bims-moremu Biomed News
on Molecular regulators of muscle mass
Issue of 2021‒03‒07
38 papers selected by
Anna Vainshtein
Craft Science Inc.

  1. Cell Rep. 2021 Mar 02. pii: S2211-1247(21)00123-6. [Epub ahead of print]34(9): 108809
      Muscle differentiation is a multifaceted and tightly controlled process required for the formation of skeletal muscle fibers. Satellite cells are the direct cellular contributors to muscle repair in injuries or disorders. Here, we show that autotaxin (Atx) expression and activity is required for satellite cell differentiation. Conditional ablation of Atx or its pharmacological inhibition impairs muscle repair. Mechanistically, we identify LPAR1 as the key receptor in Atx-LPA signaling. Myogenic gene array and pathway analysis identified that Atx-LPA signaling activates ribosomal protein S6 kinase (S6K), an mTOR-dependent master regulator of muscle cell growth via LPAR1. Furthermore, Atx transgenic mice show muscle hypertrophic effects and accelerated regeneration. Intramuscular injections of Atx/LPA show muscle hypertrophy. In addition, the regulatory effects of Atx on differentiation are conserved in human myoblasts. This study identifies Atx as a critical master regulator in murine and human muscles, identifying a promising extracellular ligand in muscle formation, regeneration, and hypertrophy.
    Keywords:  LPA; autotaxin; differentiation; hypertrophy; regeneration; satellite cells; skeletal muscle
  2. Aging Cell. 2021 Mar 06. e13322
      The causes of the decline in skeletal muscle mass and function with age, known as sarcopenia, are poorly understood. Nutrition (calorie restriction) interventions impact many cellular processes and increase lifespan and preserve muscle mass and function with age. As we previously observed an increase in life span and muscle function in aging mice on a ketogenic diet (KD), we aimed to investigate the effect of a KD on the maintenance of skeletal muscle mass with age and the potential molecular mechanisms of this action. Twelve-month-old mice were assigned to an isocaloric control or KD until 16 or 26 months of age, at which time skeletal muscle was collected for evaluating mass, morphology, and biochemical properties. Skeletal muscle mass was significantly greater at 26 months in the gastrocnemius of mice on the KD. This result in KD mice was associated with a shift in fiber type from type IIb to IIa fibers and a range of molecular parameters including increased markers of NMJ remodeling, mitochondrial biogenesis, oxidative metabolism, and antioxidant capacity, while decreasing endoplasmic reticulum (ER) stress, protein synthesis, and proteasome activity. Overall, this study shows the effectiveness of a long-term KD in mitigating sarcopenia. The diet preferentially preserved oxidative muscle fibers and improved mitochondrial and antioxidant capacity. These adaptations may result in a healthier cellular environment, decreasing oxidative and ER stress resulting in less protein turnover. These shifts allow mice to better maintain muscle mass and function with age.
    Keywords:  aging; ketogenic diet; mice; sarcopenia; skeletal muscle
  3. Nat Commun. 2021 03 03. 12(1): 1418
      Sarcopenia is a degenerative condition that consists in age-induced atrophy and functional decline of skeletal muscle cells (myofibers). A common hypothesis is that inducing myofiber hypertrophy should also reinstate myofiber contractile function but such model has not been extensively tested. Here, we find that the levels of the ubiquitin ligase UBR4 increase in skeletal muscle with aging, and that UBR4 increases the proteolytic activity of the proteasome. Importantly, muscle-specific UBR4 loss rescues age-associated myofiber atrophy in mice. However, UBR4 loss reduces the muscle specific force and accelerates the decline in muscle protein quality that occurs with aging in mice. Similarly, hypertrophic signaling induced via muscle-specific loss of UBR4/poe and of ESCRT members (HGS/Hrs, STAM, USP8) that degrade ubiquitinated membrane proteins compromises muscle function and shortens lifespan in Drosophila by reducing protein quality control. Altogether, these findings indicate that these ubiquitin ligases antithetically regulate myofiber size and muscle protein quality control.
  4. Physiol Rep. 2021 Mar;9(5): e14789
      Mechanistic/mammalian target of rapamycin (mTOR) is a central factor of protein synthesis signaling and plays an important role in the resistance training-induced increase in skeletal muscle mass and subsequent skeletal muscle hypertrophy response. In particular, mTOR complex 1 (mTORC1) promotes protein synthesis in ribosomes by activating the downstream effectors, p70S6K and 4EBP1, in skeletal muscle and is highly sensitive to rapamycin, an mTOR inhibitor. Recently, resistance training has also been shown to affect mitochondrial dynamics, which is coupled with mitochondrial function. In skeletal muscle, mitochondria dynamically change their morphology through repeated fusion and fission, which may be key for controlling the quality of skeletal muscle. However, how the mechanisms of mitochondrial dynamics function during hypertrophy in skeletal muscle remains unclear. The aim of this study was to examine the impact of mTOR inhibition on mitochondrial dynamics during skeletal muscle hypertrophy. Consistent with previous studies, functional overload by synergist (gastrocnemius and soleus) ablation-induced progressive hypertrophy (increase in protein synthesis and fiber cross-sectional area) of the plantaris muscle was observed in mice. Moreover, these hypertrophic responses were significantly inhibited by rapamycin administration. Fourteen days of functional overload increased levels of MFN2 and OPA1, which regulate mitochondrial fusion, whereas this enhancement was inhibited by rapamycin administration. Additionally, overload decreased the levels of DRP1, which regulates mitochondrial fission and oxidative phosphorylation, regardless of rapamycin administration. These observations suggest that the relative reduction in mitochondrial function or content is complemented by enhancement of mitochondrial fusion and that this complementary response may be regulated by mTORC1.
