Glycolytic Fibers Research Articles

Sodium danshensu modulates skeletal muscle fiber type formation and metabolism by inhibiting pyruvate kinase M1.

Sodium Danshensu (SDSS) is extracted from Salvia miltiorrhiza and has many pharmacological effects. However, little is known about its effects on muscle fiber formation and metabolism. Here, we aimed to investigated the role and molecular mechanisms of SDSS in modulating the formation of skeletal muscle fiber. C2C12 cells were incubated in differentiation medium with or without SDSS for 4days. C57BL/6 mice were orally administered SDSS by gavage once a day for 8weeks. Grip strength, treadmill, muscle weight, western blotting, qPCR, immunofluorescence staining and H&E staining were performed. SDSS target proteins were searched through drug affinity responsive target stability (DARTS) and mass spectrometry analysis. Furthermore, molecular docking was carried out for Pyruvate kinase M1 (PKM1). The effect of PKM1 on myosin heavy chain (MyHCs) gene expression was verified by knockdown of PKM1 experiment. SDSS induced oxidative muscle fiber-related gene expression, and inhibited glycolytic fiber-related gene expression in C2C12 cells. Muscle mass, the percentage of slow oxidative fibers, succinic dehydrogenase activity, muscle endurance, glucose tolerance, and the expression of the MyHC1 and MyHC2a genes increased while MyHC2b expression, lactate dehydrogenase activity, and the percentage of glycolytic muscle fibers decreased in SDSS-treated mice. Mechanistically, SDSS bound to the pyruvate kinase PKM1 and significantly repressed its activity. PKM1 inhibited MyHC1 and MyHC2a expression but promoted MyHC2b expression. SDSS also significantly attenuated the effects of PKM1 on muscle fiber-related gene expression in C2C12 cells. Our findings indicate that SDSS promotes muscle fiber transformation from the glycolytic type to the oxidative type by inhibiting PKM1 activity, which provide a new idea for treating muscle atrophy, muscle metabolism diseases and improving animal meat production.

Inhibition of CILP2 Improves Glucose Metabolism and Mitochondrial Dysfunction in Sarcopenia via the Wnt Signalling Pathway.

Skeletal muscle is the primary organ involved in insulin-mediated glucose metabolism. Elevated levels of CILP2 are a significant indicator of impaired glucose tolerance and are predominantly expressed in skeletal muscle. It remains unclear whether CILP2 contributes to age-related muscle atrophy through regulating the glucose homeostasis and insulin sensitivity. Initially, the expression levels of CILP2 were assessed in elderly mice and patients with sarcopenia. Lentiviral vectors were used to induce either silencing or overexpression of CILP2 in C2C12 myoblast cells. The effects of CILP2 on proliferation, myogenic differentiation, insulin sensitivity and glucose uptake were evaluated using immunofluorescence, western blotting, real-time quantitative polymerase chain reaction, RNA sequencing, glucose uptake experiments, dual-luciferase reporter assays and co-immunoprecipitation (CO-IP). An adeno-associated virus-9 containing a muscle-specific promoter was injected into SAMP8 senile mice to observe the efficacy of CILP2 knockout. We found that there was more CLIP2 expressed in the skeletal muscle of ageing mice (+1.1-fold, p < 0.01) and in patients with sarcopenia (+2.5-fold, p < 0.01) compared to the control group. Following the overexpression of CILP2, Ki67 (-65%, p < 0.01), PCNA (-32%, p < 0.05), MyoD1 (-89%, p < 0.001), MyoG (-31%, p < 0.05) and MyHC (-85%, p < 0.001), which indicate proliferation and differentiation potential, were significantly reduced. In contrast, MuRF-1 (+59%, p < 0.05), atrogin-1 (+43%, p < 0.05) and myostatin (+31%, p < 0.05), the markers of muscular atrophy, were significantly increased. Overexpression of CILP2 decreased insulin sensitivity, glucose uptake (-18%, p < 0.001), GLUT4 translocation to the membrane and the maximum respiratory capacity of mitochondria. Canonical Wnt signalling was identified through RNA sequencing as a potential pathway for CILP2 regulation in C2C12, and Wnt3a was confirmed as an interacting protein of CILP2 in the CO-IP assay. The addition of recombinant Wnt3a protein reversed the inhibitory effects on myogenesis and glucose metabolism caused by CILP2 overexpression. Conversely, CILP2 knockdown promoted myogenesis and glucose metabolism. CILP2 knockdown improved muscle atrophy in mice, characterized by significant increases in time to exhaustion (+42%, p < 0.001), grip strength (+19%, p < 0.01), muscle mass (+15%, p < 0.001) and mean muscle cross-sectional area (+37%, p < 0.01). CILP2 knockdown enhanced glycogen synthesis (+83%, p < 0.001) and the regeneration of oxidative and glycolytic muscle fibres in SAMP8 ageing mice via the Wnt/β-catenin signalling pathway. Our results indicate that CILP2 interacts with Wnt3a to suppress the Wnt/β-catenin signalling pathway and its downstream cascade, leading to impaired insulin sensitivity and glucose metabolism in skeletal muscle. Targeting CILP2 inhibition could offer potential therapeutic benefits for sarcopenia.

