Fast Fibers Research Articles

Adeno-Associated Virus 8 and 9 Myofibre Type/Size Tropism Profiling Reveals Therapeutic Effect of Microdystrophin in Canines.

Adeno-associated virus (AAV) 8 and 9 are in clinical trials for treating neuromuscular diseases such as Duchenne muscular dystrophy (DMD). Muscle consists of myofibres of different types and sizes. However, little is known about the fibre type and fibre size tropism of AAV in large mammals. We evaluated fibre type- and size-specific transduction properties of AAV8 and AAV9 in 17 dogs that received systemic gene transfer (dose 1.94 ± 0.52 × 1014 vg/kg; injected at 2.86 ± 0.30 months; harvested at 20.79 ± 3.30 months). For AAV8, two DMD dogs and three carrier dogs received an alkaline phosphatase (AP) reporter vector, and five DMD dogs received a four-repeat microdystrophin (uDys) vector. For AAV9, one normal and one DMD dog received the AP vector, and five DMD dogs received a five-repeat uDys vector. Association between AAV transduction and the fibre type/size was studied in three muscles that showed mosaic transgene expression, including the biceps femoris, teres major and latissimus dorsi. Transgene expression was detected in 30%-45% of myofibres. In the AP reporter vector-injected dogs, neither AAV8 nor AAV9 showed a statistically significant fibre type preference. Interestingly, AP expression was enriched in smaller fibres. In uDys-treated DMD dogs, slow and fast myofibres were equally transduced. Notably, uDys-expressing myofibres were significantly larger than uDys-negative myofibres irrespective of the AAV serotype (p < 0.0001). In AAV8 uDys vector-injected dogs, the mini-Feret diameter was 15%, 16% and 23% larger in uDys-positive slow, fast and hybrid fibres, respectively; the cross-sectional area was 30%, 34% and 46% larger in uDys-positive slow, fast and hybrid fibres, respectively. In AAV9 uDys vector-injected dogs, the mini-Feret diameter was 12%, 13% and 25% larger in uDys-positive slow, fast and hybrid fibres, respectively; the cross-sectional area was 25%, 28% and 59% larger in uDys-positive slow, fast and hybrid fibres, respectively. Our studies suggest that AAV8 and AAV9 transduce fast and slow myofibres at equivalent efficiency. Importantly, uDys therapy effectively prevented dystrophic myofibre atrophy. Our study provides important insight into systemic muscle AAV delivery in large mammals and supports further development of uDys gene therapy for DMD.

Aptamer-Conjugated Exosomes Ameliorate Diabetes-Induced Muscle Atrophy by Enhancing SIRT1/FoxO1/3a-Mediated Mitochondrial Function.

Muscle atrophy is associated with Type 2 diabetes mellitus, which reduces the quality of life and lacks effective treatment strategies. Previously, it was determined that human umbilical cord mesenchymal stromal cell (hucMSC)-derived exosomes (EXOs) ameliorate diabetes-induced muscle atrophy. However, the systemic application of EXOs is less selective for diseased tissues, which reduces their efficacy and safety associated with their nonspecific biological distribution invivo. Therefore, improving exosomal targeting is imperative. In this study, a skeletal muscle-specific aptamer (Apt) was used to explore the effects of Apt-functionalized EXOs derived from hucMSCs in diabetes-associated muscle atrophy and its specific mechanisms. Diabetic db/db mice and C2C12 myotubes were used to explore the effects of MSC-EXOs or Apt-EXOs in alleviating muscle atrophy. Grip strength, muscle weight and muscle fibre cross-sectional area (CSA) were used to evaluate skeletal muscle strength and muscle mass. Western blot analysis of muscle atrophy signalling, including MuRF1 and Atrogin 1 and the mitochondrial complex and Seahorse analysis were performed to investigate the underlying mechanisms of MSC-EXOs or Apt-EXOs on muscle atrophy. MSC-EXOs increased grip strength (p = 0.0002) and muscle mass (p = 0.0044 for tibialis anterior (TA) muscle, p = 0.002 for soleus (SO) muscle) in db/db mice. It also increased the CSA of muscle fibres (p = 0.0011 for all fibres, p = 0.0036 for slow muscle fibres and p = 0.0089 for fast muscle fibres) and the percentage of slow-to-fast muscle fibres (p = 0.0109). However, Atrogin 1 (p = 0.0455) and MuRF1 expression (p = 0.0168) was reduced. MSC-EXOs activated SIRT1/FoxO1/3a signalling and enhanced mitochondrial function in db/db mice and C2C12 myotubes. SIRT1 knockdown decreased the beneficial antiatrophic effects of MSC-EXOs. Additionally, Apt conjugation increased the effect of MSC-EXOs on muscle atrophy and myofiber-type transition (p = 0.0133 for grip strength, p = 0.0124 for TA muscle weight, p = 0.0008 for SO muscle weight, p < 0.0001 for CSA of all muscle fibres, p = 0.0198 for CSA of slow muscle fibres, p = 0.0213 for CSA of fast muscle fibres, p = 0.011 for percentage of slow-fast muscle fibres, p = 0.0141 for Atrogin 1 expression and p = 0.005 for MuRF1 expression). The results suggest that hucMSC-derived exosomes ameliorate diabetes-associated muscle atrophy by enhancing SIRT1/FoxO1/3a-mediated mitochondrial function and that Apt conjugation strengthens the effects of MSC-EXOs on muscle atrophy. These findings demonstrate the therapeutic potential of muscle-targeted MSC-EXOs for the treatment of muscle atrophy.

