bims-moremu 2024-04-21 papers

bims-moremu

Biomed News

on Molecular regulators of muscle mass

Issue of 2024‒04‒21
23 papers selected by
Anna Vainshtein, Craft Science Inc.

Delineating the contribution of ageing and physical activity to changes in mitochondrial characteristics across the lifespan.
RANKL signaling drives skeletal muscle into the oxidative profile.
Tracking of Nascent Matrix Deposition during Muscle Stem Cell Activation across Lifespan Using Engineered Hydrogels.
Human skeletal muscle aging atlas.
Anti-apoptotic protein Bcl-2 contributes to the determination of reserve cells during myogenic differentiation of C2C12 cells.
Differential effects of cholecalciferol and calcitriol on muscle proteolysis and oxidative stress in angiotensin II-induced C2C12 myotube atrophy.
Autophagy gene expression in skeletal muscle of older individuals is associated with physical performance, muscle volume and mitochondrial function in the study of muscle, mobility and aging (SOMMA).
Plasma metabolomic markers underlying skeletal muscle mitochondrial function relationships with cognition and motor function.
Seeding competent TDP-43 persists in human patient and mouse muscle.
APOBEC2 safeguards skeletal muscle cell fate through binding chromatin and regulating transcription of non-muscle genes during myoblast differentiation.
HDACi vorinostat protects muscle from degeneration after acute rotator cuff injury in mice.
Skeletal muscle mitochondria: A potential target for postmenopausal hormone replacement therapy.
Transcutaneous carbon dioxide suppresses skeletal muscle atrophy in a mouse model of oral squamous cell carcinoma.
Sex differences in cachexia and branched-chain amino acid metabolism following chemotherapy in mice.
On the role of dysferlin in striated muscle: membrane repair, t-tubules and Ca2+ handling.
Characterization of NEB pathogenic variants in patients reveals novel nemaline myopathy disease mechanisms and omecamtiv mecarbil force effects.
Hepatic signal transducer and activator of transcription-3 signalling drives early-stage pancreatic cancer cachexia via suppressed ketogenesis.
Effect of 2-aminoethoxydiphenyl borate on the functions of mouse skeletal muscle mitochondria.
HOIL-1L deficiency induces cell cycle alteration which causes immaturity of skeletal muscle and cardiomyocytes.
Age-Dependent Changes in the Production of Mitochondrial Reactive Oxygen Species in Human Skeletal Muscle.
Molecular Transducers of Physical Activity Consortium (MoTrPAC): Human Studies Design and Protocol.
Uremia Impedes Skeletal Myocyte Myomixer Expression and Fusogenic Activity: Implication for Uremic Sarcopenia.
DNA methylation of skeletal muscle function-related secretary factors identifies FGF2 as a potential biomarker for sarcopenia.

Mol Aspects Med. 2024 Apr 15. pii: S0098-2997(24)00031-1. [Epub ahead of print]97 101272

Delineating the contribution of ageing and physical activity to changes in mitochondrial characteristics across the lifespan.

Matthew J-C Lee, Nicholas J Saner, Alessandra Ferri, Esther García-Domínguez, James R Broatch, David J Bishop.

Ageing is associated with widespread physiological changes prominent within all tissues, including skeletal muscle and the brain, which lead to a decline in physical function. To tackle the growing health and economic burdens associated with an ageing population, the concept of healthy ageing has become a major research priority. Changes in skeletal muscle mitochondrial characteristics have been suggested to make an important contribution to the reductions in skeletal muscle function with age, and age-related changes in mitochondrial content, respiratory function, morphology, and mitochondrial DNA have previously been reported. However, not all studies report changes in mitochondrial characteristics with ageing, and there is increasing evidence to suggest that physical activity (or inactivity) throughout life is a confounding factor when interpreting age-associated changes. Given that physical activity is a potent stimulus for inducing beneficial adaptations to mitochondrial characteristics, delineating the influence of physical activity on the changes in skeletal muscle that occur with age is complicated. This review aims to summarise our current understanding and knowledge gaps regarding age-related changes to mitochondrial characteristics within skeletal muscle, as well as to provide some novel insights into brain mitochondria, and to propose avenues of future research and targeted interventions. Furthermore, where possible, we incorporate discussions of the modifying effects of physical activity, exercise, and training status, to purported age-related changes in mitochondrial characteristics.

