bims-moremu Biomed News
on Molecular regulators of muscle mass
Issue of 2022‒02‒27
forty-two papers selected by
Anna Vainshtein
Craft Science Inc.

  1. PeerJ. 2022 ;10 e12856
      Background: Exercise elicits a range of adaptive responses in skeletal muscle, which include changes in mRNA expression. To better understand the health benefits of exercise training, it is important to investigate the underlying molecular mechanisms of skeletal muscle adaptation to exercise. However, most studies have assessed the molecular events at only a few time-points within a short time frame post-exercise, and the variations of gene expression kinetics have not been addressed systematically.Methods: We assessed the mRNA expression of 23 gene isoforms implicated in the adaptive response to exercise at six time-points (0, 3, 9, 24, 48, and 72 h post exercise) over a 3-day period following a single session of high-intensity interval exercise.
    Results: The temporal patterns of target gene expression were highly variable and the expression of mRNA transcripts detected was largely dependent on the timing of muscle sampling. The largest fold change in mRNA expression of each tested target gene was observed between 3 and 72 h post-exercise.
    Discussion and Conclusions: Our findings highlight an important gap in knowledge regarding the molecular response to exercise, where the use of limited time-points within a short period post-exercise has led to an incomplete understanding of the molecular response to exercise. Muscle sampling timing for individual studies needs to be carefully chosen based on existing literature and preliminary analysis of the molecular targets of interest. We propose that a comprehensive time-course analysis on the exercise-induced transcriptional response in humans will significantly benefit the field of exercise molecular biology.
    Keywords:  Exercise; Exercise-induced adaptation; Gene expression; Skeletal muscle; Time-course
  2. FASEB J. 2022 03;36(3): e22211
      Metabolic dysfunction and insulin resistance are emerging as hallmarks of cancer and cachexia, and impair cancer prognosis. Yet, the molecular mechanisms underlying impaired metabolic regulation are not fully understood. To elucidate the mechanisms behind cancer-induced insulin resistance in muscle, we isolated extensor digitorum longus (EDL) and soleus muscles from Lewis Lung Carcinoma tumor-bearing mice. Three weeks after tumor inoculation, muscles were isolated and stimulated with or without a submaximal dose of insulin (1.5 nM). Glucose transport was measured using 2-[3 H]Deoxy-Glucose and intramyocellular signaling was investigated using immunoblotting. In soleus muscles from tumor-bearing mice, insulin-stimulated glucose transport was abrogated concomitantly with abolished insulin-induced TBC1D4 and GSK3 phosphorylation. In EDL, glucose transport and TBC1D4 phosphorylation were not impaired in muscles from tumor-bearing mice, while AMPK signaling was elevated. Anabolic insulin signaling via phosphorylation of the mTORC1 targets, p70S6K thr389, and ribosomal-S6 ser235, were decreased by cancer in soleus muscle while increased or unaffected in EDL. In contrast, the mTOR substrate, pULK1 ser757, was reduced in both soleus and EDL by cancer. Hence, cancer causes considerable changes in skeletal muscle insulin signaling that is dependent on muscle-type, which could contribute to metabolic dysregulation in cancer. Thus, the skeletal muscle could be a target for managing metabolic dysfunction in cancer.
    Keywords:  AMPK; Akt; Lewis lung carcinoma; TBC1D4; cachexia; cancer; glucose metabolism; insulin resistance; mTORC1; muscle
  3. Acta Pharmacol Sin. 2022 Feb 25.
      Canagliflozin is an antidiabetic medicine that inhibits sodium-glucose cotransporter 2 (SGLT2) in proximal tubules. Recently, it was reported to have several noncanonical effects other than SGLT2 inhibiting. However, the effects of canagliflozin on skeletal muscle regeneration remain largely unexplored. Thus, in vivo muscle contractile properties recovery in mice ischemic lower limbs following gliflozins treatment was evaluated. The C2C12 myoblast differentiation after gliflozins treatment was also assessed in vitro. As a result, both in vivo and in vitro data indicate that canagliflozin impairs intrinsic myogenic regeneration, thus hindering ischemic limb muscle contractile properties, fatigue resistance recovery, and tissue regeneration. Mitochondrial structure and activity are both disrupted by canagliflozin in myoblasts. Single-cell RNA sequencing of ischemic tibialis anterior reveals a decrease in leucyl-tRNA synthetase 2 (LARS2) in muscle stem cells attributable to canagliflozin. Further investigation explicates the noncanonical function of LARS2, which plays pivotal roles in regulating myoblast differentiation and muscle regeneration by affecting mitochondrial structure and activity. Enhanced expression of LARS2 restores the differentiation of canagliflozin-treated myoblasts, and accelerates ischemic skeletal muscle regeneration in canagliflozin-treated mice. Our data suggest that canagliflozin directly impairs ischemic skeletal muscle recovery in mice by downregulating LARS2 expression in muscle stem cells, and that LARS2 may be a promising therapeutic target for injured skeletal muscle regeneration.
    Keywords:  leucyl-tRNA synthetase 2; limb ischemia; mitochondria; muscle stem cell; myogenesis; sodium-glucose cotransporter 2 inhibitor
  4. Diabetes. 2022 Feb 22. pii: db210855. [Epub ahead of print]
      Insulin-stimulated muscle glucose uptake is a key process in glycemic control. This process depends on the redistribution of glucose transporters to the surface membrane, a process which involves regulatory proteins such as TBC1D1 and TBC1D4. Accordingly, a TBC1D4 loss-of-function mutation in human skeletal muscle is associated with increased risk of type 2 diabetes, and observations from carriers of a TBC1D1 variant associate this protein to a severe obesity phenotype. Here, we identified interactors of the endogenous TBC1D4 in human skeletal muscle by an unbiased proteomics approach. We detected 76 proteins as candidate TBC1D4 interactors. The binding of 12 of these interactors were regulated by insulin, including proteins known to be involved in glucose metabolism (e.g. 14-3-3 proteins and ACTN4). TBC1D1 also co-precipitated with TBC1D4 and vice versa in both human and mouse skeletal muscle. This interaction was not regulated by insulin nor exercise in young, healthy, lean individuals. Similarly, the exercise- and insulin-regulated phosphorylation of the TBC1D1-TBC1D4 complex was intact. In contrast, we observed an altered interaction as well as compromised insulin-stimulated phospho-regulation of the TBC1D1-TBC1D4 complex in muscle of obese individuals with type 2 diabetes. Altogether, we provide a repository of TBC1D4 interactors in human and mouse skeletal muscle, which serve as potential regulators of TBC1D4 function and, thus, insulin-stimulated glucose uptake in human skeletal muscle.
  5. J Cachexia Sarcopenia Muscle. 2022 Feb 22.
      BACKGROUND: Interventions to preserve functional capacities at advanced age are becoming increasingly important. So far, exercise provides the only means to counteract age-related decrements in physical performance and muscle function. Unfortunately, the effectiveness of exercise interventions in elderly populations is hampered by reduced acceptance and compliance as well as disuse complications. We therefore studied whether application of interleukin-6 (IL-6), a pleiotropic myokine that is induced by skeletal muscle activity and exerts broad systemic effects in response to exercise, affects physical performance and muscle function alone or in combination with training in aged mice.METHODS: Sedentary old male mice (Sed+Saline, n = 15) were compared with animals that received recombinant IL-6 (rIL-6) in an exercise-mimicking pulsatile manner (Sed+IL-6, n = 16), were trained with a moderate-intensity, low-volume endurance exercise regimen (Ex+Saline, n = 13), or were exposed to a combination of these two interventions (Ex+IL-6, n = 16) for 12 weeks. Before and at the end of the intervention, mice underwent a battery of tests to quantify endurance performance, muscle contractility in situ, motor coordination, and gait and metabolic parameters.