    Keywords:  mTOR signaling; mitochondrial dynamics; skeletal muscle hypertrophy
  5. J Biol Chem. 2021 Feb 26. pii: S0021-9258(21)00256-8. [Epub ahead of print] 100482
      Skeletal muscle is responsible for the majority of glucose disposal following meals, and this is achieved by insulin-mediated trafficking of glucose transporter type 4 (GLUT4) to the cell membrane. The eight-protein exocyst trafficking complex facilitates targeted docking of membrane-bound vesicles, a process underlying the regulated delivery of fuel transporters. We previously demonstrated the role of exocyst subunit EXOC5 in insulin-stimulated GLUT4 exocytosis and glucose uptake in cultured rat skeletal myoblasts. However, the in vivo role of EXOC5 in skeletal muscle remains unclear. Using mice with inducible, skeletal muscle-specific knockout of exocyst subunit EXOC5 (Exoc5-SMKO), we examined how muscle-specific disruption of the exocyst would affect glucose homeostasis in vivo. We found that both male and female Exoc5-SMKO mice displayed elevated fasting glucose levels. Additionally, male Exoc5-SMKO mice had impaired glucose tolerance and lower serum insulin levels. Using indirect calorimetry, we observed that male Exoc5-SMKO mice have a reduced respiratory exchange ratio during the light period and lower energy expenditure. Using the hyperinsulinemic-euglycemic clamp method, we further showed that insulin-stimulated skeletal muscle glucose uptake is reduced in Exoc5-SMKO males compared to wild-type controls. Overall, our findings indicate that EXOC5 and the exocyst are necessary for insulin-stimulated glucose uptake in skeletal muscle and regulate glucose homeostasis in vivo.
    Keywords:  diabetes; exocyst complex; glucose transporter; insulin resistance; skeletal muscle
  6. Int J Mol Sci. 2021 Feb 16. pii: 1939. [Epub ahead of print]22(4):
      Muscular regeneration is a complex biological process that occurs during acute injury and chronic degeneration, implicating several cell types. One of the earliest events of muscle regeneration is the inflammatory response, followed by the activation and differentiation of muscle progenitor cells. However, the process of novel neuromuscular junction formation during muscle regeneration is still largely unexplored. Here, we identify by single-cell RNA sequencing and isolate a subset of vessel-associated cells able to improve myogenic differentiation. We termed them 'guide' cells because of their remarkable ability to improve myogenesis without fusing with the newly formed fibers. In vitro, these cells showed a marked mobility and ability to contact the forming myotubes. We found that these cells are characterized by CD44 and CD34 surface markers and the expression of Ng2 and Ncam2. In addition, in a murine model of acute muscle injury and regeneration, injection of guide cells correlated with increased numbers of newly formed neuromuscular junctions. Thus, we propose that guide cells modulate de novo generation of neuromuscular junctions in regenerating myofibers. Further studies are necessary to investigate the origin of those cells and the extent to which they are required for terminal specification of regenerating myofibers.
    Keywords:  guide cells; mesoangioblasts; muscle injury; neuro-muscular junctions; scRNA-seq
  7. Biomedicines. 2021 Feb 20. pii: 219. [Epub ahead of print]9(2):
      In skeletal muscle, long noncoding RNAs (lncRNAs) are involved in dystrophin protein stabilization but also in the regulation of myocytes proliferation and differentiation. Hence, they could represent promising therapeutic targets and/or biomarkers for Duchenne and Becker muscular dystrophy (DMD/BMD). DMD and BMD are X-linked myopathies characterized by a progressive muscular dystrophy with or without dilatative cardiomyopathy. Two-thirds of DMD gene mutations are represented by deletions, and 63% of patients carrying DMD deletions are eligible for 45 to 55 multi-exons skipping (MES), becoming BMD patients (BMDΔ45-55). We analyzed the genomic lncRNA presence in 38 BMDΔ45-55 patients and characterized the lncRNA localized in introns 44 and 55 of the DMD gene. We highlighted that all four lncRNA are differentially expressed during myogenesis in immortalized and primary human myoblasts. In addition, the lncRNA44s2 was pointed out as a possible accelerator of differentiation. Interestingly, lncRNA44s expression was associated with a favorable clinical phenotype. These findings suggest that lncRNA44s2 could be involved in muscle differentiation process and become a potential disease progression biomarker. Based on these results, we support MES45-55 therapy and propose that the design of the CRISPR/Cas9 MES45-55 assay consider the lncRNA sequences bordering the exonic 45 to 55 deletion.
    Keywords:  Becker muscular dystrophy (BMD); Duchenne muscular dystrophy (DMD); lncRNA; long noncoding RNA; ncRNA
  8. Bio Protoc. 2020 Jul 20. 10(14): e3686
      The skeletal muscle is key for body mobility and motor performance, but aging and diseases often lead to progressive loss of muscle mass due to wasting or degeneration of muscle cells. Muscle satellite cells (MuSCs) represent a population of tissue stem cells residing in the skeletal muscles and are responsible for homeostatic maintenance and regeneration of skeletal muscles. Growth, injury, and degenerative signals activate MuSCs, which then proliferate (proliferating MuSCs are called myoblasts), differentiate and fuse with existing multinuclear muscle cells (myofibers) to mediate muscle growth and repair. Here, we describe a protocol for isolating MuSCs from skeletal muscles of mice for in vitro analysis. In addition, we provide a detailed protocol on how to culture and differentiate primary myoblasts into myotubes and an immunofluorescent staining procedure to characterize the cells. These methods are essential for modeling regenerative myogenesis in vitro to understand the dynamics, function and molecular regulation of MuSCs.
    Keywords:  Differentiation; MyoG; Myoblast; Pax7; Satellite Cell; Self-renewal; Skeletal Muscle
  9. J Cachexia Sarcopenia Muscle. 2021 Mar 05.
      BACKGROUND: Muscle atrophy is a common pathology associated with disuse, such as prolonged bed rest or spaceflight, and is associated with detrimental health outcomes. There is emerging evidence that disuse atrophy may differentially affect males and females. Cellular mechanisms contributing to the development and progression of disuse remain elusive, particularly protein turnover cascades. The purpose of this study was to investigate the initial development and progression of disuse muscle atrophy in male and female mice using the well-established model of hindlimb unloading (HU).METHODS: One hundred C57BL/6J mice (50 male and 50 female) were hindlimb suspended for 0 (control), 24, 48, 72, or 168 h to induce disuse atrophy (10 animals per group). At designated time points, animals were euthanized, and tissues (extensor digitorum longus, gastrocnemius, and soleus for mRNA analysis, gastrocnemius and extensor digitorum longus for protein synthesis rates, and tibialis anterior for histology) were collected for analysis of protein turnover mechanisms (protein anabolism and catabolism).
    RESULTS: Both males and females lost ~30% of tibialis anterior cross-sectional area after 168 h of disuse. Males had no statistical difference in MHCIIB fibre area, whereas unloaded females had ~33% lower MHCIIB cross-sectional area by 168 h of unloading. Both males and females had lower fractional protein synthesis rates (FSRs) within 24-48 h of HU, and females appeared to have a greater reduction compared with males within 24 h of HU (~23% lower FSRs in males vs. 40% lower FSRs in females). Males and females exhibited differential patterns and responses in multiple markers of protein anabolism, catabolism, and myogenic capacity during the development and progression of disuse atrophy. Specifically, females had greater mRNA inductions of catabolic factors Ubc and Gadd45a (~4-fold greater content in females compared with ~2-fold greater content in males) and greater inductions of anabolic inhibitors Redd1 and Deptor with disuse across multiple muscle tissues exhibiting different fibre phenotypes.