DOI: https://doi.org/10.1016/j.mam.2024.101272
J Bone Miner Res. 2024 Apr 15. pii: zjae058. [Epub ahead of print]

RANKL signaling drives skeletal muscle into the oxidative profile.

Paulo Henrique Cavalcanti de Araújo, Maria Eduarda Ramos Cezine, Anderson Vulczak, Luiz Carlos Vieira, Flávia Sayuri Matsuo, Júlia Maranghetti Remoto, Audrei Dos Reis Santos, Elen Haruka Miyabara, Luciane Carla Alberici, Mariana Kiomy Osako.

  The bone-muscle unit refers to the reciprocal regulation between bone and muscle by mechanical interaction and tissue communication via soluble factors. The receptor activator of NF-κB ligand (RANKL) stimulation induces mitochondrial biogenesis and increases the oxidative capacity in osteoclasts and adipocytes. RANKL may bind to the membrane bound receptor activator of NF-κB (RANK) or to osteoprotegerin (OPG), a decoy receptor that inhibits RANK-RANKL activation. RANK is highly expressed in skeletal muscle, but the contribution of RANKL to healthy skeletal muscle fiber remains elusive. Here we show that RANKL stimulation in C2C12-derived myotubes induced activation of mitochondrial biogenesis pathways as detected by RNA-seq and western blot. RANKL expanded the mitochondrial reticulum, as shown by mitochondrial DNA quantification and MitoTracker staining, and boosted the spare respiratory capacity. Using MEK and MAPK inhibitors, we found that RANKL signals via ERK and p38 to induce mitochondrial biogenesis. The soleus from OPG-/- and OPG+/- mice showed higher respiratory rates compared to C57BL6/J wild-type (WT) mice, which correlates with high serum RANKL levels. RANKL infusion using a mini-osmotic pump in WT mice increased the number of mitochondria, boosted the respiratory rate, increased succinate dehydrogenase (SDH) activity in skeletal muscle, and improved the fatigue resistance of gastrocnemius. Therefore, our findings reveal a new role of RANKL as an osteokine-like protein that impacts muscle fiber metabolism.

Keywords:  OPG; RANKL; fiber type switch; mitochondrial biogenesis; skeletal muscle

DOI:  https://doi.org/10.1093/jbmr/zjae058
Adv Biol (Weinh). 2024 Apr 14. e2400091

Tracking of Nascent Matrix Deposition during Muscle Stem Cell Activation across Lifespan Using Engineered Hydrogels.

Pamela Duran, Benjamin A Yang, Eleanor Plaster, Madeline Eiken, Claudia Loebel, Carlos A Aguilar.

  Adult stem cells occupy a niche that contributes to their function, but how stem cells rebuild their microenvironment after injury remains an open-ended question. Herein, biomaterial-based systems and metabolic labeling are utilized to evaluate how skeletal muscle stem cells deposit extracellular matrix. Muscle stem cells and committed myoblasts are observed to generate less nascent matrix than muscle resident fibro-adipogenic progenitors. When cultured on substrates that matched the stiffness of physiological uninjured and injured muscles, muscle stem cells increased nascent matrix deposition with activation kinetics. Reducing the ability to deposit nascent matrix by an inhibitor of vesicle trafficking (Exo-1) attenuated muscle stem cell function and mimicked impairments observed from muscle stem cells isolated from old muscles. Old muscle stem cells are observed to deposit less nascent matrix than young muscle stem cells, which is rescued with therapeutic supplementation of insulin-like growth factors. These results highlight the role of nascent matrix production with muscle stem cell activation.

Keywords:  biomaterials; extracellular matrix; hyaluronic acid; protein labeling; skeletal muscle

DOI:  https://doi.org/10.1002/adbi.202400091
Nat Aging. 2024 Apr 15.

Human skeletal muscle aging atlas.