    RESULTS: Mice exposed to enhanced levels of IL-6 during endurance exercise bouts showed superior improvements in endurance performance (33% more work and 12% greater peak power compared with baseline), fatigue resistance in situ (P = 0.0014 vs. Sed+Saline; P = 0.0199 vs. Sed+IL-6; and P = 0.0342 vs. Ex+Saline), motor coordination (rotarod performance, P = 0.0428), and gait (gait speed, P = 0.0053) following training. Pulsatile rIL-6 treatment in sedentary mice had only marginal effects on glucose tolerance and some gait parameters. No increase in adverse events or mortality related to rIL-6 treatment was observed.
    CONCLUSIONS: Administration of rIL-6 paired with treadmill running bouts potentiates the adaptive response to a moderate-intensity low-volume endurance exercise regimen in old mice, while being safe and well tolerated.
    Keywords:  Ageing; Exercise; Fatigue; Frailty; Interleukin-6; Muscle; Myokine
  6. J Physiol. 2022 Feb 25.
      Store-operated Ca2+ entry (SOCE) is critical to cell function. In skeletal muscle, SOCE has evolved alongside excitation-contraction coupling (EC coupling); as a result, it displays unique properties compared to SOCE in other cells. The plasma membrane of skeletal muscle is mostly internalized as the tubular (t-) system, with the tubules meeting the sarcoplasmic reticulum (SR) terminal cisternae, forming junctions where the proteins that regulate EC coupling and SOCE are positioned. In this review, we describe the properties and roles of SOCE based on direct measurements of Ca2+ influx during SR Ca2+ release and leak. SOCE is activated immediately and locally as the [Ca2+ ] of the junctional SR terminal cisternae ([Ca2+ ]jSR ) depletes. [Ca2+ ]jSR changes rapidly and steeply with increasing activity of the SR ryanodine receptor isoform 1 (RyR1). The high fidelity of [Ca2+ ]jSR with RyR1 activity likely depends on the SR Ca2+ -buffer calsequestrin (CSQ) that is located immediately behind the RyR1s inside the SR. This arrangement provides in-phase activation and deactivation of SOCE with a large dynamic range, allowing precise grading of SOCE flux. The in-phase activation of SOCE as the SR partially depletes traps Ca2+ in the cytoplasm, preventing net Ca2+ loss. Mild presentation of RyR1 leak can occur under physiological conditions, providing fibre Ca2+ redistribution without changing fibre Ca2+ content. This condition preserves normal contractile function while increasing basal metabolic rate. However, a tipping point at higher RyR1 leak driving excess cytoplasmic and mitochondrial Ca2+ load and setting a deleterious intracellular environment that compromises the integrity of the skeletal muscle. Abstract figure legend. The skeletal muscle plasma membrane invaginates into the fibre as the tubular (t-) system, to form a permanent junction with the sarcoplasmic reticulum terminal cisternae. At these junctional membranes, SR ryanodine receptors isoform 1 (RyR1) directly interact with the t-system dihydropyridine receptor (DHPR). Increased RyR1 Ca2+ activity causes a near membrane Ca2+ -depletion in the SR that activates store-operated Ca2+ entry (SOCE) and an increase in junctional space [Ca2+ ] that activates plasma membrane Ca2+ -ATPase (PMCA). In a resting fibre with chronically increased RyR1 Ca2+ leak this mechanism raises [Ca2+ ]cyto to compensate for the lowered SR Ca2+ content to relieve the SR Ca2+ release load required for normal force generation, while the raised [Ca2+ ]cyto also increases the basal Ca2+ content of the mitochondria to favour increasing ATP resynthesis rates. These events can happen in healthy or diseased cells, which may be defined by RyR1 leak magnitude. This article is protected by copyright. All rights reserved.
    Keywords:  Ca2+; SOCE; confocal; mechanically skinned fibre; ryanodine receptor; skeletal muscle; store-operated Ca2+ entry
  7. Metabolites. 2022 Feb 19. pii: 193. [Epub ahead of print]12(2):
      Taurine (2-aminoethanesulfonic acid) is required for ensuring proper muscle functioning. Knockout of the taurine transporter in mice results in low taurine concentrations in the muscle and associates with myofiber necrosis and diminished exercise capacity. Interestingly, regulation of taurine and its transporter is altered in the mdx mouse, a model for Duchenne Muscular Dystrophy (DMD). DMD is a genetic disorder characterized by progressive muscle degeneration and weakness due to the absence of dystrophin from the muscle membrane, causing destabilization and contraction-induced muscle cell damage. This review explores the physiological role of taurine in skeletal muscle and the consequences of a disturbed balance in DMD. Its potential as a supportive treatment for DMD is also discussed. In addition to genetic correction, that is currently under development as a curative treatment, taurine supplementation has the potential to reduce muscle inflammation and improve muscle strength in patients.
    Keywords:  Duchenne Muscular Dystrophy; mdx; muscle; osmolytes; taurine
  8. iScience. 2022 Feb 18. 25(2): 103863
      In clinical trials, oral supplementation with nicotinamide riboside (NR) fails to increase muscle mitochondrial respiratory capacity and insulin sensitivity but also does not increase muscle NAD+ levels. This study tests the feasibility of chronically elevating skeletal muscle NAD+ in mice and investigates the putative effects on mitochondrial respiratory capacity, insulin sensitivity, and gene expression. Accordingly, to improve bioavailability to skeletal muscle, we developed an experimental model for administering NR repeatedly through a jugular vein catheter. Mice on a Western diet were treated with various combinations of NR, pterostilbene (PT), and voluntary wheel running, but the metabolic effects of NR and PT treatment were modest. We conclude that the chronic elevation of skeletal muscle NAD+ by the intravenous injection of NR is possible but does not affect muscle respiratory capacity or insulin sensitivity in either sedentary or physically active mice. Our data have implications for NAD+ precursor supplementation regimens.
    Keywords:  Drugs; Molecular physiology; Transcriptomics
  9. Methods Mol Biol. 2022 ;2434 235-243
      Alternative pre-mRNA splicing can be cell-type specific and results in the generation of different protein isoforms from a single gene. Deregulation of canonical pre-mRNA splicing by disease-associated variants can result in genetic disorders. Antisense oligonucleotides (AONs) offer an attractive solution to modulate endogenous gene expression through alteration of pre-mRNA splicing events. Relevant in vitro models are crucial for appropriate evaluation of splicing modifying drugs. In this chapter, we describe how to investigate the splicing modulating activity of AONs in an in vitro skeletal muscle model, applied to Pompe disease. We also provide a detailed description of methods to visualize and analyze gene expression in differentiated skeletal muscle cells for the analysis of muscle differentiation and splicing outcome. The methodology described here is relevant to develop treatment options using AONs for other genetic muscle diseases as well, including Duchenne muscular dystrophy, myotonic dystrophy, and facioscapulohumeral muscular dystrophy.
    Keywords:  Antisense oligonucleotides; Human iPSC; In vitro models; Skeletal muscle; Splicing
  10. Eur J Appl Physiol. 2022 Feb 21.
      PURPOSE: Our aim was to determine the effect of repeated sprint exercise in hypoxia on HIF-1 and HIF-1-regulated genes involved in glycolysis, mitochondrial turnover and oxygen transport. We also determined whether genes upregulated by exercise in hypoxia were dependent on the activation of HIF-1 in an in vitro model of exercise in hypoxia.METHODS: Eight endurance athletes performed bouts of repeated sprint exercise in control and hypoxic conditions. Skeletal muscle was sampled pre, post and 3 h post-exercise. HIF-1α protein and HIF1A, PDK1, GLUT4, VEGFA, BNIP3, PINK1 and PGC1A mRNA were measured. C2C12 myotubes were exposed to hypoxia and muscle contraction following treatment with a HIF-1α inhibitor to determine whether hypoxia-sensitive gene expression was dependent on HIF-1α.
