Fast Glycolytic Fibers Research Articles

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

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.

Melatonin Improves Skeletal Muscle Structure and Oxidative Phenotype by Regulating Mitochondrial Dynamics and Autophagy in Zücker Diabetic Fatty Rat.

Obesity-induced skeletal muscle (SKM) inflexibility is closely linked to mitochondrial dysfunction. The present study aimed to evaluate the effects of melatonin on the red vastus lateralis (RVL) muscle in obese rat models at the molecular and morphological levels. Five-week-old male Zücker diabetic fatty (ZDF) rats and their age-matched lean littermates (ZL) were orally treated either with melatonin (10 mg/kg body weight (BW)/24 h) (M-ZDF and M-ZL) or non-treated (control) (C-ZDF and C-ZL) for 12 weeks. Western blot analysis showed that mitochondrial fission, fusion, and autophagy were altered in the C-ZDF group, accompanied by reduced SIRT1 levels. Furthermore, C-ZDF rats exhibited depleted ATP production and nitro-oxidative stress, as indicated by increased nitrites levels and reduced SOD activity. Western blotting of MyH isoforms demonstrated a significant decrease in both slow and fast oxidative fiber-specific markers expression in the C-ZDF group, concomitant with an increase in the fast glycolytic fiber markers. At the tissue level, marked fiber atrophy, less oxidative fibers, and excessive lipid deposition were noted in the C-ZDF group. Interestingly, melatonin treatment partially restored mitochondrial fission/fusion imbalance in the RVL muscle by enhancing the expression of fission (Fis1 and DRP1) markers and decreasing that of fusion (OPA1 and Mfn2) markers. It was also found to restore autophagy, as indicated by increased p62 protein level and LC3BII/I ratio. In addition, melatonin treatment increased SIRT1 protein level, mitochondrial ATP production, and SOD activity and decreased nitrites production. These effects were associated with enhanced oxidative phenotype, as evidenced by amplified oxidative fiber-specific markers expression, histochemical reaction for NADH enzyme, and muscular lipid content. In this study, we showed that melatonin might have potential therapeutic implications for obesity-induced SKM metabolic inflexibility among patients with obesity and T2DM.

Cyclin D3 deficiency promotes a slower, more oxidative skeletal muscle phenotype and ameliorates pathophysiology in the mdx mouse model of Duchenne muscular dystrophy.

We previously reported that cyclin D3-null mice display a shift toward the slow, oxidative phenotype in skeletal muscle, improved exercise endurance, and increased energy expenditure. Here, we explored the role of cyclin D3 in the physiologic response of skeletal muscle to external stimuli and in a model of muscle degenerative disease. We show that cyclin D3-null mice exhibit a further transition from glycolytic to oxidative muscle fiber type in response to voluntary exercise and an improved response to fasting. Since fast glycolytic fibers are known to be more susceptible to degeneration in Duchenne muscular dystrophy (DMD), we examined the effects of cyclin D3 inactivation on skeletal muscle phenotype in the mdx mouse model of DMD. Compared with control mdx mice, cyclin D3-deficient mdx mice display a higher proportion of slower and more oxidative myofibers, reduced muscle degenerative/regenerative processes, and reduced myofiber size variability, indicating an attenuation of dystrophic histopathology. Furthermore, mdx muscles lacking cyclin D3 exhibit reduced fatigability during repeated electrical stimulations. Notably, cyclin D3-null mdx mice show enhanced performance during recurrent trials of endurance treadmill exercise, and post-exercise muscle damage results decreased while the regenerative capacity is boosted. In addition, muscles from exercised cyclin D3-deficient mdx mice display increased oxidative capacity and increased mRNA expression of genes involved in the regulation of oxidative metabolism and the response to oxidative stress. Altogether, our findings indicate that depletion of cyclin D3 confers benefits to dystrophic muscle, suggesting that cyclin D3 inhibition may represent a promising therapeutic strategy against DMD.

Effect of hypobaric hypoxia on the fiber type transition of skeletal muscle: a synergistic therapy of exercise preconditioning with a nanocurcumin formulation.