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

Altered muscle fibre activation in an antagonistic muscle pair due to perturbed afferent feedback caused by blood flow restriction

PurposeThis study aimed to better understand the coping strategy of the neuromuscular system under perturbed afferent feedback. To this end, the neuromechanical effects of transient blood flow restriction (BFR) compared to atmospheric pressure were investigated in an antagonistic muscle pair. MethodsPerceived discomfort and neuromechanical parameters (torque and high-density electromyography) were recorded during submaximal isometric ankle dorsiflexion before, during and after BFR. The tibialis anterior and gastrocnemius lateralis muscles were studied in 14 healthy young adults. ResultsDiscomfort increased during BFR and decreased to baseline level afterwards. The exerted torque and the co-activation index remained constant, whereas the EMG signal energy increased significantly during BFR. Coherence analysis of the delta band remained constant, whereas the alpha band shows an increase during BFR. Median frequency and muscle fibre conduction velocity showed a positive trend during the first minutes of BFR before significantly decreasing. Both parameters exceeded baseline values after cuff deflation. ConclusionPerturbed afferent feedback leads to altered neuromechanical parameters. We assume that increased central drive is required to maintain force output, resulting in changed muscle fibre activity. Glycolytic fast-switch fibres are only active for a short time due to oxygen deprivation and hyperacidity, but fatigue effects predominate in the long term.

Consistent changes in muscle phenotype and mitochondrial abundance underlie dive performance across multiple lineages of diving ducks.

Diving animals must sustain high muscle activity with finite oxygen (O2) to forage underwater. Studies have shown that some diving mammals exhibit changes in the metabolic phenotype of locomotory muscles compared with non-divers, but the pervasiveness of such changes across diving animals is unclear, particularly among diving birds. Here, we examined whether changes in muscle phenotype and mitochondrial abundance are associated with dive capacity across 17 species of ducks from three distinct evolutionary clades (tribes) in the subfamily Anatinae: the longest diving sea ducks, the mid-tier diving pochards and the non-diving dabblers. In the gastrocnemius (the primary swimming and diving muscle), mitochondrial volume density in both oxidative and glycolytic fiber types was 70% and 30% higher in sea ducks compared with dabblers, respectively. These differences were associated with preferential proliferation of the subsarcolemmal subfraction, the mitochondria adjacent to the cell membrane and nearest to capillaries, relative to the intermyofibrillar subfraction. Capillary density and capillary-to-fiber ratio were positively correlated with mitochondrial volume density, with no variation in the density of oxidative fiber types across tribes. In the pectoralis, sea ducks had greater abundance of oxidative fiber types than dabblers, whereas pochards were intermediate between the two. These data suggest that skeletal muscles of sea ducks have a heightened capacity for aerobic metabolism and an enhanced ability to utilize O2 stores in the blood and muscle while diving.