Veronika R Kedlian, Yaning Wang, Tianliang Liu, Xiaoping Chen, Liam Bolt, Catherine Tudor, Zhuojian Shen, Eirini S Fasouli, Elena Prigmore, Vitalii Kleshchevnikov, Jan Patrick Pett, Tong Li, John E G Lawrence, Shani Perera, Martin Prete, Ni Huang, Qin Guo, Xinrui Zeng, Lu Yang, Krzysztof Polański, Nana-Jane Chipampe, Monika Dabrowska, Xiaobo Li, Omer Ali Bayraktar, Minal Patel, Natsuhiko Kumasaka, Krishnaa T Mahbubani, Andy Peng Xiang, Kerstin B Meyer, Kourosh Saeb-Parsy, Sarah A Teichmann, Hongbo Zhang.

Skeletal muscle aging is a key contributor to age-related frailty and sarcopenia with substantial implications for global health. Here we profiled 90,902 single cells and 92,259 single nuclei from 17 donors to map the aging process in the adult human intercostal muscle, identifying cellular changes in each muscle compartment. We found that distinct subsets of muscle stem cells exhibit decreased ribosome biogenesis genes and increased CCL2 expression, causing different aging phenotypes. Our atlas also highlights an expansion of nuclei associated with the neuromuscular junction, which may reflect re-innervation, and outlines how the loss of fast-twitch myofibers is mitigated through regeneration and upregulation of fast-type markers in slow-twitch myofibers with age. Furthermore, we document the function of aging muscle microenvironment in immune cell attraction. Overall, we present a comprehensive human skeletal muscle aging resource ( https://www.muscleageingcellatlas.org/ ) together with an in-house mouse muscle atlas to study common features of muscle aging across species.

DOI: https://doi.org/10.1038/s43587-024-00613-3
In Vitro Cell Dev Biol Anim. 2024 Apr 15.

Anti-apoptotic protein Bcl-2 contributes to the determination of reserve cells during myogenic differentiation of C2C12 cells.

Yosuke Nagata, Jun Tomimori, Tomoharu Hagiwara.

  Skeletal muscle's regenerative ability is vital for maintaining muscle function, but chronic diseases like Duchenne muscular dystrophy can deplete this capacity. Muscle satellite cells, quiescent in normal situations, are activated during muscle injury, expressing myogenic regulatory factors, and producing myogenic progenitor cells. It was reported that muscle stem cells in primary culture and reserve cells in C2C12 cells express anti-apoptotic protein Bcl-2. Although the role of Bcl-2 expressed in myogenic cells has been thought to be to enhance cell viability, we hypothesized that Bcl-2 may promote the formation of reserve cells. The expression pattern analysis showed the expression of Bcl-2 in undifferentiated mononucleated cells, emphasizing its usefulness as a reserve cell marker and reminding us that cells expressing Bcl-2 have low proliferative potential. Silencing of Bcl-2 by transfection with siRNA decreased cell viability and the number of reserve cells, while overexpression of Bcl-2 not only increases cell viability but also inhibits muscle differentiation and proliferation. These results emphasize dual roles of Bcl-2 in protecting cells from apoptosis and contributing to reserve cell formation by regulating myoblast proliferation and/or differentiation. Overall, the study sheds light on the multifaceted role of Bcl-2 in the maintenance of skeletal muscle regeneration.

Keywords:  Bcl-2; Muscle satellite cells; Quiescence; Reserve cells; Skeletal muscle regeneration

DOI:  https://doi.org/10.1007/s11626-024-00905-3
Physiol Rep. 2024 Apr;12(8): e16011

Differential effects of cholecalciferol and calcitriol on muscle proteolysis and oxidative stress in angiotensin II-induced C2C12 myotube atrophy.

Muthita Hirunsai, Ratchakrit Srikuea.