    RESULTS: Sprint exercise in hypoxia increased HIF-1α protein expression immediately post-exercise [fold change (FC) = 3.5 ± 2.0]. Gene expression of PDK1 (FC = 2.1 ± 1.2), BNIP3 (FC = 2.4 ± 1.4) and VEGFA (FC = 2.7 ± 1.7) increased 3 h post-exercise in hypoxia but not control. PGC1A mRNA increased 3 h post-exercise in control (FC = 5.16) and hypoxia (FC = 5.7 ± 4.1) but there was no difference between the trials. Results from the in vitro experiment showed that hypoxia plus contraction also increased PDK1, BNIP3, and VEGFA gene expression. These responses were inhibited when HIF-1 protein activity was suppressed.
    CONCLUSION: Repeated sprint exercise in hypoxia upregulates some genes involved in glycolytic metabolism, mitochondrial turnover, and oxygen transport. HIF-1α is necessary for the expression of these genes in skeletal muscle cells.
    Keywords:  BNIP3; C2C12; Gene expression; LLTH; PGC-1; RSH
  11. Biomedicines. 2022 Feb 21. pii: 507. [Epub ahead of print]10(2):
      Muscular dystrophies (MDs) are a heterogeneous group of congenital neuromuscular disorders whose clinical signs include myalgia, skeletal muscle weakness, hypotonia, and atrophy that leads to progressive muscle disability and loss of ambulation. MDs can also affect cardiac and respiratory muscles, impairing life-expectancy. MDs in clude Duchenne muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy and limb-girdle muscular dystrophy. These and other MDs are caused by mutations in genes that encode proteins responsible for the structure and function of skeletal muscles, such as components of the dystrophin-glycoprotein-complex that connect the sarcomeric-actin with the extracellular matrix, allowing contractile force transmission and providing stability during muscle contraction. Consequently, in dystrophic conditions in which such proteins are affected, muscle integrity is disrupted, leading to local inflammatory responses, oxidative stress, Ca2+-dyshomeostasis and muscle degeneration. In this scenario, dysregulation of connexin hemichannels seem to be an early disruptor of the homeostasis that further plays a relevant role in these processes. The interaction between all these elements constitutes a positive feedback loop that contributes to the worsening of the diseases. Thus, we discuss here the interplay between inflammation, oxidative stress and connexin hemichannels in the progression of MDs and their potential as therapeutic targets.
    Keywords:  connexin hemichannels; inflammation; muscular dystrophies; oxidative stress; resveratrol
  12. Int J Mol Sci. 2022 Feb 12. pii: 2036. [Epub ahead of print]23(4):
      The proliferation and differentiation of skeletal muscle cells are usually controlled by serum components. Myogenic differentiation is induced by a reduction of serum components in vitro. It has been recently reported that serum contains not only various growth factors with specific actions on the proliferation and differentiation of myogenic cells, but also exogenous exosomes, the function of which is poorly understood in myogenesis. We have found that exosomes in fetal bovine serum are capable of exerting an inhibitive effect on the differentiation of C2C12 myogenic cells in vitro. In this process of inhibition, the downregulation of Tceal5 and Tceal7 genes was observed. Expression of these genes is specifically increased in direct proportion to myogenic differentiation. Loss- or gain- of function studies with Tceal5 and Tceal7 indicated that they have the potential to regulate myogenic differentiation via exosomes in fetal bovine serum.
    Keywords:  Tceal5; Tceal7; exosome; myogenic differentiation; serum
  13. Stem Cell Rev Rep. 2022 Feb 21.
      BACKGROUND: Bone marrow stromal cells (BMSCs) form a perivascular cell population in the bone marrow. These cells do not present naïve myogenic potential. However, their myogenic identity could be induced experimentally in vitro or in vivo. In vivo, after transplantation into injured muscle, BMSCs rarely fused with myofibers. However, BMSC participation in myofiber reconstruction increased if they were modified by NICD or PAX3 overexpression. Nevertheless, BMSCs paracrine function could play a positive role in skeletal muscle regeneration. Previously, we showed that SDF-1 treatment and coculture with myofibers increased BMSC ability to reconstruct myofibers. We also noticed that SDF-1 treatment changed selected miRNAs expression, including miR151 and miR5100.METHODS: Mouse BMSCs were transfected with miR151 and miR5100 mimics and their proliferation, myogenic differentiation, and fusion with myoblasts were analyzed.
    RESULTS: We showed that miR151 and miR5100 played an important role in the regulation of BMSC proliferation and migration. Moreover, the presence of miR151 and miR5100 transfected BMSCs in co-cultures with human myoblasts increased their fusion. This effect was achieved in an IGFBP2 dependent manner.
    CONCLUSIONS: Mouse BMSCs did not present naïve myogenic potential but secreted proteins could impact myogenic cell differentiation. miR151 and miR5100 transfection changed BMSC migration and IGFBP2 and MMP12 expression in BMSCs. miR151 and miR5100 transfected BMSCs increased myoblast fusion in vitro.
    Keywords:  SDF-1; bone marrow-derived mesenchymal stem/stromal cells; fusion; miR151; miR5100; migration; myoblasts; proliferation
  14. Elife. 2022 Feb 21. pii: e72792. [Epub ahead of print]11
      Myofibers are the main components of skeletal muscle, which is the largest tissue in the body. Myofibers are highly adaptive and can be altered under different biological and disease conditions. Therefore, transcriptional and epigenetic studies on myofibers are crucial to discover how chromatin alterations occur in the skeletal muscle under different conditions. However, due to the heterogenous nature of skeletal muscle, studying myofibers in isolation proves to be a challenging task. Single cell sequencing has permitted the study of the epigenome of isolated myonuclei. While this provides sequencing with high dimensionality, the sequencing depth is lacking, which makes comparisons between different biological conditions difficult. Here we report the first implementation of single myofiber ATAC-Seq, which allows for the sequencing of an individual myofiber at a depth sufficient for peak calling and for comparative analysis of chromatin accessibility under various physiological and disease conditions. Application of this technique revealed significant differences in chromatin accessibility between resting and regenerating myofibers, as well as between myofibers from a mouse model of Duchenne Muscular Dystrophy (mdx) and wild type (WT) counterparts. This technique can lead to a wide application in the identification of chromatin regulatory elements and epigenetic mechanisms in muscle fibers during development and in muscle-wasting diseases.
    Keywords:  genetics; genomics; mouse
  15. EMBO Rep. 2022 Feb 24. e53746
      Cachexia is a wasting syndrome characterized by devastating skeletal muscle atrophy that dramatically increases mortality in various diseases, most notably in cancer patients with a penetrance of up to 80%. Knowledge regarding the mechanism of cancer-induced cachexia remains very scarce, making cachexia an unmet medical need. In this study, we discovered strong alterations of iron metabolism in the skeletal muscle of both cancer patients and tumor-bearing mice, characterized by decreased iron availability in mitochondria. We found that modulation of iron levels directly influences myotube size in vitro and muscle mass in otherwise healthy mice. Furthermore, iron supplementation was sufficient to preserve both muscle function and mass, prolong survival in tumor-bearing mice, and even rescues strength in human subjects within an unexpectedly short time frame. Importantly, iron supplementation refuels mitochondrial oxidative metabolism and energy production. Overall, our findings provide new mechanistic insights in cancer-induced skeletal muscle wasting, and support targeting iron metabolism as a potential therapeutic option for muscle wasting diseases.