Transcriptome profiling of fast/glycolytic and slow/oxidative muscle fibers in aging and obesity

Aging and obesity pose significant threats to public health and are major contributors to muscle atrophy. The trends in muscle fiber types under these conditions and the transcriptional differences between different muscle fiber types remain unclear. Here, we demonstrate distinct responses of fast/glycolytic fibers and slow/oxidative fibers to aging and obesity. We found that in muscles dominated by oxidative fibers, the proportion of oxidative fibers remains unchanged during aging and obesity. However, in muscles dominated by glycolytic fibers, despite the low content of oxidative fibers, a significant decrease in proportion of oxidative fibers was observed. Consistently, our study uncovered that during aging and obesity, fast/glycolytic fibers specifically increased the expression of genes associated with muscle atrophy and inflammation, including Dkk3, Ccl8, Cxcl10, Cxcl13, Fbxo32, Depp1, and Chac1, while slow/oxidative fibers exhibit elevated expression of antioxidant protein Nqo-1 and downregulation of Tfrc. Additionally, we noted substantial differences in the expression of calcium-related signaling pathways between fast/glycolytic fibers and slow/oxidative fibers in response to aging and obesity. Treatment with a calcium channel inhibitor thapsigargin significantly increased the abundance of oxidative fibers. Our study provides additional evidence to support the transcriptomic differences in muscle fiber types under pathophysiological conditions, thereby establishing a theoretical basis for modulating muscle fiber types in disease treatment.

Effects of thyroid hormone on monochromatic light combinations mediate skeletal muscle fiber pattern in broilers

It has been shown that monochromatic green light and blue light promote skeletal muscle development in early (P0-P26) and later growth stages (P27-P42), respectively. This study further investigated the effects of monochromatic light combinations on myogenesis and myofiber types transformation in broilers. Here, a total of 252 chicks were exposed to monochromatic light [red (R), green (G), blue (B), or white light (W)], and monochromatic light combination [green and blue light combination (GB), blue and green light combination (BG), red and blue combination (RB)] until P42. Compared with other groups, GB significantly increased body weight, and muscle organ index, both proportions of larger-size myofibers and oxidative myofibers in the pectoralis major (PM) and gastrocnemius muscle (GAS). Meanwhile, GB up-regulated the abundance of oxidative genes MYH7B and MYH1B, transcription factors PAX7 and Myf5, antioxidant proteins Nrf2, HO-1, and GPX4, and the activities of antioxidant enzymes CAT, GPx, and T-AOC, but down-regulated the abundance of glycolytic related genes MYH 1A, MyoD, MyoG, Mstn, Keap1, TNFa, and MDA levels. Consistent with the change of myofiber pattern, GB significantly reduced serum thyroid hormone (TH) levels, up-regulated skeletal muscle deiodinase DIO3 expression and down-regulated deiodinase DIO2 expression, which may directly lead to the reduction of intramuscular TH levels to affect myofiber types transformation. In contrast, the proportion of fast glycolytic muscle fibers increased in the RR with increasing TH levels. After thyroidectomy, the above parameters were inversed and resulted in no significant difference of each color light treatment group. These data suggested that GB significantly increased the proportion of oxidative muscle fibers and antioxidant capacity in skeletal muscle of broilers, which was regulated by TH-DIO2/DIO3 signaling pathway.

Exercise in Chronic Kidney Disease Patients Does Not Significantly Alter Fibrosis in Quadriceps Muscle

