bims-moremu Biomed News
on Molecular regulators of muscle mass
Issue of 2021‒03‒21
fifty-two papers selected by
Anna Vainshtein
Craft Science Inc.

  1. Skelet Muscle. 2021 Mar 15. 11(1): 6
      BACKGROUND: Sarcopenia is a common skeletal disease related to myogenic disorders and muscle atrophy. Current clinical management has limited effectiveness. We sought to investigate the role of miR-1290 in myoblast differentiation and muscle atrophy.METHODS: By transfecting miR-1290 into C2C12 cells, we investigated whether miR-1290 regulates myogenesis and myotube atrophy via AKT/P70 signaling pathway. MHC staining was performed to assess myoblast differentiation. Differentiation-related MHC, Myod, and Myog protein levels, and atrophy-related MuRF1 and atrogin-1 were explored by western blot. An LPS-induced muscle atrophy rat model was developed. RT-PCR was conducted to analyze miR-1290 serum levels in muscle atrophy patients and normal controls (NCs).
    RESULTS: The miR-1290 transfection increased MHC-positive cells and MHC, Myod, and Myog protein levels in the miR-1290 transfection group, demonstrating that miR-1290 promoted C2C12 myoblast differentiation. Myotube diameter in the miR-1290 transfection group was higher than in the TNF-α-induced model group. Western blot analysis showed decreased MuRF1 and atrogin-1 levels in the miR-1290 transfection group compared with the model group, demonstrating that miR-1290 protected against myoblast cellular atrophy. Luciferase assay and western blot analysis showed that miR-1290 regulation was likely caused by AKT/p70/FOXO3 phosphorylation activation. In the LPS-induced muscle atrophy rat model, miR-1290 mimics ameliorated gastrocnemius muscle loss and increased muscle fiber cross-sectional area. Clinically, miR-1290 serum level was significantly decreased in muscle atrophy patients.
    CONCLUSIONS: We found that miR-1290 enhances myoblast differentiation and inhibits myotube atrophy through Akt/p70/FoxO3 signaling in vitro and in vivo. In addition, miR-1290 may be a potential therapeutic target for sarcopenia treatment.
    Keywords:  Akt/P70/FoxO3; Atrophy; Myoblast differentiation; miR-1290
  2. Biochim Biophys Acta Mol Basis Dis. 2021 Mar 12. pii: S0925-4439(21)00059-4. [Epub ahead of print] 166126
      Mitochondrial-derived peptide (MOTS-c) has gained increasing attention as a promising therapeutic or prevention strategy for obesity and diabetes mellitus. MOTS-c targets the folate cycle, leading to an accumulation of 5-aminomidazole-4-carboxamide ribonucleotide (AICAR) as well as AMPK activation. AMPK is a well-known upstream regulator of the proliferation-activated receptor co-activator 1 (PGC-1α), which can improve mitochondrial biogenesis via co-transcriptional modifications. We hypothesized that AMPK can induce the expression of MOTS-c through PGC-1α. Our study aimed to explore whether MOTS-c and/or exercise can regulate MOTS-c expression, attenuate insulin resistance and enhance glucose metabolism both in vitro and in vivo. It was found that C2C12 myotubes exposed to Compound C (an AMPK inhibitor) had deceases in the protein and mRNA expressions of PGC-1α and MOTS-c. PGC-1α knockdown downregulated the protein and mRNA expressions of MOTS-c in C2C12 myotubes, whereas both PGC-1α overexpression and recombinant MOTS-c supplementation upregulated the protein and mRNA expressions of MOTS-c in C2C12 myotubes. Furthermore, the skeletal muscle and plasma levels of MOTS-c were markedly reduced in high-fat diet-induced obese mice. Treadmill training remarkably upregulated the protein levels of MOTS-c, PGC-1α and GLUT4, along with the phosphorylation levels of AMPK and ACC. Altogether, these results indicate that AMPK/PGC-1α pathway can mediate the secretion and/or production of MOTS-c in skeletal muscle, implying the possible roles of exercise intervention and recombinant MOTS-c in treating obesity and diabetes mellitus.
    Keywords:  AMPK; Exercise; Insulin resistance; MOTS-c; PGC-1α; Skeletal muscle
  3. Nat Biomed Eng. 2021 Mar 18.
      Muscle loss and impairment resulting from traumatic injury can be alleviated by therapies using muscle stem cells. However, collecting sufficient numbers of autologous myogenic stem cells and expanding them efficiently has been challenging. Here we show that myogenic stem cells (predominantly Pax7+ cells)-which were selectively expanded from readily obtainable dermal fibroblasts or skeletal muscle stem cells using a specific cocktail of small molecules and transplanted into muscle injuries in adult, aged or dystrophic mice-led to functional muscle regeneration in the three animal models. We also show that sustained release of the small-molecule cocktail in situ through polymer nanoparticles led to muscle repair by inducing robust activation and expansion of resident satellite cells. Chemically induced stem cell expansion in vitro and in situ may prove to be advantageous for stem cell therapies that aim to regenerate skeletal muscle and other tissues.
  4. Biochem Biophys Res Commun. 2021 Mar 16. pii: S0006-291X(21)00418-6. [Epub ahead of print]552 52-58
      METTL3 increasing the mature miRNA levels via N6-Methyladenosine (m6A) modification of primary miRNA (pri-miRNA) transcripts has emerged as an important post-transcriptional regulation of miRNA biogenesis. Our previous studies and others have showed that muscle specific miRNAs are essential for skeletal muscle differentiation. Whether these miRNAs are also regulated by METTL3 is still unclear. Here, we found that m6A motifs were present around most of these miRNAs, which were indeed m6A modified as confirmed by m6A-modified RNA immunoprecipitation (m6A RIP). However, we surprisingly found that these muscle specific miRNAs were repressed instead of increased by METTL3 in C2C12 in vitro differentiation and mouse skeletal muscle regeneration after injury in vivo model. To elucidate the underlined mechanism, we performed reporter assays in 293T cells and validated METTL3 increasing these miRNAs at post-transcriptional level as expected. Furthermore, in myogenic C2C12 cells, we found that METTL3 not only repressed the expression of myogenic transcription factors (TFs) which can enhance the muscle specific miRNAs, but also increased the expression of epigenetic regulators which can repress these miRNAs. Thus, METTL3 could repress the muscle specific miRNAs at transcriptional level indirectly. Taken together, our results demonstrated that skeletal muscle specific miRNAs were repressed by METTL3 and such repression is likely synthesized transcriptional and post-transcriptional regulations.
    Keywords:  HDAC; MEF2C; METTL3; Skeletal muscle differentiation; m6A; miRNA
  5. Stem Cell Rev Rep. 2021 Mar 19.
      Stem cell antigen-1 (Sca-1) is a glycosyl-phosphatidylinositol-anchored membrane protein that is expressed in a sub-population of muscle stem and progenitor cell types. Reportedly, Sca-1 regulates the myogenic property of myoblasts and Sca-1-/- mice exhibited defective muscle regeneration. Although the role of Sca-1 in muscle development and maintenance is well-acknowledged, molecular composition of muscle derived Sca-1+ cells is not characterized. Here, we applied a high-resolution mass spectrometry-based workflow to characterize the proteomic landscape of mouse hindlimb skeletal muscle derived Sca-1+ cells. Furthermore, we characterized the impact of the cellular microenvironments on the proteomes of Sca-1+ cells. The proteome component of freshly isolated Sca-1+ cells (ex vivo) was compared with that of Sca-1+ cells expanded in cell culture (in vitro). The analysis revealed significant differences in the protein abundances in the two conditions reflective of their functional variations. The identified proteins were enriched in various biological pathways. Notably, we identified proteins related to myotube differentiation, myotube cell development and myoblast fusion. We also identified a panel of cell surface marker proteins that can be leveraged in future to enrich Sca-1+ cells using combinatorial strategies. Comparative analysis implicated the activation of various pathways leading to increased protein synthesis under in vitro condition. We report here the most comprehensive proteome map of Sca-1+ cells that provides insights into the molecular networks operative in Sca-1+ cells. Importantly, through our work we generated the proteomic blueprint of protein abundances significantly altered in Sca-1+ cells under ex vivo and in vitro conditions. The curated data can also be visualized at .
    Keywords:  Mass spectrometry‐based proteomics; Regenerative stem cells; Stem cell antigen-1 (Sca-1); Stem cells proteomics
  6. Front Physiol. 2021 ;12 619710
      Skeletal muscle possesses remarkable plasticity that permits functional adaptations to a wide range of signals such as motor input, exercise, and disease. Small animal models have been pivotal in elucidating the molecular mechanisms regulating skeletal muscle adaptation and plasticity. However, these small animal models fail to accurately model human muscle disease resulting in poor clinical success of therapies. Here, we review the potential of in vitro three-dimensional tissue-engineered skeletal muscle models to study muscle function, plasticity, and disease. First, we discuss the generation and function of in vitro skeletal muscle models. We then discuss the genetic, neural, and hormonal factors regulating skeletal muscle fiber-type in vivo and the ability of current in vitro models to study muscle fiber-type regulation. We also evaluate the potential of these systems to be utilized in a patient-specific manner to accurately model and gain novel insights into diseases such as Duchenne muscular dystrophy (DMD) and volumetric muscle loss. We conclude with a discussion on future developments required for tissue-engineered skeletal muscle models to become more mature, biomimetic, and widely utilized for studying muscle physiology, disease, and clinical use.
