bims-moremu Biomed News
on Molecular regulators of muscle mass
Issue of 2022‒05‒08
thirty-one papers selected by
Anna Vainshtein
Craft Science Inc.

  1. J Appl Physiol (1985). 2022 May 05.
      The strategy of gene delivery into skeletal muscles has provided exciting avenues in identifying new potential therapeutics towards muscular disorders and addressing basic research questions in muscle physiology through overexpression and knockdown studies. In vivo electroporation methodology offers a simple, rapidly effective technique for the delivery of plasmid DNA into post-mitotic skeletal muscle fibers and the ability to easily explore the molecular mechanisms of skeletal muscle plasticity. The purpose of this review is to describe how to robustly electroporate plasmid DNA into different hindlimb muscles of rodent models. Further, key parameters (e.g., voltage, hyaluronidase, plasmid concentration) which contribute to the successful introduction of plasmid DNA into skeletal muscle fibers will be discussed. In addition, details on processing tissue for immunohistochemistry and fiber cross-sectional area (CSA) analysis will be outlined. The overall goal of this review is to provide the basic and necessary information needed for successful implementation of in vivo electroporation of plasmid DNA and thus open new avenues of discovery research in skeletal muscle physiology.
    Keywords:  Atrophy; Electroporation; Gene transfer; Hypertrophy; Protein Synthesis
  2. Methods Mol Biol. 2022 ;2429 455-471
      Culturing primary muscle stem cells ex vivo is a useful method for studying this cell population in controlled environments. Primary muscle stem cells respond to external stimuli differently than immortalized myoblasts (C2C12 cells), making ex vivo culture of muscle stem cells an important tool in understanding cell responses to stimuli. Primary muscle stem cells cultured ex vivo retain a majority of the characteristics they possess in vivo such as the abilities to differentiate into multinucleated structures, and self-renew a stem cell-like population. In this chapter, we describe methods for isolating primary muscle stem cells, controlled differentiation into myotubes, and quantification of differentiation using IncuCyte live cell imaging and analysis software.
    Keywords:  Differentiation; Eccentricity; IncuCyte ZOOM; Muscle stem cells; Myotubes; Satellite cells
  3. J Cachexia Sarcopenia Muscle. 2022 May 03.
      BACKGROUND: Critical illness myopathy (CIM) is a debilitating condition characterized by the preferential loss of the motor protein myosin. CIM is a by-product of critical care, attributed to impaired recovery, long-term complications, and mortality. CIM pathophysiology is complex, heterogeneous and remains incompletely understood; however, loss of mechanical stimuli contributes to critical illness-associated muscle atrophy and weakness. Passive mechanical loading and electrical stimulation (ES) therapies augment muscle mass and function. While having beneficial outcomes, the mechanistic underpinning of these therapies is less known. Therefore, here we aimed to assess the mechanism by which chronic supramaximal ES ameliorates CIM in a unique experimental rat model of critical care.METHODS: Rats were subjected to 8 days of critical care conditions entailing deep sedation, controlled mechanical ventilation, and immobilization with and without direct soleus ES. Muscle size and function were assessed at the single cell level. RNAseq and western blotting were employed to understand the mechanisms driving ES muscle outcomes in CIM.
    RESULTS: Following 8 days of controlled mechanical ventilation and immobilization, soleus muscle mass, myosin : actin ratio, and single muscle fibre maximum force normalized to cross-sectional area (CSA; specific force) were reduced by 40-50% (P < 0.0001). ES significantly reduced the loss of soleus muscle fibre CSA and myosin : actin ratio by approximately 30% (P < 0.05) yet failed to effect specific force. RNAseq pathway analysis revealed downregulation of insulin signalling in the soleus muscle following critical care, and GLUT4 trafficking was reduced by 55% leading to an 85% reduction of muscle glycogen content (P < 0.01). ES promoted phosphofructokinase and insulin signalling pathways to control levels (P < 0.05), consistent with the maintenance of GLUT4 translocation and glycogen levels. AMPK, but not AKT, signalling pathway was stimulated following ES, where the downstream target TBC1D4 increased 3 logFC (P = 0.029) and AMPK-specific P-TBC1D4 levels were increased approximately two-fold (P = 0.06). Reduction of muscle protein degradation rather than increased synthesis promoted soleus CSA, as ES reduced E3 ubiquitin proteins, Atrogin-1 (P = 0.006) and MuRF1 (P = 0.08) by approximately 50%, downstream of AMPK-FoxO3.
    CONCLUSIONS: ES maintained GLUT4 translocation through increased AMPK-TBC1D4 signalling leading to improved muscle glucose homeostasis. Soleus CSA and myosin content was promoted through reduced protein degradation via AMPK-FoxO3 E3 ligases, Atrogin-1 and MuRF1. These results demonstrate chronic supramaximal ES reduces critical care associated muscle wasting, preserved glucose signalling, and reduced muscle protein degradation in CIM.
    Keywords:  Critical illness myopathy; E3 ligase; GLUT4 signalling; Muscle wasting; TBC1D4
  4. Biochem Biophys Res Commun. 2022 Apr 19. pii: S0006-291X(22)00600-3. [Epub ahead of print]612 22-29
      Carnosine and anserine are abundant peptides found in the skeletal muscle and nervous system in many vertebrates. Several in vitro and in vivo studies have demonstrate that exogenously administered carnosine improves exercise performance. Furthermore, carnosine is an antioxidant and antifatigue supplement. However, the physiological functions of endogenous carnosine and its related histidine-containing dipeptides in a living organism remain unclear. We aimed to clarify the physiological roles of endogenous carnosine by investigating the characteristics of carnosine synthase gene-deficient mice and the effects of carnosine on skeletal muscle protein metabolism. We discovered that carnosine and anserine were undetectable in the skeletal muscle of carnosine synthase knockout mice. We also quantified protein gene expression and enzyme levels in muscle protein metabolism. Gene and protein levels of the muscle protein synthesizer insulin-like growth factor-1 (IGF-1) and the degrading enzyme cathepsin B were markedly lower in carnosine synthase gene-deficient mice than those in wild-type mice. The amount of 3-methylhistidine (a marker for muscle proteolysis) in forced exercise and the weight of the gastrocnemius muscle were considerably lower in carnosine synthase gene-deficient mice than in wild-type mice. Consequently, we showed that carnosine deficiency affects weight maintenance and protein metabolism in skeletal muscle, suggesting that carnosine regulates skeletal muscle protein metabolism.