Background: Chronic kidney disease (CKD) is commonly associated with decreased physical activity and muscle dysfunction. Muscles in CKD become fibrotic, containing excess extracellular matrix (ECM), particularly collagen. Collagen is cross-linked in the ECM, and increased cross-linking is often associated with fibrosis, stiffness, and reduced ECM remodeling. However, it is not known how exercise in patients with CKD may impact the extent of collagen cross-linking or fibrosis. Additionally, muscle fibrosis may impair muscle adaptations to exercise including, fiber size, fiber type, or vasculature. We predicted that collagen content and cross-linking would decrease, and there would be more Type I fibers and larger fiber size in the patients that were in the exercise group. Methods: Participants were randomized to exercise (n=15) or no exercise (n=5) groups in the Exercise Study Testing Enhanced Energetics of Mitochondria (ESTEEM) clinical trial of non-dialysis patients with CKD. The exercise program consisted of 35 min sessions, 3 times a week for 12 weeks consisting of high-intensity interval training, strength training, and power walking. Vastus lateralis biopsies were obtained before and after the 12 weeks of exercise. A bundle of muscle tissue was powdered and separated into a pepsin soluble and insoluble fraction before applying a hydroxyproline assay for collagen content, with the insoluble fraction representing more cross-linked collagen. For histological analysis 10 μm thick cryosections were stained with Picrosirius Red, or immunofluorescent stains to investigate satellite cells, vasculature, fibro-adipogenic progenitors (FAPs), and muscle fiber types. Picrosirius red sections were imaged under polarized light to look at birefringence as a measure of collagen density. Sections that were stained for fiber type and vasculature were analyzed using semi-automatic muscle analysis using segmentation of histology (SMASH). Results: The overall amount of collagen and collagen cross-linking were not altered in either the exercise or no exercise groups. Picrosirius red stained sections did not reveal a change in ECM area or collagen packing density with exercise. Surprisingly minimum ferret diameter and percentage of type I (slow twitch) fibers decreased in most patients that underwent the exercise program. Type I and type IIa (fast oxidative) fiber size also significantly decreased, while there was no change in type IIx (fast glycolytic) fiber size. Conclusion: This study shows that exercise does not dramatically reduce ECM content or muscle fibrosis in patients with CKD. Surprisingly, the response to exercise was opposite of that anticipated in healthy subject with exercise in CKD producing smaller muscle fibers and a shift away from more oxidative type I fibers. This shows that exercise may have limited capacity to enhance function in CKD muscle, potentially due to irreversible fibrosis. Work supported by mitochondrial bioenergetics in persons with chronic kidney disease (NIH R03DK114502). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Deletion of Myostatin Significantly and Preferentially Improves Muscle Function in a Sex-Dependent Manner in Both an Obese and Aging Mouse Model

Skeletal muscle mass and function are essential components of health and homeostasis. In addition to its role in balance and locomotion, skeletal muscle is the body’s largest metabolic reservoir, absorbing significant amounts of glucose and amino acids from the blood stream. The loss of muscle mass is associated with impaired glucose tolerance, threatens vascular health and contributes to cardiovascular disease in obese and aging (sarcopenic) populations, along with driving overall mortality. The myokine myostatin (GDF-8) is a potent negative regulator of skeletal muscle growth that is upregulated in humans and animal models of obesity and aging. Loss of myostatin is known to increase the number of glycolytic muscle fibers and has been shown to correct indices that govern cardiometabolic health in obesity and aging, improving endothelial function and glucose homeostasis, and protecting against renal injury and hypertension. However, these studies have largely been conducted using male mice, leaving the question regarding the effectiveness of myostatin deletion on skeletal muscle function, independent of sex, largely unexplored. As such, our hypothesis is that the deletion of myostatin will improve skeletal muscle function in both male and female mice, regardless of the disease phenotype being obesity or aging. Young (3-5 mo.) lean and obese mice were used, along with aged (18-24 mo.) mice. The obese db/db model was utilized as it is a well characterized model of hyperphagia that results in obesity and subsequent metabolic disease, including significant whole-body adiposity and reduced muscle mass. On this background we have crossed mice harboring constitutive genetic deletion of myostatin to reverse muscle loss. Thus, myostatin deletion was crossed onto the relevant backgrounds in combination with aging or obesity and included both sexes. Muscle function was assessed in all groups using in vivo plantarflexion. Young male mice demonstrated an ~20% increase in force production compared to the females. Results show that aging and obesity inhibited skeletal muscle function in both sexes, with female mice showing a lessened impact of obesity or aging. Myostatin deletion improved muscle function in male mice, returning both obesity-derived weakness and aging-induced sarcopenia back to levels of young control. However, female mice without myostatin experienced a relative gain-of-function, with force production significantly exceeding that of the young control. The implication of these results is that weight loss may be more effective in protecting muscle function in diseases in the male population whereas myostatin inhibition, either pharmaceutically or with prescription exercise, may result in greater improvements in the female sex comparatively. This research gratefully acknowledges support from the NIA (K01 AG064121), OCAST (HR21-045-1) and the Niblack Research Scholars Program. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identification of different myofiber types in pigs muscles and construction of regulatory networks