Hypobaric hypoxia (HH) leads to various adverse effects on skeletal muscles, including atrophy and reduced oxidative work capacity. However, the effects of HH on muscle fatigue resistance and myofiber remodeling are largely unexplored. Therefore, the present study aimed to explore the impact of HH on slow-oxidative fibers and to evaluate the ameliorative potential of exercise preconditioning and nanocurcumin formulation on muscle anti-fatigue ability. C2C12 cells (murine myoblasts) were used to assess the effect of hypoxia (0.5%, 24h) with and without the nanocurcumin formulation (NCF) on myofiber phenotypic conversion. To further validate this hypothesis, male Sprague Dawley rats were exposed to a simulated HH (7620m) for 7days, along with NCF administration and/or exercise training. Both in vitro and in vivo studies revealed a significant reduction in slow-oxidative fibers (p < 0.01, 61% vs. normoxia control) under hypoxia. There was also a marked decrease in exhaustion time (p < 0.01, 65% vs. normoxia) in hypoxia control rats, indicating a reduced work capacity. Exercise preconditioning along with NCF supplementation significantly increased the slow-oxidative fiber proportion and exhaustion time while maintaining mitochondrial homeostasis. These findings suggest that HH leads to an increased transition of slow-oxidative fibers to fast glycolytic fibers and increased muscular fatigue. Administration of NCF in combination with exercise preconditioning restored this myofiber remodeling and improved muscle anti-fatigue ability.

Sex differences in isolated skeletal muscle force production: a spectrum of responses

Whether sex differences in skeletal muscle microvascular responses differ in response to muscle contraction is currently unknown. To determine vascular responses to muscle contraction in vivo we use intravital microscopy using thin skeletal muscle models of mixed fibre types (i.e. hamster retractor muscle). However, to effectively quantify this relationship in vivo, the initial muscle contraction stimulus must be constant across the sexes and thus, we must first determine whether there are sex differences in skeletal muscle force generation. We hypothesized that sexual dimorphisms in force production would not be apparent in muscles of any fibre type composition. To test this hypothesis, we used skeletal muscles with unique fibre type characteristics and oxidative capacities. Mouse soleus (SOL: primarily type 1 &gt;75%), extensor digitorum longus (EDL: primarily type IIB ~60%), and costal diaphragm (DIA: primarily type IIA ~55%) muscles were isolated from adult male and female CD-1 mice. The retractor muscle (RET: primarily type IIX ~58%) was also isolated from adult male and female Syrian hamsters. Muscles were electrically stimulated in vitro and force production was quantified at optimal length during a force-frequency test (1-120Hz) and twitch (1Hz) contractions. In mice, females generated greater force during the force-frequency test in SOL (10-120Hz) and EDL (30-120Hz) and generated greater twitch forces compared to males (SOL: female 34.7 ± 2.4 mN/mm 2 , n=21 vs male 27.8 ± 2.0 mN/mm 2 , n=17; EDL: female 41.2 ± 2.2 mN/mm 2 , n=35 vs male 32.5 ± 2.1 mN/mm 2 , n=29). Females generated greater force during the force frequency test in DIA at 30Hz only and no significant sex differences were observed in DIA twitch force (female 0.24 ± 0.02 g/mg, n=19 vs male 0.20 ± 0.02 g/mg, n=21). In hamster RET no significant sex differences in force were observed during the force frequency test or during the twitch contraction (female 0.13 ± 0.01 g/mg, n=19 vs male 0.14 ± 0.02 g/mg, n=20). Collectively, these findings demonstrate that sex differences in force production were observed in muscles primarily comprised of slow oxidative (type 1) and fast glycolytic (type IIB) fibres but not in muscles primarily comprised of intermediate fibres (type IIA and type IIX). These data show that on a spectrum of muscle fibres from slow oxidative to fast glycolytic, sex differences are most apparent on the extremes of the spectrum, whereas sexual dimorphisms in the middle of the spectrum are not apparent. This work is supported by NSERC. NSERC This is the full abstract presented at the American Physiology Summit 2023 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.

Prevalence and Mechanisms of Skeletal Muscle Atrophy in Metabolic Conditions.