    Keywords:  Anserine; Carnosine; Knockout mice; Skeletal muscle metabolism
  5. Nat Commun. 2022 May 02. 13(1): 2370
      Decline in skeletal muscle cell size (myofiber atrophy) is a key feature of cancer-induced wasting (cachexia). In particular, atrophy of the diaphragm, the major muscle responsible for breathing, is an important determinant of cancer-associated mortality. However, therapeutic options are limited. Here, we have used Drosophila transgenic screening to identify muscle-secreted factors (myokines) that act as paracrine regulators of myofiber growth. Subsequent testing in mouse myotubes revealed that mouse Fibcd1 is an evolutionary-conserved myokine that preserves myofiber size via ERK signaling. Local administration of recombinant Fibcd1 (rFibcd1) ameliorates cachexia-induced myofiber atrophy in the diaphragm of mice bearing patient-derived melanoma xenografts and LLC carcinomas. Moreover, rFibcd1 impedes cachexia-associated transcriptional changes in the diaphragm. Fibcd1-induced signaling appears to be muscle selective because rFibcd1 increases ERK activity in myotubes but not in several cancer cell lines tested. We propose that rFibcd1 may help reinstate myofiber size in the diaphragm of patients with cancer cachexia.
  6. Elife. 2022 May 04. pii: e75529. [Epub ahead of print]11
      Age-dependent loss of body wall muscle function and impaired locomotion occur within 2 weeks in C. elegans; however, the underlying mechanism has not been fully elucidated. In humans, age-dependent loss of muscle function occurs at about 80 years of age and has been linked to dysfunction of ryanodine receptor (RyR)/intracellular calcium (Ca2+) release channels on the sarcoplasmic reticulum (SR). Mammalian skeletal muscle RyR1 channels undergo age-related remodeling due to oxidative overload, leading to loss of the stabilizing subunit calstabin1 (FKBP12) from the channel macromolecular complex. This destabilizes the closed state of the channel resulting in intracellular Ca2+ leak, reduced muscle function, and impaired exercise capacity. We now show that the C. elegans RyR homolog, UNC-68, exhibits a remarkable degree of evolutionary conservation with mammalian RyR channels and similar age-dependent dysfunction. Like RyR1 in mammals UNC-68 encodes a protein that comprises a macromolecular complex which includes the calstabin1 homolog FKB-2 and is immunoreactive with antibodies raised against the RyR1 complex. Further, as in aged mammals, UNC-68 is oxidized and depleted of FKB-2 in an age-dependent manner, resulting in 'leaky' channels, depleted SR Ca2+ stores, reduced body wall muscle Ca2+ transients, and age-dependent muscle weakness. FKB-2 (ok3007)-deficient worms exhibit reduced exercise capacity. Pharmacologically induced oxidization of UNC-68 and depletion of FKB-2 from the channel independently caused reduced body wall muscle Ca2+ transients. Preventing FKB-2 depletion from the UNC-68 macromolecular complex using the Rycal drug S107 improved muscle Ca2+ transients and function. Taken together, these data suggest that UNC-68 oxidation plays a role in age-dependent loss of muscle function. Remarkably, this age-dependent loss of muscle function induced by oxidative overload, which takes ~2 years in mice and ~80 years in humans, occurs in less than 2-3 weeks in C. elegans, suggesting that reduced antioxidant capacity may contribute to the differences in life span amongst species.
    Keywords:  C. elegans; biochemistry; cell biology; chemical biology
  7. Life Sci Alliance. 2022 Sep;pii: e202101215. [Epub ahead of print]5(9):
      miR-486 is a muscle-enriched microRNA, or "myomiR," that has reduced expression correlated with Duchenne muscular dystrophy (DMD). To determine the function of miR-486 in normal and dystrophin-deficient muscles and elucidate miR-486 target transcripts in skeletal muscle, we characterized mir-486 knockout mice (mir-486 KO). mir-486 KO mice developed disrupted myofiber architecture, decreased myofiber size, decreased locomotor activity, increased cardiac fibrosis, and metabolic defects were exacerbated in mir-486 KO:mdx 5cv (DKO) mice. To identify direct in vivo miR-486 muscle target transcripts, we integrated RNA sequencing and chimeric miRNA eCLIP sequencing to identify key transcripts and pathways that contribute towards mir-486 KO and dystrophic disease pathologies. These targets included known and novel muscle metabolic and dystrophic structural remodeling factors of muscle and skeletal muscle contractile transcript targets. Together, our studies identify miR-486 as essential for normal muscle function, a driver of pathological remodeling in dystrophin-deficient muscle, a useful biomarker for dystrophic disease progression, and highlight the use of multiple omic platforms to identify in vivo microRNA target transcripts.
  8. Eur J Endocrinol. 2022 May 01. pii: EJE-21-0776. [Epub ahead of print]
      OBJECTIVE: Skeletal muscle is the major site of insulin action. There are limited data on the relationship between insulin action and skeletal muscle myogenic/regenerative potential. RUNX1 is a transcription factor which plays a role in muscle development and regeneration. The aim of our study was to assess the role of skeletal muscle myogenic/regenerative potential in the development of insulin resistance through the studies on RUNX1 transcription factor.DESIGN: Cross-sectional study. Experimental part with myoblast cell line culture.
    METHODS: We examined 41 young healthy volunteers, 21 normal-weight and 20 with overweight or obesity. Hyperinsulinemic-euglycemic clamp and vastus lateralis muscle biopsy were performed. In L6 myoblast and human skeletal muscle myoblasts (hSkMM) cell cultures, Runx1 was silenced at 2 stages of development. Cell growth, the expression of markers of myogenesis, nuclei fusion index, Akt phosphorylation and glucose uptake were measured.