    CONCLUSIONS: These results suggest that the aetiology of disuse muscle atrophy is more complicated and nuanced than previously thought, with different responses based on muscle phenotypes and between males and females, with females having greater inductions of atrophic markers early in the development of disuse atrophy.
    Keywords:  Females; Males; Muscle loss; Protein anabolism; Protein catabolism; Sex differences
  10. Cells. 2021 Feb 17. pii: 417. [Epub ahead of print]10(2):
      IgLON5 is a cell adhesion protein belonging to the immunoglobulin superfamily and has important cellular functions. The objective of this study was to determine the role played by IgLON5 during myogenesis. We found IgLON5 expression progressively increased in C2C12 myoblasts during transition from the adhesion to differentiation stage. IgLON5 knockdown (IgLON5kd) cells exhibited reduced cell adhesion, myotube formation, and maturation and reduced expressions of different types of genes, including those coding for extracellular matrix (ECM) components (COL1a1, FMOD, DPT, THBS1), cell membrane proteins (ITM2a, CDH15), and cytoskeletal protein (WASP). Furthermore, decreased IgLON5 expression in FMODkd, DPTkd, COL1a1kd, and ITM2akd cells suggested that IgLON5 and these genes mutually control gene expression during myogenesis. IgLON5 immunoneutralization resulted in significant reduction in the protein level of myogenic markers (MYOD, MYOG, MYL2). IgLON5 expression was higher in the CTX-treated gastrocnemius mice muscles (day 7), which confirmed increase expression of IgLON5 during muscle. Collectively, these results suggest IgLON5 plays an important role in myogenesis, muscle regeneration, and that proteins in ECM and myoblast membranes form an interactive network that establishes an essential microenvironment that ensures muscle stem cell survival.
    Keywords:  IgLON5; extracellular matrix; muscle stem (satellite) cell; myoblast; myogenesis; skeletal muscle
  11. Physiol Rep. 2021 Mar;9(5): e14763
      The effects of malnutrition on skeletal muscle result in not only the loss of muscle mass but also fatigue intolerance. It remains unknown whether the metabolic capacity is related to the fiber type composition of skeletal muscle under malnourished condition although malnutrition resulted in preferential atrophy in fast muscle. The purpose of the present study was to investigate the effects of metabolic capacity in fast and slow muscles via the energy-sensing of AMPK and SIRT1 in malnutrition. Wistar rats were randomly divided into control and malnutrition groups. The rats in the malnutrition group were provided with a low-protein diet, and daily food intake was limited to 50% for 12 weeks. Malnutrition with hypoalbuminemia decreased the body weight and induced the loss of plantaris muscle mass, but there was little change in the soleus muscle. An increase in the superoxide level in the plasma and a decrease in SOD-2 protein expression in both muscles were observed in the malnutrition group. In addition, the expression level of AMPK in the malnutrition group increased in both muscles. Conversely, the expression level of SIRT1 decreased in both muscles of the malnutrition group. In addition, malnutrition resulted in a decrease in the expression levels of PGC-1α and PINK protein, and induced a decrease in the levels of two key mitochondrial enzymes (succinate dehydrogenase and citrate synthase) and COX IV protein expression in both muscles. These results indicate that malnutrition impaired the metabolic capacity in both fast and slow muscles via AMPK-independent SIRT1 inhibition induced by increased oxidative stress.
    Keywords:  SIRT1; malnutrition; metabolic capacity; oxidative stress
  12. Trends Cell Biol. 2021 Mar 02. pii: S0962-8924(21)00031-3. [Epub ahead of print]
      Muscle stem cells (MuSCs) are tissue-resident stem cells required for growth and repair of skeletal muscle, that are otherwise maintained in a cell-cycle-arrested state called quiescence. While quiescence was originally believed to be a state of cellular inactivity, increasing evidence suggests that quiescence is dynamically regulated and contributes to stemness, the long-term capacity to maintain regenerative functions. Here, we review the current understanding of MuSC quiescence and highlight recently discovered molecular markers, which differentiate depth of quiescence and influence self-renewal capacity. We also discuss how quiescent MuSCs integrate paracrine factors from their niche and dynamically regulate cell signaling, metabolism and proteostasis as they anticipate physiological needs, and how perturbing these cues during aging impairs muscle regeneration.
  13. Physiol Rep. 2021 Mar;9(5): e14790
      Prostaglandin (PG) E2  has been linked to increased inflammation and attenuated resistance exercise adaptations in skeletal muscle. Nonaspirin cyclooxygenase (COX) inhibitors have been shown to reduce these effects. This study examined the effect of low-dose aspirin on skeletal muscle COX production of PGE2 at rest and following resistance exercise. Skeletal muscle (vastus lateralis) biopsies were taken from six individuals (4 M/2 W) before and 3.5 hr after a single bout of resistance exercise for ex vivo PGE2 production under control and low (10 μM)- or standard (100 μM)-dose aspirin conditions. Sex-specific effects of aspirin were also examined by combining the current findings with our previous similar ex vivo skeletal muscle investigations (n = 20, 10 M/10 W). Low-dose aspirin inhibited skeletal muscle PGE2 production (p < 0.05). This inhibition was similar to standard-dose aspirin (p > 0.05) and was not influenced by resistance exercise (p > 0.05) (overall effect: -18 ± 5%). Men and women had similar uninhibited skeletal muscle PGE2 production at rest (men: 1.97 ± 0.33, women: 1.96 ± 0.29 pg/mg wet weight/min; p > 0.05). However, skeletal muscle of men was 60% more sensitive to aspirin inhibition than women (p < 0.05). In summary, the current findings 1) confirm low-dose aspirin inhibits the PGE2 /COX pathway in human skeletal muscle, 2) show that resistance exercise does not alter aspirin inhibitory efficacy, and 3) suggest the skeletal muscle of men and women could respond differently to long-term consumption of low-dose aspirin, one of the most common chronically consumed drugs in the world.
