showed a non-linear stress-strain relationship. Our data support the idea that a highly oxidizing environment promotes non-linear fibre stiffening and confirms that our MyoRobot platform is a suitable tool for investigating redox-related changes in muscle biomechanics.