Fast and high-resolution multimode fiber speckle imaging via optical coherence control

Fast and high-resolution multimode fiber speckle imaging via optical coherence control

Spatially ordered recruitment of fast muscles in accordance with movement strengths in larval zebrafish

In vertebrates, skeletal muscle comprises fast and slow fibers. Slow and fast muscle cells in fish are spatially segregated; slow muscle cells are located only in a superficial region, and comprise a small fraction of the total muscle cell mass. Slow muscles support low-speed, low-force movements, while fast muscles are responsible for high-speed, high-force movements. However, speed and strength of movement are not binary states, but rather fall on a continuum. This raises the question of whether any recruitment patterns exist within fast muscles, which constitute the majority of muscle cell mass. In the present study, we investigated activation patterns of trunk fast muscles during movements of varying speeds and strengths using larval zebrafish. We employed two complementary methods: calcium imaging and electrophysiology. The results obtained from both methods supported the conclusion that there are spatially-ordered recruitment patterns in fast muscle cells. During weaker/slower movements, only the lateral portion of fast muscle cells is recruited. As the speed or strength of the movements increases, more fast muscle cells are recruited in a spatially-ordered manner, progressively from lateral to medial. We also conducted anatomical studies to examine muscle fiber size. The results of those experiments indicated that muscle fiber size increases systematically from lateral to medial. Therefore, the spatially ordered recruitment of fast muscle fibers, progressing from lateral to medial, correlates with an increase in fiber size. These findings provide significant insights into the organization and function of fast muscles in larval zebrafish, illustrating how spatial recruitment and fiber size interact to optimize movement performance.

Fourier Neural Operator Accelerated Fast and Accurate Optical Fiber Transmission Modeling

Fourier Neural Operator Accelerated Fast and Accurate Optical Fiber Transmission Modeling

Transcriptomic and lipidomic profiling reveals distinct bioactive lipid signatures in slow and fast muscles and highlights the role of resolvin-D2 in fiber type determination during myogenesis.

Skeletal muscles are predominantly composed of long, multinucleated muscle fibers, classified according to their metabolic and contractile phenotype. The determination of fiber types is influenced by various factors (e.g., innervation, hormones, physical demand). Our laboratory and others showed that resolvins, lipid mediators derived from omega-3 fatty acids, promote muscle regeneration and function after an injury or in models of muscular dystrophies; however, the effect of resolvins on the determination of muscle phenotype remains unknown. Here, we investigated the impact of lipid mediators on muscle phenotype during myogenesis. Transcriptomics analysis of single-nuclei RNAseq data sets revealed that the enzymes responsible for bioactive lipids biosynthesis are differentially expressed in slow fibers versus fast fibers. Lipidomics analysis of slow-twitch muscle (soleus) versus fast-twitch muscle (tibialis anterior) showed that the levels of lipids derived from arachidonic acid are similar between muscle groups, but lipids derived from alpha-linolenic acid, linoleic acid, eicosapentaenoic acid, and docosahexaenoic acid are enriched in slow-twitch muscle. Screening for different lipids invitro showed that resolvin-D2 enhances the formation of myotubes expressing the slow myosin heavy chain isoform. Invivo, the administration of resolvin-D2 enhances muscle strength, increases myofiber size, and affects fiber typing in injured muscles but not in uninjured muscles. Resolvin-D2 promoted the transition toward the dominant fiber types in regenerating muscle (i.e., type I in the slow-twitch soleus and type IIB in the fast-twitch tibialis anterior muscle), suggesting its participation in fiber typing in conjunction with other factors. Overall, these findings identified new roles of bioactive lipids in the regulation of fiber typing, which could have therapeutic applicability in muscle injuries or dystrophies.