BackgroundSkeletal muscle is composed of muscle fibers with different physiological characteristics, which plays an important role in regulating skeletal muscle metabolism, movement and body homeostasis. The type of skeletal muscle fiber directly affects meat quality. However, the transcriptome and gene interactions between different types of muscle fibers are not well understood.ResultsIn this paper, we selected 180-days-old Large White pigs and found that longissimus dorsi (LD) muscle was dominated by fast-fermenting myofibrils and soleus (SOL) muscle was dominated by slow-oxidizing myofibrils by frozen sections and related mRNA and protein assays. Here, we selected LD muscle and SOL muscle for transcriptomic sequencing, and identified 312 differentially expressed mRNA (DEmRs), 30 differentially expressed miRNA (DEmiRs), 183 differentially expressed lncRNA (DElRs), and 3417 differentially expressed circRNA (DEcRs). The ceRNA network included ssc-miR-378, ssc-miR-378b-3p, ssc-miR-24-3p, XR_308817, XR_308823, SMIM8, MAVS and FOS as multiple core nodes that play important roles in muscle development. Moreover, we found that different members of the miR-10 family expressed differently in oxidized and glycolytic muscle fibers, among which miR-10a-5p was highly expressed in glycolytic muscle fibers (LD) and could target MYBPH gene mRNA. Therefore, we speculate that miR-10a-5p may be involved in the transformation of muscle fiber types by targeting the MYHBP gene. In addition, PPI analysis of differentially expressed mRNA genes showed that ACTC1, ACTG2 and ACTN2 gene had the highest node degree, suggesting that this gene may play a key role in the regulatory network of muscle fiber type determination.ConclusionsWe can conclude that these genes play a key role in regulating muscle fiber type transformation. Our study provides transcriptomic profiles and ceRNA interaction networks for different muscle fiber types in pigs, providing reference for the transformation of pig muscle fiber types and the improvement of meat quality.

Type selective ablation of postnatal slow and fast fatigue-resistant motor neurons in mice induces late onset kinetic and postural tremor following fiber-type transition and myopathy

Animals on Earth need to hold postures and execute a series of movements under gravity and atmospheric pressure. VAChT-Cre is a transgenic Cre driver mouse line that expresses Cre recombinase selectively in motor neurons of S-type (slow-twitch fatigue-resistant) and FR-type (fast-twitch fatigue-resistant). Sequential motor unit recruitment is a fundamental principle for fine and smooth locomotion; smaller-diameter motor neurons (S-type, FR-type) first contract low-intensity oxidative type I and type IIa muscle fibers, and thereafter larger-diameter motor neurons (FInt-type, FF-type) are recruited to contract high-intensity glycolytic type IIx and type IIb muscle fibers. To selectively eliminate S- and FR-type motor neurons, VAChT-Cre mice were crossbred with NSE-DTA mice in which the cytotoxic diphtheria toxin A fragment (DTA) was expressed in Cre-expressing neurons. The VAChT-Cre;NSE-DTA mice were born normally but progressively manifested various characteristics, including body weight loss, kyphosis, kinetic and postural tremor, and muscular atrophy. The progressive kinetic and postural tremor was remarkable from around 20 weeks of age and aggravated. Muscular atrophy was apparent in slow muscles, but not in fast muscles. The increase in motor unit number estimation was detected by electromyography, reflecting compensatory re-innervation by remaining FInt- and FF-type motor neurons to the orphaned slow muscle fibers. The muscle fibers gradually manifested fast/slow hybrid phenotypes, and the remaining FInt-and FF-type motor neurons gradually disappeared. These results suggest selective ablation of S- and FR-type motor neurons induces progressive muscle fiber-type transition, exhaustion of remaining FInt- and FF-type motor neurons, and late-onset kinetic and postural tremor in mice.