    RESULTS: Skeletal muscle RUNX1 expression was decreased in overweight/obese individuals in comparison with normal-weight individuals and was positively related to insulin sensitivity, independently of BMI. Runx1 loss-of-function at the stage of myoblast inhibited myoblast proliferation and differentiation, reduced insulin-stimulated Akt phosphorylation and insulin-stimulated glucose uptake. In contrast, Runx1 knockdown in myotubes did not affect Akt phosphorylation, glucose uptake and other parameters studied.
    CONCLUSIONS: Myogenic/regenerative potential of adult skeletal muscle may be an important determinant of insulin action. Our data suggest that muscle RUNX1 may play a role in the modulation of insulin action through its effect on myogenesis.
  9. Mol Cell Neurosci. 2022 Apr 27. pii: S1044-7431(22)00036-7. [Epub ahead of print]120 103730
      The sympathetic nervous system (SNS) regulates skeletal muscle motor innervation and stabilizes the NMJ in health, disease and aging. Previous studies using both chemical (6-hydroxydopamine, 6-OHDA) and microsurgically-induced sympathetic denervation examined the NMJ organization and transmission in the mouse; however, a detailed quantification of the postterminal on larger hindlimb muscles involved in gait mechanics and posture is lacking. The purpose of this study was to determine whether targets of the sympathetic neuron (SN) exhibiting different intrinsic composition such as the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus muscles differ in their response to SN deprivation, and to develop a strategy to accurately quantify the impact of sympathectomy on the NMJ postterminal including those fibers located deeper in the muscle. This approach included muscle fixed ex vivo or through transcardial perfusion in mice treated with 6-OHDA or control ascorbic acid. We measured NMJ postterminal mean terminal total area, number of postterminal fragments, mean fragment area, and mean distance between fragments in free-floating alpha-bungarotoxin-stained in 1038 isolated muscle fibers. We found that muscle fiber sympathetic innervation plays a crucial role in the structural organization of the motorneuron-myofiber synapse postterminal and its deprivation leads to AChR cluster dispersion or shrinking as described in various neuromuscular diseases and aging.
    Keywords:  Neuromuscular junction postterminal; Skeletal muscle; Sympathetic neuron
  10. Eur J Pharmacol. 2022 May 03. pii: S0014-2999(22)00256-4. [Epub ahead of print] 174995
      The skeletal muscle (SkM) is the largest organ, which plays a vital role in controlling musculature, locomotion, body heat regulation, physical strength, and metabolism of the body. A sedentary lifestyle, aging, cachexia, denervation, immobilization, etc. Can lead to an imbalance between protein synthesis and degradation, which is further responsible for SkM atrophy (SmA). To date, the understanding of the mechanism of SkM mass loss is limited which also restricted the number of drugs to treat SmA. Thus, there is an urgent need to develop novel approaches to regulate muscle homeostasis. Presently, some natural products attained immense attraction to regulate SkM homeostasis. The natural products, i.e., polyphenols (resveratrol, curcumin), terpenoids (ursolic acid, tanshinone IIA, celastrol), flavonoids, alkaloids (tomatidine, magnoflorine), vitamin D, etc. exhibit strong potential against SmA. Some of these natural products have been reported to have equivalent potential to standard treatments to prevent body lean mass loss. Indeed, owing to the large complexity, diversity, and slow absorption rate of bioactive compounds made their usage quite challenging. Moreover, the use of natural products is controversial due to their partially known or elusive mechanism of action. Therefore, the present review summarizes various experimental and clinical evidence of some important bioactive compounds that shall help in the development of novel strategies to counteract SmA elicited by various causes.
    Keywords:  Mitochondrial biogenesis; Muscle mass; Myogenesis; Natural products; Protein synthesis; Skeletal muscle
  11. Front Physiol. 2022 ;13 872745
      Metformin and statins are currently the focus of large clinical trials testing their ability to counter age-associated declines in health, but recent reports suggest that both may negatively affect skeletal muscle response to exercise. However, it has also been suggested that metformin may act as a possible protectant of statin-related muscle symptoms. The potential impact of combined drug use on the hypertrophic response to resistance exercise in healthy older adults has not been described. We present secondary statin analyses of data from the MASTERS trial where metformin blunted the hypertrophy response in healthy participants (>65 years) following 14 weeks of progressive resistance training (PRT) when compared to identical placebo treatment (n = 94). Approximately one-third of MASTERS participants were taking prescribed statins. Combined metformin and statin resulted in rescue of the metformin-mediated impaired growth response to PRT but did not significantly affect strength. Improved muscle fiber growth may be associated with medication-induced increased abundance of CD11b+/CD206+ M2-like macrophages. Sarcopenia is a significant problem with aging and this study identifies a potential interaction between these commonly used drugs which may help prevent metformin-related blunting of the beneficial effects of PRT. Trial Registration:, NCT02308228, Registered on 25 November 2014.
    Keywords:  cellular features; metformin; muscle hypertrophy; resistance training; statin
  12. Acta Neuropathol. 2022 May 06.
      Androgens and androgen-related molecules exert a plethora of functions across different tissues, mainly through binding to the transcription factor androgen receptor (AR). Despite widespread therapeutic use and misuse of androgens as potent anabolic agents, the molecular mechanisms of this effect on skeletal muscle are currently unknown. Muscle mass in adulthood is mainly regulated by the bone morphogenetic protein (BMP) axis of the transforming growth factor (TGF)-β pathway via recruitment of mothers against decapentaplegic homolog 4 (SMAD4) protein. Here we show that, upon activation, AR forms a transcriptional complex with SMAD4 to orchestrate a muscle hypertrophy programme by modulating SMAD4 chromatin binding dynamics and enhancing its transactivation activity. We challenged this mechanism of action using spinal and bulbar muscular atrophy (SBMA) as a model of study. This adult-onset neuromuscular disease is caused by a polyglutamine expansion (polyQ) in AR and is characterized by progressive muscle weakness and atrophy secondary to a combination of lower motor neuron degeneration and primary muscle atrophy. Here we found that the presence of an elongated polyQ tract impairs AR cooperativity with SMAD4, leading to an inability to mount an effective anti-atrophy gene expression programme in skeletal muscle in response to denervation. Furthermore, adeno-associated virus, serotype 9 (AAV9)-mediated muscle-restricted delivery of BMP7 is able to rescue the muscle atrophy in SBMA mice, supporting the development of treatments able to fine-tune AR-SMAD4 transcriptional cooperativity as a promising target for SBMA and other conditions associated with muscle loss.