Molecular pathways involved in the control of contractile and metabolic properties of skeletal muscle fibers as potential therapeutic targets for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin, a subsarcolemmal protein whose absence results in increased susceptibility of the muscle fiber membrane to contraction-induced injury. This results in increased calcium influx, oxidative stress, and mitochondrial dysfunction, leading to chronic inflammation, myofiber degeneration, and reduced muscle regenerative capacity. Fast glycolytic muscle fibers have been shown to be more vulnerable to mechanical stress than slow oxidative fibers in both DMD patients and DMD mouse models. Therefore, remodeling skeletal muscle toward a slower, more oxidative phenotype may represent a relevant therapeutic approach to protect dystrophic muscles from deterioration and improve the effectiveness of gene and cell-based therapies. The resistance of slow, oxidative myofibers to DMD pathology is attributed, in part, to their higher expression of Utrophin; there are, however, other characteristics of slow, oxidative fibers that might contribute to their enhanced resistance to injury, including reduced contractile speed, resistance to fatigue, increased capillary density, higher mitochondrial activity, decreased cellular energy requirements. This review focuses on signaling pathways and regulatory factors whose genetic or pharmacologic modulation has been shown to ameliorate the dystrophic pathology in preclinical models of DMD while promoting skeletal muscle fiber transition towards a slower more oxidative phenotype.

Anatomical and histological analyses of ankle plantar flexors: insights into connective tissue composition and muscle architecture.

The tibialis posterior (TP), flexor digitorum longus (FDL), and flexor hallucis longus (FHL) are muscles that contribute to the stability of foot and ankle movements, playing a crucial role in achieving optimal gait. However, a comprehensive examination of the anatomical characteristics and histological variances of each muscle has not been conclusively established. A total of 10 un-embalmed cadavers were dissected, and muscles from each cadaver were consistently harvested from the musculotendon junction. The ratio of collagen and elastic fibers was assessed through three immunohistological analyses, focusing on distinct histological characteristics in type I (slow twitch) and type II (fast twitch) fibers. Additionally, Ultrasonography was utilized to compare and analyze the thickness, fascicle angle, and muscle fiber length of each muscle. Concerning the relative proportion of elastic fibers to collagen, the TP exhibited the highest collagen content (21.9 ± 0.30%, mean ± standard deviation), while the FHL had the highest elastic fiber proportion (48.4 ± 0.44%). The TP predominantly comprised slow type muscle fibers (36.88 ± 0.83%), whereas the FHL contained a higher density of fast type muscle fibers (32.46 ± 4.02%). US analysis indicated that the thickness of the TP was relatively greater (2.0 ± 0.2mm) compared to the FDL (1.2 ± 0.1mm) and FHL (1.1 ± 0.1mm). Additionally, the fascicle length was notably longer in the TP (25.6 ± 4.1mm). Our anatomical and histological findings indicate that the tibialis posterior (TP) is the thickest with a significant physiological angle and a high collagen content. This characteristic enables the TP to provide stability by transmitting a constant force to the calf. On the other hand, the flexor hallucis longus (FHL) exhibits the highest elastic fiber content, confirming its ability to exert instantaneous, swift, and powerful force.