    Keywords:  cachexia; iron; metabolism; mitochondria; muscle
  16. Oncogenesis. 2022 Feb 22. 11(1): 9
      Rhabdomyosarcoma (RMS) is a deadly cancer of skeletal muscle origin. Pannexin 1 (PANX1) is down-regulated in RMS and increasing its levels drastically inhibits RMS progression. PANX1 upregulation thus represents a prospective new treatment strategy for this malignancy. However, the mechanisms regulating PANX1 expression, in RMS and other contexts, remain largely unknown. Here we show that both RMS and normal skeletal muscle express a comparable amount of PANX1 mRNAs, but surprisingly the canonical 5' untranslated region (5' UTR) or 5' leader of the transcript is completely lost in RMS. We uncover that quercetin, a natural plant flavonoid, increases PANX1 protein levels in RMS by inducing re-expression of a 5' leader-containing PANX1 transcript variant that is efficiently translated. This particular PANX1 mRNA variant is also present in differentiated human skeletal muscle myoblasts (HSMM) that highly express PANX1. Mechanistically, abolishing ETV4 transcription factor binding sites in the PANX1 promoter significantly reduced the luciferase reporter activities and PANX1 5' UTR levels, and both quercetin treatment in RMS cells and induction of differentiation in HSMM enriched the binding of ETV4 to its consensus element in the PANX1 promoter. Notably, quercetin treatment promoted RMS differentiation in a PANX1-dependent manner. Further showing its therapeutic potential, quercetin treatment prevented RMS in vitro tumor formation while inducing complete regression of established spheroids. Collectively, our results demonstrate the tumor-suppressive effects of quercetin in RMS and present a hitherto undescribed mechanism of PANX1 regulation via ETV4-mediated transcription of a translationally functional 5' leader-containing PANX1 mRNA.
  17. Cells. 2022 Feb 17. pii: 710. [Epub ahead of print]11(4):
      Nicotinamide adenine dinucleotide (NAD) is a versatile chemical compound serving as a coenzyme in metabolic pathways and as a substrate to support the enzymatic functions of sirtuins (SIRTs), poly (ADP-ribose) polymerase-1 (PARP-1), and cyclic ADP ribose hydrolase (CD38). Under normal physiological conditions, NAD+ consumption is matched by its synthesis primarily via the salvage pathway catalyzed by nicotinamide phosphoribosyltransferase (NAMPT). However, aging and muscular contraction enhance NAD+ utilization, whereas NAD+ replenishment is limited by cellular sources of NAD+ precursors and/or enzyme expression. This paper will briefly review NAD+ metabolic functions, its roles in regulating cell signaling, mechanisms of its degradation and biosynthesis, and major challenges to maintaining its cellular level in skeletal muscle. The effects of aging, physical exercise, and dietary supplementation on NAD+ homeostasis will be highlighted based on recent literature.
    Keywords:  NAD+; aging; exercise; mitochondria; sirtuin; skeletal muscle
  18. Biomolecules. 2022 Feb 05. pii: 261. [Epub ahead of print]12(2):
      BMAL1 is a core mammalian circadian clock transcription factor responsible for the regulation of the expression of thousands of genes. Previously, male skeletal-muscle-specific BMAL1-inducible-knockout (iMS-BMAL1 KO) mice have been described as a model that exhibits an aging-like phenotype with an altered gait, reduced mobility, muscle weakness, and impaired glucose uptake. Given this aging phenotype and that chronic kidney disease is a disease of aging, the goal of this study was to determine if iMS-BMAL1 KO mice exhibit a renal phenotype. Male iMS-BMAL1 KO and control mice were challenged with a low potassium diet for five days. Both genotypes responded appropriately by conserving urinary potassium. The iMS-BMAL1 KO mice excreted less potassium during the rest phase during the normal diet but there was no genotype difference during the active phase. Next, iMS-BMAL1 KO and control mice were used to compare markers of kidney injury and assess renal function before and after a phase advance protocol. Following phase advance, no differences were detected in renal mitochondrial function in iMS-BMAL1 KO mice compared to control mice. Additionally, the glomerular filtration rate and renal morphology were similar between groups in response to phase advance. Disruption of the clock in skeletal muscle tissue activates inflammatory pathways within the kidney of male mice, and there is evidence of this affecting other organs, such as the lungs. However, there were no signs of renal injury or altered function following clock disruption of skeletal muscle under the conditions tested.
    Keywords:  circadian clock genes; integrative physiology; renal function
  19. Front Physiol. 2021 ;12 779121
      Insulin and IGF-1, acting through the insulin receptor (IR) and IGF-1 receptor (IGF1R), maintain muscle mass and mitochondrial function, at least part of which occurs via their action to regulate gene expression. Here, we show that while muscle-specific deletion of IR or IGF1R individually results in only modest changes in the muscle transcriptome, combined deletion of IR/IGF1R (MIGIRKO) altered > 3000 genes, including genes involved in mitochondrial dysfunction, fibrosis, cardiac hypertrophy, and pathways related to estrogen receptor, protein kinase A (PKA), and calcium signaling. Functionally, this was associated with decreased mitochondrial respiration and increased ROS production in MIGIRKO muscle. To determine the role of FoxOs in these changes, we performed RNA-Seq on mice with muscle-specific deletion of FoxO1/3/4 (M-FoxO TKO) or combined deletion of IR, IGF1R, and FoxO1/3/4 in a muscle quintuple knockout (M-QKO). This revealed that among IR/IGF1R regulated genes, >97% were FoxO-dependent, and their expression was normalized in M-FoxO TKO and M-QKO muscle. FoxO-dependent genes were related to oxidative phosphorylation, inflammatory signaling, and TCA cycle. Metabolomic analysis showed accumulation of TCA cycle metabolites in MIGIRKO, which was reversed in M-QKO muscle. Likewise, calcium signaling genes involved in PKA signaling and sarcoplasmic reticulum calcium homeostasis were markedly altered in MIGIRKO muscle but normalized in M-QKO. Thus, combined loss of insulin and IGF-1 action in muscle transcriptionally alters mitochondrial function and multiple regulatory and signaling pathways, and these changes are mediated by FoxO transcription factors.
    Keywords:  FoxO transcription factors; RNA sequencing; calcium signaling; diabetes; insulin/IGF-1 receptors; mitochondrial dysfunction; muscle transcription
  20. J Cell Physiol. 2022 Feb 22.
      Exercise is important for the prevention and treatment of sarcopenia and osteoporosis. Although the interactions between skeletal muscles and bone have recently been reported, the myokines linking muscle to bone during exercise remain unknown. We previously revealed that chronic exercise using treadmill running blunts ovariectomy-induced osteopenia in mice. We herein performed an RNA sequence analysis of the gastrocnemius and soleus muscles of male mice with or without chronic exercise to identify the myokines responsible for the effects of chronic exercise on the muscle/bone relationship. We extracted peripheral myelin protein 22 (PMP22) as a humoral factor that was putatively induced by chronic exercise in the soleus and gastrocnemius muscles of mice from the RNA sequence analysis. Chronic exercise significantly enhanced the expression of PMP22 in the gastrocnemius and soleus muscles of female mice. PMP22 suppressed macrophage-colony stimulating factor and receptor activator factor κB ligand-induced increases in the expression of osteoclast-related genes and osteoclast formation from mouse bone marrow cells. Moreover, PMP22 significantly inhibited osteoblast differentiation, alkaline phosphatase activity, and mineralization in mouse osteoblast cultures; however, the overexpression of PMP22 did not affect muscle phenotypes in mouse muscle C2C12 cells. A simple regression analysis revealed that PMP22 mRNA levels in the gastrocnemius and soleus muscles were positively related to cortical bone mineral density at the femurs of mice with or without chronic exercise. In conclusion, we identified PMP22 as a novel myokine induced by chronic exercise in mice. We first showed that PMP22 suppresses osteoclast formation and the osteoblast phenotype in vitro.