Skeletal muscle atrophy is prevalent in a myriad of pathological conditions, such as diabetes, denervation, long-term immobility, malnutrition, sarcopenia, obesity, Alzheimer's disease, and cachexia. This is a critically important topic that has significance in the health of the current society, particularly older adults. The most damaging effect of muscle atrophy is the decreased quality of life from functional disability, increased risk of fractures, decreased basal metabolic rate, and reduced bone mineral density. Most skeletal muscle in humans contains slow oxidative, fast oxidative, and fast glycolytic muscle fiber types. Depending on the pathological condition, either oxidative or glycolytic muscle type may be affected to a greater extent. This review article discusses the prevalence of skeletal muscle atrophy and several mechanisms, with an emphasis on high-fat, high-sugar diet patterns, obesity, and diabetes, but including other conditions such as sarcopenia, Alzheimer's disease, cancer cachexia, and heart failure.

FHL3 promotes the formation of fast glycolytic muscle fibers by interacting with YY1 and muscle glycolytic metabolism.

The proportions of the various muscle fiber types are important in the regulation of skeletal muscle metabolism, as well as animal meat production. Four-and-a-half LIM domain protein 3 (FHL3) is highly expressed in fast glycolytic muscle fibers and differentially regulates the expression of myosin heavy chain (MyHC) isoforms at the cellular level. Whether FHL3 regulates the transformation of muscle fiber types in vivo and the regulatory mechanism is unclear. In this study, muscle-specific FHL3 transgenic mice were generated by random integration, and lentivirus-mediated gene knockdown or overexpression in muscles of mice or pigs was conducted. Functional analysis showed that overexpression of FHL3 in muscles significantly increased the proportion of fast-twitch myofibers and muscle mass but decreased muscle succinate dehydrogenase (SDH) activity and whole-body oxygen consumption. Lentivirus-mediated FHL3 knockdown in muscles significantly decreased muscle mass and the proportion of fast-twitch myofibers. Mechanistically, FHL3 directly interacted with the Yin yang 1 (YY1) DNA-binding domain, repressed the binding of YY1 to the fast glycolytic MyHC2b gene regulatory region, and thereby promoted MyHC2b expression. FHL3 also competed with EZH2 to bind the repression domain of YY1 and reduced H3K27me3 enrichment in the MyHC2b regulatory region. Moreover, FHL3 overexpression reduced glucose tolerance by affecting muscle glycolytic metabolism, and its mRNA expression in muscle was positively associated with hemoglobin A1c (HbA1c) in patients with type 2 diabetes. Therefore, FHL3 is a novel potential target gene for the treatment of muscle metabolism-related diseases and improvement of animal meat production.

SKELETAL MUSCLE SENSITIVITY TO WASTING INDUCED BY UROTHELIAL CARCINOMA.

Skeletal muscle wasting is a common phenotypic feature of several types of cancer, and it is associated with functional impairment, respiratory complications, and fatigue. However, equivocal evidence remains regarding the impact of cancer-induced muscle wasting on the different fiber types. The aim of this study was to investigate the impact of urothelial carcinoma induced in mice on the histomorphometric features and collagen deposition in different skeletal muscles. Thirteen ICR (CD1) male mice were randomly assigned into two groups: exposed to 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in drinking water for 12 weeks, plus 8 weeks of tap water (BBN, n = 8) or with access to tap water for 20 weeks (CONT, n = 5). Tibialis anterior, soleus, and diaphragm muscles were collected from all animals. For cross-sectional area and myonuclear domain analysis, muscle sections were stained with hematoxylin and eosin, and for collagen deposition assessment, muscle sections were stained with picrosirius red. All animals from the BBN group developed urothelial preneoplastic and neoplastic lesions, and the tibialis anterior from these animals presented a reduced cross-sectional area (p < 0.001), with a decreased proportion of fibers with a higher cross-sectional area, increased collagen deposition (p = 0.017), and higher myonuclear domain (p = 0.031). BBN mice also showed a higher myonuclear domain in the diaphragm (p = 0.015). Urothelial carcinoma induced muscle wasting of the tibialis anterior, expressed by a decreased cross-sectional area, higher infiltration of fibrotic tissue, and increased myonuclear domain, which also increased in the diaphragm, suggesting that fast glycolytic muscle fibers are more susceptible to be affected by cancer development.

Nicotinamide riboside kinases regulate skeletal muscle fiber-type specification and are rate-limiting for metabolic adaptations during regeneration.