Fast tunable single-frequency Yb-doped fiber laser

Fast tunable single-frequency Yb-doped fiber laser

Axial muscle‐fibre orientations in larval zebrafish

AbstractMost teleost fish propel themselves with lateral body waves powered by their axial muscles. These muscles also power suction feeding through rapid expansion of the mouth cavity. They consist of muscle segments (myomeres), separated by connective tissue sheets (myosepts). In adult teleosts, the fast axial muscle fibres follow pseudo‐helical trajectories, which are thought to distribute strain (relative fibre length change) approximately evenly across transverse sections during swimming, thereby optimizing power generation. To achieve strain equalization, a significant angle to the longitudinal axis on the frontal plane (azimuth) is necessary near the medial plane, increasing strain. Additionally, a deviation from longitudinal orientation on the sagittal plane (elevation) is required laterally to decrease strain. Despite several detailed morphological studies, our understanding of muscle‐fibre orientations in the entire axial musculature of fish remains incomplete. Furthermore, most research has been done in post‐larval stages, leaving a knowledge gap regarding the changing axial muscle architecture during larval development. Larval fish exhibit different body size, body shape and swimming kinematics compared to adults. They experience relatively high viscous forces, requiring higher tail‐beat amplitudes to overcome increased drag. Additionally, larval fish swim with higher tail‐beat frequencies. Histological studies have shown that in larval fish, muscle fibres in the anal region transition from an almost longitudinal orientation to a pseudo‐helical pattern by 3 dpf (days post‐fertilization). However, these studies were limited to a few sections of the body and were prone to shrinkage and tissue damage. Here, we introduce a novel methodology for quantifying muscle‐fibre orientations along the entire axial muscles. We selected 4 dpf larval zebrafish for our analyses, a stage where larvae are actively swimming but not yet free‐feeding. High‐resolution confocal 3D scans were obtained from four genetically modified zebrafish expressing green fluorescent protein in fast muscle fibres. Fluorescence variation allowed segmentation of individual muscle fibres, which were then converted to fish‐bound coordinates by correcting for the fish's position and orientation in the scan, and normalized to pool results across individuals. We show that at 4 dpf, muscle‐fibre trajectories exhibit a helical pattern tapering towards the tail. Average fibre angles decrease from anterior to posterior, with azimuth varying over the dorsoventral axis and elevation varying over the mediolateral axis. Notably, only the anteriormost 20% of the body displayed higher azimuth angles near the medial plane. Angles between neighbouring fibres were substantial, particularly at the rim of the epaxial and hypaxial muscles. The revealed muscle‐fibre architecture at this age presumably contributes to the swimming performance of these larvae, but that swimming performance is probably not the only driving factor for the fibre pattern. Our methodology offers a promising avenue for exploring muscle‐fibre orientations across ontogenetic series and provides a foundation for in‐depth functional studies on the role of muscle architecture in facilitating swimming performance of larval fish.

Cumulative effects of H+ and Pi on force and power of skeletal muscle fibres from young and older adults.

The cellular causes of the age-related loss in power output and increased fatigability are unresolved. We previously observed that the depressive effects of hydrogen (H+) (pH 6.2) and inorganic phosphate (Pi) (30mm) did not differ in muscle fibres from young and older men. However, the effects may have been saturated in the severe fatigue-mimicking condition, potentially masking age differences in the sensitivity of the cross-bridge to these metabolites. Thus, we compared the contractile mechanics of muscle fibres from the vastus lateralis of 13 young (20-32years, seven women) and 12 older adults (70-90years, six women) in conditions mimicking quiescent muscle and a range of elevated H+ (pH 6.8-6.6-6.2) and Pi (12-20-30mm). The older adult knee extensor muscles showed hallmark signs of ageing, including 19% lower thigh lean mass, 60% lower power and a greater fatigability compared to young adult muscles. Progressively increasing concentrations of H+ and Pi in the chemically-permeabilized fibre experiments caused a linear decrease in fibre force, velocity and power; however, the effects did not differ with age or sex. Fast fibre cross-sectional area was 41% smaller in older compared to young adults, which corresponded with lower absolute power. Size-specific power was greater in fibres from older compared to young adults, indicating the age-related decline in absolute power was explained by differences in fibre size. These data suggest the age-related loss in power is determined primarily by fast fibre atrophy in men and women, but the age-related increase in fatigability cannot be explained by an increased sensitivity of the cross-bridge to H+ and Pi. KEY POINTS: The causes of the age-related loss in muscle power output and the increase in fatigability during dynamic exercise remain elusive. We show that progressively increasing concentrations of hydrogen (H+) and inorganic phosphate (Pi) causes a linear decrease in muscle fibre force, velocity and power, but the depressive effects of these metabolites on cross-bridge function did not differ in fibres from older compared to young adults across a range of fatigue-mimicking conditions. We also found peak absolute power did not differ in slow fibres from young and older adults but it was ∼33% lower in older adult fast fibres, which was explained entirely by age differences in fibre size. These data suggest that fast fibre atrophy is a major factor contributing to the loss in power of older men and women, but that the age-related increase in fatigability cannot be explained by an increased sensitivity of the cross-bridge to H+ and Pi.