    Keywords:  PMP22; bone; exercise; muscle
  21. J Cachexia Sarcopenia Muscle. 2022 Feb 22.
      BACKGROUND: The cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards MNs, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Because MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle-secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology.METHODS: Sporadic ALS patients were confirmed to be ALS according to El Escorial criteria and were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS-R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n = 27) or deltoids of aged-matched healthy subjects (n = 30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RT-qPCR, and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human-derived iPSC MNs and on healthy human myotubes, with untreated cells used as controls.
    RESULTS: An accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing. Compared with vesicles from healthy control myotubes, when administered to healthy MNs the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. The RNA-processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient MNs was abolished by anti-CD63 immuno-blocking of vesicle uptake.
    CONCLUSIONS: ALS muscle vesicles are shown to be toxic to MNs, which establishes the skeletal muscle as a potential source of vesicle-mediated toxicity in ALS.
    Keywords:  Cell-cell communication; MND; Secreted vesicles; sporadic ALS
  22. Membranes (Basel). 2022 Jan 26. pii: 153. [Epub ahead of print]12(2):
      Defects in membrane repair contribute to the development of muscular dystrophies, such as Miyoshi muscular dystrophy 1, limb girdle muscular dystrophy (LGMD), type R2 or R12. Deciphering membrane repair dysfunctions in the development of muscular dystrophies requires precise and detailed knowledge of the membrane repair machinery in healthy human skeletal muscle cells. Using correlative light and electron microscopy (CLEM), we studied the trafficking of four members of the annexin (ANX) family, in myotubes damaged by laser ablation. Our data support a model in which ANXA4 and ANXA6 are recruited to the disruption site by propagating as a wave-like motion along the sarcolemma. They may act in membrane resealing by proceeding to sarcolemma remodeling. On the other hand, ANXA1 and A2 exhibit a progressive cytoplasmic recruitment, likely by interacting with intracellular vesicles, in order to form the lipid patch required for membrane resealing. Once the sarcolemma has been resealed, ANXA1 is released from the site of the membrane injury and returns to the cytosol, while ANXA2 remains accumulated close to the wounding site on the cytoplasmic side. On the other side of the repaired sarcolemma are ANXA4 and ANXA6 that face the extracellular milieu, where they are concentrated in a dense structure, the cap subdomain. The proposed model provides a basis for the identification of cellular dysregulations in the membrane repair of dystrophic human muscle cells.
    Keywords:  annexin; correlative light and electron microscopy; membrane repair; skeletal muscle
  23. Cells. 2022 Feb 17. pii: 713. [Epub ahead of print]11(4):
      BACKGROUND: Adiponectin (Adn), released by adipocytes and other cell types such as skeletal muscle, has insulin-sensitizing and anti-inflammatory properties. Sphingosine 1-phosphate (S1P) is reported to act as effector of diverse biological actions of Adn in different tissues. S1P is a bioactive sphingolipid synthesized by the phosphorylation of sphingosine catalyzed by sphingosine kinase (SK) 1 and 2. Consolidated findings support the key role of S1P in the biology of skeletal muscle.METHODS AND RESULTS: Here we provide experimental evidence that S1P signalling is modulated by globular Adn treatment being able to increase the phosphorylation of SK1/2 as well as the mRNA expression levels of S1P4 in C2C12 myotubes. These findings were confirmed by LC-MS/MS that showed an increase of S1P levels after Adn treatment. Notably, the involvement of S1P axis in Adn action was highlighted since, when SK1 and 2 were inhibited by PF543 and ABC294640 inhibitors, respectively, not only the electrophysiological changes but also the increase of oxygen consumption and of aminoacid levels induced by the hormone, were significantly inhibited.
    CONCLUSION: Altogether, these findings show that S1P biosynthesis is necessary for the electrophysiological properties and oxidative metabolism of Adn in skeletal muscle cells.
    Keywords:  NMR metabolomics; S1P receptors; adiponectin; electrophysiological properties; oxygen consumption; skeletal muscle; sphingosine 1-phosphate; sphingosine kinase
  24. Nat Commun. 2022 Feb 24. 13(1): 1039
      The contractile properties of adult myofibers are shaped by their Myosin heavy chain isoform content. Here, we identify by snATAC-seq a 42 kb super-enhancer at the locus regrouping the fast Myosin genes. By 4C-seq we show that active fast Myosin promoters interact with this super-enhancer by DNA looping, leading to the activation of a single promoter per nucleus. A rainbow mouse transgenic model of the locus including the super-enhancer recapitulates the endogenous spatio-temporal expression of adult fast Myosin genes. In situ deletion of the super-enhancer by CRISPR/Cas9 editing demonstrates its major role in the control of associated fast Myosin genes, and deletion of two fast Myosin genes at the locus reveals an active competition of the promoters for the shared super-enhancer. Last, by disrupting the organization of fast Myosin, we uncover positional heterogeneity within limb skeletal muscles that may underlie selective muscle susceptibility to damage in certain myopathies.
  25. Bone Res. 2022 Feb 25. 10(1): 22
      The cells of origin of neurogenic heterotopic ossifications (NHOs), which develop frequently in the periarticular muscles following spinal cord injuries (SCIs) and traumatic brain injuries, remain unclear because skeletal muscle harbors two progenitor cell populations: satellite cells (SCs), which are myogenic, and fibroadipogenic progenitors (FAPs), which are mesenchymal. Lineage-tracing experiments using the Cre recombinase/LoxP system were performed in two mouse strains with the fluorescent protein ZsGreen specifically expressed in either SCs or FAPs in skeletal muscles under the control of the Pax7 or Prrx1 gene promoter, respectively. These experiments demonstrate that following muscle injury, SCI causes the upregulation of PDGFRα expression on FAPs but not SCs and the failure of SCs to regenerate myofibers in the injured muscle, with reduced apoptosis and continued proliferation of muscle resident FAPs enabling their osteogenic differentiation into NHOs. No cells expressing ZsGreen under the Prrx1 promoter were detected in the blood after injury, suggesting that the cells of origin of NHOs are locally derived from the injured muscle. We validated these findings using human NHO biopsies. PDGFRα+ mesenchymal cells isolated from the muscle surrounding NHO biopsies could develop ectopic human bones when transplanted into immunocompromised mice, whereas CD56+ myogenic cells had a much lower potential. Therefore, NHO is a pathology of the injured muscle in which SCI reprograms FAPs to undergo uncontrolled proliferation and differentiation into osteoblasts.
  26. Aging Cell. 2022 Feb 24. e13569
      Age-related muscle atrophy and weakness, or sarcopenia, are significant contributors to compromised health and quality of life in the elderly. While the mechanisms driving this pathology are not fully defined, reactive oxygen species, neuromuscular junction (NMJ) disruption, and loss of innervation are important risk factors. The goal of this study is to determine the impact of mitochondrial hydrogen peroxide on neurogenic atrophy and contractile dysfunction. Mice with muscle-specific overexpression of the mitochondrial H2 O2  scavenger peroxiredoxin3 (mPRDX3) were crossed to Sod1KO mice, an established mouse model of sarcopenia, to determine whether reduced mitochondrial H2 O2 can prevent or delay the redox-dependent sarcopenia. Basal rates of H2 O2  generation were elevated in isolated muscle mitochondria from Sod1KO, but normalized by mPRDX3 overexpression. The mPRDX3 overexpression prevented the declines in maximum mitochondrial oxygen consumption rate and calcium retention capacity in Sod1KO. Muscle atrophy in Sod1KO was mitigated by ~20% by mPRDX3 overexpression, which was associated with an increase in myofiber cross-sectional area. With direct muscle stimulation, maximum isometric specific force was reduced by ~20% in Sod1KO mice, and mPRDX3 overexpression preserved specific force at wild-type levels. The force deficit with nerve stimulation was exacerbated in Sod1KO compared to direct muscle stimulation, suggesting NMJ disruption in Sod1KO. Notably, this defect was not resolved by overexpression of mPRDX3. Our findings demonstrate that muscle-specific PRDX3 overexpression reduces mitochondrial H2 O2  generation, improves mitochondrial function, and mitigates loss of muscle quantity and quality, despite persisting NMJ impairment in a murine model of redox-dependent sarcopenia.