Nicotinamide riboside kinases (NRKs) control the conversion of dietary Nicotinamide Riboside (NR) to NAD+, but little is known about their contribution to endogenous NAD+ turnover and muscle plasticity during skeletal muscle growth and remodeling. Using NRK1/2 double KO (NRKdKO) mice, we investigated the influence of NRKs on NAD+ metabolism and muscle homeostasis, and on the response to neurogenic muscle atrophy and regeneration following muscle injury. Muscles from NRKdKO animals have altered nicotinamide (NAM) salvage and a decrease in mitochondrial content. In single myonuclei RNAseq of skeletal muscle, NRK2 mRNA expression is restricted to type IIx muscle fibers, and perturbed NAD+ turnover and mitochondrial metabolism shifts the fiber type composition of NRKdKO muscle to fast glycolytic IIB fibers. NRKdKO does not influence muscle atrophy during denervation but alters muscle repair after myofiber injury. During regeneration, muscle stem cells (MuSCs) from NRKdKO animals hyper-proliferate but fail to differentiate. NRKdKO also alters the recovery of NAD+ during muscle regeneration as well as mitochondrial adaptations and extracellular matrix remodeling required for tissue repair. These metabolic perturbations result in a transient delay of muscle regeneration which normalizes during myofiber maturation at late stages of regeneration via over-compensation of anabolic IGF1-Akt signaling. Altogether, we demonstrate that NAD+ synthesis controls mitochondrial metabolism and fiber type composition via NRK1/2 and is rate-limiting for myogenic commitment and mitochondrial maturation during skeletal muscle repair.

The Role of PRMT1 in Skeletal Muscle Hypertrophy: A Research Protocol

Introduction: Protein arginine methyltransferases (PRMTs) are an important class of enzymes that catalyze post-translational modification of arginine residues on histone or nonhistone substrates. In skeletal muscle, PRMT1 expression has been linked to muscle homeostasis, regeneration, metabolism, and synthesis. The expression of PRMT1 in skeletal muscle has already been shown to be induced by treadmill exercise, however, the relationship between PRMT1 and skeletal muscle hypertrophy remains unexplored. This study aims to determine the significance of PRMT1 in muscle hypertrophy by identifying whether wild-type mice (WT) experience elevated levels of exercise-induced muscle hypertrophy compared to PRMT1-knockout mice (KO). Methods: 12-month-old mice will be split into male (M) and female (F) groups. Both M and F groups will then be randomly distributed to WT and KO groups. WT M, KO M, WT F, and KO F groups will be further randomly divided into sedentary (SED) and exercise (EX) groups. EX groups will undergo a resistance training program for 8 weeks while SED groups will be at rest. All mice will be euthanized by cervical dislocation while at rest by the end of the 8 weeks, and their tissue samples will be collected. The progression of muscle hypertrophy between groups will be assessed weekly using Bioseb grip strength tests in grams normalized to body mass in grams (g/g). Following the 3 months, muscle biopsies of the gastrocnemius (GAS) muscle will be taken to perform fluorescence staining of the myofibers. Primary and secondary antibodies against myosin heavy chains I, IIa, IIx, and IIb will be used to stain cross-sectional areas (CSA). The CSA of the fast-glycolytic fibers will be measured to quantify muscle hypertrophy between WT and KO groups. Anticipated Results: The Bioseb grip strength tests and GAS immunostaining should demonstrate that WT mice have greater grip strength and myofibers with larger CSAs compared to KO mice. Conclusion: These results would demonstrate the importance of PRMT1 in facilitating exercise-induced muscle hypertrophy. Future directions may involve exploring downstream signaling molecules of PRMT1 to identify potential PRMT1 up regulators, accelerating muscle hypertrophy and combating pathological muscle atrophy.

Effect of dietary L-theanine supplementation on skeletal muscle fiber type transformation in weaning piglets

The aim of this study was to explore the effect of dietary L-theanine (LT) supplementation on skeletal muscle fiber type transformation in weaning piglets. Our data showed that LT significantly increased the slow-twitch fiber-related genes expression and the percentage of slow oxidative fiber, and decreased the MyHC IIb mRNA expression and the percentage of fast glycolytic fiber. In addition, LT significantly increased the succinic dehydrogenase (SDH) and malate dehydrogenase (MDH) activities and increased the LDH activities. In addition, LT significantly affected mitochondrial biogenesis and function and antioxidative related genes expression, and increased the protein expression of p-adenosine monophosphate (AMP)-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear factor E2-related factor 2 (Nrf2), NADPH quinone oxidoreductase-1 (NQO1) and heme oxygenase-1 (HO-1) and decreased the Keap1 protein levels. Furthermore, our data indicated that LT significantly increased the mRNA and protein expression of prospero-related homeobox 1 (Prox1), calcineurin A (CnA), and NFATc1, suggesting that dietary LT supplementation promoted skeletal muscle fiber transition from types II to I might be via activation of calcineurin signaling pathway. Taken together, these findings suggested that LT promoted the transformation of muscle fiber types from slow oxidative to fast glycolytic by increasing antioxidant capacity and improving mitochondrial biogenesis and function and activation of calcineurin signaling pathway.