Skeletal muscle cell transformation and its influencing factors

Abstract. The composition of muscle fiber type is closely related to pork quality, and the pork quality with high proportion of slow muscle fiber is relatively good. In the study, various regulatory factors were found to act on key genes that determine the type of skeletal muscle fibers. The results show that there are many transcription factors affecting the transformation of muscle fiber types, including calcineurin-NFAT signaling pathway, MEF2-HDACs signaling, AMPK-PGC1 and nuclear receptor PPAR /PPAR . However, the underlying mechanism of individual regulators is still unclear. The mechanism of action of PGC-1 , MicroRNA and PPAR () and the synergistic effects of MEF2 and PPAR with PGC-1 and PPAR () and ERR were analyzed. Relevant results were obtained such as PGC-1 gene expression can induce the conversion of type IIx muscle fibers to type I muscle fibers, and miR-133a regulates the conversion of slow muscle fibers to fast muscle fibers by targeting TEAD1; MiR-208b regulates the transformation of slow muscle fibers by targeting and inhibiting METTL8. PPAR () drives the formation of functional type I muscle fibers. In this paper, there is still a gap in the deep molecular mechanism, in the follow-up study, we can further clarify the factors of the key genes to determine the type of muscle fibers, as well as the deep influence mechanism between each factor. Future research and exploration can focus on the regulation factor proposed in this paper.

THE EFFECT OF BALLISTIC EXERCISES USING RUBBER BANDS ON DEVELOPING SPECIAL ENDURANCE AND ACHIEVING 400M PERFORMANCE FOR ATHLETES UNDER 19 YEARS OLD

The speed events are exciting activities that have specific physical requirements to achieve performance and outstanding results and compete internationally. This is particularly important in individual sports, especially for youth categories in athletics. The 400m event is considered one of the most challenging sprint events due to the special physical capabilities required to achieve performance. This race demands a huge amount of speed, endurance, and strength, in addition to the requirements of willpower, determination, and perseverance. Achieving the highest levels in this race necessitates a high level of important fitness components and characteristics. Training for this group of runners focuses on speed endurance and strength endurance through ballistic exercises using rubber bands, which help develop the working muscles of the athletes and enhance their physical capacity, subsequently improving performance. Ballistic exercises with rubber bands are considered modern training methods in athletics, aiming to develop muscular strength through strength training. This type of training forces the athlete's body to recruit and stimulate fast muscle fibers, which is crucial because fast muscle fibers have the potential for growth and development in strength training. The importance of this research lies in using ballistic training with rubber bands to develop special capacities and its impact on performance.

Neurotization of Decellularized Muscle Graft Increases de novo Type I Slow Muscle Fiber Formation and Large Fiber Size Frequency

Volumetric muscle loss (VML) injuries are the result of extreme trauma from battlefield injuries, tumor ablations, and other physical traumas such as car crash injuries. The abrupt loss of muscle restricts the tissue's remaining regenerative capacity, leading to loss of satellite cells, peripheral nerve connections, and aberrant fibrosis. Prior research from our lab demonstrated that decellularized muscle matrix (DMM) supported regeneration of de novo fibers within the graft. The goal of this study was to determine whether DMM treated with a peripheral nerve using neurotization surgeries would enhance muscle regeneration and innervation. Forty-eight male Sprague Dawley rats were randomized and received a 1.5×1 cm defect treated with no treatment empty defect (ED), DMM, or autograft with a direct peroneal (antagonist) neurotization or tibial via end to side graft (agonist) neurotization. DMM grafts treated with neurotization utilizing either peroneal or tibial nerve axons increased fast twitch fibers within the grafted area compared to untreated DMM or ED. Additionally, the frequency distribution of myofiber size shifted toward a healthier morphology in the tibial nerve axon neurotized DMM compared to the uninjured medial head. Lastly, Nanostring gene results showed DMM treated with a neurotization shifted expression towards a more regenerative phenotype with some myogenic markers returning to sham levels. These data indicate that injured muscle treated with DMM and neurotization becomes pro-regenerative and can contribute to the functionalization of DMM. Statement of SignificanceExtremity soft tissue trauma like volumetric muscle loss (VML) can result in permanent loss of skeletal muscle mass, denervation, and ischemia posing a significant clinical challenge. VML injuries disrupt normal tissue architecture in addition to intramuscular axons which are critical elements in muscle function and regeneration. The overall objective of this study was to enhance axon growth into a VML injury treated with decellularized muscle matrix (DMM) using neurotization. DMM is an acellular biomaterial capable of regenerating skeletal muscle; however, without bona fide neuromuscular connections, functional gains are small. This study demonstrates that introducing motor axons into an acellular regenerative material using neurotization enhanced muscle regeneration and promoted slow twitch fiber formation.

Anatomy and Immunohistochemistry of Woodpecker Tail Muscles.