  Renin-angiotensin system activation contributes to skeletal muscle atrophy in aging individuals with chronic diseases. We aimed to explore the effects of cholecalciferol (VD3) and calcitriol (1,25VD3) on signaling of muscle proteolysis and oxidative stress in myotubes challenged with angiotensin II (AII). The mouse C2C12 myotubes were assigned to vehicle, AII, AII + VD3, AII + 1,25VD3, and AII + losartan groups. The expression levels of muscle-specific E3 ubiquitin ligase proteins, autophagy-related proteins, and oxidative stress markers were investigated. We demonstrated the diverse effects of VD3 and 1,25VD3 on AII-induced myotube atrophy. The myotube diameter was preserved by treatment with 100 nM VD3 and losartan, while 1 and 10 nM 1,25VD3 increased levels of FoxO3a, MuRF1, and atrogin-1 protein expression in myotubes exposed to AII. Treatment with AII + 10 nM 1,25VD3 resulted in the upregulation of LC3B-II, LC3B-II/LC3B-I, and mature cathepsin L, which are autophagic marker proteins. The p62/SQSTM1 protein was downregulated and vitamin D receptor was upregulated after treatment with AII + 10 nM 1,25VD3. A cellular redox imbalance was observed as AII + 10 nM 1,25VD3-induced reactive oxygen species and NADPH oxidase-2 overproduction, and these changes were associated with an inadequate response of antioxidant superoxide dismutase-1 and catalase proteins. Collectively, these findings provide a translational perspective on the role of vitamin D3 in alleviating muscle atrophy related to high levels of AII.

Keywords:  autophagy; cathepsin; muscle atrophy; p62/SQSTM1; reactive oxygen species; vitamin D

DOI:  https://doi.org/10.14814/phy2.16011
Aging Cell. 2024 Apr 16. e14118

Autophagy gene expression in skeletal muscle of older individuals is associated with physical performance, muscle volume and mitochondrial function in the study of muscle, mobility and aging (SOMMA).

Paul M Coen, Zhiguang Huo, Gregory J Tranah, Haley N Barnes, Xiping Zhang, Christopher A Wolff, Kevin Wu, Peggy M Cawthon, Russell T Hepple, Frederico G S Toledo, Daniel S Evans, Olaya Santiago-Fernández, Ana Maria Cuervo, Stephen B Kritchevsky, Anne B Newman, Steven R Cummings, Karyn A Esser.

  Autophagy is essential for proteostasis, energetic balance, and cell defense and is a key pathway in aging. Identifying associations between autophagy gene expression patterns in skeletal muscle and physical performance outcomes would further our knowledge of mechanisms related with proteostasis and healthy aging. Muscle biopsies were obtained from participants in the Study of Muscle, Mobility, and Aging (SOMMA). For 575 participants, RNA was sequenced and expression of 281 genes related to autophagy regulation, mitophagy, and mTOR/upstream pathways was determined. Associations between gene expression and outcomes including mitochondrial respiration in muscle fiber bundles (MAX OXPHOS), physical performance (VO2 peak, 400 m walking speed, and leg power), and thigh muscle volume, were determined using negative binomial regression models. For autophagy, key transcriptional regulators including TFE3 and NFKB-related genes (RELA, RELB, and NFKB1) were negatively associated with outcomes. On the contrary, regulators of oxidative metabolism that also promote overall autophagy, mitophagy, and pexophagy (PPARGC1A, PPARA, and EPAS1) were positively associated with multiple outcomes. In line with this, several mitophagy, fusion, and fission-related genes (NIPSNAP2, DNM1L, and OPA1) were also positively associated with outcomes. For mTOR pathway and related genes, expression of WDR59 and WDR24, both subunits of GATOR2 complex (an indirect inhibitor of mTORC1), and PRKAG3, which is a regulatory subunit of AMPK, were negatively correlated with multiple outcomes. Our study identifies autophagy and selective autophagy such as mitophagy gene expression patterns in human skeletal muscle related to physical performance, muscle volume, and mitochondrial function in older persons which may lead to target identification to preserve mobility and independence.

Keywords:  aging; autophagy; gene expression; mTor; mitochondria; mobility; oxidative metabolism

DOI:  https://doi.org/10.1111/acel.14118
Age Ageing. 2024 Apr 01. pii: afae079. [Epub ahead of print]53(4):

Plasma metabolomic markers underlying skeletal muscle mitochondrial function relationships with cognition and motor function.

Qu Tian, Erin E Greig, Keenan A Walker, Kenneth W Fishbein, Richard G Spencer, Susan M Resnick, Luigi Ferrucci.