Skeletal Muscle Dysfunction in Experimental Pulmonary Hypertension Kosmas Kosmas1,2, Zoe Michael2,3, Fotios Spyropoulos1,2, Jeffrey Widrick2,4, Ravi Jasuja2, Aimilia Papathanasiou1,2, Helen Christou 1,21Department of Pediatric Newborn Medicine, Brigham and Women’s Hospital, Boston, MA 02115, USA 2Harvard Medical School, Boston, MA 02215, USA 3Department of Pediatrics, Boston Children’s Hospital, Boston, MA 02215, USA 4Department of Genetics, Boston Children’s Hospital, Boston, MA 02215,

BackgroundPulmonary arterial hypertension (PAH), is a progressive disease that seriously compromises quality of life due to dyspnea and fatigue on exertion that worsens over time such that patients are ultimately immobile. Although hemodynamic compromise is the main contributor to exercise intolerance in PAH, accumulating evidence supports that intrinsic skeletal muscle dysfunction also occurs and may contribute to this symptomatology. We previously showed that treatment with the carbonic anhydrase inhibitor Acetazolamide (ACTZ) ameliorates the hemodynamic components of experimental PAH but its effects on skeletal muscle function and exercise tolerance are unknown. Our objective is to characterize the mechanisms of skeletal muscle dysfunction in a preclinical model of severe PAH and evaluate the effect of ACTZ treatment.HypothesesWe hypothesized that reduced exercise endurance in experimental PAH is associated with increased proportion of fast glycolytic (Type II) fibers to slow oxidative (Type I) fibers, and sarcomeric dysfunction in skeletal muscles and that treatment with ACTZ, alters muscle fiber specificity, improves skeletal muscle contractile properties and exercise tolerance.MethodsWe used the adult rat Sprague Dawley sugen/hypoxia (Su/Hx) model of severe pulmonary hypertension and performed hemodynamic and exercise tolerance studies (treadmill). We evaluated the abundance of Type I and Type II fiber specific markers and sarcomeric organization in muscles from Su/Hx and ACTZ treated rats and measured twitch and tetanic forces, contraction and relaxation times in diaphragm, soleus and extensor digitorum longus (EDL). We used the rat myoblast cell line (L6) to evaluate the molecular mechanisms of muscle fiber switch using immunofluorescence, RNA and protein expression studies.ResultsWe found that diaphragm and soleus muscle from the adult rat sugen/hypoxia (Su/Hx) model of severe PAH have increased abundance of type II fiber markers and thick sarcomeric aggregates and these changes are accompanied by decreased twitch and tetanic forces, contraction and half‐relaxation time and exercise endurance despite absence of muscle atrophy. Acetazolamide (ACTZ), treatment restored the above derangements. In vitro rat myotube differentiation was associated with a predominance of type II fiber markers in conjunction with increased abundance of the transcription factor FoxO1. This pattern of increased abundance of FoxO1 and increased type II markers was also seen in Su/Hx soleus muscles and was reversed in response to ACTZ treatment.ConclusionsWe conclude that a shift in skeletal muscle fiber type specification may underlie skeletal muscle dysfunction in experimental PAH and that treatment with ACTZ may ameliorate skeletal muscle dysfunction through an inhibitory effect on FoxO1‐mediated fiber type specification.