Histotopographic Organization of Levator Ani Muscle in Laboratory Rats

Morphological organization of the levator ani muscle still remains unclear due to methodological problems in the study of the pelvic floor muscles. Therefore, the study of the histological structure of muscle tissue m. levator ani in animals is of not only theoretical but also practical importance for the study and modeling of pelvic floor dysfunction conditions in animal models. The aim is to study the enzymohistochemical and ultramicroscopic organization of the levator ani muscle in white laboratory rats. Material and methods. The study was performed on 10 laboratory Wistar rats (5 females and 5 males) aged 12–14 months. The metabolic profile of muscle fibers was determined using the Nachlass succinate dehydrogenase test. Imaging and linear measurements were carried out on a Leica UC 7 microscope (Germany), using its software. Ultrathin sections were counterstained with uranyl acetate and lead citrate, viewed nd photographed using a Hitachi HT 7700 Exalens (Japan) electron microscope. Results. All types of muscle fibers are present in levator ani muscle: slow fibers and rapid muscle fibers of typs IIA and IIB. It has been established that the total cross-sectional area of white type fibers is 3 times greater than the total area of other fibers. Significant sex differences were found in relative square parameters of glycolytic fibers in m. levator ani (p=0,009). At the ultrastructural level, it was found that in some muscle fibers the mitochondrial apparatus is well developed, in others, mitochondria are few in number, have smaller sizes, are located singly between myofibrils, without forming clusters. In such fibers, there are large accumulations of glycogen between the myofibrils. Conclusion. In the laboratory rat, the levator ani muscle is heterogeneous both in metabolic activity of muscle fibers and in ultrastructural organization. This feature of the muscle involves not only static work in creating intra-abdominal pressure and retaining the pelvic organs, but also isotonic contraction, acting as a synergist to ensure the movement of the tail.

Downregulation of mitochondrial metabolism is a driver for fast skeletal muscle loss during mouse aging

Skeletal muscle aging is characterized by the loss of muscle mass, strength and function, mainly attributed to the atrophy of glycolytic fibers. Underlying mechanisms driving the skeletal muscle functional impairment are yet to be elucidated. To unbiasedly uncover its molecular mechanisms, we recurred to gene expression and metabolite profiling in a glycolytic muscle, Extensor digitorum longus (EDL), from young and aged C57BL/6JRj mice. Employing multi-omics approaches we found that the main age-related changes are connected to mitochondria, exhibiting a downregulation in mitochondrial processes. Consistent is the altered mitochondrial morphology. We further compared our mouse EDL aging signature with human data from the GTEx database, reinforcing the idea that our model may recapitulate muscle loss in humans. We are able to show that age-related mitochondrial downregulation is likely to be detrimental, as gene expression signatures from commonly used lifespan extending interventions displayed the opposite direction compared to our EDL aging signature.

A novel protein encoded by circKANSL1L regulates skeletal myogenesis via the Akt-FoxO3 signaling axis

Skeletal muscle is one the largest organs of the body and is involved in animal production and human health. Circular RNAs (circRNAs) have been implicated in skeletal myogenesis through largely unknown mechanisms. Herein, we report the phenotypic and metabolomic analysis of porcine longissimus dorsi muscles in Lantang and Landrace piglets, revealing a high-content of slow-oxidative fibers responsible for high-quality meat product in Lantang piglets. Using single-cell transcriptomics, we identified four myogenesis-related cell types, and the Akt-FoxO3 signaling axis was the most significantly enriched pathway in each subpopulation in the different pig breeds, as well as in fast-twitch glycolytic fibers. Using the multi-dimensional bioinformatic tools of circRNAome-seq and Ribo-seq, we identified a novel circRNA, circKANSL1L, with a protein-coding ability in porcine muscles, whose expression level correlated with myoblast proliferation and differentiation in vitro, as well as the transformation between distinct mature myofibers in vivo. The protein product of circKANSL1L could interact with Akt to decrease the phosphorylation level of FoxO3, which subsequently promoted FoxO3 transcriptional activity to regulate skeletal myogenesis. Our results established the existence of a protein encoded by circKANSL1L and demonstrated its potential functions in myogenesis.

Impact of cooking temperature on pork longissimus, and muscle fibre type, on quality traits and protein denaturation of four pork muscles

Variations in pork quality impact consumer acceptance, and fibre type differences between muscles contribute to this variation. The aim was to investigate the influence of variations in muscle fibre types and protein denaturation peaks across four pork muscles and the influence of ageing and cooking temperature on longissimus quality traits. The longissimus, masseter, cutaneous trunci, and psoas major from 13 carcases were removed 1-day postmortem and subjected to 0- or 14-days ageing (d0, d14). Quality traits, protein denaturation peak temperature (DSC), fibre diameter and fibre type proportions were measured. Cook loss for longissimus was similar on d0 and d14, but was higher on d14 for masseter, cutaneous trunci, and psoas major. Warner-Bratzler shear force was highest, and ultimate pH was lowest, for longissimus, and similar among cutaneous trunci, masseter, and psoas major. Masseter had lowest L* and highest a* and longissimus and cutaneous trunci had highest L* and lowest a*. The DSC temperature peaks for longissimus occurred at lower temperatures relative to the other muscles. Fibre diameter was largest for type-IIb fibres relative to type-IIa and type-I. Longissimus and cutaneous trunci had predominantly type-IIb glycolytic (71%, 51% respectively), masseter had predominantly type-IIa intermediate (50%) and psoas major had predominantly type-I oxidative (48%) fibres. The glycolytic longissimus had the lowest DSC temperature peaks and the lowest quality meat. Masseter had the highest proportion of type-I fibres but was generally similar in quality traits to psoas major, and also similar to cutaneous trunci which had more glycolytic fibres than masseter.