    Keywords:  cyclooxygenase; low-dose aspirin; prostaglandin E2; resistance exercise; skeletal muscle
  14. JCI Insight. 2021 Mar 04. pii: 143469. [Epub ahead of print]
      During denervation induced muscle atrophy, the loss of neuro-muscular junction (NMJ) integrity and the consequent cessation of nerve signal transmission to muscle, lead to a decline in myofiber size mass and contractile activity. However, the identity of the cell types implicated in the muscle response to nerve injury has not been clearly defined. Here, we describe a subpopulation of muscle resident glial cells activated by loss of NMJ integrity. Gene expression analysis at bulk and single cell level revealed the existence of a population of Itga7-expressing cells, which are distinct from muscle satellite cells and are selectively activated upon nerve injury. Upon nerve lesion, these cells expanded and activated a neurotrophic gene program, including the expression of a prospective selection marker - Ngfr - and a number of neurotrophic genes as well as ECM components. Among them, we observed that Tenascin C (Tnc) was specifically produced by muscle glial cells activated by nerve injury and preferentially localized to NMJ. Activation of muscle-resident glial cells by nerve injury induced a neurotrophic phenotype, which was reversible upon recovery of NMJ integrity; by contrast, muscle-resident glial cells in skeletal muscles of a mouse model of Amyotrophic Lateral Sclerosis (ALS) steadily increased over the course of the disease and exhibited an impaired neurotrophic activity, suggesting that pathogenic activation of glial cells may be implicated in ALS progression.
    Keywords:  Muscle Biology; Neuromuscular disease; Skeletal muscle
  15. Cell Rep. 2021 Mar 02. pii: S2211-1247(21)00110-8. [Epub ahead of print]34(9): 108796
      Mechanical signals, such as those evoked by maximal-intensity contractions (MICs), can induce an increase in muscle mass. Rapamycin-sensitive signaling events are widely implicated in the regulation of this process; however, recent studies indicate that rapamycin-insensitive signaling events are also involved. Thus, to identify these events, we generate a map of the MIC-regulated and rapamycin-sensitive phosphoproteome. In total, we quantify more than 10,000 unique phosphorylation sites and find that more than 2,000 of these sites are significantly affected by MICs, but remarkably, only 38 of the MIC-regulated events are mediated through a rapamycin-sensitive mechanism. Further interrogation of the rapamycin-insensitive phosphorylation events identifies the S473 residue on Tripartite Motif-Containing 28 (TRIM28) as one of the most robust MIC-regulated phosphorylation sites, and extensive follow-up studies suggest that TRIM28 significantly contributes to the homeostatic regulation of muscle size and function as well as the hypertrophy that occurs in response to increased mechanical loading.
    Keywords:  atrophy; contraction; exercise; growth; hypertrophy; mTOR; mechanical loading; phosphorylation; rapamycin; signal transduction
  16. Physiol Rep. 2021 Mar;9(5): e14770
      Athletes train to improve strength and endurance to demonstrate maximum performance during competitions. Training methods vary but most focus on strength, endurance, or both. Concurrent training is a combination of two different modes of training. In this study, we combined resistance exercise (RE) and high-intensity interval exercise (HIIE) to investigate the influence of the order of the concurrent training on signal molecules on hypertrophy and glycolysis in the skeletal muscle. The phosphorylation levels of mechanistic target of rapamycin (mTOR) signals, p70 S6 kinase (p70S6 K), ribosomal protein S6 (S6), and glycogen synthase kinase beta (GSK-3β) were significantly increased in the HIIE first group compared with the control group. The combined training course did not affect the glycogen content and expression levels of proteins concerning glycolytic and metabolic capacity, suggesting that a combination of HIIE and RE on the same day, with HIIE prior to RE, improves hypertrophy response and glycolysis enhancement.
    Keywords:  concurrent training; glycolysis; high-intensity interval exercise; mTOR signaling; resistance exercise
  17. Antioxidants (Basel). 2021 Feb 25. pii: 343. [Epub ahead of print]10(3):
      Doxorubicin (DOX) is an anthracycline antibiotic used to treat a wide variety of hematological and solid tumor cancers. While DOX is highly effective at reducing tumor burden, its clinical use is limited by the development of adverse effects to both cardiac and skeletal muscle. The detrimental effects of DOX to muscle tissue are associated with the increased incidence of heart failure, dyspnea, exercise intolerance, and reduced quality of life, which have been reported in both patients actively receiving chemotherapy and cancer survivors. A variety of factors elevate the probability of DOX-related morbidity in patients; however, the role of sex as a biological variable to calculate patient risk remains unclear. Uncertainty regarding sexual dimorphism in the presentation of DOX myotoxicity stems from inadequate study design to address this issue. Currently, the majority of clinical data on DOX myotoxicity come from studies where the ratio of males to females is unbalanced, one sex is omitted, and/or the patient cohort include a broad age range. Furthermore, lack of consensus on standard outcome measures, difficulties in long-term evaluation of patient outcomes, and other confounding factors (i.e., cancer type, drug combinations, adjuvant therapies, etc.) preclude a definitive answer as to whether differences exist in the incidence of DOX myotoxicity between sexes. This review summarizes the current clinical and preclinical literature relevant to sex differences in the incidence and severity of DOX myotoxicity, the proposed mechanisms for DOX sexual dimorphism, and the potential for exercise training to serve as an effective therapeutic countermeasure to preserve muscle strength and function in males and females.
    Keywords:  cancer; cardiac muscle; chemotherapy; sexual dimorphism; skeletal muscle
  18. J Physiol. 2021 Mar 01.
    Keywords:  exercise; human physiology; metabolism; nicotinamide riboside; skeletal muscle
  19. Mol Genet Metab. 2021 Feb 20. pii: S1096-7192(21)00048-2. [Epub ahead of print]
      Osteogenesis imperfecta (OI) is a heritable connective tissue disorder with patients exhibiting bone fragility and muscle weakness. The synergistic biochemical and biomechanical relationship between bone and muscle is a critical potential therapeutic target, such that muscle weakness should not be ignored. Previous studies demonstrated mitochondrial dysfunction in the skeletal muscle of oim/oim mice, which model a severe human type III OI. Here, we further characterize this mitochondrial dysfunction and evaluate several parameters of whole body and skeletal muscle metabolism. We demonstrate reduced mitochondrial respiration in female gastrocnemius muscle, but not in liver or heart mitochondria, suggesting that mitochondrial dysfunction is not global in the oim/oim mouse. Myosin heavy chain fiber type distributions were altered in the oim/oim soleus muscle with a decrease (-33 to 50%) in type I myofibers and an increase (+31%) in type IIa myofibers relative to their wildtype (WT) littermates. Additionally, altered body composition and increased energy expenditure were observed oim/oim mice relative to WT littermates. These results suggest that skeletal muscle mitochondrial dysfunction is linked to whole body metabolic alterations and to skeletal muscle weakness in the oim/oim mouse.