Tideglusib inhibition of GSK3 promotes the oxidative muscle phenotype and reduces serum creatine kinase in D2 mdx mice

Introduction Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by an absence of dystrophin that compromises membrane integrity, ultimately resulting in muscle weakness, wasting, and premature death. The fast glycolytic muscle fibres are known to be most susceptible to dystrophic pathology, while slow oxidative fibres are less affected. Thus, promoting the slow oxidative phenotype has become a viable therapeutic strategy. Recent work from our lab has shown that inhibiting the enzyme glycogen synthase kinase 3 (GSK3) can promote the slow oxidative phenotype leading to enhancements in fatigue resistance. Furthermore, we have shown that inhibiting GSK3 augments muscle specific force production and myoblast fusion. Therefore, inhibiting GSK3 may aid in alleviating dystrophic pathology. Tideglusib is a potent GSK3 inhibitor currently undergoing clinical trials for myotonic dystrophy, another form of muscular dystrophy. Here, we tested whether treating the DMD preclinical mdx mouse with tideglusib would alter muscle oxidative phenotype, improve muscle function, and reduce serum creatine kinase (CK) levels, a marker of cellular damage. Methods Male DBA/2J wild type (WT) and mdx mice were ordered from Jackson Laboratories at 5-6 weeks of age. Three groups were included in this study; 1) WT healthy control, 2) mdx tideglusib (10 mg/kg/day, via oral gavage), and 3) mdx vehicle (26% peg400, 15% Chremaphor EL and water). The mdx-tideglusib and vehicle mice underwent their respective treatments for 2 weeks with ad libitum access to food and water. Subsequently, all mice were subjected to a hangwire test to assess muscle function. Mice were then euthanized and their serum extracted for CK activity analyses using a commercially available kit that was fitted onto a 96-well plate. Extensor digitorum longus (EDL) muscles were collected and homogenized for Western blotting to investigate phosphorylated (serine 9) and total GSK3b content along with myosin heavy chain (MHC) I and IIa levels. Results The hangwire test results showed that mdx mice treated with tideglusib were able to sustain hanging on the wire for a significantly longer period of time compared with the mdx vehicle group (p = 0.03). Serum CK analyses revealed that while the mdx vehicle group had significantly higher levels of activity compared with WT (p = 0.009), there was a 30% reduction in the mdx tideglusib mice that was no longer significantly different from WT. Western blotting revealed that though phosphorylated GSK3b levels were unaltered, tideglusib treatment led to a significant reduction in GSK3b content compared with vehicle (p = 0.04). Additionally, there was a significant increase in the oxidative MHC isoforms (I and IIa, p = 0.02) in the mdx tideglusib group compared with vehicle. Conclusions Our results demonstrate the potential use of tideglusib for DMD. We show that tideglusib treatment inhibited GSK3 in mdx mice through a reduction in total GSK3 content. In turn, we also found a significant increase in oxidative MHC isoforms (I and IIa), which was associated with enhanced muscle performance and reduced muscle damage.

Lycopene promotes a fast-to-slow fiber type transformation through Akt/FoxO1 signaling pathway and miR-22-3p

Abstract This study investigated molecular mechanisms of lycopene regulating muscle fiber type transformation in vitro and in vivo. Remarkably, TNNI1 protein level was increased, while TNNI2 protein level was decreased in C2C12 myotubes and in mice after lycopene treatment. Lycopene also enhanced enzyme activities of succinic dehydrogenase and malate dehydrogenase and reduced lactate dehydrogenase activity. Meanwhile, lycopene markedly upregulated the protein expressions of P-Akt and P-FoxO1 and decreased FoxO1 protein level. However, specific blocking Akt/FoxO1 signaling by wortmannin effectively eliminated lycopene-induced upregulation of slow MyHC and downregulation of fast MyHC in C2C12 myotubes. In addition, lycopene effectively inhibited miR-22-3p expression in C2C12 myotubes and in mice, whereas miR-22-3p mimics attenuated transformation from fast glycolytic fiber to slow oxidative fiber caused by lycopene. Our findings provided the first evidence that lycopene promoted muscle fiber type transformation from fast-twitch to slow-twitch through Akt/FoxO1 pathway and miR-22-3p.

Functional morphology and fiber types of the masseter muscles of two caviomorph rodents with contrasting lifestyles, Ctenomys talarum (Ctenomyidae) and Cavia aperea (Caviidae).