  BACKGROUND: Lower skeletal muscle mitochondrial function is associated with future cognitive impairment and mobility decline, but the biological underpinnings for these associations are unclear. We examined metabolomic markers underlying skeletal muscle mitochondrial function, cognition and motor function.METHODS: We analysed data from 560 participants from the Baltimore Longitudinal Study of Aging (mean age: 68.4 years, 56% women, 28% Black) who had data on skeletal muscle oxidative capacity (post-exercise recovery rate of phosphocreatine, kPCr) via 31P magnetic resonance spectroscopy and targeted plasma metabolomics using LASSO model. We then examined which kPCr-related markers were also associated with cognition and motor function in a larger sample (n = 918, mean age: 69.4, 55% women, 27% Black).
RESULTS: The LASSO model revealed 24 metabolites significantly predicting kPCr, with the top 5 being asymmetric dimethylarginine, lactic acid, lysophosphatidylcholine a C18:1, indoleacetic acid and triacylglyceride (17:1_34:3), also significant in multivariable linear regression. The kPCr metabolite score was associated with cognitive or motor function, with 2.5-minute usual gait speed showing the strongest association (r = 0.182). Five lipids (lysophosphatidylcholine a C18:1, phosphatidylcholine ae C42:3, cholesteryl ester 18:1, sphingomyelin C26:0, octadecenoic acid) and 2 amino acids (leucine, cystine) were associated with both cognitive and motor function measures.
CONCLUSION: Our findings add evidence to the hypothesis that mitochondrial function is implicated in the pathogenesis of cognitive and physical decline with aging and suggest that targeting specific metabolites may prevent cognitive and mobility decline through their effects on mitochondria. Future omics studies are warranted to confirm these findings and explore mechanisms underlying mitochondrial dysfunction in aging phenotypes.

Keywords:  cognition; metabolomics; mitochondria; motor; older people; skeletal muscle

DOI:  https://doi.org/10.1093/ageing/afae079
bioRxiv. 2024 Apr 04. pii: 2024.04.03.587918. [Epub ahead of print]

Seeding competent TDP-43 persists in human patient and mouse muscle.

Eileen M Lynch, Sara Pittman, Jil Daw, Chiseko Ikenaga, Sheng Chen, Dhruva D Dhavale, Meredith E Jackrel, Yuna M Ayala, Paul Kotzbauer, Cindy V Ly, Alan Pestronk, Thomas E Lloyd, Conrad C Weihl.

TAR DNA-binding protein 43 (TDP-43) is an RNA binding protein that accumulates as aggregates in the central nervous system of some neurodegenerative diseases. However, TDP-43 aggregation is also a sensitive and specific pathologic feature found in a family of degenerative muscle diseases termed inclusion body myopathy (IBM). TDP-43 aggregates from ALS and FTD brain lysates may serve as self-templating aggregate seeds in vitro and in vivo, supporting a prion-like spread from cell to cell. Whether a similar process occurs in IBM patient muscle is not clear. We developed a mouse model of inducible, muscle-specific cytoplasmic localized TDP-43. These mice develop muscle weakness with robust accumulation of insoluble and phosphorylated sarcoplasmic TDP-43, leading to eosinophilic inclusions, altered proteostasis and changes in TDP-43-related RNA processing that resolve with the removal of doxycycline. Skeletal muscle lysates from these mice also have seeding competent TDP-43, as determined by a FRET-based biosensor, that persists for weeks upon resolution of TDP-43 aggregate pathology. Human muscle biopsies with TDP-43 pathology also contain TDP-43 aggregate seeds. Using lysates from muscle biopsies of patients with IBM, IMNM and ALS we found that TDP-43 seeding capacity was specific to IBM. Surprisingly, TDP-43 seeding capacity anti-correlated with TDP-43 aggregate and vacuole abundance. These data support that TDP-43 aggregate seeds are present in IBM skeletal muscle and represent a unique TDP-43 pathogenic species not previously appreciated in human muscle disease.Summary: TDP-43 aggregate seeds persist in mouse and human skeletal muscle independent of large TDP-43 inclusions.

DOI: https://doi.org/10.1101/2024.04.03.587918
Proc Natl Acad Sci U S A. 2024 Apr 23. 121(17): e2312330121

APOBEC2 safeguards skeletal muscle cell fate through binding chromatin and regulating transcription of non-muscle genes during myoblast differentiation.