    Keywords:  Genetic mouse models; Mitochondrial dysfunction; Osteogenesis imperfecta; Skeletal muscle
  20. Nutrients. 2021 Feb 23. pii: 706. [Epub ahead of print]13(2):
      Globally, people 65 years of age and older are the fastest growing segment of the population. Physiological manifestations of the aging process include undesirable changes in body composition, declines in cardiorespiratory fitness, and reductions in skeletal muscle size and function (i.e., sarcopenia) that are independently associated with mortality. Decrements in muscle protein synthetic responses to anabolic stimuli (i.e., anabolic resistance), such as protein feeding or physical activity, are highly characteristic of the aging skeletal muscle phenotype and play a fundamental role in the development of sarcopenia. A more definitive understanding of the mechanisms underlying this age-associated reduction in anabolic responsiveness will help to guide promyogenic and function promoting therapies. Recent studies have provided evidence in support of a bidirectional gut-muscle axis with implications for aging muscle health. This review will examine how age-related changes in gut microbiota composition may impact anabolic response to protein feeding through adverse changes in protein digestion and amino acid absorption, circulating amino acid availability, anabolic hormone production and responsiveness, and intramuscular anabolic signaling. We conclude by reviewing literature describing lifestyle habits suspected to contribute to age-related changes in the microbiome with the goal of identifying evidence-informed strategies to preserve microbial homeostasis, anabolic sensitivity, and skeletal muscle with advancing age.
    Keywords:  aging; muscle protein synthesis; protein; sarcopenia; skeletal muscle
  21. Mol Med Rep. 2021 May;pii: 306. [Epub ahead of print]23(5):
      Muscle atrophy, a side effect from administration of the anti‑inflammatory medication dexamethasone (DEX), is preventable by concomitant administration of the major monomeric constituent of Panax ginseng C.A. Meyer, 20(S)‑ginsenoside Rg3 (S‑Rg3). Putative S‑Rg3‑associated prevention of DEX‑induced muscle atrophy may involve S‑Rg3 mitigation of DEX‑induced mitochondrial dysfunction. In the present study, MTT assays revealed enhanced cell viability following S‑Rg3 treatment of DEX‑injured C2C12 myotubes. Subsequent PCR and western blotting results demonstrated S‑Rg3‑induced reduction of expression of muscle atrophy F‑box protein (atrogin‑1) and muscle RING‑finger protein‑1, proteins previously linked to muscle atrophy. Additionally, S‑Rg3 treatment of DEX‑injured myotubes led to aggregation of Rg3 monomers in cells and dose‑dependent increases in cellular mitochondrial basal respiratory oxygen consumption rate and intracellular ATP levels compared with their levels in untreated DEX‑injured myotubes. In addition, S‑Rg3 treatment significantly reversed DEX‑induced reductions of expression of key mitochondrial respiratory electron transport chain subunits of protein complexes II, III and V in DEX‑injured myotube cells. Furthermore, S‑Rg3 alleviation of mitochondrial dysfunction associated with DEX‑induced injury of C2C12 myotubes was linked to S‑Rg3‑associated decreases in both forkhead box O3 (FoxO3) protein expression and phosphorylation of AMP‑activated protein kinase (AMPK). Collectively, these results implicate S‑Rg3 modulation of signaling within the AMPK‑FoxO3 pathway as a putative mechanism underlying S‑Rg3 alleviation of DEX‑induced muscle atrophy.
  22. Int J Mol Sci. 2021 Feb 23. pii: 2225. [Epub ahead of print]22(4):
      The muscle-specific ubiquitin ligase MuRF1 regulates muscle catabolism during chronic wasting states, although its roles in general metabolism are less-studied. Here, we metabolically profiled MuRF1-deficient knockout mice. We also included knockout mice for MuRF2 as its closely related gene homolog. MuRF1 and MuRF2-KO (knockout) mice have elevated serum glucose, elevated triglycerides, and reduced glucose tolerance. In addition, MuRF2-KO mice have a reduced tolerance to a fat-rich diet. Western blot and enzymatic studies on MuRF1-KO skeletal muscle showed perturbed FoxO-Akt signaling, elevated Akt-Ser-473 activation, and downregulated oxidative mitochondrial metabolism, indicating potential mechanisms for MuRF1,2-dependent glucose and fat metabolism regulation. Consistent with this, the adenoviral re-expression of MuRF1 in KO mice normalized Akt-Ser-473, serum glucose, and triglycerides. Finally, we tested the MuRF1/2 inhibitors MyoMed-205 and MyoMed-946 in a mouse model for type 2 diabetes mellitus (T2DM). After 28 days of treatment, T2DM mice developed progressive muscle weakness detected by wire hang tests, but this was attenuated by the MyoMed-205 treatment. While MyoMed-205 and MyoMed-946 had no significant effects on serum glucose, they did normalize the lymphocyte-granulocyte counts in diabetic sera as indicators of the immune response. Thus, small molecules directed to MuRF1 may be useful in attenuating skeletal muscle strength loss in T2DM conditions.
    Keywords:  MuRF1; MuRF2; chemical biology; diabetes mellitus; glucose and muscle metabolism
  23. BMC Endocr Disord. 2021 Feb 27. 21(1): 32
      BACKGROUND: Insulin resistance (IR) in skeletal muscle is a key feature of the pre-diabetic state, hypertension, dyslipidemia, cardiovascular diseases and also predicts type 2 diabetes. However, the underlying molecular mechanisms are still poorly understood.METHODS: To explore these mechanisms, we related global skeletal muscle gene expression profiling of 38 non-diabetic men to a surrogate measure of insulin sensitivity, i.e. homeostatic model assessment of insulin resistance (HOMA-IR).
    RESULTS: We identified 70 genes positively and 110 genes inversely correlated with insulin sensitivity in human skeletal muscle, identifying autophagy-related genes as positively correlated with insulin sensitivity. Replication in an independent study of 9 non-diabetic men resulted in 10 overlapping genes that strongly correlated with insulin sensitivity, including SIRT2, involved in lipid metabolism, and FBXW5 that regulates mammalian target-of-rapamycin (mTOR) and autophagy. The expressions of SIRT2 and FBXW5 were also positively correlated with the expression of key genes promoting the phenotype of an insulin sensitive myocyte e.g. PPARGC1A.