    Keywords:  Androgen receptor; Androgens; Muscle atrophy; SBMA; TGFβ pathway; Transcriptional cooperativity
  13. Front Physiol. 2022 ;13 845504
      Fundamental aspects underlying downstream processes of skeletal muscle regeneration, such as myonuclear positioning and transcription are poorly understood. This investigation begins to address deficiencies in knowledge by examining the kinetics of myonuclear accretion, positioning, and global transcription during injury-induced muscle regeneration in mice. We demonstrate that myonuclear accretion plateaus within 7 days of an injury and that the majority (∼70%) of myonuclei are centrally aligned in linear arrays (nuclear chains) throughout the course of regeneration. Relatively few myonuclei were found in a peripheral position (∼20%) or clustered (∼10%) together during regeneration. Importantly, transcriptional activity of individual myonuclei in nuclear chains was high, and greater than that of peripheral or clustered myonuclei. Transcription occurring primarily in nuclear chains elevated the collective transcriptional activity of regenerating myofibers during the later stage of regeneration. Importantly, the number of myonuclei in chains and their transcriptional activity were statistically correlated with an increase in myofiber size during regeneration. Our findings demonstrate the positional context of transcription during regeneration and highlight the importance of centralized nuclear chains in facilitating hypertrophy of regenerating myofibers after injury.
    Keywords:  myogenic cell fusion; myonuclear accretion; myonuclear positioning; myonuclear transcription; regenerating myofibers; satellite cells
  14. Skelet Muscle. 2022 May 06. 12(1): 10
      Skeletal muscle wasting is a clinically remarkable phenotypic feature of pulmonary arterial hypertension (PAH) that increases the risk of mortality. Growth differentiation factor 11 (GDF11), centrally involved in PAH pathogenesis, has an inhibitory effect on skeletal muscle growth in other conditions. However, whether GDF11 is involved in the pathogenesis of skeletal muscle wasting in PAH remains unknown. We showed that serum GDF11 levels in patients were increased following PAH. Skeletal muscle wasting in the MCT-treated PAH model is accompanied by an increase in circulating GDF11 levels and local catabolic markers (Fbx32, Trim63, Foxo1, and protease activity). In vitro GDF11 activated phosphorylation of STAT3. Antagonizing STAT3, with Stattic, in vitro and in vivo, could partially reverse proteolytic pathways including STAT3/socs3 and iNOS/NO in GDF11-meditated muscle wasting. Our findings demonstrate that GDF11 contributes to muscle wasting and the inhibition of its downstream molecule STAT3 shows promise as a therapeutic intervention by which muscle atrophy may be directly prevented in PAH.
    Keywords:  GDF11; Pulmonary arterial hypertension; STAT3; Skeletal muscle atrophy
  15. Eur J Appl Physiol. 2022 May 05.
      The 70-kDa heat shock protein (HSP70) is a ubiquitous molecular chaperone which is highly inducible by cellular stress such as exercise. To investigate the role of muscle glycogen content on the HSP70 expression, muscle glycogen was manipulated by consumption of either water (H2O) or a carbohydrate-enriched diet (CHO) during recovery from 4 h of glycogen-depleting cycling exercise in fourteen elite endurance athletes. Muscle biopsies were obtained pre- and post-exercise, and after 4 and 24 h of recovery, and analyzed for HSP70 mRNA expression, as well as HSP70 protein expression and muscle glycogen within the same skeletal muscle fibers using immunohistochemistry. Exercise reduced glycogen by 59 ± 10% (P < 0.0001). After 4 h of recovery, glycogen approached resting levels in the CHO group (86% of pre, P = 0.28) but remained suppressed in the H2O group (41% of pre, P < 0.001) (group × time interaction: P = 0.002). Importantly, both the HSP70 mRNA (+ 1.6-fold (+ 0.28/- 0.24), P = 0.02) and protein expression (+ 147 ± 99%, P < 0.0001) was substantially increased after exercise and remained elevated in both groups after 4 h of recovery, despite clear differences in muscle glycogen content. Thus, muscle glycogen content was not related to the variation in single fiber HSP70 expression at the 4-h time-point (r2 = 0.004). In conclusion, muscle HSP70 expression remained elevated during recovery from prolonged exercise in highly trained skeletal muscle, irrespective of muscle glycogen availability.
    Keywords:  Endurance exercise; Glycogen; HSP70; Metabolic stress; Recovery
  16. Proc Natl Acad Sci U S A. 2022 May 10. 119(19): e2201136119
      SignificanceVertebrate skeletal muscle excitation-contraction coupling (ECC) is based on Ca2+-influx-independent interchannel cross-talk between DHPR and RyR1. The skeletal muscle DHPR complex consists of the main, voltage-sensing, and pore-forming α1S subunit, the auxiliary β1a, α2δ-1, γ1 subunits, and Stac3. The DHPRβ1a subunit plays an essential role in full triad targeting of DHPRα1S, voltage sensing, and tetrad formation (grouping of four DHPRs)-the three prerequisites for skeletal muscle ECC. Hence, a lack of DHPRβ1a results in a lethal phenotype in both β1-null mice and zebrafish. Here, we identified the nonconserved, distal C terminus of DHPRβ1a as playing a pivotal role in the formation of DHPR tetrads, and thus allosteric DHPR-RyR1 coupling, essential for proper skeletal muscle ECC.