    Keywords:  Duchenne Muscle dystrophy; disease modeling; fiber-type; innervation; myosin heavy chain; satellite cell; skeletal muscle; tissue engineering
  7. Front Cell Dev Biol. 2021 ;9 636498
      Cachexia is a complex wasting syndrome that overwhelmingly affects the majority of late-stage cancer patients. Additionally, there are currently no efficacious therapeutic agents to treat the muscle atrophy induced by the cancer. While several preclinical studies have investigated the molecular signals orchestrating cachexia, very little information exists pertaining to ovarian cancer and the associated cachexia. Work from our lab has recently demonstrated that the steroidal lactone Withaferin A (WFA) is capable of attenuating the atrophying effects of ovarian cancer in a preclinical mouse model. However, it remained to be determined whether WFA's effect was in response to its anti-tumorigenic properties, or if it was capable of targeting skeletal muscle directly. The purpose of this study was to uncover whether WFA was capable of regulating muscle mass under tumor-free and tumor-bearing conditions. Treatment with WFA led to an improvement in functional muscle strength and mass under tumor-bearing and naïve conditions. WFA and ovarian cancer were observed to act antagonistically upon critical skeletal muscle regulatory systems, notably myogenic progenitors and proteolytic degradation pathways. Our results demonstrated for the first time that, while WFA has anti-tumorigenic properties, it also exerts hypertrophying effects on skeletal muscle mass, suggesting that it could be an anti-cachectic agent in the settings of ovarian cancer.
    Keywords:  atrophy; cachexia; catabolism; ovary; satellite cells
  8. Autophagy. 2021 Mar 17.
      CREG1 (cellular repressor of E1A-stimulated genes 1) is involved in tissue homeostasis and influences macroautophagy/autophagy to protect cardiovascular function. However, the physiological and pathological role of CREG1 in the skeletal muscle is not clear. Here, we established a skeletal muscle-specific creg1 knockout mouse model (creg1;Ckm-Cre) by crossing the Creg1-floxed mice (Creg1fl/fl) with a transgenic line expressing Cre recombinase under the muscle-specific Ckm (creatine kinase, muscle) promoter. In creg1;Ckm-Cre mice, the exercise time to exhaustion and running distance were significantly reduced compared to Creg1fl/fl mice at the age of 9 months. In addition, the administration of recombinant (re)CREG1 protein improved the motor function of 9-month-old creg1;Ckm-Cre mice. Moreover, electron microscopy images of 9-month-old creg1;Ckm-Cre mice showed that the mitochondrial quality and quantity were abnormal and associated with increased levels of PINK1 (PTEN induced putative kinase 1) and PRKN/PARKIN (parkin RBR E3 ubiquitin protein ligase) but reduced levels of the mitochondrial proteins PTGS2/COX2, COX4I1/COX4, and TOMM20. These results suggested that CREG1 deficiency accelerated the induction of mitophagy in the skeletal muscle. Mechanistically, gain-and loss-of-function mutations of Creg1 altered mitochondrial morphology and function, impairing mitophagy in C2C12 cells. Furthermore, HSPD1/HSP60 (heat shock protein 1) (401-573 aa) interacted with CREG1 (130-220 aa) to antagonize the degradation of CREG1 and was involved in the regulation of mitophagy. To the best of our knowledge, this was the first time to demonstrate that CREG1 localized to the mitochondria and played an important role in mitophagy modulation that determined skeletal muscle wasting during the growth process or disease conditions.
    Keywords:  CREG1; HSPD1; mitochondria; mitophagy; skeletal muscle
  9. Aging (Albany NY). 2021 Mar 10. 13
      In this study, we investigated the beneficial effects of high endogenous levels of n-3 polyunsaturated fatty acids (PUFAs) on skeletal muscle repair and regeneration using a mouse cardiotoxin (CTX, 20 μM/200 μL) -induced gastrocnemius muscle injury model. Transgenic fat-1 mice expressing the Caenorhabditis elegans fat-1 gene, encoding n-3 fatty acid desaturase, showed higher n-3 PUFA levels and lower n-6/n-3 PUFA ratios in gastrocnemius muscle tissues. Hematoxylin and eosin and Masson's trichrome staining of gastrocnemius sections revealed increased muscle fiber size and reduced fibrosis in fat-1 mice on days 7 and 14 after CTX injections. Gastrocnemius muscle tissues from fat-1 mice showed reduced inflammatory responses and increased muscle fiber regeneration reflecting enhanced activation of satellite cells on day 3 after cardiotoxin injections. Gastrocnemius muscle tissues from cardiotoxin-treated fat-1 mice showed reduced levels of pro-apoptotic proteins (Caspase 3 and Bax) and increased levels of anti-apoptotic proteins (Bcl-2 and Survivin). Moreover, eicosapentaenoic acid (EPA) reduced the incidence of apoptosis among cardiotoxin-treated C2C12 mouse myoblasts. These findings demonstrate that higher endogenous n-3 PUFA levels in fat-1 mice enhances skeletal muscle repair and regeneration following cardiotoxin-induced injury.
    Keywords:  fat-1 mice; injury; n-3 PUFAs; skeletal muscle
  10. J Exp Biol. 2021 Mar 18. pii: jeb234237. [Epub ahead of print]224(Pt 6):
      Regular exercise induces a broad spectrum of adaptation reactions in a variety of tissues and organs. However, the respective mechanisms are incompletely understood. In the context of their analysis, animal model systems, specifically rodent treadmill running protocols, play an important role. However, few researchers have studied different aspects of adaptation, such as cardiorespiratory and skeletal muscle training effects, within one set of experiments. Here, we analyzed physiological adaptation to 10 weeks of regular, moderate-intensity, uphill treadmill running in mice, a widely used model for endurance exercise training. To study the effects of reactive oxygen species (ROS), which have been suggested to be major regulators of training adaptation, a subgroup of mice was treated with the ROS scavenger PDTC (pyrrolidine dithiocarbamate). We found that mass gain in mice that exercised under PDTC treatment lagged behind that of all other experimental groups. In addition, both exercise and PDTC significantly and additively decreased resting heart rate. Furthermore, there was a trend towards an enhanced proportion of type 2A skeletal muscle fibers and differential expression of metabolism-associated genes, indicating metabolic and functional adaptation of skeletal muscle fibers. By contrast, there were no effects on grip strength and relative mass of individual muscles, suggesting that our protocol of uphill running did not increase skeletal muscle hypertrophy and strength. Taken together, our data suggest that a standard protocol of moderate-intensity uphill running induces adaptation reactions at multiple levels, part of which might be modulated by ROS, but does not enhance skeletal muscle hypertrophy and force.
    Keywords:  Cardiorespiratory adaptation; Exercise; Metabolic adaptation; Reactive oxygen species
  11. Biosci Biotechnol Biochem. 2021 Mar 19. pii: zbab049. [Epub ahead of print]
      It has been reported that orange peel extract (OPE) and the four major polymethoxyflavones (PMFs) in OPE have a protective effect against downhill running (DR)-induced skeletal muscle inflammation. However, the mechanism is not well understood. We investigated the potential of OPE and PMF compounds for increasing anti-inflammatory cytokine levels. The plasma interleukin-1 receptor antagonist (IL-1RA) level was increased 1 and 8 h after OPE administration in rats. Nobiletin induced the secretion of IL-1RA from C2C12 myotubes. In the inflammatory state of skeletal muscle after DR, OPE administration reduced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) expression, NF-κB-DNA binding, and monocyte chemotactic protein-1 mRNA levels, but these effects were all abrogated by the intravenous administration of IL-1RA neutralizing antibody. These results indicated that OPE reduces skeletal muscle inflammatory state after DR via an increase in IL-1RA, and that IL-1 receptor signaling is important for skeletal muscle inflammation after DR.
    Keywords:  IL-1RA; downhill running; inflammation; nobiletin; skeletal muscle
  12. Front Cell Dev Biol. 2021 ;9 635063
      Tubular Aggregate Myopathy (TAM) is a hereditary ultra-rare muscle disorder characterized by muscle weakness and cramps or myasthenic features. Biopsies from TAM patients show the presence of tubular aggregates originated from sarcoplasmic reticulum due to altered Ca2+ homeostasis. TAM is caused by gain-of-function mutations in STIM1 or ORAI1, proteins responsible for Store-Operated-Calcium-Entry (SOCE), a pivotal mechanism in Ca2+ signaling. So far there is no cure for TAM and the mechanisms through which STIM1 or ORAI1 gene mutation lead to muscle dysfunction remain to be clarified. It has been established that post-natal myogenesis critically relies on Ca2+ influx through SOCE. To explore how Ca2+ homeostasis dysregulation associated with TAM impacts on muscle differentiation cascade, we here performed a functional characterization of myoblasts and myotubes deriving from patients carrying STIM1 L96V mutation by using fura-2 cytofluorimetry, high content imaging and real-time PCR. We demonstrated a higher resting Ca2+ concentration and an increased SOCE in STIM1 mutant compared with control, together with a compensatory down-regulation of genes involved in Ca2+ handling (RyR1, Atp2a1, Trpc1). Differentiating STIM1 L96V myoblasts persisted in a mononuclear state and the fewer multinucleated myotubes had distinct morphology and geometry of mitochondrial network compared to controls, indicating a defect in the late differentiation phase. The alteration in myogenic pathway was confirmed by gene expression analysis regarding early (Myf5, Mef2D) and late (DMD, Tnnt3) differentiation markers together with mitochondrial markers (IDH3A, OGDH). We provided evidences of mechanisms responsible for a defective myogenesis associated to TAM mutant and validated a reliable cellular model usefull for TAM preclinical studies.