Woodpeckers (Order Piciformes) belong to a group of birds characterized by their hammering capabilities in which the bill is utilized as a tool to probe for food and to excavate nest cavities. They have numerous specializations for this behavior, including their bill and tongue, feet for gripping vertical tree trunks, and tail feathers with thickened shafts to provide stability as a postural appendage. We hypothesized that (1) woodpecker tail musculature is also modified for clinging behaviors with a heterogeneous distribution of fast and slow muscle fibers, and that (2) the tree-trunk foraging Hairy Woodpeckers would have more slow muscle fibers in their M. depressor caudae than Northern Flickers, which forage on the ground where they probe the substrate for insects. We performed immunohistochemistry to identify the fiber type distributions for tail muscles Mm. caudofemoralis pars caudalis, lateralis caudae, levator caudae, and depressor caudae in four Hairy Woodpeckers and five Northern Flickers. Our results show that these tail muscles in the two woodpecker species are comprised of a majority of fast muscle fibers common among dynamic locomotor muscles. Interestingly, we report a functionally-significant distribution of slow muscle fibers in M. depressor caudae predicted to be utilized in propping of the tail during tree climbing and support. Further, we found more slow fibers (13.80% ± 4.49%) in the trunk-foraging Hairy Woodpeckers compared with the ground-foraging Northern Flicker (7.40% ± 4.95%), which we interpret to be related to the trunk-foraging habits of Hairy Woodpeckers.

Molecular determinants of skeletal muscle force loss in response to 5days of dry immersion in human.

Astronauts in Earth's orbit experience microgravity, resulting in a decline of skeletal muscle mass and function. On Earth, models simulating microgravity have shown that the extent of the loss in muscle force is greater than the loss in muscle mass. The reasons behind this disproportionate loss of muscle force are still poorly understood. In the present study, we hypothesize that alongside the loss in skeletal muscle mass, modifications in the expression profile of genes encoding critical determinants of resting membrane potential, excitation-contraction coupling and Ca2+ handling contribute to the decline in skeletal muscle force. Healthy male volunteers (n=18) participated in a 5-day dry immersion (DI) study, an Earth-based model of simulated microgravity. Muscle force measurement and MRI analysis of the cross-sectional area of thigh muscles were performed before and after DI. Biopsies of the vastus lateralis skeletal muscle performed before and after DI were used for the determination Ca2+ properties of isolated muscle fibres, molecular and biochemical analyses. The extent of the decline in force, measured as maximal voluntary contraction of knee extensors (-11.1%, P<0.01) was higher than the decline in muscle mass (-2.5%, P<0.01). The decline in muscle mass was molecularly supported by a significant repression of the anabolic IGF-1/Akt/mTOR pathway (-19.9% and -40.9% in 4E-BP1 and RPS6 phosphorylation, respectively), a transcriptional downregulation of the autophagy-lysosome pathway and a downregulation in the mRNA levels of myofibrillar protein slow isoforms. At the single fibre level, biochemical and tension-pCa curve analyses showed that the loss in force was independent of fibre type (-11% and -12.3% in slow and fast fibres, respectively) and Ca2+ activation properties. Finally, we showed a significant remodelling in the expression of critical players of resting membrane potential (aquaporin 4: -24.9%, ATP1A2: +50.4%), excitation-contraction coupling (CHRNA1: +75.1%, CACNA2D1: -23.5%, JPH2: -24.2%, TRDN: -15.6%, S100A1: +27.2%), and Ca2+ handling (ATP2A2: -32.5%, CASQ1: -15%, ORAI1: -36.2%, ATP2B1: -19.1%). These findings provide evidence that a deregulation in the expression profile of critical molecular determinants of resting membrane potential, excitation-contraction coupling, and Ca2+ handling could be involved in the loss of muscle force induced by DI. They also provide the paradigm for the understanding of muscle force loss during prolonged bed rest periods as those encountered in intensive care unit.