    Keywords:  aging; hydrogen peroxide; mitochondria; peroxiredoxin3; sarcopenia
  27. Adv Physiol Educ. 2022 Feb 24.
      There is a widely variable breadth of coverage of skeletal muscle content across both undergraduate human anatomy and undergraduate anatomy and physiology (A&P) courses. In response to the need for a more global understanding of the content taught in undergraduate anatomy courses, we developed an online survey (administered through Qualtrics) where both human Anatomy and Anatomy & Physiology faculty could report skeletal muscle coverage in their courses. The survey also collected comparative demographic institutional data such as the type of institution (community college versus 4-year), course format, and geographic location of the undergraduate institution. Skeletal muscles surveyed included those listed and described in a typical undergraduate human anatomy text (1). The data indicated some interesting instructional trends regarding muscular system coverage. First, both the identification and action of specific muscles are taught at a higher frequency than the teaching of either attachments or innervation. Innervation of specific skeletal muscles is the least taught concept. In each body region, certain muscles were taught with higher frequency than others. This research shows there is a global trend in teaching identification of specific skeletal muscles within each body region and often this is accompanied by teaching actions of said muscles. These general instructional trends may increase our understanding of the anatomical and physiological education our undergraduate students are receiving and will lead to further critical conversations about content development and curriculum.
    Keywords:  anatomy and physiology curriculum; anatomy curriculum; curricular comparison; muscular system
  28. Int J Mol Sci. 2022 Feb 11. pii: 1983. [Epub ahead of print]23(4):
      FSHD is caused by loss of silencing of the DUX4 gene, but the DUX4 protein has not yet been directly detected immunohistologically in affected muscle, raising the possibility that DUX4 expression may occur at time points prior to obtaining adult biopsies for analysis, with consequent perturbations of muscle being responsible for disease progression. To test the extent to which muscle can regenerate following DUX4-mediated degeneration, we employed an animal model with reversible DUX4 expression, the iDUX4pA;HSA mouse. We find that muscle histology does recover substantially after DUX4 expression is switched off, with the extent of recovery correlating inversely with the duration of prior DUX4 expression. However, despite fairly normal muscle histology, and recovery of most cytological parameters, the fibroadipogenic progenitor compartment, which is significantly elevated during bouts of fiber-specific DUX4 expression, does not return to basal levels, even many weeks after a single burst of DUX4 expression. We find that muscle that has recovered from a DUX4 burst acquires a propensity for severe fibrosis, which can be revealed by subsequent cardiotoxin injuries. These results suggest that a past history of DUX4 expression leads to maintained pro-fibrotic alterations in the cellular physiology of muscle, with potential implications for therapeutic approaches.
    Keywords:  DUX4; facioscapulohumeral muscular dystrophy; fibroadiopgenic progenitors; fibrosis
  29. Cell Rep. 2022 Feb 22. pii: S2211-1247(22)00133-4. [Epub ahead of print]38(8): 110409
      Thyroid hormones (THs) are key metabolic regulators coordinating short- and long-term energy needs. In skeletal muscle, THs modulate energy metabolism in pathophysiological conditions. Indeed, hypo- and hyperthyroidism are leading causes of muscle weakness and strength; however, the metabolic pathways underlying these effects are still poorly understood. Using molecular, biochemical, and isotope-tracing approaches combined with mass spectrometry and denervation experiments, we find that THs regulate glutamine metabolism and anaplerotic fluxes by up-regulating the glutamate pyruvate transaminase 2 (GPT2) gene. In humans, GPT2 autosomal recessive mutations cause a neurological syndrome characterized by intellectual disability, microcephaly, and progressive motor symptoms. Here, we demonstrate a role of the TH/GPT2 axis in skeletal muscle in which it regulates muscle weight and fiber diameter in resting and atrophic conditions and results in protection from muscle loss during atrophy. These results describe an anabolic route by which THs rewire glutamine metabolism toward the maintenance of muscle mass.
    Keywords:  GPT2; glutamine metabolism; skeletal muscle; thyroid hormone; type 2 deiodinase
  30. Bone Joint Res. 2022 Feb;11(2): 121-133
      AIMS: The decrease in the number of satellite cells (SCs), contributing to myofibre formation and reconstitution, and their proliferative capacity, leads to muscle loss, a condition known as sarcopenia. Resistance training can prevent muscle loss; however, the underlying mechanisms of resistance training effects on SCs are not well understood. We therefore conducted a comprehensive transcriptome analysis of SCs in a mouse model.METHODS: We compared the differentially expressed genes of SCs in young mice (eight weeks old), middle-aged (48-week-old) mice with resistance training intervention (MID+ T), and mice without exercise (MID) using next-generation sequencing and bioinformatics.
    RESULTS: After the bioinformatic analysis, the PI3K-Akt signalling pathway and the regulation of actin cytoskeleton in particular were highlighted among the top ten pathways with the most differentially expressed genes involved in the young/MID and MID+ T/MID groups. The expression of Gng5, Atf2, and Rtor in the PI3K-Akt signalling pathway was higher in the young and MID+ T groups compared with the MID group. Similarly, Limk1, Arhgef12, and Araf in the regulation of the actin cytoskeleton pathway had a similar bias. Moreover, the protein expression profiles of Atf2, Rptor, and Ccnd3 in each group were paralleled with the results of NGS.
    CONCLUSION: Our results revealed that age-induced muscle loss might result from age-influenced genes that contribute to muscle development in SCs. After resistance training, age-impaired genes were reactivated, and age-induced genes were depressed. The change fold in these genes in the young/MID mice resembled those in the MID + T/MID group, suggesting that resistance training can rejuvenate the self-renewing ability of SCs by recovering age-influenced genes to prevent sarcopenia. Cite this article: Bone Joint Res 2022;11(2):121-133.
    Keywords:  Age-induced sarcopenia; RNA; Resistance training; Satellite cells; actin; antibodies; cancer; grip strength; mouse model; muscle fibre; muscle strength loss; resistance training; western blotting
  31. Biomedicines. 2022 Jan 28. pii: 302. [Epub ahead of print]10(2):
      Pompe disease is an inherited neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). The most severe form is infantile-onset Pompe disease, presenting shortly after birth with symptoms of cardiomyopathy, respiratory failure and skeletal muscle weakness. Late-onset Pompe disease is characterized by a slower disease progression, primarily affecting skeletal muscles. Despite recent advancements in enzyme replacement therapy management several limitations remain using this therapeutic approach, including risks of immunogenicity complications, inability to penetrate CNS tissue, and the need for life-long therapy. The next wave of promising single therapy interventions involves gene therapies, which are entering into a clinical translational stage. Both adeno-associated virus (AAV) vectors and lentiviral vector (LV)-mediated hematopoietic stem and progenitor (HSPC) gene therapy have the potential to provide effective therapy for this multisystemic disorder. Optimization of viral vector designs, providing tissue-specific expression and GAA protein modifications to enhance secretion and uptake has resulted in improved preclinical efficacy and safety data. In this review, we highlight gene therapy developments, in particular, AAV and LV HSPC-mediated gene therapy technologies, to potentially address all components of the neuromuscular associated Pompe disease pathology.