    Keywords:  STIM1; calcium homeostasis; high content imaging; myogenesis (in vitro); tubular aggregate myopathy
  13. Commun Biol. 2021 Mar 19. 4(1): 355
      Mechanical stress induced by contractions constantly threatens the integrity of muscle Z-disc, a crucial force-bearing structure in striated muscle. The PDZ-LIM proteins have been proposed to function as adaptors in transducing mechanical signals to preserve the Z-disc structure, however the underlying mechanisms remain poorly understood. Here, we show that LDB3, a well-characterized striated muscle PDZ-LIM protein, modulates mechanical stress signaling through interactions with the mechanosensing domain in filamin C, its chaperone HSPA8, and PKCα in the Z-disc of skeletal muscle. Studies of Ldb3Ala165Val/+ mice indicate that the myopathy-associated LDB3 p.Ala165Val mutation triggers early aggregation of filamin C and its chaperones at muscle Z-disc before aggregation of the mutant protein. The mutation causes protein aggregation and eventually Z-disc myofibrillar disruption by impairing PKCα and TSC2-mTOR, two important signaling pathways regulating protein stability and disposal of damaged cytoskeletal components at a major mechanosensor hub in the Z-disc of skeletal muscle.
  14. Dent Mater J. 2021 Mar 13.
      In the present study, we investigated the possible involvement of the TGF-β/Smad signaling pathway in the osteopontin-derived SVVYGLR (SV) peptide-mediated migratory activities of myogenic cells and evaluated the facilitative effects of the SV peptide on the differentiation of myogenic cells in vitro. The SV peptide-induced migration in both human-derived satellite cells and myoblasts was substantially suppressed by the TGF-β1 receptor inhibitor SB431542 or SB505124. Besides, the expression level of the Smad3 phosphorylation was further enhanced by the addition of the SV peptide in comparison with control groups. Furthermore, an increase in the expression of myogenin-positive nuclei and a higher number of nascent myotubes with myosin heavy chain expression was confirmed in cultured myoblasts supplemented with the SV peptide. These results suggest that the involvement of the TGF-β/Smad signaling pathway in the SV peptide-mediated migration and the facilitative effect of the SV peptide on the differentiation of myogenic cells into myotubes.
    Keywords:  Myotube; Osteopontin; SVVYGLR; Skeletal muscle; TGF-β
  15. FASEB J. 2021 Apr;35(4): e21462
      Muscle may contribute to the systemic inflammatory environment during critical illness, but leukocyte interaction and cytokine influence on muscle and its response has not been fully explored in this context. Using an in vivo model of intratracheal lipopolysaccharide (IT LPS)-induced acute lung injury, we show that skeletal muscle rapidly responds with expression of proinflammatory genes, which may be explained by migration of LPS into the circulation. Treatment of mature C2C12 myotubes with LPS at a level achieved in the circulation following IT LPS elicited a proinflammatory cytokine expression profile similar to that of in vivo murine muscle following IT LPS. Stimulation with toll-like receptor (TLR) 2 and 3 agonists provoked comparable responses in C2C12 myotubes. Additionally, co-cultures of C2C12 myotubes and bone marrow-derived macrophages (BMDM) identified the capacity of macrophages to increase myotube proinflammatory gene expression, with tumor necrosis factor-α (TNFα) gene and protein expression largely attributable to BMDM. To investigate the contribution of TNFα in the synergy of the co-culture environment, C2C12 myotubes were treated with recombinant TNFα, co-cultures were established using TNF-deficient BMDM, and co-cultures were also depleted of TNFα using antibodies. To determine whether the in vitro observations were relevant in vivo, mice received intramuscular administration of LPS ± TNFα or TNFα-neutralizing antibodies and showed that TNFα is both sufficient and necessary to induce synergistic cytokine release from muscle. Taken together, these data demonstrate how skeletal muscle tissue may contribute proinflammatory cytokines following acute endotoxin injury and the potential of leukocytes to augment this response via TNFα secretion.
    Keywords:  acute lung injury; cytokines; inflammation; macrophage; skeletal muscle
  16. J Extracell Vesicles. 2021 Mar;10(5): e12060
      Cachexia, characterized by loss of skeletal muscle mass and function, is estimated to inflict the majority of patients with oesophageal squamous cell carcinoma (ESCC) and associated with their poor prognosis. However, its underlying mechanisms remain elusive. Here, we developed an ESCC-induced cachexia mouse model using human xenograft ESCC cell lines and found that ESCC-derived extracellular vesicles (EVs) containing prolyl 4-hydroxylase subunit beta (P4HB) induced apoptosis of skeletal muscle cells. We further identified that P4HB promoted apoptotic response through activating ubiquitin-dependent proteolytic pathway and regulated the stability of phosphoglycerate dehydrogenase (PHGDH) and subsequent antiapoptotic protein Bcl-2. Additionally, we proved that the P4HB inhibitor, CCF642, not only rescued apoptosis of muscle cells in vitro, but also prevented body weight loss and muscle wasting in ESCC-induced cachexia mouse model. Overall, these findings demonstrate a novel pathway for ESCC-induced muscle wasting and advocate for the development of P4HB as a potential intervention target for cachexia in patients with ESCC.
    Keywords:  apoptosis; cachexia; extracellular vesicles; inhibitor; muscle wasting; oesophageal cancer
  17. Skelet Muscle. 2021 Mar 17. 11(1): 7
      Satellite cells (SCs) are muscle stem cells capable of regenerating injured muscle. The study of their functional potential depends on the availability of methods for the isolation and expansion of pure SCs with preserved myogenic properties after serial passages in vitro. Here, we describe the ice-cold treatment (ICT) method, which is a simple, economical, and efficient method for the isolation and in vitro expansion of highly pure mouse and human SCs. It involves a brief (15-30 min) incubation on ice (0 °C) of a dish containing a heterogeneous mix of adherent muscle mononuclear cells, which leads to the detachment of only the SCs, and gives rise to cultures of superior purity compared to other commonly used isolation methods. The ICT method doubles up as a gentle passaging technique, allowing SC expansion over extended periods of time without compromising their proliferation and differentiation potential. Moreover, SCs isolated and expanded using the ICT method are capable of regenerating injured muscle in vivo. The ICT method involves minimal cell manipulation, does not require any expertise or expensive reagents, it is fast, and highly reproducible, and greatly reduces the number of animals or human biopsies required in order to obtain sufficient number of SCs. The cost-effectiveness, accessibility, and technical simplicity of this method, as well as its remarkable efficiency, will no doubt accelerate SC basic and translational research bringing their therapeutic use closer to the clinic.
    Keywords:  Satellite cell isolation; Satellite cells in vitro expansion; Skeletal muscle regeneration
  18. J Biochem. 2021 Mar 17. pii: mvab030. [Epub ahead of print]
      Although skeletal muscle cells and adipocytes are derived from the same mesoderm, they do not transdifferentiate in vivo and are strictly distinct at the level of gene expression. To elucidate some of the regulatory mechanisms underlying this strict distinction, Pax7, a myogenic factor, was ectopically expressed in 3T3-L1 adipose progenitor cells to perturb their adipocyte differentiation potential. Transcriptome analysis showed that ectopic expression of Pax7 repressed the expression of some adipocyte genes and induced expression of some skeletal muscle cell genes. We next profiled the epigenomic state altered by Pax7 expression using H3K27ac, an activating histone mark, and H3K27me3, a repressive histone mark, as indicators. Our results show that ectopic expression of Pax7 did not result in the formation of H3K27ac at loci of skeletal muscle-related genes, but instead resulted in the formation of H3K27me3 at adipocyte-related gene loci. These findings suggest that the primary function of ectopic Pax7 expression is the formation of H3K27me3, and muscle gene expression results from secondary regulation.
    Keywords:  Adipogenesis; Epigenome; Myogenesis; Transcription Factor; Transcriptome
  19. Med Sci Sports Exerc. 2021 Feb 19.
      PURPOSE: To determine the role of mammalian target of rapamycin (mTORC1) activation and catabolic markers in resistance training's (RT) anti-atrophy effect during cachexia-induced muscle loss.METHODS: Myofiber atrophy was induced by injecting Walker 256 tumor cells into rats exposed or not exposed to the RT protocol of ladder climbing. The role of RT-induced anabolic stimulation was investigated in tumor-bearing rats with the mTORC1 inhibitor rapamycin, and cross-sectional areas of skeletal muscle were evaluated to identify atrophy or hypertrophy. Components of the mTORC1 and ubiquitin-proteasome pathways were assessed by real-time PCR or immunoblotting.
    RESULTS: While RT prevented myofiber atrophy and impaired the strength of tumor-bearing rats, in healthy rats it promoted activated mTORC1, as demonstrated by p70S6K's increased phosphorylation and myofiber's enlarged cross-sectional area. However, RT promoted no changes in the ratio of p70S6K to phospho-p70S6K protein expression while prevented myofiber atrophy in tumor-bearing rats. Beyond that, treatment with rapamycin did not preclude RT's preventive effect on myofiber atrophy in tumor-bearing rats. Thus, RT's ability to prevent cancer-induced myofiber atrophy seems to be independent of mTORC1's and p70S6K's activation. Indeed, RT's preventive effect on cancer-induced myofiber atrophy was associated with its capacity to attenuate elevated TNF-α and IL-6 as well as to prevent oxidative damage in muscles and an elevated abundance of atrogin-1.
    CONCLUSION: By inducing attenuated myofiber atrophy independent of mTORC1's signaling activation, RT prevents muscle atrophy during cancer by reducing inflammation, oxidative damage, and atrogin-1 expression.
  20. Immunometabolism. 2021 ;pii: e210013. [Epub ahead of print]3(2):
      In this commentary we discuss new findings presented by Shang et al. regarding the role of macrophage-derived glutamine in skeletal muscle repair. Loss-of-function of glutamate dehydrogenase in macrophages led to an upregulation of glutamine synthesis which sustained glutamine levels in muscle tissue and facilitated satellite cell proliferation and differentiation.