GADD45A is a mediator of mitochondrial loss, atrophy, and weakness in skeletal muscle.

Aging and many illnesses and injuries impair skeletal muscle mass and function, but the molecular mechanisms are not well understood. To better understand the mechanisms, we generated and studied transgenic mice with skeletal muscle-specific expression of growth arrest and DNA damage inducible α (GADD45A), a signaling protein whose expression in skeletal muscle rises during aging and a wide range of illnesses and injuries. We found that GADD45A induced several cellular changes that are characteristic of skeletal muscle atrophy, including a reduction in skeletal muscle mitochondria and oxidative capacity, selective atrophy of glycolytic muscle fibers, and paradoxical expression of oxidative myosin heavy chains despite mitochondrial loss. These cellular changes were at least partly mediated by MAP kinase kinase kinase 4, a protein kinase that is directly activated by GADD45A. By inducing these changes, GADD45A decreased the mass of muscles that are enriched in glycolytic fibers, and it impaired strength, specific force, and endurance exercise capacity. Furthermore, as predicted by data from mouse models, we found that GADD45A expression in skeletal muscle was associated with muscle weakness in humans. Collectively, these findings identify GADD45A as a mediator of mitochondrial loss, atrophy, and weakness in mouse skeletal muscle and a potential target for muscle weakness in humans.

383 Effect of Feeding Ractopamine to Finisher Lard-Type Mangalica Pigs on Longissimus Muscle Fiber Myosin-Heavy Chain Isoform Expression and Cross-Sectional Area

Abstract Heritage breeds such as Mangalica pigs are gaining popularity in US niche markets and currently sold as high-quality meat due to greater meat quality characteristics. Research has demonstrated that feeding the β-adrenergic agonist ractopamine hydrochloride (RAC), stimulates hypertrophic growth of type-2, fast-twitch glycolytic muscle fibers via mTOR signaling resulting in greater myofibrillar protein synthesis. Therefore, the current study evaluated the effect of feeding RAC to Mangalica pigs during the finisher phase on longissimus muscle fiber myosin heavy-chain (MHC) isoform expression and cross-sectional area. Mangalica pigs were randomly assigned finisher diets for 21 d with 20 ppm (RAC; n = 8) or without (CTL; n = 6). Longissimus muscle samples were collected during carcass fabrication, flash-frozen in liquid nitrogen and stored at -80 °C. Immunofluorescence staining of 5-µm-thick cryosections to identify Type-1 (Developmental Studies Hybridoma Bank (DSHB) antibody Ba-D5), Type-2a (DSHB SC-71), and 2x (DSHB BF-35) MHC positive fibers. Type 2b fibers were determined by subtraction. Fibers in each image (≥ 150) were assessed for fiber type and their cross-sectional area (CSA) at 200X magnification. Data were analyzed as a one-way ANOVA using the GLIMMIX procedure of SAS V9.4. Means were separated with the PDIFF option and declared different when P ≤ 0.05. Tendencies were declared when 0.0501 ≤ P ≤ 0.1000. Feeding RAC decreased the density of Type 2a (P = 0.0136) and Type 2b (P = 0.0186) fibers in the longissimus muscle of Mangalica pigs. A tendency for RAC to reduce the density of Type 1, slow-twitch (6.61 vs. 16.14 fiber per mm2; P = 0.0505) fibers and increase the density of primarily oxidative fibers expressing a combination of Type 1 + Type 2a MHC isoforms (10.23 vs. 2.88%; P = 0.0524) was also observed. When analyzed as a proportion of total fibers, RAC reduced the proportion of fibers expressing only Type 2a (11.8 vs. 6.70%; P = 0.0468) and Type 2b (0.19 vs. 1.32%; P = 0.0101) MHC isoforms but increased the percentage of fibers expressing both Type 1 + Type 2a MHC isoforms (4.66 vs. 1.13%; P = 0.0280). Feeding RAC did not impact the density or proportion of fibers expressing Type 2x MHC (P ≥ 0.386). Mean longissimus muscle fiber CSA was not impacted by feeding Mangalica pigs RAC for 21 d (P = 0.4815). The CSA of the smallest and largest fibers nor fiber distribution were influenced by RAC inclusion in the diet of Mangalica pigs (P ≥ 0.484). In conclusion, feeding RAC Mangalica pigs impacted fiber MHC isoform expression in the longissimus muscle without altering fiber CSA. Future studies will explore the impact of RAC on the CSA of the various fiber types as well as the impact on other muscles in the Mangalica pig.