9291 Hypothyroidism impairs skeletal muscle regeneration after injury

Abstract Disclosure: P. Aguiari: None. V. Villani: None. K.Y. Liu: None. G.A. Brent: None. L. Perin: None. A. Milanesi: None. Thyroid hormone (TH) signaling plays an essential role in muscle development and function, in the maintenance of muscle mass, and in regeneration after injury. Disruption of TH signaling results in an abnormal skeletal muscle phenotype, impaired regeneration, and sarcopenia with aging, reflecting the myopathic changes described in hypothyroid patients. Muscle stem cells (MuSCs) are the mediators of post-natal skeletal muscle plasticity. Normally quiescent, in response to injury they enter the cell cycle (myoblasts) and proliferate. Myoblasts then exit the cell cycle and differentiate into myocytes that will fuse with existing myofibers to restore tissue architecture and function. Via TH receptor alpha, TH regulates all the stages of the regeneration program and regulates the expression of multiple myogenic genes. Despite all this evidence, to date there are no studies investigating MuSCs behavior in response to injury during hypothyroidism. We characterized injury-induced regeneration of the tibialis anterior muscle (TAM) in euthyroid and hypothyroid mice, fed a low iodine + 0.15% propylthiouracil diet. To investigate the cell cycle dynamics of MuSCs, we generated Pax7-Fucci mice carrying the fluorescent ubiquitination-based cell-cycle indicator (Fucci) system under the Pax7 promoter. Muscle injury was induced via cardiotoxin injection in the TAM of 3-month-old Pax7-Fucci mice. Hypothyroid mice present signs of impaired regeneration compared to euthyroid mice. From histological analysis, at different time points, we observed smaller fiber diameters, myofibers with central nuclei, and a significantly reduced number of fast glycolytic type IIb fibers, the prevalent muscle fiber type in the TA muscle. ScRNAseq analysis shows that at a late phase of regeneration (14 days after injury) hypothyroid MuSCs and myoblasts are still in the activation state and overexpress genes related to DNA replication and cell proliferation, while genes related to myogenic differentiation and cell cycle exit are downregulated. Differentiated myocytes in the hypothyroid fibers present an immature phenotype and are characterized by overexpression of embryonic/temporary and slow myosin genes and downregulation of mature fast myosins. Cell cycle analysis of the Fucci signal through Flow Cytometry confirmed a higher number of activated hypothyroid MuSCs at 4, 7, and 28 days after injury. These cells accumulate in the S phase and are unable to exit the cell cycle and differentiate towards myocytes. These data demonstrate that hypothyroidism impairs muscle tissue regeneration halting MuSCs progression through the cell cycle, myoblast cell cycle exit, and fiber maturation. Investigating muscle stem cell behavior and response to injury during hypothyroidism is crucial to understanding the mechanism behind thyroid hormone-induced myopathies and identifying possible therapeutical targets. Presentation: 6/2/2024

8670 Hypothyroidism impairs skeletal muscle regeneration after injury

Abstract Disclosure: P. Aguiari: None. V. Villani: None. K.Y. Liu: None. G.A. Brent: None. L. Perin: None. A. Milanesi: None. Thyroid hormone (TH) signaling plays an essential role in muscle development and function, in the maintenance of muscle mass, and in regeneration after injury. Disruption of TH signaling results in an abnormal skeletal muscle phenotype, impaired regeneration, and sarcopenia with aging, reflecting the myopathic changes described in hypothyroid patients. Muscle stem cells (MuSCs) are the mediators of post-natal skeletal muscle plasticity. Normally quiescent, in response to injury they enter the cell cycle (myoblasts) and proliferate. Myoblasts then exit the cell cycle and differentiate into myocytes that will fuse with existing myofibers to restore tissue architecture and function. Via TH receptor alpha, TH regulates all the stages of the regeneration program and regulates the expression of multiple myogenic genes. Despite all this evidence, to date there are no studies investigating MuSCs behavior in response to injury during hypothyroidism. We characterized injury-induced regeneration of the tibialis anterior muscle (TAM) in euthyroid and hypothyroid mice, fed a low iodine + 0.15% propylthiouracil diet. To investigate the cell cycle dynamics of MuSCs, we generated Pax7-Fucci mice carrying the fluorescent ubiquitination-based cell-cycle indicator (Fucci) system under the Pax7 promoter. Muscle injury was induced via cardiotoxin injection in the TAM of 3-month-old Pax7-Fucci mice. Hypothyroid mice present signs of impaired regeneration compared to euthyroid mice. From histological analysis, at different time points, we observed smaller fiber diameters, myofibers with central nuclei, and a significantly reduced number of fast glycolytic type IIb fibers, the prevalent muscle fiber type in the TA muscle. ScRNAseq analysis shows that at a late phase of regeneration (14 days after injury) hypothyroid MuSCs and myoblasts are still in the activation state and overexpress genes related to DNA replication and cell proliferation, while genes related to myogenic differentiation and cell cycle exit are downregulated. Differentiated myocytes in the hypothyroid fibers present an immature phenotype and are characterized by overexpression of embryonic/temporary and slow myosin genes and downregulation of mature fast myosins. Cell cycle analysis of the Fucci signal through Flow Cytometry confirmed a higher number of activated hypothyroid MuSCs at 4, 7, and 28 days after injury. These cells accumulate in the S phase and are unable to exit the cell cycle and differentiate towards myocytes. These data demonstrate that hypothyroidism impairs muscle tissue regeneration halting MuSCs progression through the cell cycle, myoblast cell cycle exit, and fiber maturation. Investigating muscle stem cell behavior and response to injury during hypothyroidism is crucial to understanding the mechanism behind thyroid hormone-induced myopathies and identifying possible therapeutical targets. Presentation: 6/2/2024