    Keywords:  amino acids; inflammation; macrophage metabolism; muscle repair; satellite cells
  21. Am J Physiol Cell Physiol. 2021 Mar 17.
      The dystrophin-glycoprotein complex (DGC) is a multi-protein structure required to maintain muscle fiber membrane integrity, transmit force by linking the actin cytoskeleton with the extracellular matrix, and maintain muscle homeostasis. Membrane localization of dystrophin is perturbed in muscles wasting as a consequence of cancer cachexia, tenotomy and advanced ageing, which are all associated with low level, chronic inflammation. Strategies to preserve dystrophin expression at the sarcolemma might therefore combat muscle wasting. Phosphorylation of dystrophin serine 3059 (S3059) enhances the interaction between dystrophin and β-dystroglycan. To test the contribution of amino acid phosphorylation to muscle fiber size changes, dystrophin constructs with phospho-null and phosphomimetic mutations were transfected into C2C12 muscle cells or AAV-293 cells in the presence or absence of kinase inhibitors/activators to assess effects on myotube diameter and protein function. Over-expression of a dystrophin construct with a phospho-null mutation at S3059 in vitro reduced myotube size in healthy C2C12 cells. Conversely over-expression of a phosphomimetic mutation at S3059 attenuated inflammation-induced myotube atrophy. Increased ERK activation by addition of phorbol myristate acetate (PMA) also reduced inflammation-associated myotube atrophy, and increased the interaction between dystrophin and β-dystroglycan, which was partially attenuated in the presence of a phosphomimetic mutation at dystrophin S3059. These findings demonstrate a link between increased ERK activation, dystrophin S3059 phosphorylation, stabilization of the DGC, and the regulation of muscle fiber size. Interventions that increase dystrophin S3059 phosphorylation to promote stronger binding of dystrophin to β-dystroglycan may have therapeutic potential for attenuation of inflammation-associated muscle wasting.
    Keywords:  dystrophin; inflammation; muscle atrophy; phosphorylation
  22. Dev Cell. 2021 Mar 10. pii: S1534-5807(21)00165-9. [Epub ahead of print]
      Negative elongation factor (NELF) is a critical transcriptional regulator that stabilizes paused RNA polymerase to permit rapid gene expression changes in response to environmental cues. Although NELF is essential for embryonic development, its role in adult stem cells remains unclear. In this study, through a muscle-stem-cell-specific deletion, we showed that NELF is required for efficient muscle regeneration and stem cell pool replenishment. In mechanistic studies using PRO-seq, single-cell trajectory analyses and myofiber cultures revealed that NELF works at a specific stage of regeneration whereby it modulates p53 signaling to permit massive expansion of muscle progenitors. Strikingly, transplantation experiments indicated that these progenitors are also necessary for stem cell pool repopulation, implying that they are able to return to quiescence. Thus, we identified a critical role for NELF in the expansion of muscle progenitors in response to injury and revealed that progenitors returning to quiescence are major contributors to the stem cell pool repopulation.
    Keywords:  NELF; PEDF signaling; muscle regeneration; muscle stem cells; nascent transcript stability; p53 signaling; promoter proximal pausing; stem cell niche; stem cell self-renewal; transcriptional regulation
  23. Front Physiol. 2021 ;12 633058
      Sarcolipin (SLN) is a regulator of sarco/endo plasmic reticulum Ca2+-ATPase (SERCA) pump and has been shown to be involved in muscle nonshivering thermogenesis (NST) and energy metabolism. Interestingly, SLN expression is significantly upregulated both during muscle development and in several disease states. However, the significance of altered SLN expression in muscle patho-physiology is not completely understood. We have previously shown that transgenic over-expression of SLN in skeletal muscle is not detrimental, and can promote oxidative metabolism and exercise capacity. In contrast, some studies have suggested that SLN upregulation in disease states is deleterious for muscle function and ablation of SLN can be beneficial. In this perspective article, we critically examine both published and some new data to determine the relevance of SLN expression to disease pathology. The new data presented in this paper show that SLN levels are induced in muscle during systemic bacterial (Salmonella) infection or lipopolysaccharides (LPS) treatment. We also present data showing that SLN expression is significantly upregulated in different types of muscular dystrophies including myotubular myopathy. These data taken together reveal that upregulation of SLN expression in muscle disease is progressive and increases with severity. Therefore, we suggest that increased SLN expression should not be viewed as the cause of the disease; rather, it is a compensatory response to meet the higher energy demand of the muscle. We interpret that higher SLN/SERCA ratio positively modulate cytosolic Ca2+ signaling pathways to promote mitochondrial biogenesis and oxidative metabolism to meet higher energy demand in muscle.
    Keywords:  Ca2+-handling proteins; muscle disease; sarco/endo plasmic reticulum Ca2+ ATPase; sarcolipin; skeletal muscle
  24. FASEB J. 2021 Apr;35(4): e21223
      The role of Sidt2 in the process of glucose and lipid metabolism has been recently reported. However, whether Sidt2 is involved in the metabolic regulation in skeletal muscle remains unknown. In this study, for the first time, using skeletal muscle-selective Sidt2 knockout mice, we found that Sidt2 was vital for the quality control of mitochondria in mouse skeletal muscle. These mice showed significantly reduced muscle tolerance and structurally abnormal mitochondria. Deletion of the Sidt2 gene resulted in decreased expression of mitochondrial fusion protein 2 (Mfn2) and Dynamin-related protein 1 (Drp1), as well as peroxisome proliferator-activated receptor γ coactivator-1 (PGC1-α). In addition, the clearance of damaged mitochondria in skeletal muscle was inhibited upon Sidt2 deletion, which was caused by blockade of autophagy flow. Mechanistically, the fusion of autophagosomes and lysosomes was compromised in Sidt2 knockout skeletal muscle cells. In summary, the deletion of the Sidt2 gene not only interfered with the quality control of mitochondria, but also inhibited the clearance of mitochondria and caused the accumulation of a large number of damaged mitochondria, ultimately leading to the abnormal structure and function of skeletal muscle.
    Keywords:  Sidt2; autophagy; mitochondria; myopathy; quality control
  25. FASEB J. 2021 Apr;35(4): e21489
      Psychosocial stressors can cause physical inactivity, cardiac damage, and hypotension-induced death in the mdx mouse model of Duchenne muscular dystrophy (DMD). Because repeated exposure to mild stress can lead to habituation in wild-type mice, we investigated the response of mdx mice to a mild, daily stress to determine whether habituation occurred. Male mdx mice were exposed to a 30-sec scruff restraint daily for 12 weeks. Scruff restraint induced immediate physical inactivity that persisted for at least 60 minutes, and this inactivity response was just as robust after 12 weeks as it was after one day. Physical inactivity in the mdx mice was not associated with acute skeletal muscle contractile dysfunction. However, skeletal muscle of mdx mice that were repeatedly stressed had slow-twitch and tetanic relaxation times and trended toward high passive stiffness, possibly due to a small but significant increase in muscle fibrosis. Elevated urinary corticosterone secretion, adrenal hypertrophy, and a larger adrenal cortex indicating chronic activation of the hypothalamic-pituitary-adrenal (HPA) axis were measured in 12-week stressed mdx mice relative to those unstressed. However, pharmacological inhibition of the HPA axis did not affect scruff-induced physical inactivity and acute corticosterone injection did not recapitulate the scruff-induced phenotype, suggesting the HPA axis is not the driver of physical inactivity. Our results indicate that the response of mdx mice to an acute mild stress is non-habituating and that when that stressor is repeated daily for weeks, it is sufficient to exacerbate some phenotypes associated with dystrophinopathy in mdx mice.
    Keywords:  Duchenne muscular dystrophy; Dystrophin; fibrosis; hypothalamic-pituitary-adrenal axis; skeletal muscle
  26. Stem Cell Res Ther. 2021 Mar 16. 12(1): 183
      BACKGROUND: Peripheral arterial disease (PAD) affects millions of people and compromises quality of life. Critical limb ischemia (CLI), which is the most advanced stage of PAD, can cause nonhealing ulcers and strong chronic pain, and it shortens the patients' life expectancy. Cell-based angiogenic therapies are becoming a real therapeutic approach to treat CLI. Pericytes are cells that surround vascular endothelial cells to reinforce vessel integrity and regulate local blood pressure and metabolism. In the past decade, researchers also found that pericytes may function as stem or progenitor cells in the body, showing the potential to differentiate into several cell types. We investigated the gene expression profiles of pericytes during the early stages of limb ischemia, as well as the alterations in pericyte subpopulations to better understand the behavior of pericytes under ischemic conditions.METHODS: In this study, we used a hindlimb ischemia model to mimic CLI in C57/BL6 mice and explore the role of pericytes in regeneration. To this end, muscle pericytes were isolated at different time points after the induction of ischemia. The phenotypes and transcriptomic profiles of the pericytes isolated at these discrete time points were assessed using flow cytometry and RNA sequencing.
    RESULTS: Ischemia triggered proliferation and migration and upregulated the expression of myogenesis-related transcripts in pericytes. Furthermore, the transcriptomic analysis also revealed that pericytes induce or upregulate the expression of a number of cytokines with effects on endothelial cells, leukocyte chemoattraction, or the activation of inflammatory cells.
    CONCLUSIONS: Our findings provide a database that will improve our understanding of skeletal muscle pericyte biology under ischemic conditions, which may be useful for the development of novel pericyte-based cell and gene therapies.