    CONCLUSIONS: The muscle expression of 180 genes were correlated with insulin sensitivity. These data suggest that activation of genes involved in lipid metabolism, e.g. SIRT2, and genes regulating autophagy and mTOR signaling, e.g. FBXW5, are associated with increased insulin sensitivity in human skeletal muscle, reflecting a highly flexible nutrient sensing.
  24. Bio Protoc. 2020 Feb 20. 10(4): e3525
      Whole transcriptome analysis is a key method in biology that allows researchers to determine the effect a condition has on gene regulation. One difficulty in RNA sequencing of muscle is that traditional methods are performed on the whole muscle, but this captures non-myogenic cells that are part of the muscle. In order to analyze only the transcriptome of myofibers we combine single myofiber isolation with SMART-Seq to provide high resolution genome wide expression of a single myofiber.
    Keywords:  Gene expression analysis; SMART-Seq; Single myofiber; Skeletal muscle; Transcriptomics
  25. Cell Tissue Res. 2021 Mar 03.
      Skeletal muscle fibers are classified as slow-twitch and fast-twitch fibers, which have different reactive oxygen species (ROS) metabolism and mitochondrial biogenesis. Recently, Attractin (Atrn), which encodes secreted (sAtrn) and transmembrane (mAtrn)-type proteins, has been shown to be involved in free radical scavenging. Although Atrn has been found in skeletal muscle, little is known about the expression levels and function of Atrn in each muscle fiber type. Therefore, we investigate sAtrn and mAtrn expression levels in the slow-twitch soleus (sol) and fast-twitch extensor digitorum longus (EDL) muscles as well as the morphology and expression levels of antioxidant enzymes and functional mitochondrial markers using Atrn-deficient muscles. Both types of Atrn were expressed in the sol and EDL. mAtrn was mainly expressed in the adult sol, whereas sAtrn expression levels did not differ between muscle types. Moreover, mAtrn in the sol was abundantly localized in the subsarcolemmal area, especially in the myoplasm near mitochondria. Atrn-deficient Zitter rats showed muscle fiber atrophy, myofibril misalignment, mitochondrial swelling and vacuolation in the sol but not EDL. Furthermore, the Atrn-deficient sol exhibited a marked reduction in antioxidant enzyme SOD1, GPx1, catalase and Prx6 and mitochondrial functional protein, UCP2, expression. Even Atrn-deficient EDL showed a significant reduction in Prx3, Prx6, UCP2 and UCP3 expression. These data indicate that Atrn-deficiency disturbs ROS metabolism in skeletal muscles. In particular, mAtrn is involved in metabolism in the slow-twitch sol muscle and mAtrn-deficiency may cause ROS imbalance, resulting in morphological abnormalities in the muscle.
    Keywords:  Mitochondria; Sarcomere; Type I muscle fiber; Type II muscle fiber; Zitter rat
  26. Food Funct. 2021 Mar 05.
      This study aimed to investigate the role and underlying molecular mechanism of quercetin in regulating skeletal muscle fiber type transition. We found that dietary quercetin supplementation in mice significantly increased oxidative fiber-related gene expression, slow-twitch fiber percentage and succinic dehydrogenase (SDH) activity. By contrast, quercetin decreased lactate dehydrogenase (LDH) activity, fast MyHC protein expression, fast-twitch fiber percentage, and MyHC IIb mRNA expression. Furthermore, quercetin significantly increased serum adiponectin (AdipoQ) concentration, and the expression levels of AdipoQ and AdipoR1. However, inhibition of adiponectin signaling by AdipoR1 siRNA significantly attenuated the effects of quercetin on muscle fiber type-related gene expression, the percentages of slow MyHC-positive and fast MyHC-positive fibers, and metabolic enzyme activity in C2C12 myotubes. Together, our data indicated that quercetin could promote skeletal fiber switching from glycolytic type II to oxidative type I through AdipoQ signaling.
  27. Int J Mol Sci. 2021 Feb 16. pii: 1937. [Epub ahead of print]22(4):
      The accumulation of damaged mitochondria due to insufficient autophagy has been implicated in the pathophysiology of skeletal muscle aging. Ulk1 is an autophagy-related kinase that initiates autophagosome assembly and may also play a role in autophagosome degradation (i.e., autophagy flux), but the contribution of Ulk1 to healthy muscle aging is unclear. Therefore, the purpose of this study was to investigate the role of Ulk1-mediated autophagy in skeletal muscle aging. At age 22 months (80% survival rate), muscle contractile and metabolic function were assessed using electrophysiology in muscle-specific Ulk1 knockout mice (MKO) and their littermate controls (LM). Specific peak-isometric torque of the ankle dorsiflexors (normalized by tibialis anterior muscle cross-sectional area) and specific force of the fast-twitch extensor digitorum longus muscles was reduced in MKO mice compared to LM mice (p < 0.03). Permeabilized muscle fibers from MKO mice had greater mitochondrial content, yet lower mitochondrial oxygen consumption and greater reactive oxygen species production compared to fibers from LM mice (p ≤ 0.04). Alterations in neuromuscular junction innervation patterns as well as changes to autophagosome assembly and flux were explored as possible contributors to the pathological features in Ulk1 deficiency. Of primary interest, we found that Ulk1 phosphorylation (activation) to total Ulk1 protein content was reduced in older muscles compared to young muscles from both human and mouse, which may contribute to decreased autophagy flux and an accumulation of dysfunctional mitochondria. Results from this study support the role of Ulk1-mediated autophagy in aging skeletal muscle, reflecting Ulk1's dual role in maintaining mitochondrial integrity through autophagosome assembly and degradation.
    Keywords:  aging; autophagy flux; mitophagy; neuromuscular junction; sarcopenia
  28. Bio Protoc. 2019 Oct 05. 9(19): e3378
      Skeletal muscle is composed of different cells and myofiber types, with distinct metabolic and structural features. Generally, transcriptomic analysis of skeletal muscle is performed using whole muscle, resulting in average information as all cells composing the organ contribute to the expression value detected for each gene with the loss of information about the distinctive features of each specific myofiber type. Since myofibers are the smallest complete contractile system of skeletal muscle influencing its contraction velocity and metabolism, it would be beneficial to have fiber-specific information about gene expression. Here, we describe a protocol for the isolation and the transcriptomic analysis of single individual myofibers. The protocol was set up using single myofibers isolated from soleus and Extensor Digitorum Longus (EDL) muscles, but it can be applied to all skeletal muscles. Briefly, muscles are enzymatically dissociated and individually collected. Long RNAs (> 200 nt) and short RNAs (< 200 nt) are separately purified from each myofiber and used to produce libraries for microarray or sequencing analysis. Through this approach, myofiber-specific transcriptional profiles can be produced, free from transcripts from other non-contractile cell types, in order to identify mRNA-miRNA-lncRNA regulatory networks specific for each myofiber type.