Angiogenic potential of skeletal muscle derived extracellular vesicles differs between oxidative and glycolytic muscle tissue in mice

Skeletal muscle fibers regulate surrounding endothelial cells (EC) via secretion of numerous angiogenic factors, including extracellular vesicles (SkM-EV). Muscle fibers are broadly classified as oxidative (OXI) or glycolytic (GLY) depending on their metabolic characteristics. OXI fibers secrete more pro-angiogenic factors and have greater capillary densities than GLY fibers. OXI muscle secretes more EV than GLY, however it is unknown whether muscle metabolic characteristics regulate EV contents and signaling potential. EVs were isolated from primarily oxidative or glycolytic muscle tissue from mice. MicroRNA (miR) contents were determined and endothelial cells were treated with OXI- and GLY-EV to investigate angiogenic signaling potential. There were considerable differences in miR contents between OXI- and GLY-EV and pathway analysis identified that OXI-EV miR were predicted to positively regulate multiple endothelial-specific pathways, compared to GLY-EV. OXI-EV improved in vitro angiogenesis, which may have been mediated through nitric oxide synthase (NOS) related pathways, as treatment of endothelial cells with a non-selective NOS inhibitor abolished the angiogenic benefits of OXI-EV. This is the first report to show widespread differences in miR contents between SkM-EV isolated from metabolically different muscle tissue and the first to demonstrate that oxidative muscle tissue secretes EV with greater angiogenic signaling potential than glycolytic muscle tissue.

Skeletal muscle proteome analysis underpins multifaceted mitochondrial dysfunction in Friedreich's ataxia.

Friedreich's ataxia (FRDA) is a severe multisystemic disorder caused by a deficiency of the mitochondrial protein frataxin. While some aspects of FRDA pathology are developmental, the causes underlying the steady progression are unclear. The inaccessibility of key affected tissues to sampling is a main hurdle. Skeletal muscle displays a disease phenotype and may be sampled in vivo to address open questions on FRDA pathophysiology. Thus, we performed a quantitative mass spectrometry-based proteomics analysis in gastrocnemius skeletal muscle biopsies from genetically confirmed FRDA patients (n = 5) and controls. Obtained data files were processed using Proteome Discoverer and searched by Sequest HT engine against a UniProt human reference proteome database. Comparing skeletal muscle proteomics profiles between FRDA and controls, we identified 228 significant differentially expressed (DE) proteins, of which 227 were downregulated in FRDA. Principal component analysis showed a clear separation between FRDA and control samples. Interactome analysis revealed clustering of DE proteins in oxidative phosphorylation, ribosomal elements, mitochondrial architecture control, and fission/fusion pathways. DE findings in the muscle-specific proteomics suggested a shift toward fast-twitching glycolytic fibers. Notably, most DE proteins (169/228, 74%) are target of the transcription factor nuclear factor-erythroid 2. Our data corroborate a mitochondrial biosignature of FRDA, which extends beyond a mere oxidative phosphorylation failure. Skeletal muscle proteomics highlighted a derangement of mitochondrial architecture and maintenance pathways and a likely adaptive metabolic shift of contractile proteins. The present findings are relevant for the design of future therapeutic strategies and highlight the value of skeletal muscle-omics as disease state readout in FRDA.