    Keywords:  Limb ischemia; Muscle; Pericytes; Peripheral arterial disease; RNA-seq
  27. Mol Ther Nucleic Acids. 2021 Jun 04. 24 67-78
      Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant disease that results from an alanine expansion in the N-terminal domain of Poly-A Binding Protein Nuclear-1 (PABPN1). We have recently demonstrated that a two-vector gene therapy strategy significantly ameliorated the pathology in a mouse model of OPMD. This approach entailed intramuscular injection of two recombinant adeno-associated viruses (AAVs), one expressing three short hairpin RNAs (shRNAs) to silence both mutant and wild-type PABPN1 and one expressing a codon-optimized version of PABPN1 that is insensitive to RNA interference. Here we report the continued development of this therapeutic strategy by delivering "silence and replace" sequences in a single AAV vector named BB-301. This construct is composed of a modified AAV serotype 9 (AAV9) capsid that expresses a unique single bifunctional construct under the control of the muscle-specific Spc5-12 promoter for the co-expression of both the codon-optimized PABPN1 protein and two small inhibitory RNAs (siRNAs) against PABPN1 modeled into microRNA (miRNA) backbones. A single intramuscular injection of BB-301 results in robust inhibition of mutant PABPN1 and concomitant replacement of the codon-optimized PABPN1 protein. The treatment restores muscle strength and muscle weight to wild-type levels as well as improving other physiological hallmarks of the disease in a mouse model of OPMD.
    Keywords:  AAV; OPMD; PABPN1; gene therapy; oculopharyngeal muscular dystrophy; siRNA; silence and replace
  28. Med Sci Sports Exerc. 2021 Mar 12.
      PURPOSE: We aimed to investigate the hypothesis that type I collagen plays a role in increasing bone mineral density (BMD) and muscle stiffness, leading to low and high risks of fatigue fracture and muscle injury, respectively, in athletes. As a potential mechanism, we focused on the effect of the type I collagen alpha 1 chain gene (COL1A1) variant associated with transcriptional activity on bone and skeletal muscle properties.METHODS: The association between COL1A1 rs1107946 and fatigue fracture/muscle injury was evaluated in Japanese athletes. Effects of the polymorphism on tissue properties (BMD and muscle stiffness) and type I collagen α1/α2 chain ratios in muscles were examined in Japanese non-athletes.
    RESULTS: The C allele carrier frequency was greater in female athletes with fatigue fracture than in those without (odds ratio [OR]: 2.44, 95% confidence interval [CI]: 1.17-5.77) and lower in female athletes with muscle injury than in those without (OR: 0.46, 95% CI: 0.24-0.91). Prospective validation analysis confirmed that in female athletes, muscle injury was less frequent in C allele carriers than in AA genotype carriers (Multivariable adjusted hazard ratio: 0.27, 95% CI: 0.08-0.96). Among female non-athletes, the C allele of rs1107946 was associated with lower BMD and lower muscle stiffness. Muscle biopsy revealed that C allele carriers tended to have a larger type I collagen α1/α2 chain ratio than AA genotype carriers (2.24 vs. 2.05, P = 0.056), suggesting a higher proportion of type I collagen α1 homotrimers.
    CONCLUSION: The COL1A1 rs1107946 polymorphism exerts antagonistic effects on fatigue fracture and muscle injury among female athletes by altering the properties of these tissues, potentially owing to increased levels of type I collagen α1 chain homotrimers.
  29. Front Cell Dev Biol. 2021 ;9 640399
      Glucose is a major energy source consumed by proliferating mammalian cells. Therefore, in general, proliferating cells have the preference of high glucose contents in extracellular environment. Here, we showed that high glucose concentrations impede the proliferation of satellite cells, which are muscle-specific stem cells, under adherent culture conditions. We found that the proliferation activity of satellite cells was higher in glucose-free DMEM growth medium (low-glucose medium with a glucose concentration of 2 mM) than in standard glucose DMEM (high-glucose medium with a glucose concentration of 19 mM). Satellite cells cultured in the high-glucose medium showed a decreased population of reserve cells, identified by staining for Pax7 expression, suggesting that glucose concentration affects cell fate determination. In conclusion, glucose is a factor that decides the cell fate of skeletal muscle-specific stem cells. Due to this unique feature of satellite cells, hyperglycemia may negatively affect the regenerative capability of skeletal muscle myofibers and thus facilitate sarcopenia.
    Keywords:  glucose; primary culture; proliferation; satellite cell; self-renewal
  30. Life Sci. 2021 Mar 11. pii: S0024-3205(21)00325-8. [Epub ahead of print] 119340
      AIMS: Hypoxic training promotes human cardiopulmonary function and exercise performance efficiently, but the myocellular mechanism has been less studied. We aimed to examine the effects of hypoxic trainings on mitochondrial turnover and vascular remodeling of skeletal muscle.MAIN METHODS: C57BL/6 J mice were divided into control, hypoxic exposure, exercise training, "live high-train low" (LHTL), and "live low-train high" (LLTH) groups (n = 8/group). Western blot and immunohistochemistry were used to evaluate mitochondrial turnover of gastrocnemius and angiogenesis of quadriceps after six weeks interventions.
    KEY FINDINGS: Compared with control group, both LHTL and LLTH increased phosphorylation levels of p38 MAPK markedly (p < 0.05). LLTH also elevated PGC-1α protein expression significantly (p < 0.05). All interventions did not influence Bnip3 and Drp-1 proteins levels (p > 0.05), while LLTH enhanced Parkin and Mff protein contents significantly (p < 0.05). Immunohistochemical analysis showed both LHTL and LLTH promoted CD31 and VEGF expressions (p < 0.05). ATP content, citrate synthase activities of gastrocnemius were robustly elevated in LHTL and LLTH groups (p < 0.01). The exercise training increased Mff protein and ATP content in gastrocnemius as well as VEGF expression in quadriceps (p < 0.05). The hypoxic exposure also increased ATP content, citrate synthase, and ATP synthase activities in gastrocnemius as well as VEGF expression in quadriceps (p < 0.01).
    SIGNIFICANCE: Our results suggested that hypoxic trainings, especially LLTH, promoted mitochondrial turnover and angiogenesis of skeletal muscle, which may be an underlying mechanism of hypoxic training-induced exercise capacity.
    Keywords:  Exercise; Hypoxic training; Mitochondrial biogenesis; Mitophagy
  31. FASEB J. 2021 Apr;35(4): e21346
      Dynamin 2 (DNM2) is a ubiquitously expressed protein involved in many functions related to trafficking and remodeling of membranes and cytoskeleton dynamics. Mutations in the DNM2 gene cause the autosomal dominant centronuclear myopathy (AD-CNM), characterized mainly by muscle weakness and central nuclei. Several defects have been identified in the KI-Dnm2R465W/+ mouse model of the disease to explain the muscle phenotype, including reduction of the satellite cell pool in muscle, but the functional consequences of this depletion have not been characterized until now. Satellite cells (SC) are the main source for muscle growth and regeneration of mature tissue. Here, we investigated muscle regeneration in the KI-Dnm2R465W/+ mouse model for AD-CNM. We found a reduced number of Pax7-positive SCs, which were also less activated after induced muscle injury. The muscles of the KI-Dnm2R465W/+ mouse regenerated more slowly and less efficiently than wild-type ones, formed fewer new myofibers, and did not recover its normal mass 15 days after injury. Altogether, our data provide evidence that the muscle regeneration is impaired in the KI-Dnm2R465W/+ mouse and contribute with one more layer to the comprehension of the disease, by identifying a new pathomechanism linked to DNM2 mutations which may be involved in the muscle-specific impact occurring in AD-CNM.
    Keywords:  GTPase; congenital; injury; muscle; stem cell
  32. Sci Adv. 2021 Mar;pii: eabf7412. [Epub ahead of print]7(12):
      Understanding the mechanisms of myogenesis in human induced pluripotent stem cells (hiPSCs) is a prerequisite to achieving patient-specific therapy for diseases of skeletal muscle. hiPSCs of different origin show distinctive kinetics and ability to differentiate into myocytes. To address the unique cellular and temporal context of hiPSC differentiation, we perform a longitudinal comparison of the transcriptomic profiles of three hiPSC lines that display differential myogenic specification, one robust and two blunted. We detail temporal differences in mechanisms that lead to robust myogenic specification. We show gene expression signatures of putative cell subpopulations and extracellular matrix components that may support myogenesis. Furthermore, we show that targeted knockdown of ZIC3 at the outset of differentiation leads to improved myogenic specification in blunted hiPSC lines. Our study suggests that β-catenin transcriptional cofactors mediate cross-talk between multiple cellular processes and exogenous cues to facilitate specification of hiPSCs to mesoderm lineage, leading to robust myogenesis.
  33. Exp Mol Med. 2021 Mar 17.
      Cancer cachexia is a highly debilitating condition characterized by weight loss and muscle wasting that contributes significantly to the morbidity and mortality of pancreatic cancer. The factors that induce cachexia in pancreatic cancer are largely unknown. We previously showed that pancreatic adenocarcinoma upregulated factor (PAUF) secreted by pancreatic cancer cells is responsible for tumor growth and metastasis. Here, we analyzed the relation between pancreatic cancer-derived PAUF and cancer cachexia in mice and its clinical significance. Body weight loss and muscle weight loss were significantly higher in mice with Panc-1/PAUF tumors than in those with Panc-1/Mock tumors. Direct administration of rPAUF to muscle recapitulated tumor-induced atrophy, and a PAUF-neutralizing antibody abrogated tumor-induced muscle wasting in Panc-1/PAUF tumor-bearing mice. C2C12 myotubes treated with rPAUF exhibited rapid inactivation of Akt-Foxo3a signaling, resulting in Atrogin1/MAFbx upregulation, myosin heavy chain loss, and muscle atrophy. The neutrophil-to-lymphocyte ratio and body weight loss were significantly higher in pancreatic cancer patients with high PAUF expression than in those with low PAUF expression. Analysis of different pancreatic cancer datasets showed that PAUF expression was significantly higher in the pancreatic cancer group than in the nontumor group. Analysis of The Cancer Genome Atlas data found associations between high PAUF expression or a high DNA copy number and poor overall survival. Our data identified tumor-secreted circulating PAUF as a key factor of cachexia, causing muscle wasting in mice. Neutralizing PAUF may be a useful therapeutic strategy for the treatment of pancreatic cancer-induced cachexia.