    Keywords:  Non-coding RNAs; Regulatory network; Single myofiber; Skeletal muscle; Transcriptomic analysis; miRNAs
  29. Front Cell Dev Biol. 2021 ;9 627409
      Mutations in the CCN6 (WISP3) gene are linked with a debilitating musculoskeletal disorder, termed progressive pseudorheumatoid dysplasia (PPRD). Yet, the functional significance of CCN6 in the musculoskeletal system remains unclear. Using zebrafish as a model organism, we demonstrated that zebrafish Ccn6 is present partly as a component of mitochondrial respiratory complexes in the skeletal muscle of zebrafish. Morpholino-mediated depletion of Ccn6 in the skeletal muscle leads to a significant reduction in mitochondrial respiratory complex assembly and activity, which correlates with loss of muscle mitochondrial abundance. These mitochondrial deficiencies are associated with notable architectural and functional anomalies in the zebrafish muscle. Taken together, our results indicate that Ccn6-mediated regulation of mitochondrial respiratory complex assembly/activity and mitochondrial integrity is important for the maintenance of skeletal muscle structure and function in zebrafish. Furthermore, this study suggests that defects related to mitochondrial respiratory complex assembly/activity and integrity could be an underlying cause of muscle weakness and a failed musculoskeletal system in PPRD.
    Keywords:  CCN6; PPRD; mitochondria; muscle; respiratory complex; zebrafish
  30. Nat Commun. 2021 03 01. 12(1): 1451
      Proprioceptive feedback mainly derives from groups Ia and II muscle spindle (MS) afferents and group Ib Golgi tendon organ (GTO) afferents, but the molecular correlates of these three afferent subtypes remain unknown. We performed single cell RNA sequencing of genetically identified adult proprioceptors and uncovered five molecularly distinct neuronal clusters. Validation of cluster-specific transcripts in dorsal root ganglia and skeletal muscle demonstrates that two of these clusters correspond to group Ia MS afferents and group Ib GTO afferent proprioceptors, respectively, and suggest that the remaining clusters could represent group II MS afferents. Lineage analysis between proprioceptor transcriptomes at different developmental stages provides evidence that proprioceptor subtype identities emerge late in development. Together, our data provide comprehensive molecular signatures for groups Ia and II MS afferents and group Ib GTO afferents, enabling genetic interrogation of the role of individual proprioceptor subtypes in regulating motor output.
  31. Antioxidants (Basel). 2021 Feb 26. pii: 350. [Epub ahead of print]10(3):
      The aim of this study was to investigate the effects of resistance training (RT) on the redox status of skeletal muscle in older adults. Thirteen males aged 64 ± 9 years performed full-body RT 2x/week for 6 weeks. Muscle biopsies were obtained from the vastus lateralis prior to and following RT. The mRNA, protein, and enzymatic activity levels of various endogenous antioxidants were determined. In addition, skeletal muscle 4-hydroxynonenal and protein carbonyls were determined as markers of oxidative damage. Protein levels of heat shock proteins (HSPs) were also quantified. RT increased mRNA levels of all assayed antioxidant genes, albeit protein levels either did not change or decreased. RT increased total antioxidant capacity, catalase, and glutathione reductase activities, and decreased glutathione peroxidase activity. Lipid peroxidation also decreased and HSP60 protein increased following RT. In summary, 6 weeks of RT decreased oxidative damage and increased antioxidant enzyme activities. Our results suggest the older adult responses to RT involve multi-level (transcriptional, post-transcriptional, and post-translational) control of the redox status of skeletal muscle.
    Keywords:  antioxidants; exercise; oxidative damage; oxidative stress; redox homeostasis
  32. Nutrients. 2021 Feb 18. pii: 661. [Epub ahead of print]13(2):
      Although animal protein is usually considered to be a more potent stimulator of muscle protein synthesis than plant protein, the effect of protein source on lean mass and muscle strength needs to be systematically reviewed. This study aimed to examine potential differences in the effect of animal vs. plant protein on lean mass and muscle strength, and the possible influence of resistance exercise training (RET) and age. The following databases were searched: PubMed, Embase, Scopus and CINAHL Plus with Full Text, and 3081 articles were screened. A total of 18 articles were selected for systematic review, of which, 16 were used for meta-analysis. Total protein intakes were generally above the recommended dietary allowance at the baseline and end of intervention. Results from the meta-analyses demonstrated that protein source did not affect changes in absolute lean mass or muscle strength. However, there was a favoring effect of animal protein on percent lean mass. RET had no influence on the results, while younger adults (<50 years) were found to gain absolute and percent lean mass with animal protein intake (weighted mean difference (WMD), 0.41 kg; 95% confidence interval (CI) 0.08 to 0.74; WMD 0.50%; 95% CI 0.00 to 1.01). Collectively, animal protein tends to be more beneficial for lean mass than plant protein, especially in younger adults.
    Keywords:  body composition; muscle mass; muscular strength; protein source
  33. Nutrients. 2021 Feb 26. pii: 745. [Epub ahead of print]13(3):
      Sarcopenia, defined as age-related reduction in muscle mass, strength, and physical performance, is associated with other age-related health conditions such as osteoporosis, osteosarcopenia, sarcopenic obesity, physical frailty, and cachexia. From a healthy aging perspective, lifestyle interventions that may help overcome characteristics and associated comorbidities of sarcopenia are clinically important. One possible intervention is creatine supplementation (CR). Accumulating research over the past few decades shows that CR, primarily when combined with resistance training (RT), has favourable effects on aging muscle, bone and fat mass, muscle and bone strength, and tasks of physical performance in healthy older adults. However, research is very limited regarding the efficacy of CR in older adults with sarcopenia or osteoporosis and no research exists in older adults with osteosarcopenia, sarcopenic obesity, physical frailty, or cachexia. Therefore, the purpose of this narrative review is (1) to evaluate and summarize current research involving CR, with and without RT, on properties of muscle and bone in older adults and (2) to provide a rationale and justification for future research involving CR in older adults with osteosarcopenia, sarcopenic obesity, physical frailty, or cachexia.