    Keywords:  excitation–contraction coupling; skeletal muscle; tetrad formation; voltage-gated Ca2+ channel; β subunit
  17. Appl Physiol Nutr Metab. 2022 May 04.
      The interest in a diet with a higher proportion of plant-based foods to animal-based foods is a global food pattern trend. However, there are concerns regarding adopting plants as the main dietary protein source to support muscle protein synthesis and muscle mass. These concerns are centred on three issues: lower protein bioavailability due to antinutritional compounds in plants, lower per-serve scores of protein at similar energy intake, and amino acid scores of plants being lower than optimal. We aimed here to synthesize and discuss evidence around plant protein in human nutrition focusing on the capacity of these proteins to stimulate muscle protein synthesis as a key part of gaining or maintaining muscle mass. In this review, we address the issues of plant protein quality and provide evidence for how plant proteins can be made more effective to stimulate muscle protein synthesis and support muscle mass in partial or total replacement of consumption of products of animal origin. Novelty: ● Plant proteins are known, in general, to have lower protein quality scores than animal proteins, and this may have important implications, especially for those aiming to increase their skeletal muscle mass through exercise. ● A plant-based diet has been postulated to have lower protein quality limiting MPS responses and potentially compromising exercise-induced gains in muscle mass. ● Current evidence shows that plant proteins can stimulate MPS, as can whole foods, especially when combining food groups, increasing portion sizes, and optimizing amino acid bioavailability through processing or common preparation methods.
  18. Cell Death Discov. 2022 Apr 30. 8(1): 236
      Skeletal muscle myogenesis is a sophisticated process controlled by genetic and epigenetic regulators. In animals, one of the key enzymes for the DNA demethylation of 5-methylcytosine is TET2. Although TET2 is essential for muscle development, the mechanisms by which TET2 regulates myogenesis, particularly the implication for muscle stem cells, remains unclear. In the present study, we employed the TET2 knockout mouse model to investigate the function of TET2 in muscle development and regeneration. We observed that TET2 deficiency caused impaired muscle stem cell proliferation and differentiation, resulting in the reduction in both myofiber number and muscle tissue size. Specifically, TET2 maintains calcium homeostasis in muscle stem cells by controlling the DNA methylation levels of the calcium pathway genes. Forced expression of the sodium/calcium exchanger protein SLC8A3 could rescue the myogenic defects in TET2 knockout cells. Our data not only illustrated the vital function of TET2 during myogenesis but also identified novel targets that contribute to calcium homeostasis for enhancing muscle function.
  19. Front Cardiovasc Med. 2022 ;9 789331
      Background: Oxidative stress and impaired autophagic flux play important roles in the development of peripheral artery disease (PAD). SS31 is considered an important antioxidant peptide and autophagy regulator. We aimed to investigate the role of SS31 in PAD myopathy and its possible mechanism both in vivo and in vitro.Methods: A hind limb ischemia (HLI) model was established with old C57BL/6 (14-month-old) mice. Mice in the SS31 group were intraperitoneally injected with SS31 (3 mg/kg) for 4 weeks. We examined skeletal muscle function and histomorphology, autophagy-related protein levels and reactive oxygen species (ROS) content. For the in vitro experiments, after C2C12 myotubes were treated with CoCl2, SS31, and chloroquine (CQ) or rapamycin (RAPA), we measured ROS content, autophagy-related protein levels and antioxidant enzyme expression.
    Results: SS31 treatment effectively enhanced the recovery of skeletal muscle function, alleviated skeletal muscle injury and suppressed mitochondrial ROS production in ischemic limbs. SS31 reduced apoptosis and oxidative stress, and SS31 restored impaired autophagic flux by inhibiting the AKT-mTOR pathway. In vitro studies showed that SS31 restored autophagic flux and improved oxidative stress in C2C12 cells. Moreover, phosphorylated AKT (p-AKT) and phosphorylated mTOR (p-mTOR) levels were reduced.
    Conclusion: These experiments indicated that SS31 can inhibit oxidative stress by restoring autophagic flux to reverse hypoxia-induced injury in vivo and in vitro.
    Keywords:  AKT-mTOR pathway; Szeto-Schiller peptide; autophagic flux; oxidative stress; peripheral artery disease (PAD)
  20. Hum Mutat. 2022 May 05.
      Nemaline myopathies (NEM) are genetically and clinically heterogenous. Biallelic or monoallelic variants in TNNT1, encoding slow skeletal troponin T1 (TnT1), cause NEM. We report a 2-year-old patient and his mother carrying the heterozygous TNNT1 variant c.194A>C/p.(Asp65Ala) that occurred de novo in the mother. Both had muscle hypotrophy and muscle weakness. Muscle pathology in the proband's mother revealed slow twitch type 1 fiber hypotrophy and fast twitch type 2 fiber hypertrophy that was confirmed by a reduced ratio of slow skeletal myosin to fast skeletal myosin type 2a. RT-PCR and immunoblotting data demonstrated increased levels of high-molecular-weight TnT1 isoforms in skeletal muscle of the proband's mother that were also observed in some controls. In an overexpression system, complex formation of TnT1-D65A with tropomyosin 3 (TPM3) was enhanced. The previously reported TnT1-E104V and TnT1-L96P mutants showed reduced or no co-immunoprecipitation with TPM3. Our studies support pathogenicity of the TNNT1 p.(Asp65Ala) variant. This article is protected by copyright. All rights reserved.