  34. Clin Transl Sci. 2021 Mar 20.
      Mechanical ventilation (MV) is a life-saving instrument used to provide ventilatory support for critically ill patients and patients undergoing surgery. Unfortunately, an unintended consequence of prolonged MV is the development of inspiratory weakness due to both diaphragmatic atrophy and contractile dysfunction; this syndrome is labeled ventilator-induced diaphragm dysfunction (VIDD). VIDD is clinically important because diaphragmatic weakness is an important contributor to problems in weaning patients from MV. Investigations into the pathogenesis of VIDD reveal that oxidative stress is essential for the rapid development of VIDD as redox disturbances in diaphragm fibers promote accelerated proteolysis. Currently, no standard treatment exists to prevent VIDD and therefore, developing a strategy to avert VIDD is vital. Guided by evidence indicating that activation of the classical axis of the renin-angiotensin system (RAS) in diaphragm fibers promotes oxidative stress and VIDD, we hypothesized that activation of the non-classical RAS signaling pathway via angiotensin1-7 (Ang1-7) will protect against VIDD. Using an established animal model of prolonged MV, our results disclose that infusion of Ang1-7 protects the diaphragm against MV-induced contractile dysfunction and fiber atrophy in both fast and slow muscle fibers. Further, Ang1-7 shielded diaphragm fibers against MV-induced mitochondrial damage, oxidative stress, and protease activation. Collectively, these results reveal that treatment with Ang1-7 protects against VIDD, in part, due to diminishing oxidative stress and protease activation. These important findings provide robust evidence that Ang 1-7 has the therapeutic potential to protect against VIDD by preventing MV-induced contractile dysfunction and atrophy of both slow and fast muscle fibers.
    Keywords:  mechanical ventilation; mitochondrial dysfunction; oxidative stress; proteolysis; renin-angiotensin system; respiratory muscles; skeletal muscles
  35. J Cell Physiol. 2021 Mar 19.
      Fatty acids (FA) exert physiological and pathophysiological effects leading to changes in skeletal muscle metabolism and function, however, in vitro models to investigate these changes are limited. These experiments sought to establish the effects of physiological and pathophysiological concentrations of exogenous FA upon the function of tissue engineered skeletal muscle (TESkM). Cultured initially for 14 days, C2C12 TESkM was exposed to FA-free bovine serum albumin alone or conjugated to a FA mixture (oleic, palmitic, linoleic, and α-linoleic acids [OPLA] [ratio 45:30:24:1%]) at different concentrations (200 or 800 µM) for an additional 4 days. Subsequently, TESkM morphology, functional capacity, gene expression and insulin signaling were analyzed. There was a dose response increase in the number and size of lipid droplets within the TESkM (p < .05). Exposure to exogenous FA increased the messenger RNA expression of genes involved in lipid storage (perilipin 2 [p < .05]) and metabolism (pyruvate dehydrogenase lipoamide kinase isozyme 4 [p < .01]) in a dose dependent manner. TESkM force production was reduced (tetanic and single twitch) (p < .05) and increases in transcription of type I slow twitch fiber isoform, myosin heavy chain 7, were observed when cultured with 200 µM OPLA compared to control (p < .01). Four days of OPLA exposure results in lipid accumulation in TESkM which in turn results in changes in muscle function and metabolism; thus, providing insight ito the functional and mechanistic changes of TESkM in response to exogenous FA.
    Keywords:  contraction; insulin sensitivity; lipids
  36. Geroscience. 2021 Mar 19.
      Mitochondrial DNA (mtDNA) quality and quantity relate to two hallmarks of aging-genomic instability and mitochondrial dysfunction. Physical performance relies on mitochondrial integrity and declines with age, yet the interactions between mtDNA quantity, quality, and physical performance are unclear. Using a validated digital PCR assay specific for mtDNA deletions, we tested the hypothesis that skeletal muscle mtDNA deletion mutation frequency (i.e., a measure of mtDNA quality) or mtDNA copy number predicts physical performance in older adults. Total DNA was isolated from vastus lateralis muscle biopsies and used to quantitate mtDNA copy number and mtDNA deletion frequency by digital PCR. The biopsies were obtained from a cross-sectional cohort of 53 adults aged 50 to 86 years. Before the biopsy procedure, physical performance measurements were collected, including VO2max, modified physical performance test score, 6-min walk distance, gait speed, grip strength, and total lean and leg mass. Linear regression models were used to evaluate the relationships between age, sex, and the outcomes. We found that mtDNA deletion mutation frequency increased exponentially with advancing age. On average from ages 50 to 86, deletion frequency increased from 0.008 to 0.15%, an 18-fold increase. Females may have lower deletion frequencies than males at older ages. We also measured declines in VO2max and mtDNA copy number with age in both sexes. The mtDNA deletion frequency measured from single skeletal muscle biopsies predicted 13.3% of the variation in VO2max. Copy number explained 22.6% of the variation in mtDNA deletion frequency and 10.4% of the lean mass variation. We found predictive relationships between age, mtDNA deletion mutation frequency, mtDNA copy number, and physical performance. These data are consistent with a role for mitochondrial function and genome integrity in maintaining physical performance with age. Analyses of mtDNA quality and quantity in larger cohorts and longitudinal studies could extend our understanding of the importance of mitochondrial DNA in human aging and longevity.
    Keywords:  Aging; Mitochondria; Mitochondrial DNA; Mutation; Physical performance; Skeletal muscle
  37. Med Sci Sports Exerc. 2021 Feb 18.
      INTRODUCTION: Exercise-induced microRNAs (miRNAs) expression has been implicated in the regulation of skeletal muscle plasticity. However, the specificity and acute time course in miRNA expression following divergent exercise modes are unknown. In a randomized cross-over design, we compared the acute expression profile of eight skeletal muscle miRNAs previously reported to be involved in skeletal muscle development, growth and maintenance following a bout of either resistance exercise (RE), high intensity interval exercise (HIIE) and concurrent resistance and high intensity interval exercises (CE).METHODS: Nine untrained young men (23.9±2.8y, 70.1±14.9kg, 177.2±3.0cm, 41.4±5.2ml·kg-1·min-1) underwent a counter-balanced cross-over design in which they performed bouts of RE (2x10 repetitions maximum 45°Leg Press and Leg Extension exercises), HIEE (12x1 min sprints at VO2peak with 1min rest intervals between sprints) and CE (RE followed by HIIE), separated by one week. Vastus lateralis biopsies were harvested immediately before (Pre), and immediately (0h), 4h and 8h after each exercise bout.
    RESULTS: There were similar increases (main effect of time; P<0.05) in miR-1-3p,-133a-3p,-133b, -181a-3p, and -486 expression at 8h from Pre with all exercise modes. Besides a main effect of time, miR-23a-3p and -206 presented a main effect of condition with lower expression after HIIE compared to RE and CE.
    CONCLUSIONS: Select miRNAs (miR-1-3p, -133a-3p,-133b,-23a-3p,-181a-3p,-206,-486) do not exhibit an expression specificity in the acute recovery period following a single bout of either RE, HIIE or CE in skeletal muscle. Our data also indicate that RE has a higher effect on the expression of miR-23a-3p and -206 than HIIE. As upregulation of these miRNAs appears to be confined to the 8h period post-exercise, this may subsequently impact the expression patterns of target mRNAs forming the basis of exercise-induced adaptive responses.
  38. Aging (Albany NY). 2021 Mar 18. 13
      Declines in mitochondrial mass are thought to be a hallmark of mammalian aging, and a ketogenic diet (KD) may prevent the age-related decreases in mitochondrial content. The objective of this study was to investigate the impact of a KD on markers of mitochondrial mass. Mice were fed an isocaloric control diet (CD) or KD from 12 months of age. Tissues were collected after 1 month and 14 months of intervention, and a panel of commonly used markers of mitochondrial mass (mitochondrial enzyme activities and levels, mitochondrial to nuclear DNA ratio, and cardiolipin content) were measured. Our results showed that a KD stimulated activities of marker mitochondrial enzymes including citrate synthase, Complex I, and Complex IV in hindlimb muscle in aged mice. KD also increased the activity of citrate synthase and prevented an age-related decrease in Complex IV activity in aged brain. No other markers were increased in these tissues. Furthermore, the impacts of a KD on liver and kidney were mixed with no pattern indicative of a change in mitochondrial mass. In conclusion, results of the present study suggest that a KD induces tissue-specific changes in mitochondrial enzyme activities, or structure, rather than global changes in mitochondrial mass across tissues.
    Keywords:  brain; diet; kidney; liver; skeletal muscle
  39. Am J Sports Med. 2021 Mar;49(4): 1073-1085
      BACKGROUND: Skeletal muscle injuries represent a major concern in sports medicine. Cell therapy has emerged as a promising therapeutic strategy for muscle injuries, although the preclinical data are still inconclusive and the potential clinical use of cell therapy has not yet been established.PURPOSE: To evaluate the effects of muscle precursor cells (MPCs) on muscle healing in a small animal model.
    STUDY DESIGN: Controlled laboratory study.