    Keywords:  Pompe disease; adeno-associated viral vector; enzyme replacement therapy; hematopoietic stem cell; lentiviral vectors
  32. Proc Natl Acad Sci U S A. 2022 Mar 01. pii: e2109576119. [Epub ahead of print]119(9):
      The mechanoenzyme dynamin 2 (DNM2) is crucial for intracellular organization and trafficking. DNM2 is mutated in dominant centronuclear myopathy (DNM2-CNM), a muscle disease characterized by defects in organelle positioning in myofibers. It remains unclear how the in vivo functions of DNM2 are regulated in muscle. Moreover, there is no therapy for DNM2-CNM to date. Here, we overexpressed human amphiphysin 2 (BIN1), a membrane remodeling protein mutated in other CNM forms, in Dnm2 RW/+ and Dnm2 RW/RW mice modeling mild and severe DNM2-CNM, through transgenesis or with adeno-associated virus (AAV). Increasing BIN1 improved muscle atrophy and main histopathological features of Dnm2 RW/+ mice and rescued the perinatal lethality and survival of Dnm2 RW/RW mice. In vitro experiments showed that BIN1 binds and recruits DNM2 to membrane tubules, and that the BIN1-DNM2 complex regulates tubules fission. Overall, BIN1 is a potential therapeutic target for dominant centronuclear myopathy linked to DNM2 mutations.
    Keywords:  amphiphysin; congenital myopathy; dynamin; gene therapy; membrane remodeling
  33. Nat Metab. 2022 Feb 24.
      Extensive research has shown that interleukin 6 (IL-6) is a multifunctional molecule that is both proinflammatory and anti-inflammatory, depending on the context. Here, we combine an evolutionary perspective with physiological data to propose that IL-6's context-dependent effects on metabolism reflect its adaptive role for short-term energy allocation. This energy-allocation role is especially salient during physical activity, when skeletal muscle releases large amounts of IL-6. We predict that during bouts of physical activity, myokine IL-6 fulfills the three main characteristics of a short-term energy allocator: it is secreted from muscle in response to an energy deficit, it liberates somatic energy through lipolysis and it enhances muscular energy uptake and transiently downregulates immune function. We then extend this model of energy allocation beyond myokine IL-6 to reinterpret the roles that IL-6 plays in chronic inflammation, as well as during COVID-19-associated hyperinflammation and multiorgan failure.
  34. Int J Mol Sci. 2022 Feb 20. pii: 2339. [Epub ahead of print]23(4):
      Myotonic dystrophy (DM) is a dominantly inherited multisystemic disorder affecting various organs, such as skeletal muscle, heart, the nervous system, and the eye. Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are caused by expanded CTG and CCTG repeats, respectively. In both forms, the mutant transcripts containing expanded repeats aggregate as nuclear foci and sequester several RNA-binding proteins, resulting in alternative splicing dysregulation. Although certain alternative splicing events are linked to the clinical DM phenotypes, the molecular mechanisms underlying multiple DM symptoms remain unclear. Interestingly, multi-systemic DM manifestations, including muscle weakness, cognitive impairment, cataract, and frontal baldness, resemble premature aging. Furthermore, cellular senescence, a critical contributor to aging, is suggested to play a key role in DM cellular pathophysiology. In particular, several senescence inducers including telomere shortening, mitochondrial dysfunction, and oxidative stress and senescence biomarkers such as cell cycle inhibitors, senescence-associated secretory phenotype, chromatin reorganization, and microRNA have been implicated in DM pathogenesis. In this review, we focus on the clinical similarities between DM and aging, and summarize the involvement of cellular senescence in DM and the potential application of anti-aging DM therapies.
    Keywords:  accelerated aging; alternative splicing; repeat expansion
  35. PLoS One. 2022 ;17(2): e0264146
      Triadin, a protein of the sarcoplasmic reticulum (SR) of striated muscles, anchors the calcium-storing protein calsequestrin to calcium release RyR channels at the junction with t-tubules, and modulates these channels by conformational effects. Triadin ablation induces structural SR changes and alters the expression of other proteins. Here we quantify alterations of calcium signaling in single skeletal myofibers of constitutive triadin-null mice. We find higher resting cytosolic and lower SR-luminal [Ca2+], 40% lower calsequestrin expression, and more CaV1.1, RyR1 and SERCA1. Despite the increased CaV1.1, the mobile intramembrane charge was reduced by ~20% in Triadin-null fibers. The initial peak of calcium release flux by pulse depolarization was minimally altered in the null fibers (revealing an increase in peak calcium permeability). The "hump" phase that followed, attributable to calcium detaching from calsequestrin, was 25% lower, a smaller change than expected from the reduced calsequestrin content and calcium saturation. The exponential decay rate of calcium transients was 25% higher, consistent with the higher SERCA1 content. Recovery of calcium flux after a depleting depolarization was faster in triadin-null myofibers, consistent with the increased uptake rate and lower SR calsequestrin content. In sum, the triadin knockout determines an increased RyR1 channel openness, which depletes the SR, a substantial loss of calsequestrin and gains in other couplon proteins. Powerful functional compensations ensue: activation of SOCE that increases [Ca2+]cyto; increased SERCA1 activity, which limits the decrease in [Ca2+]SR and a restoration of SR calcium storage of unknown substrate. Together, they effectively limit the functional loss in skeletal muscles.
  36. J Vis Exp. 2022 Feb 07.
      With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.
  37. PLoS One. 2022 ;17(2): e0264171
      We investigated whether time-of-day dependent changes in the rat soleus (SOL) muscle size, after eccentric exercises, operate via the mechanistic target of rapamycin (mTOR) signaling pathway. For our first experiment, we assigned 9-week-old male Wistar rats randomly into four groups: light phase (zeitgeber time; ZT6) non-trained control, dark phase (ZT18) non-trained control, light phase-trained, and dark phase-trained. Trained animals performed 90 min of downhill running once every 3 d for 8 weeks. The second experiment involved dividing 9-week-old male Wistar rats to control and exercise groups. The latter were subjected to 15 min of downhill running at ZT6 and ZT18. The absolute (+12.8%) and relative (+9.4%) SOL muscle weights were higher in the light phase-trained group. p70S6K phosphorylation ratio was 42.6% higher in the SOL muscle of rats that had exercised only in light (non-trained ZT6). Collectively, the degree of muscle hypertrophy in SOL is time-of-day dependent, perhaps via the mTOR/p70S6K signaling.
  38. Metabolites. 2022 Jan 28. pii: 125. [Epub ahead of print]12(2):
      Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein implicated in various functions, including metabolism, tissue regeneration, and functional homeostasis. SPARC/Sparc declines with ageing but increases with exercise. We aim to verify two hypotheses: (1) SPARC deficiency leads to an ageing-like phenotype (metabolic decline, muscle loss, etc.), and (2) SPARC overexpression would mimic exercise, counteract ageing, and improve age-related changes. Our mice experiments are divided into two parts. First, we explore the consequences of Sparc knockout (KO) and compare them to the ageing effects. We also observe the effects of exercise. In the second part, we study the effects of SPARC overexpression and compare them to the exercise benefits. At the end, we make an analysis of the results to point out the analogies between Sparc KO and the ageing-like phenotype on the one hand and make comparisons between SPARC overexpression and exercise in the context of exercise counteracting ageing. The measurements were mainly related to tissue weights, adiposity, metabolism, and muscle strength. The main findings are that Sparc KO reduced glucose tolerance, muscle glucose transporter expression, and abdominal adipose tissue weight but increased glycogen content in the muscle. SPARC overexpression increased muscle strength, muscle mass, and expressions of the muscle glucose transporter and mitochondrial oxidative phosphorylation but lowered the glycemia and the adiposity, especially in males. Collectively, these findings, and the data we have previously reported, show that Sparc KO mice manifest an ageing-like phenotype, whereas SPARC overexpression and exercise generate similar benefits. The benefits are towards counteracting both the SPARC deficiency-induced ageing-like phenotype as well as reversing the age-related changes. The potential applications of these findings are to build/optimize Sparc KO-based animal models of various health conditions and, on the other hand, to develop therapies based on introducing SPARC or targeting SPARC-related pathways to mimic exercise against age-related and metabolic disorders.