    Keywords:  Nemaline myopathy; actin; autosomal recessive; loss of function; muscle biopsy; nemaline rods
  21. J Muscle Res Cell Motil. 2022 May 07.
      Mutations in the sialic acid biosynthesis enzyme GNE lead to a late-onset, debilitating neuromuscular disorder, GNE myopathy, characterized by progressive skeletal muscle weakness. The mechanisms responsible for skeletal muscle specificity, late-onset, and disease progression are unknown. Our main aim is to understand the reason for skeletal muscle-specific phenotype. To answer this question, we have analyzed the expression profile of the GNE gene and its multiple mRNA variants in different human tissues. A combinatorial approach encompassing bioinformatics tools and molecular biology techniques was used. NCBI, Ensembl, and GTEx were used for data mining. The expression analysis of GNE and its variants was performed with cDNA tissue panel using PCR and targeted RNA-seq. Among nine different GNE isoforms reported in this study, transcript variants 1, X1, and X2 were not tissue specific. Transcript variants 1, 6, X1, and X2, were found in skeletal muscles suggesting their possible role in GNE myopathy. In the current study, we present new data about GNE expression patterns in human tissues. Our results suggest that there may be a link between tissue-specific pathology and isoform pattern in skeletal muscles, which could provide clues for the development of new treatment strategies for GNE myopathy.
    Keywords:  GNE myopathy; Gene expression; Isoforms; Sialic acid; Skeletal muscles
  22. Nat Commun. 2022 May 06. 13(1): 2503
      Selenoprotein N (SELENON), a selenocysteine (Sec)-containing protein with high reductive activity, maintains redox homeostasis, thereby contributing to skeletal muscle differentiation and function. Loss-of-function mutations in SELENON cause severe neuromuscular disorders. In the early-to-middle stage of myoblast differentiation, SELENON maintains redox homeostasis and modulates endoplasmic reticulum (ER) Ca2+ concentration, resulting in a gradual reduction from the middle-to-late stages due to unknown mechanisms. The present study describes post-transcriptional mechanisms that regulate SELENON expression during myoblast differentiation. Part of an Alu element in the second intron of SELENON pre-mRNA is frequently exonized during splicing, resulting in an aberrant mRNA that is degraded by nonsense-mediated mRNA decay (NMD). In the middle stage of myoblast differentiation, ADAR1-mediated A-to-I RNA editing occurs in the U1 snRNA binding site at 5' splice site, preventing Alu exonization and producing mature mRNA. In the middle-to-late stage of myoblast differentiation, the level of Sec-charged tRNASec decreases due to downregulation of essential recoding factors for Sec insertion, thereby generating a premature termination codon in SELENON mRNA, which is targeted by NMD.
  23. Stem Cells. 2022 Mar 03. 40(1): 74-87
      The diminished ability for muscle to regenerate is associated with aging, diabetes, and cancers. Muscle regeneration depends on the activation and differentiation of satellite cells (SCs). Inactivation of Mst1/2 promotes cell proliferation by activating Yap, and that has been reported as a potential therapeutic target for improving many organ regeneration. However, the function of Mst1/2 in SCs fate decision and that effect on muscle regeneration remain unknown. By using inducible conditional knockout Mst1/2 in the SCs of mice and an inhibitor of Mst1/2, we found that inhibition of Mst1/2 in SCs significantly decrease Yap phosphorylation, thus causing Yap to accumulate in the nucleus and impairing SC differentiation; Mst1/2 were slightly elevated by irisin stimulation during SC differentiation; but inhibiting Mst1/2 in SCs significantly impaired irisin-induced muscle regeneration. These results indicate that Mst1/2 is necessary for SC differentiation and inhibiting Mst1/2 as a therapeutic target has potential risks for muscle regeneration.
    Keywords:  Irisin; Mst1/2; Yap; muscle regeneration; satellite cell
  24. J Cachexia Sarcopenia Muscle. 2022 May 05.
      BACKGROUND: Cachexia is frequent, deadly, and untreatable for patients with pancreatic ductal adenocarcinoma (PDAC). The reproductive hormone and cytokine Activin is a mediator of PDAC cachexia, and Activin receptor targeting was clinically tested for cancer cachexia therapy. However, sex-specific manifestations and mechanisms are poorly understood, constraining development of effective treatments.METHODS: Cachexia phenotypes, muscle gene/protein expression, and effects of the Activin blocker ACVR2B/Fc were assessed in LSL-KrasG12D/+ , LSL-Trp53R172H/+ , and Pdx-1-Cre (KPC) mice with autochthonic PDAC. Effects of PDAC and sex hormones were modelled by treating C2C12 myotubes with KPC-cell conditioned medium (CM) and estradiol. Muscle gene expression by RNAseq and change in muscle from serial CT scans were measured in patients with PDAC.
    RESULTS: Despite equivalent tumour latency (median 17 weeks) and mortality (24.5 weeks), male KPC mice showed earlier and more severe cachexia than females. In early PDAC, male gastrocnemius, quadriceps, and tibialis anterior muscles were reduced (-21.7%, -18.9%, and -20.8%, respectively, all P < 0.001), with only gastrocnemius reduced in females (-16%, P < 0.01). Sex differences disappeared in late PDAC. Plasma Activin A was similarly elevated between sexes throughout, while oestrogen and testosterone levels suggested a virilizing effect of PDAC in females. Estradiol partially protected myotubes from KPC-CM induced atrophy and promoted expression of the potential Activin inhibitor Fstl1. Early-stage female mice showed greater muscle expression of Activin inhibitors Fst, Fstl1, and Fstl3; this sex difference disappeared by late-stage PDAC. ACVR2B/Fc initiated in early PDAC preserved muscle and fat only in male KPC mice, with increases of 41.2%, 52.6%, 39.3%, and 348.8%, respectively, in gastrocnemius, quadriceps, tibialis, and fat pad weights vs. vehicle controls, without effect on tumour. No protection was observed in females. At protein and RNA levels, pro-atrophy pathways were induced more strongly in early-stage males, with sex differences less evident in late-stage disease. As with mass, ACVR2B/Fc blunted atrophy-associated pathways only in males. In patients with resectable PDAC, muscle expression of Activin inhibitors FSTL1, FSLT3, and WFIKKN2/GASP2 were higher in women than men. Overall, among 124 patients on first-line gemcitabine/nab-paclitaxel for PDAC, only men displayed muscle loss (P < 0.001); average muscle wasting in men was greater (-6.63 ± 10.70% vs. -1.62 ± 12.00% mean ± SD, P = 0.038) and more rapid (-0.0098 ± 0.0742%/day vs. -0.0466 ± 0.1066%/day, P = 0.017) than in women.