    METHODS: A total of 27 rats were used in the study. MPCs were isolated from rat (n = 3) medial gastrocnemius muscles and expanded in primary culture. Skeletal muscle injury was induced in 24 rats, and the animals were assigned to 3 groups. At 36 hours after injury, animals received treatment based on a single ultrasound-guided MPC (105 cells) injection (Cells group) or MPC injection in combination with 2 weeks of daily exercise training (Cells+Exercise group). Animals receiving intramuscular vehicle injection were used as controls (Vehicle group). Muscle force was determined 2 weeks after muscle injury, and muscles were collected for histological and immunofluorescence evaluation.
    RESULTS: Red fluorescence-labeled MPCs were successfully transplanted in the site of the injury by ultrasound-guided injection and were localized in the injured area after 2 weeks. Transplanted MPCs participated in the formation of regenerating muscle fibers as corroborated by the co-localization of red fluorescence with developmental myosin heavy chain (dMHC)-positive myofibers by immunofluorescence analysis. A strong beneficial effect on muscle force recovery was detected in the Cells and Cells+Exercise groups (102.6% ± 4.0% and 101.5% ± 8.5% of maximum tetanus force of the injured vs healthy contralateral muscle, respectively) compared with the Vehicle group (78.2% ± 5.1%). Both Cells and Cells+Exercise treatments stimulated the growth of newly formed regenerating muscles fibers, as determined by the increase in myofiber cross-sectional area (612.3 ± 21.4 µm2 and 686.0 ± 11.6 µm2, respectively) compared with the Vehicle group (247.5 ± 10.7 µm2), which was accompanied by a significant reduction of intramuscular fibrosis in Cells and Cells+Exercise treated animals (24.2% ± 1.3% and 26.0% ± 1.9% of collagen type I deposition, respectively) with respect to control animals (40.9% ± 4.1% in the Vehicle group). MPC treatment induced a robust acceleration of the muscle healing process as demonstrated by the decreased number of dMHC-positive regenerating myofibers (enhanced replacement of developmental myosin isoform by mature myosin isoforms) (4.3% ± 2.6% and 4.1% ± 1.5% in the Cells and Cells+Exercise groups, respectively) compared with the Vehicle group (14.8% ± 13.9%).
    CONCLUSION: Single intramuscular administration of MPCs improved histological outcome and force recovery of the injured skeletal muscle in a rat injury model that imitates sports-related muscle injuries. Cell therapy showed a synergistic effect when combined with an early active rehabilitation protocol in rats, which suggests that a combination of treatments can generate novel therapeutic strategies for the treatment of human skeletal muscle injuries.
    CLINICAL RELEVANCE: Our study demonstrates the strong beneficial effect of MPC transplant and the synergistic effect when the cell therapy is combined with an early active rehabilitation protocol for muscle recovery in rats; this finding opens new avenues for the development of effective therapeutic strategies for muscle healing and clinical trials in athletes undergoing MPC transplant and rehabilitation protocols.
    Keywords:  MPCs; cell therapy; muscle healing; physical exercise therapy; rat model; skeletal muscle injury
  40. Anim Sci J. 2021 Jan;92(1): e13544
      Muscle-fiber type in livestock skeletal muscles influences meat quality, but the underlying mechanisms remain unclear. We previously showed that Homeobox A11 (Hoxa11) and Homeobox A13 (Hoxa13) are differentially expressed in fast- and slow-twitch muscles, but their effects on the formation of muscle-fiber types and intramuscular fat deposition have not been investigated. Here, our results revealed that overexpression of Hoxa11 and Hoxa13 delayed cell-cycle progression in C2C12 myoblasts, reduced their proliferation, and promoted their differentiation into slow-twitch muscle fibers. Knockdown experiments produced the opposite results. The conditioned media of differentiated C2C12 cells with Hoxa11/Hoxa13 overexpression or knockdown were harvested. Staining results showed that adipogenesis of preadipocytes was significantly promoted by Hoxa13 knockdown C2C12 cell culture medium. Changes in lipid accumulation were due to a reduction in lipid decomposition and an increase in triglyceride synthesis; genes related to fatty-acid synthesis were decreased. In conclusion, our study showed that Hoxa11 and Hoxa13 promote slow-twitch muscle formation and indirectly regulate preadipocyte adipogenesis, which may facilitate meat-quality improvement in the future.
    Keywords:   Hoxa11 ; Hoxa13 ; intramuscular fat; muscle fiber types; pigs
  41. Front Cell Dev Biol. 2021 ;9 632303
      Brown adipose tissue (BAT) plays critical thermogenic, metabolic and endocrine roles in mammals, and aberrant BAT function is associated with metabolic disorders including obesity and diabetes. The major BAT depots are clustered at the neck and forelimb levels, and arise largely within the dermomyotome of somites, from a common progenitor with skeletal muscle. However, many aspects of BAT embryonic development are not well understood. Hoxa5 patterns other tissues at the cervical and brachial levels, including skeletal, neural and respiratory structures. Here, we show that Hoxa5 also positively regulates BAT development, while negatively regulating formation of epaxial skeletal muscle. HOXA5 protein is expressed in embryonic preadipocytes and adipocytes as early as embryonic day 12.5. Hoxa5 null mutant embryos and rare, surviving adults show subtly reduced iBAT and sBAT formation, as well as aberrant marker expression, lower adipocyte density and altered lipid droplet morphology. Conversely, the epaxial muscles that arise from a common dermomyotome progenitor are expanded in Hoxa5 mutants. Conditional deletion of Hoxa5 with Myf5/Cre can reproduce both BAT and epaxial muscle phenotypes, indicating that HOXA5 is necessary within Myf5-positive cells for proper BAT and epaxial muscle development. However, recombinase-based lineage tracing shows that Hoxa5 does not act cell-autonomously to repress skeletal muscle fate. Interestingly, Hoxa5-dependent regulation of adipose-associated transcripts is conserved in lung and diaphragm, suggesting a shared molecular role for Hoxa5 in multiple tissues. Together, these findings establish a role for Hoxa5 in embryonic BAT development.
    Keywords:  Hoxa5; adipose development; brown adipose tissue; differentiation; skeletal muscle development
  42. Exerc Sport Sci Rev. 2021 Apr 01. 49(2): 67-76
      Exercise stimulates the biogenesis of mitochondria in muscle. Some literature supports the use of pharmaceuticals to enhance mitochondria as a substitute for exercise. We provide evidence that exercise rejuvenates mitochondrial function, thereby augmenting muscle health with age, in disease, and in the absence of cellular regulators. This illustrates the power of exercise to act as mitochondrial medicine in muscle.
  43. Int J Sport Nutr Exerc Metab. 2021 Mar 18. pii: ijsnem.2020-0356. [Epub ahead of print] 1-10
      Branched-chain amino acids (BCAA) are one of the most popular sports supplements, marketed under the premise that they enhance muscular adaptations. Despite their prevalent consumption among athletes and the general public, the efficacy of BCAA has been an ongoing source of controversy in the sports nutrition field. Early support for BCAA supplementation was derived from extrapolation of mechanistic data on their role in muscle protein metabolism. Of the three BCAA, leucine has received the most attention because of its ability to stimulate the initial acute anabolic response. However, a substantial body of both acute and longitudinal research has now accumulated on the topic, affording the ability to scrutinize the effects of BCAA and leucine from a practical standpoint. This article aims to critically review the current literature and draw evidence-based conclusions about the putative benefits of BCAA or leucine supplementation on muscle strength and hypertrophy as well as illuminate gaps in the literature that warrant future study.
    Keywords:  BCAA; anabolism; muscle mass; muscular adaptations
  44. Curr Opin Pharmacol. 2021 Mar 12. pii: S1471-4892(21)00011-4. [Epub ahead of print]57 140-147
      Sarcopenia, the age-related decline in muscle mass and strength/function, is a prototypical geroscience condition. The dissection of muscle-specific molecular pathways through analyses of tissue biopsies has provided valuable insights into the pathophysiology of sarcopenia. However, such an approach is unsuitable for capturing the dynamic nature of the condition. Furthermore, the muscle sampling procedure may be perceived as burdensome especially by multimorbid, frail older adults. To overcome these limitations, sophisticated statistical methods have been devised for the simultaneous analysis of circulating factors related to the multiple domains of sarcopenia. This approach has shown potential for achieving a more comprehensive appraisal of the condition, unveiling new therapeutic targets, and identifying meaningful biomarkers. Here, we discuss the main pathogenetic pathways of sarcopenia, with a focus on mediators that are currently in the spotlight as biomarkers and potential treatment targets.
    Keywords:  Cytokines; Inflammation; Mitochondria; Muscle; mTOR
  45. Exp Gerontol. 2021 Mar 15. pii: S0531-5565(21)00076-0. [Epub ahead of print] 111301
      INTRODUCTION: A vast amount of research has focused on the effects of physical fitness (PF) on mortality, with little research evaluating the effects of PF on future expected health related quality of life (HRQoL).AIM: To evaluate how current PF influences future HRQoL measured in a prospective 8-year study in older adults.
    METHODS: A total of 617 (157 males) older adults (>65y) participated in the study. PF was assessed with the EXERNET battery in 2008-2009 (baseline) and 2016-2017 (follow-up). HRQoL was assessed using the EQ-5D-3L questionnaire in both evaluations. PF tertiles were developed from baseline PF variables: FIT (highest PF values), REGULAR and UNFIT (lowest PF values) taking into account age and sex. Follow-up HRQoL values were compared to sex and age-specific expected values. Logistic regressions were performed to test differences between PF tertiles regarding future expected quality of life. Linear regressions were developed to test whether baseline PF could predict future HRQoL scores.
    RESULTS: The FIT group showed higher probabilities of an improved HRQoL when compared to the UNFIT group. All PF variables seemed to be important at some point of the study except upper extremities flexibility. Aerobic endurance was the variable that showed to be significant for most of the HRQoL predictions.