The aim of this work is the analysis of histochemical and morphometric properties of the masseter muscles of Ctenomys talarum and Cavia aperea. The former belongs to a subterranean rodent clade, Ctenomyidae, which has evolved a robust masticatory apparatus adapted to chisel-tooth digging and processing of abrasive grasses; C. aperea belongs to the family Caviidae, with relatively graceful jaws and mandibular musculatures, consistent with less mechanically challenging diets. Adult males were captured, immediately transported to the laboratory, and euthanized in a CO2 chamber. The musculus masseter superficialis and musculus masseter profundus on the left side of the animals were used to analyze the histochemical composition of the fiber types treated with myosin adenosine triphosphatase, succinate dehydrogenase and periodic acid Schiff. The mean fiber diameters, relative areas, and frequencies of each muscle fiber type were calculated. The mm. masseter superficialis and masseter profundus on the right side were used to measure the physiological cross-sectional area (PCSA). Based on this measurement, the internal force (F) was estimated. In the m. masseter profundus of both species and in the m. masseter superficialis of C. aperea intermediate fast oxidative-glycolytic fibers (FOGi) predominated. In the mm. masseter superficialis and masseter profundus of C. talarum the relative area of fast glycolytic (FG) fibers was greater than that of the muscles of C. aperea, whose main muscle fiber component is FOGi fibers. When corrected for body mass differences, PCSA was higher for the mm. masseter superficialis of C. talarum. This and the larger relative area of FG fibers, probably contributes to the exertion of large bite forces in C. talarum, as measured in previous studies.

Caveolin-3: A Causative Process of Chicken Muscular Dystrophy.

The etiology of chicken muscular dystrophy is the synthesis of aberrant WW domain containing E3 ubiquitin-protein ligase 1 (WWP1) protein made by a missense mutation of WWP1 gene. The β-dystroglycan that confers stability to sarcolemma was identified as a substrate of WWP protein, which induces the next molecular collapse. The aberrant WWP1 increases the ubiquitin ligase-mediated ubiquitination following severe degradation of sarcolemmal and cytoplasmic β-dystroglycan, and an erased β-dystroglycan in dystrophic αW fibers will lead to molecular imperfection of the dystrophin-glycoprotein complex (DGC). The DGC is a core protein of costamere that is an essential part of force transduction and protects the muscle fibers from contraction-induced damage. Caveolin-3 (Cav-3) and dystrophin bind competitively to the same site of β-dystroglycan, and excessive Cav-3 on sarcolemma will block the interaction of dystrophin with β-dystroglycan, which is another reason for the disruption of the DGC. It is known that fast-twitch glycolytic fibers are more sensitive and vulnerable to contraction-induced small tears than slow-twitch oxidative fibers under a variety of diseased conditions. Accordingly, the fast glycolytic αW fibers must be easy with rapid damage of sarcolemma corruption seen in chicken muscular dystrophy, but the slow oxidative fibers are able to escape from these damages.

Cytological analysis of integumentary and muscular adaptations in three sand-dwelling marine teleosts, Ammodytes tobianus (Ammodytidae), Gorgasia preclara (Congridae) and Heteroconger hassi (Congridae) (Teleostei; Actinopterygii).

Sandy bottoms are a ubiquitous environment found from sea bottoms to intertidal and freshwater zones. They are inhabited by many invertebrates and vertebrates which have developed morphological and physiological adaptations to sustain life under these particular conditions. Sandy habitats exhibit three potential constraints: abrasion, hypoxia and mechanical resistance. Here, three teleost species living in sandy environments were investigated: Ammodytes tobianus (Ammodytidae), Gorgasia preclara and Heteroconger hassi (Congridae). These teleost fishes were studied for their integument and muscular systems, which are potentially subject to sand abrasion and hypoxia, respectively. Based on histochemistry and transmission electron microscopy, we found the complex mucus system of G. preclara and H. hassi consists of two types of goblet cells and one type of sacciform cell. The secretions of both species are made of complex polysaccharides. In contrast, the scaly integument of A. tobianus has only a few goblet cells and no sacciform cells. We also highlighted, by immunohistochemistry, that the epidermal cell proliferation was much higher for this latter species, potentially resulting from the high rate of sand abrasion when A. tobianus buries itself quickly in the substrate. For all species, the major muscle fibre type was revealed by histoenzymology and corresponds to fast glycolytic fibres followed by intermediate fibres with slow fibres in the lowest proportion. Ammodytes tobianus possesses the highest fast fibre proportion (about 87% for A. tobianus and 75–78% for both garden eels). Our results provide new insights into the previously poorly studied teleost species, such as G. preclara, and allow us to highlight the complex skin histology of both garden eel species. Furthermore, the previously unknown muscle typing of these three species was determined.