J Paulo Lorenzo, Linda Molla, Elias Moris Amro, Ignacio L Ibarra, Sandra Ruf, Cedrik Neber, Christos Gkougkousis, Jana Ridani, Poorani Ganesh Subramani, Jonathan Boulais, Dewi Harjanto, Alin Vonica, Javier M Di Noia, Christoph Dieterich, Judith B Zaugg, F Nina Papavasiliou.

  The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide (APOBEC) family is composed of nucleic acid editors with roles ranging from antibody diversification to RNA editing. APOBEC2, a member of this family with an evolutionarily conserved nucleic acid-binding cytidine deaminase domain, has neither an established substrate nor function. Using a cellular model of muscle differentiation where APOBEC2 is inducibly expressed, we confirmed that APOBEC2 does not have the attributed molecular functions of the APOBEC family, such as RNA editing, DNA demethylation, and DNA mutation. Instead, we found that during muscle differentiation APOBEC2 occupied a specific motif within promoter regions; its removal from those regions resulted in transcriptional changes. Mechanistically, these changes reflect the direct interaction of APOBEC2 with histone deacetylase (HDAC) transcriptional corepressor complexes. We also found that APOBEC2 could bind DNA directly, in a sequence-specific fashion, suggesting that it functions as a recruiter of HDAC to specific genes whose promoters it occupies. These genes are normally suppressed during muscle cell differentiation, and their suppression may contribute to the safeguarding of muscle cell fate. Altogether, our results reveal a unique role for APOBEC2 within the APOBEC family.

Keywords:  APOBEC family; DNA binding; muscle differentiation; safeguard factor; transcriptional regulator

DOI:  https://doi.org/10.1073/pnas.2312330121
Bone Joint Res. 2024 Apr 15. 13(4): 169-183

HDACi vorinostat protects muscle from degeneration after acute rotator cuff injury in mice.

Lara Gil-Melgosa, Rafael Llombart-Blanco, Leire Extramiana, Isabel Lacave, Gloria Abizanda, Estibaliz Miranda, Xabier Agirre, Felipe Prósper, Antonio Pineda-Lucena, Juan Pons-Villanueva, Ana Pérez-Ruiz.

Aims: Rotator cuff (RC) injuries are characterized by tendon rupture, muscle atrophy, retraction, and fatty infiltration, which increase injury severity and jeopardize adequate tendon repair. Epigenetic drugs, such as histone deacetylase inhibitors (HDACis), possess the capacity to redefine the molecular signature of cells, and they may have the potential to inhibit the transformation of the fibro-adipogenic progenitors (FAPs) within the skeletal muscle into adipocyte-like cells, concurrently enhancing the myogenic potential of the satellite cells.Methods: HDACis were added to FAPs and satellite cell cultures isolated from mice. The HDACi vorinostat was additionally administered into a RC injury animal model. Histological analysis was carried out on the isolated supra- and infraspinatus muscles to assess vorinostat anti-muscle degeneration potential.
Results: Vorinostat, a HDACi compound, blocked the adipogenic transformation of muscle-associated FAPs in culture, promoting myogenic progression of the satellite cells. Furthermore, it protected muscle from degeneration after acute RC in mice in the earlier muscle degenerative stage after tenotomy.
Conclusion: The HDACi vorinostat may be a candidate to prevent early muscular degeneration after RC injury.

DOI: https://doi.org/10.1302/2046-3758.134.BJR-2023-0292.R1
Acta Physiol (Oxf). 2024 Apr 17. e14149

Skeletal muscle mitochondria: A potential target for postmenopausal hormone replacement therapy.

Takashi Yokota.

DOI: https://doi.org/10.1111/apha.14149
PLoS One. 2024 ;19(4): e0302194

Transcutaneous carbon dioxide suppresses skeletal muscle atrophy in a mouse model of oral squamous cell carcinoma.

Aki Sasaki, Daisuke Takeda, Hotaka Kawai, Yoshiaki Tadokoro, Aki Murakami, Nanae Yatagai, Satomi Arimoto, Hitoshi Nagatsuka, Masaya Akashi, Takumi Hasegawa.