7284 Ablation of hexose-6-phosphate dehydrogenase in mice causes skeletal muscle atrophy and dramatically impairs exercise capacity

Abstract Disclosure: M. Zogg: None. A.D. Grötsch: None. J. Birk: None. P. Pelczar: None. J. Bouitbir: None. A. Odermatt: None. Hexose-6-phosphate dehydrogenase (H6PD) catalyzes the first two steps of the pentose-phosphate pathway (PPP) within the endoplasmic reticulum (ER), generating the redox equivalent NADPH. It has been shown that mice lacking H6PD develop defects mainly in skeletal muscles [1]. Nevertheless, the mechanisms underlying the myopathy in H6PD KO mice is unknown. Here, we further assessed muscle responses to H6PD deficiency and aimed to uncover the mechanisms that may cause myopathy in mice. We conducted a detailed characterization of the muscle structure and function as well as energy metabolism in oxidative and glycolytic muscles of H6PD KO mice. At the age of 15 weeks, H6PD KO mice displayed lower body weight than WT mice, despite similar food and water intake. Grip strength was significantly lower in H6PD KO than in WT mice. This was associated with decreased weight of the mixed (gastrocnemius, quadriceps) and fast fiber type rich muscles of the hind leg (extensor digitorum longus, tibialis anterior) in H6PD KO animals. Atrogin1 and Murf1 mRNA expression, markers of atrophy, increased only in the white (glycolytic) quadriceps. In this context, fast-twitch fibers in WT mice displayed higher H6pd mRNA expression than slow-twitch fibers. Electron microscopy analysis of red and white gastrocnemius muscles displayed the presence of large intramyofibrillar vacuoles, and morphologic changes of the sarcoplasmic reticulum and mitochondria in H6PD KO muscle. Metabolically, H6PD KO muscles presented an accumulation of glycogen granules and significantly decreased mRNA expression of glucose transporter Glut4, glucose-6-phosphate transporter G6pt and glycogen phosphorylase Pygm. The exercise capacity, measured by maximal running distance, decreased by more than 50% in H6PD KO mice. Upon exercise, both WT and H6PD KO animals were able to mobilize the majority of glycogen stored in their muscles. However, immediately after exercise, there was a significant increase in plasma lactate and glucose levels in H6PD KO animals. Disturbances in glucose metabolism were further supported by in vitro experiments using stable C2C12 H6PD KO myoblasts, revealing a shift in basal state mitochondrial fuel oxidation usage from glucose to fatty acid and glutamine in metabolic dependency assays. In conclusion, our findings revealed that H6PD seems to play a key role in proper function and energy metabolism of skeletal muscles.

Identification of Giant Isoforms of Obscurin in Rat Striated Muscles Using Polyclonal Antibodies.

Using produced polyclonal antibodies specific to the N-terminal sequence (residues 61-298) of rat obscurin, we investigated the isoform composition of this protein in 4 striated muscles: myocardium of the left ventricle, diaphragm, skeletal m. gastrocnemius (containing mainly fast fibers), and m. soleus (containing mainly slow fibers). The m. gastrocnemius, m. soleus, and diaphragm were found to have 2 giant isoforms of obscurin: a smaller A-isoform and a larger B-isoform. Their molecular weights were ~870 and ~1150 kDa in the diaphragm and m. gastrocnemius and ~880 and ~1130 kDa in m. soleus, respectively. The B-isoform to A-isoform ratio was 1:3 in the diaphragm and m. soleus and 1:4 in the m. gastrocnemius. In the left-ventricular myocardium, A-isoform of obscurin with a molecular weight of ~880 kDa was found. No other obscurin isoforms or their fragments within the molecular weight range of 10 up to ~800 kDa were revealed in the investigated rat striated muscles. The antibodies produced are recommended for research into qualitative and quantitative changes of giant obscurin isoforms in rat striated muscles in the norm and during the development of pathological processes.