    CONCLUSIONS: Pancreatic ductal adenocarcinoma cachexia displays sex-specific phenotypes in mice and humans, with Activin a preferential driver of muscle wasting in males. Sex is a major modulator of cachexia mechanisms. Consideration of sexual dimorphism is essential for discovery and development of effective treatments.
    Keywords:  ACVR2B; Activin; Cachexia; Estradiol; Muscle wasting; Pancreatic cancer; Sexual dimorphism; Weight loss
  25. Sci Rep. 2022 May 06. 12(1): 7450
      Rheumatoid arthritis (RA) T cells drive autoimmune features via metabolic reprogramming that reduces oxidative metabolism. Exercise training improves cardiorespiratory fitness (i.e., systemic oxidative metabolism) and thus may impact RA T cell oxidative metabolic function. In this pilot study of RA participants, we took advantage of heterogeneous responses to a high-intensity interval training (HIIT) exercise program to identify relationships between improvements in cardiorespiratory fitness with changes in peripheral T cell and skeletal muscle oxidative metabolism. In 12 previously sedentary persons with seropositive RA, maximal cardiopulmonary exercise tests, fasting blood, and vastus lateralis biopsies were obtained before and after 10 weeks of HIIT. Following HIIT, improvements in RA cardiorespiratory fitness were associated with changes in RA CD4 + T cell basal and maximal respiration and skeletal muscle carnitine acetyltransferase (CrAT) enzyme activity. Further, changes in CD4 + T cell respiration were associated with changes in naïve CD4 + CCR7 + CD45RA + T cells, muscle CrAT, and muscle medium-chain acylcarnitines and fat oxidation gene expression profiles. In summary, modulation of cardiorespiratory fitness and molecular markers of skeletal muscle oxidative metabolism during exercise training paralleled changes in T cell metabolism. Exercise training that improves RA cardiorespiratory fitness may therefore be valuable in managing pathologically related immune and muscle dysfunction.Trial registration:, NCT02528344. Registered on 19 August 2015.
  26. J Exp Biol. 2022 May 06. pii: jeb.243732. [Epub ahead of print]
      Residual force enhancement (RFE) is the increase in steady-state force after active stretch relative to the force during isometric contraction at the same final length. The mdm mutation in mice, characterized by a small deletion in N2A titin, has been proposed to prevent N2A titin-actin interactions so that active mdm muscles are more compliant than WT. This decrease in active muscle stiffness is associated with reduced RFE. We investigated RFE in permeabilized soleus (SOL) and extensor digitorum longus (EDL) fiber bundles from wild type and mdm mice. On each fiber bundle, we performed active and passive stretches from an average sarcomere length of 2.6 - 3.0 µm at a slow rate of 0.04 µm/s, as well as isometric contractions at the initial and final lengths. One-way ANOVA showed that SOL and EDL fiber bundles from mdm mice exhibited significantly lower RFE than WT (P<0.0001). This result is consistent with previous observations in single myofibrils and intact muscles. However, it contradicts the results from a previous study which appeared to show that compensatory mechanisms could restore titin force enhancement in single fibers from mdm psoas. We suggest that residual force enhancement measured previously in mdm single fibers was an artifact of the high variability in passive tension found in degenerating fibers, which begins after ∼24 days of age. The results are consistent with the hypothesis that RFE is reduced in mdm skeletal muscles due to impaired Ca2+ dependent titin-actin interactions resulting from the small deletion in N2A titin.
    Keywords:  Active stress after stretch; Extensor digitorum longus; Isometric stress; Passive stress after stretch; Soleus; Titin
  27. Mol Cell Endocrinol. 2022 May 01. pii: S0303-7207(22)00111-3. [Epub ahead of print] 111663
      Insulin resistance (InR) is manifested in skeletal muscle by decreased insulin-stimulated glucose uptake due to impaired insulin signaling and multiple post-receptor intracellular defects. Chronic glucose-induced insulin resistance leads to the activation of Ser/Thr kinases and elevated phosphorylation of insulin receptor substrate 1 (IRS1) on Ser residues. Phosphorylation of IRS1 triggers the dissociation of IRS1 and its downstream effector, phosphatidylinositol 3-kinase. In the present study, we provide evidence for the insulin-sensitizing role of smoothelin-like protein 1 (SMTNL1) that is a ligand-dependent co-regulator of steroid receptors, predominantly the progesterone receptor. SMTNL1 was transiently overexpressed in insulin-resistant C2C12 myotubes. A proteome profiler array revealed that mTOR and Ser/Thr kinases were SMTNL1-dependent signaling pathways. In the presence of progesterone, overexpression was coupled to decreased Ser phosphorylation of IRS1 at Ser307, Ser318, and Ser612 residues. SMTNL1 also induced the expression and activity of the p85 subunit of PI3K. SMTNL1 regulated the expression of PKCε, which phosphorylates IRS1 at Ser318 residue. SMTNL1 also regulated ERK1/2 and JNK, which phosphorylate IRS1 at Ser612 and Ser307, respectively. Real-time metabolic measurements of oxygen consumption rate and extracellular acidification rate revealed that SMTNL1 improved glycolysis and promoted the utilization of alternative carbon fuels. SMTNL1 also rescued the mitochondrial respiration defect induced by chronic insulin exposure. Collectively, SMTNL1 plays a crucial role in maintaining the physiological ratio of Tyr/Ser IRS1 phosphorylation and attenuates the insulin-signaling cascade that contributes to impaired glucose disposal, which makes it a potential therapeutic target for improving InR.