    Keywords:  ageing; exercise; metabolism; secreted protein acidic and rich in cysteine (SPARC)
  39. iScience. 2022 Feb 18. 25(2): 103848
      Cellular senescence is a driver of many age-related pathologies. There is an active search for pharmaceuticals termed senolytics that can mitigate or remove senescent cells in vivo by targeting genes that promote the survival of senescent cells. We utilized single-cell RNA sequencing to identify CRYAB as a robust senescence-induced gene and potential target for senolysis. Using chemical inhibitor screening for CRYAB disruption, we identified 25-hydroxycholesterol (25HC), an endogenous metabolite of cholesterol biosynthesis, as a potent senolytic. We then validated 25HC as a senolytic in mouse and human cells in culture and in vivo in mouse skeletal muscle. Thus, 25HC represents a potential class of senolytics, which may be useful in combating diseases or physiologies in which cellular senescence is a key driver.
    Keywords:  Biological sciences; Cell biology; Molecular biology; Transcriptomics
  40. J Cachexia Sarcopenia Muscle. 2022 Feb 20.
      We performed a systematic review, meta-analysis, and meta-regression to determine if increasing daily protein ingestion contributes to gaining lean body mass (LBM), muscle strength, and physical/functional test performance in healthy subjects. A protocol for the present study was registered (PROSPERO, CRD42020159001), and a systematic search of Medline, Embase, CINAHL, and Web of Sciences databases was undertaken. Only randomized controlled trials (RCT) where participants increased their daily protein intake and were healthy and non-obese adults were included. Research questions focused on the main effects on the outcomes of interest and subgroup analysis, splitting the studies by participation in a resistance exercise (RE), age (<65 or ≥65 years old), and levels of daily protein ingestion. Three-level random-effects meta-analyses and meta-regressions were conducted on data from 74 RCT. Most of the selected studies tested the effects of additional protein ingestion during RE training. The evidence suggests that increasing daily protein ingestion may enhance gains in LBM in studies enrolling subjects in RE (SMD [standardized mean difference] = 0.22, 95% CI [95% confidence interval] 0.14:0.30, P < 0.01, 62 studies, moderate level of evidence). The effect on LBM was significant in subjects ≥65 years old ingesting 1.2-1.59 g of protein/kg/day and for younger subjects (<65 years old) ingesting ≥1.6 g of protein/kg/day submitted to RE. Lower-body strength gain was slightly higher by additional protein ingestion at ≥1.6 g of protein/kg/day during RE training (SMD = 0.40, 95% CI 0.09:0.35, P < 0.01, 19 studies, low level of evidence). Bench press strength is slightly increased by ingesting more protein in <65 years old subjects during RE training (SMD = 0.18, 95% CI 0.03:0.33, P = 0.01, 32 studies, low level of evidence). The effects of ingesting more protein are unclear when assessing handgrip strength and only marginal for performance in physical function tests. In conclusion, increasing daily protein ingestion results in small additional gains in LBM and lower body muscle strength gains in healthy adults enrolled in resistance exercise training. There is a slight effect on bench press strength and minimal effect performance in physical function tests. The effect on handgrip strength is unclear.
    Keywords:  Muscle mass; Muscle strength; Physical function; Protein quantity
  41. J Am Med Dir Assoc. 2022 Feb 17. pii: S1525-8610(22)00097-4. [Epub ahead of print]
      OBJECTIVE: This study aimed to determine the comparative effectiveness of interventions in treatment of sarcopenia. The primary outcome was the measure of treatment effect on muscle mass, and secondary outcomes were the treatment effect on muscle strength and physical performance.DESIGN: Systematic review and network meta-analysis (NMA).
    SETTING AND PARTICIPANTS: Participants with sarcopenia receiving interventions targeting sarcopenia in any setting.
    METHODS: Data sources: Relevant RCTs were identified by a systematic search of several electronic databases, including CINAHL, Embase, MEDLINE, and the Cochrane Central Registry of Controlled Trials (CENTRAL) from January 1995 to July 2019. Duplicate title and abstract and full-text screening, data extraction, and risk of bias assessment were performed.
    DATA EXTRACTION: All RCTs examining sarcopenia interventions [mixed exercise (combined aerobic and resistance exercise), aerobic exercise, resistance exercise, balance exercise, physical activity and protein or nutrition supplementation, acupuncture, whole-body vibration, protein supplement or interventions to increase protein intake, any nutritional intervention other than protein, and pharmacotherapy] were included. Comparators were standard care, placebo, or another intervention.
    DATA SYNTHESIS: We performed Bayesian NMA; continuous outcome data were pooled using the standardized mean difference effect size. Interventions were ranked using the surface under the cumulative ranking curve (SUCRA) for each outcome.
    RESULTS: A total of 59 RCTs were included after screening of 4315 citations and 313 full-text articles. Network meta-analysis of muscle mass outcome (including 46 RCTs, 3649 participants, 11 interventions) suggested that mixed exercise were the most effective intervention (SUCRA = 93.94%) to increase muscle mass. Physical activity and protein or nutrition supplementation, and aerobic exercise were the most effective interventions to improve muscle strength and physical performance, respectively. Overall, mixed exercise is the most effective intervention in increasing muscle mass and was one of the 3 most effective interventions in increasing muscle strength and physical performance.
    CONCLUSIONS AND IMPLICATIONS: Mixed exercise and physical activity with nutritional supplementation are the most effective sarcopenia interventions. Most of the included studies have a high risk of bias. More robust RCTs are needed to increase the confidence of our NMA results and the quality of evidence.
    Keywords:  Sarcopenia; evidence; exercise; muscle mass; muscle strength; network meta-analysis; older adults; physical performance
  42. Int J Mol Sci. 2022 Feb 16. pii: 2197. [Epub ahead of print]23(4):
      Mutations in either mitochondrial DNA (mtDNA) or nuclear genes that encode mitochondrial proteins may lead to dysfunctional mitochondria, giving rise to mitochondrial diseases. Some mitochondrial myopathies, however, present without a known underlying cause. Interestingly, methylation of mtDNA has been associated with various clinical pathologies. The present study set out to assess whether mtDNA methylation could explain impaired mitochondrial function in patients diagnosed with myopathy without known underlying genetic mutations. Enhanced mtDNA methylation was indicated by pyrosequencing for muscle biopsies of 14 myopathy patients compared to four healthy controls, at selected cytosines in the Cytochrome B (CYTB) gene, but not within the displacement loop (D-loop) region. The mtDNA methylation patterns of the four healthy muscle biopsies were highly consistent and showed intriguing tissue-specific differences at particular cytosines with control skin fibroblasts cultured in vitro. Within individual myopathy patients, the overall mtDNA methylation pattern correlated well between muscle and skin fibroblasts. Despite this correlation, a pilot analysis of four myopathy and five healthy fibroblast samples did not reveal a disease-associated difference in mtDNA methylation. We did, however, detect increased expression of solute carrier family 25A26 (SLC25A26), encoding the importer of S-adenosylmethionine, together with enhanced mtDNA copy numbers in myopathy fibroblasts compared to healthy controls. To confirm that pyrosequencing indeed reflected DNA methylation and not bisulfite accessibility, mass spectrometry was employed. Although no myopathy-related differences in total amount of methylated cytosines were detected at this stage, a significant contribution of contaminating nuclear DNA (nDNA) was revealed, and steps to improve enrichment for mtDNA are reported. In conclusion, in this explorative study we show that analyzing the mitochondrial genome beyond its sequence opens novel avenues to identify potential molecular biomarkers assisting in the diagnosis of unexplained myopathies.
    Keywords:  cytochrome B; mitochondrial DNA methylation; mitochondrial epigenome; myopathies