    Keywords:  cachexia; frailty; osteoporosis; osteosarcopenia; sarcopenia
  34. Int J Mol Sci. 2021 Feb 19. pii: 2070. [Epub ahead of print]22(4):
      Statins are the most prescribed and effective drugs to treat cardiovascular diseases (CVD). Nevertheless, these drugs can be responsible for skeletal muscle toxicity which leads to reduced compliance. The discontinuation of therapy increases the incidence of CVD. Thus, it is essential to assess the risk. In fact, many studies have been performed at preclinical and clinical level to investigate pathophysiological mechanisms and clinical implications of statin myotoxicity. Consequently, new toxicological aspects and new biomarkers have arisen. Indeed, these drugs may affect gene transcription and ion transport and contribute to muscle function impairment. Identifying a marker of toxicity is important to prevent or to cure statin induced myopathy while assuring the right therapy for hypercholesterolemia and counteracting CVD. In this review we focused on the mechanisms of muscle damage discovered in preclinical and clinical studies and highlighted the pathological situations in which statin therapy should be avoided. In this context, preventive or substitutive therapies should also be evaluated.
    Keywords:  biomarkers; ion channels; myopathy; skeletal muscle; statin
  35. Cells. 2021 Feb 17. pii: 423. [Epub ahead of print]10(2):
      microRNAs play an important role in the growth and development of chicken embryos, including the regulation of skeletal muscle genesis, myoblast proliferation, differentiation, and apoptosis. Our previous RNA-seq studies showed that microRNA-27b-3p (miR-27b-3p) might play an important role in regulating the proliferation and differentiation of chicken primary myoblasts (CPMs). However, the mechanism of miR-27b-3p regulating the proliferation and differentiation of CPMs is still unclear. In this study, the results showed that miR-27b-3p significantly promoted the proliferation of CPMs and inhibited the differentiation of CPMs. Then, myostatin (MSTN) was confirmed to be the target gene of miR-27b-3p by double luciferase reporter assay, RT-qPCR, and Western blot. By overexpressing and interfering with MSTN expression in CPMs, the results showed that overexpression of MSTN significantly inhibited the proliferation and differentiation of CPMs. In contrast, interference of MSTN expression had the opposite effect. This study showed that miR-27b-3p could promote the proliferation of CPMs by targeting MSTN. Interestingly, both miR-27b-3p and MSTN can inhibit the differentiation of CPMs. These results provide a theoretical basis for further understanding the function of miR-27b-3p in chicken and revealing its regulation mechanism on chicken muscle growth.
    Keywords:  MSTN; differentiation; miR-27b-3p; myoblast; proliferation
  36. J Clin Med. 2021 Feb 17. pii: 820. [Epub ahead of print]10(4):
      Duchenne muscular dystrophy (DMD) is the most common childhood muscular dystrophy affecting ~1:5000 live male births. Following the identification of pathogenic variations in the dystrophin gene in 1986, the underlining genotype/phenotype correlations emerged and the role of the dystrophin protein was elucidated in skeletal, smooth, and cardiac muscles, as well as in the brain. When the dystrophin protein is absent or quantitatively or qualitatively modified, the muscle cannot sustain the stress of repeated contractions. Dystrophin acts as a bridging and anchoring protein between the sarcomere and the sarcolemma, and its absence or reduction leads to severe muscle damage that eventually cannot be repaired, with its ultimate substitution by connective tissue and fat. The advances of an understanding of the molecular pathways affected in DMD have led to the development of many therapeutic strategies that tackle different aspects of disease etiopathogenesis, which have recently led to the first successful approved orphan drugs for this condition. The therapeutic advances in this field have progressed exponentially, with second-generation drugs now entering in clinical trials as gene therapy, potentially providing a further effective approach to the condition.
    Keywords:  Duchenne muscular dystrophy; antisense oligonucleotide chemistry; dystrophin restoration; exon-skipping; gene therapy; innovative clinical trials; stop codon reversion
  37. J Mol Biol. 2021 Feb 26. pii: S0022-2836(21)00095-4. [Epub ahead of print] 166901
      Striated muscle responds to mechanical overload by rapidly up-regulating the expression of the cardiac ankyrin repeat protein, CARP, which then targets the sarcomere by binding to titin N2A in the I-band region. To date, the role of this interaction in the stress response of muscle remainspoorly understood. Here, we characterise the molecular structure of the CARP-receptor site in titin (UN2A) and its binding of CARP. We find that titin UN2A contains a central three-helix bundle fold (ca 45 residues in length) that is joined to N- and C-terminal flanking immunoglobulin domains by long, flexible linkers with partial helical content. CARP binds titin by engaging an α-hairpin in the three-helix fold of UN2A, the C-terminal linker sequence, and the BC loop in Ig81, which jointly form a broad binding interface. Mutagenesis showed that the CARP/N2A association withstands sequence variations in titin N2A and we use this information to evaluate 85 human single nucleotide variants. In addition, actin co-sedimentation, co-transfection in C2C12 cells, proteomics on heart lysates, and the mechanical response of CARP-soaked myofibrils imply that CARP induces the cross-linking of titin and actin myofilaments, thereby increasing myofibril stiffness. We conclude that CARP acts as a regulator of force output in the sarcomere that preserves muscle mechanical performance upon overload stress.
    Keywords:  Actin cytoskeleton; Hydrogen-deuterium exchange mass spectrometry; Muscle stress response; Nuclear magnetic resonance; Sarcomere mechanics
  38. Biomolecules. 2021 Feb 13. pii: 277. [Epub ahead of print]11(2):
      Osteoporosis and other conditions associated with low bone density or quality are highly prevalent, are increasing as the population ages and with increased glucocorticoid use to treat conditions with elevated inflammation. There is an unmet need for therapeutics which can target skeletal precursors to induce osteoblast differentiation and osteogenesis. Genes associated with high bone mass represent interesting targets for manipulation, as they could offer ways to increase bone density. A damaging mutation in SMAD9 has recently been associated with high bone mass. Here we show that Smad9 labels groups of osteochondral precursor cells, which are not labelled by the other Regulatory Smads: Smad1 or Smad5. We show that Smad9+ cells are proliferative, and that the Smad9+ pocket expands following osteoblast ablation which induced osteoblast regeneration. We further show that treatment with retinoic acid, prednisolone, and dorsomorphin all alter Smad9 expression, consistent with the effects of these drugs on the skeletal system. Taken together these results demonstrate that Smad9+ cells represent an undifferentiated osteochondral precursor population, which can be manipulated by commonly used skeletal drugs. We conclude that Smad9 represents a target for future osteoanabolic therapies.
    Keywords:  BMP-signalling; Smad9; biomolecule; musculoskeletal; osteoblast; osteoporosis; zebrafish