    Keywords:  Insulin receptor substrate-1; Insulin resistance; Insulin signaling; Phosphorylation; Pregnancy; Skeletal muscle metabolism
  28. Am J Physiol Endocrinol Metab. 2022 May 06.
      The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that has spread worldwide, resulting in over 6 million deaths as of March 2022. Older people have been disproportionately affected by the disease, as they have greater risk of hospitalization, are more vulnerable to severe infection, and have higher mortality than younger patients. Although effective vaccines have been rapidly developed and administered globally, several clinical trials are ongoing to repurpose existing drugs to combat severe infection. One such drug, rapamycin, is currently under study for this purpose, given its immunosuppressant effects that are mediated by its inhibition of the mechanistic target of rapamycin (mTOR), a master regulator of cell growth. Consistent with this premise, acute rapamycin administration in young healthy humans blocks or attenuates mTOR and its downstream effectors, leading to the inhibition of muscle protein synthesis (MPS). Skeletal muscle mass declines when MPS is chronically lower than muscle protein breakdown. This is consequential for older people who are more susceptible to anabolic resistance (i.e., the blunting of MPS) due to reduced activity, sedentariness, or bed rest such as that associated with COVID-19 hospitalization, and who have also demonstrated a delayed or blunted ability to regain inactivity-induced muscle loss. The lack of studies investigating rapamycin administration on skeletal muscle in older people, and the emergence of effective antiviral medications against severe infection, may indicate the reduced relevance of drug repurposing for present or future pandemics.
    Keywords:  SARS-CoV-2; aging; anabolic resistance; mTOR; muscle protein synthesis
  29. Cell Signal. 2022 Apr 28. pii: S0898-6568(22)00103-6. [Epub ahead of print]95 110341
      Long non-coding RNAs (lncRNAs) play essential roles in myogenesis. Here, we identified a novel long non-coding RNA, named COPS3 AS lncRNA (COP9 signalosome complex subunit 3 antisense lncRNA), which was transcribed from the mouse COPS3 gene antisense strand and highly expressed in glycolytic muscle fibers. Functionally, COPS3 AS lncRNA knockdown inhibited myogenic differentiation in myoblasts, whereas its overexpression promoted the process. Moreover, COPS3 AS lncRNA maintained the fast-twitch myotubes phenotype. Mechanistically, although COPS3 AS lncRNA did not form AS lncRNA/mRNA dimer with COPS3 mRNA, it as a competing endogenous RNA (ceRNA) to sponge miR-762, promoted myogenic differentiation and Fast-MyHC expression by modulating miR-762 target gene myogenic differentiation 1 (MyoD1). Taken together, COPS3 AS lncRNA is a key candidate regulator of myogenesis and fast-MyHC myotubes specification by miR-762/MyoD signalling axis.
    Keywords:  COPS3 AS lncRNA; MyoD; Myogenesis; Myotube phenotype; miR-762
  30. Exp Gerontol. 2022 Apr 30. pii: S0531-5565(22)00131-0. [Epub ahead of print] 111823
      Sarcopenia, a geriatric syndrome that is characterized by a progressive and generalized skeletal muscle disorder, can be associated with many comorbidities, including obesity, diabetes, and fracture. Currently, the importance and urgency of sarcopenia have gained more consensus. Discovering the mechanisms of sarcopenia has been more and more important. It has been previously suggested that immune system during ageing plays an important role in the progression of sarcopenia. Immune ageing, which is often highlighted in elder individuals, may be an important contributor in sarcopenia. Immune ageing can occur in different aspects. The alteration in immune organs can affect both the innate immunity and adaptive immunity, affecting the whole condition of the body through circulation. Several kinds of immune cells, including lymphocytes, macrophages, neutrophils and other immune cells, together alters the situation of muscle fiber, causing muscle weakness, loss of muscle strength and muscle mass. Synergistic and cumulative effect of cytokines, such as TNF-α and IFN-γ, interrelates with obesity and diabetes, impairing the condition of skeletal muscle tissue and leading to deterioration of sarcopenia. Studying the relationship of sarcopenia and immune system offers great potential in future studies. Thoroughly studying these mechanisms can help to better determine an ideal scheme and better management of sarcopenia and its associated comorbidities, which tends to offer deeper insight and guidance in treating sarcopenia through alterations of food intake, exercise and medical intervention.
    Keywords:  Ageing; Cytokines; Immune ageing; Immune cells; Immune system; Sarcopenia; Treatment
  31. Physiol Res. 2021 Nov 30. 70(Suppl 1): S91-S98
      Sarcopenia is defined as an age-associated loss of skeletal muscle function and muscle mass and is common in older adults. Sarcopenia as a disease is currently of interest not only to orthopedists and surgeons but also to internists, endocrinologists, rheumatologists, cardiologists, diabetologists, gynaecologists, geriatricians and paediatricians. In cooperation with the 5th Internal Medicine Clinic, we, as a unit of clinical research, aimed to describe a sarcopenic specific miRNA expression profile for disease diagnostics and classification of the severity of muscle performance deterioration. This study included a total of 80 patients (age 55-86 years) hospitalized at the V. Internal medicine clinic of LFUK and UNB with different severity of muscle performance deterioration. The study participants were evaluated and classified according to short physical performance battery score (SPPB). In this study, we investigated the role of circulating miRNAs in sarcopenia in the elderly. We hypothesized that sarcopenia effects the expression of muscle tissue-specific miRNAs (MyomiRNAs), which could be potentially reflected in the blood plasma miRNA expression profile. The expression of specific circulating miRNAs in patients with different muscle performances was analyzed. Patients' blood plasma was evaluated for the expression of myomiRNAs: miRNA-29a, miRNA-29b, miRNA-1, miRNA-133a, miRNA-133b, miRNA-206, miRNA-208b and miRNA-499, and the data were correlated with diagnostic indicators of the disease. We showed a specific sarcopenia miRNA profile that could be considered a possible biomarker for the disease. Patients with low muscle performance showed increased miRNA-1, miRNA-29a and miRNA-29b expression and decreased for the miRNA-206, miRNA-133a, miRNA-133b, miRNA-208b and miRNA-499 expression. We show that the severity of muscle performance deterioration in sarcopenia correlates with specific miRNA expression. We also propose the profile of miRNAs expression in blood plasma as a specific biomarker for sarcopenia diagnostics. Future clinical studies will be necessary to eventually naturally have to elucidate the underlined molecular mechanism responsible for specific miRNAs expression in sarcopenia pathology and progression of the disease.