    CONCLUSION: PF influences future HRQoL in older adults who accordingly should try to remain fit to maintain an increased age-adjusted HRQoL.
    Keywords:  Aging; Cardiorespiratory fitness; Elderly; Muscle strength; Physical activity; Quality of life
  46. Physiol Rev. 2021 Mar 18.
      The design of the energy metabolism system in striated muscle remains a major area of investigation. Here, we review our current understanding and emerging hypotheses regarding the metabolic support of muscle contraction. Maintenance of ATP free energy, so called energy homeostasis, via mitochondrial oxidative phosphorylation is critical to sustained contractile activity and this major design criterion is the focus of this review. Cell volume invested in mitochondria reduces the space available for generating contractile force, and this spatial balance between mitochondria and contractile elements to meet the varying sustained power demands across muscle types is another important design criterion. This is accomplished with remarkably similar mass-specific mitochondrial protein composition across muscle types, implying that it is the organization of mitochondria within the muscle cell that is critical to supporting sustained muscle function. Beyond the production of ATP, ubiquitous distribution of ATPases throughout the muscle requires rapid distribution of potential energy across these large cells. Distribution of potential energy has long been thought to occur primarily through facilitated metabolite diffusion but recent analysis has questioned the importance of this process under normal physiological conditions. Recent structural and functional studies have supported the hypothesis that the mitochondrial reticulum provides a rapid energy distribution system via the conduction of the mitochondrial membrane potential to maintain metabolic homeostasis during contractile activity. We extensively review this aspect of the energy metabolism design contrasting it with metabolite diffusion models and how mitochondrial structure can play a role in the delivery of energy in the striated muscle.
    Keywords:  cellular energy distribution; mitochondria; mitochondrial networks; mitochondrial reticulum; oxidative phosphorylation
  47. Aging Cell. 2021 Mar 16. e13337
      Aging drives progressive loss of the ability of tissues to recover from stress, partly through loss of somatic stem cell function and increased senescent burden. We demonstrate that bone marrow-derived mesenchymal stem cells (BM-MSCs) rapidly senescence and become dysfunctional in culture. Injection of BM-MSCs from young mice prolonged life span and health span, and conditioned media (CM) from young BM-MSCs rescued the function of aged stem cells and senescent fibroblasts. Extracellular vesicles (EVs) from young BM-MSC CM extended life span of Ercc1-/- mice similarly to injection of young BM-MSCs. Finally, treatment with EVs from MSCs generated from human ES cells reduced senescence in culture and in vivo, and improved health span. Thus, MSC EVs represent an effective and safe approach for conferring the therapeutic effects of adult stem cells, avoiding the risks of tumor development and donor cell rejection. These results demonstrate that MSC-derived EVs are highly effective senotherapeutics, slowing the progression of aging, and diseases driven by cellular senescence.
    Keywords:  aging; extracellular vesicles; mesenchymal stem cells; senescence; stem cells
  48. Chronobiol Int. 2021 Mar 15. 1-9
      This study aimed to examine the effect of circadian rhythms (CR) on anaerobic performance and subsequent recovery, muscle damage, and respiratory muscle strength. Twenty diurnally active male football players (age, 22.20 ± 3.14 y) were asked to perform the Wingate anaerobic power test three times for 30 s each at 09:00, 14:00 and 19:00 h, with a minimum recovery period of 1 week between each testing day. Pretest oral temperature, respiratory muscle strength, oxygen saturation, and rating of perceived exertion were recorded at three different time of the day. To examine post-exercise recovery, heart rate (HR) and lactic acid (LA) levels were recorded before and after the tests. Blood samples were collected 20 min after each test to assess muscle damage. The body temperature taken at 19:00 h was the highest of the three (p < .01). After the tests, the LA value at 19:00 h was higher than that at 09:00 h (p < .05). According to CR, the HR values measured after anaerobic exercise were higher at 14:00 h (p < .05). The peak power value was higher at 14:00 h than at 19:00 h (p < .058). CR does not affect muscle damage and respiratory muscle strength. Further, at 14:00 h, anaerobic power was higher and recovery occurred faster compared to the other test times of 09:00 and 19:00 h. Therefore, it is recommended that anaerobic training should be performed early in the afternoon.
    Keywords:  Circadian rhythm; anaerobic power; muscle damage; recovery from exercise; respiratory muscle strength
  49. J Neuromuscul Dis. 2021 Mar 06.
      BACKGROUND: Metabolic myopathies are a heterogenous group of muscle diseases typically characterized by exercise intolerance, myalgia and progressive muscle weakness. Effective treatments for some of these diseases are available, but while our understanding of the pathogenesis of metabolic myopathies related to glycogen storage, lipid metabolism and β-oxidation is well established, evidence linking treatments with the precise causative genetic defect is lacking.OBJECTIVE: The objective of this study was to collate all published evidence on pharmacological therapies for the aforementioned metabolic myopathies and link this to the genetic mutation in a format amenable to databasing for further computational use in line with the principles of the "treatabolome" project.
    METHODS: A systematic literature review was conducted to retrieve all levels of evidence examining the therapeutic efficacy of pharmacological treatments on metabolic myopathies related to glycogen storage and lipid metabolism. A key inclusion criterion was the availability of the genetic variant of the treated patients in order to link treatment outcome with the genetic defect.
    RESULTS: Of the 1,085 articles initially identified, 268 full-text articles were assessed for eligibility, of which 87 were carried over into the final data extraction. The most studied metabolic myopathies were Pompe disease (45 articles), multiple acyl-CoA dehydrogenase deficiency related to mutations in the ETFDH gene (15 articles) and systemic primary carnitine deficiency (8 articles). The most studied therapeutic management strategies for these diseases were enzyme replacement therapy, riboflavin, and carnitine supplementation, respectively.
    CONCLUSIONS: This systematic review provides evidence for treatments of metabolic myopathies linked with the genetic defect in a computationally accessible format suitable for databasing in the treatabolome system, which will enable clinicians to acquire evidence on appropriate therapeutic options for their patient at the time of diagnosis.
  50. Sci Rep. 2021 Mar 15. 11(1): 5952
      Becker muscular dystrophy (BMD) is the milder allelic variant of Duchenne muscular dystrophy, with higher dystrophin levels. To anticipate on results of interventions targeting dystrophin expression it is important to know the natural variation of dystrophin expression between different muscles and over time. Dystrophin was quantified using capillary Western immunoassay (Wes) in the anterior tibial (TA) muscle of 37 BMD patients. Variability was studied using two samples from the same TA biopsy site in nine patients, assessing nine longitudinal TA biopsies, and eight simultaneously obtained vastus lateralis (VL) muscle biopsies. Measurements were performed in duplicate with two primary antibodies. Baseline dystrophin levels were correlated to longitudinal muscle strength and functional outcomes. Results showed low technical variability and high precision for both antibodies. Dystrophin TA levels ranged from 4.8 to 97.7%, remained stable over a 3-5 year period, and did not correlate with changes in longitudinal muscle function. Dystrophin levels were comparable between TA and VL muscles. Intra-muscle biopsy variability was low (5.2% and 11.4% of the total variability of the two antibodies). These observations are relevant for the design of clinical trials targeting dystrophin production, and may urge the need for other biomarkers or surrogate endpoints.
  51. Biochem Biophys Res Commun. 2021 Mar 11. pii: S0006-291X(21)00392-2. [Epub ahead of print]551 27-32
      Tropomyosin and troponin regulate muscle contraction by participating in a macromolecular scale steric-mechanism to control myosin-crossbridge - actin interactions and consequently contraction. At low-Ca2+, the C-terminal 30% of troponin subunit-I (TnI) is proposed to trap tropomyosin in a position on thin filaments that sterically interferes with myosin-binding, thus causing muscle relaxation. In contrast, at high-Ca2+, inhibition is released after the C-terminal domains dissociate from F-actin-tropomyosin as its component switch-peptide domain binds to the N-lobe of troponin-C (TnC). Recent, paradigm-shifting, cryo-EM reconstructions by the Namba group have revealed density attributed to TnI along cardiac muscle thin filaments at both low- and high-Ca2+ concentration. Modeling the reconstructions showed expected high-Ca2+ hydrophobic interactions of the TnI switch-peptide and TnC. However, under low-Ca2+ conditions, sparse interactions of TnI and tropomyosin, and in particular juxtaposition of non-polar switch-peptide residues and charged tropomyosin amino acids in the published model seem difficult to reconcile with an expected steric-blocking conformation. This anomaly is likely due to inaccurate fitting of tropomyosin into the cryo-EM volume. In the current study, the low-Ca2+ cryo-EM volume was fitted with a more accurate tropomyosin model and representation of cardiac TnI. Our results show that at low-Ca2+ a cluster of hydrophobic residues at the TnI switch-peptide and adjacent H4 helix (Ala149, Ala151, Met 154, Leu159, Gly160, Ala161, Ala163, Leu167, Leu169, Ala171, Leu173) draw-in tropomyosin surface residues (Ile143, Ile146, Ala151, Ile154), presumably attracting the entire tropomyosin cable to its myosin-blocking position on actin. The modeling confirms that neighboring TnI "inhibitory domain" residues (Arg145, Arg148) bind to thin filaments at actin residue Asp25, as previously suggested. ClusPro docking of TnI residues 137-184 to actin-tropomyosin, including the TnI inhibitory-domain, switch-peptide and Helix H4, verified the modeled configuration. Our residue-to-residue contact-mapping of the TnI-tropomyosin association lends itself to experimental validation and functional localization of disease-bearing mutations.
    Keywords:  Actin; Cryo-electron microscopy; Molecular modeling; Protein-protein docking; Tropomyosin; Troponin