Cancer cachexia causes skeletal muscle atrophy, impacting the treatment and prognosis of patients with advanced cancer, but no treatment has yet been established to control cancer cachexia. We demonstrated that transcutaneous application of carbon dioxide (CO2) could improve local blood flow and reduce skeletal muscle atrophy in a fracture model. However, the effects of transcutaneous application of CO2 in cancer-bearing conditions are not yet known. In this study, we calculated fat-free body mass (FFM), defined as the skeletal muscle mass, and evaluated the expression of muscle atrophy markers and uncoupling protein markers as well as the cross-sectional area (CSA) to investigate whether transcutaneous application of CO2 to skeletal muscle could suppress skeletal muscle atrophy in cancer-bearing mice. Human oral squamous cell carcinoma was transplanted subcutaneously into the upper dorsal region of nude mice, and 1 week later, CO2 gas was applied to the legs twice a week for 4 weeks and FFM was calculated by bioimpedance spectroscopy. After the experiment concluded, the quadriceps were extracted, and muscle atrophy markers (muscle atrophy F-box protein (MAFbx), muscle RING-finger protein 1 (MuRF-1)) and uncoupling protein markers (uncoupling protein 2 (UCP2) and uncoupling protein 3 (UCP3)) were evaluated by real-time polymerase chain reaction and immunohistochemical staining, and CSA by hematoxylin and eosin staining. The CO2-treated group exhibited significant mRNA and protein expression inhibition of the four markers. Furthermore, immunohistochemical staining showed decreased MAFbx, MuRF-1, UCP2, and UCP3 in the CO2-treated group. In fact, the CSA in hematoxylin and eosin staining and the FFM revealed significant suppression of skeletal muscle atrophy in the CO2-treated group. We suggest that transcutaneous application of CO2 to skeletal muscle suppresses skeletal muscle atrophy in a mouse model of oral squamous cell carcinoma.

DOI: https://doi.org/10.1371/journal.pone.0302194
Physiol Rep. 2024 Apr;12(8): e16003

Sex differences in cachexia and branched-chain amino acid metabolism following chemotherapy in mice.

Stephen Mora, Gagandeep Mann, Olasunkanmi A J Adegoke.

  Chemotherapy is a major contributor to cachexia, but studies often investigate male animals. Here, we investigated whether sex modifies the effects of chemotherapy on cachexia and BCAA metabolism. Ten-week-old CD2F1 male and female mice were treated with the chemotherapy drug cocktail folfiri (50 mg/kg 5-fluorouracil, 90 mg/kg leucovorin, and 24 mg/kg CPT11) (drug) or vehicle twice a week for 6 weeks. Insulin tolerance tests were conducted and BCAA levels and metabolism were measured in plasma and tissues. Drug treatment reduced body and skeletal muscle weights and anabolic signaling in both sexes, with females showing worsened outcomes (p < 0.05 for all). Drug treatment increased plasma BCAA only in males, but BCAA concentrations in the skeletal muscle of both sexes were decreased; this decrease was more profound in males (p = 0.0097). In addition, muscle expression of the BCAA transporter LAT1 was reduced; this reduction was more severe in females (p = 0.0264). In both sexes, the (inhibitory) phosphorylation of BCKD-E1αser293 was increased along with decreased BCKD activity. In the liver, drug treatment increased BCAA concentrations and LAT1 expression, but BCKD activity was suppressed in both sexes (p < 0.05 for all). Our results demonstrate that altered BCAA metabolism may contribute to chemotherapy-induced cachexia in a sex-dependent manner.

Keywords:  branched‐chain amino acid; cachexia; chemotherapy; metabolism; sex

DOI:  https://doi.org/10.14814/phy2.16003
J Physiol. 2024 Apr 14.

On the role of dysferlin in striated muscle: membrane repair, t-tubules and Ca2+ handling.

C J Quinn, E J Cartwright, A W Trafford, K M Dibb.

  Dysferlin is a 237 kDa membrane-associated protein characterised by multiple C2 domains with a diverse role in skeletal and cardiac muscle physiology. Mutations in DYSF are known to cause various types of human muscular dystrophies, known collectively as dysferlinopathies, with some patients developing cardiomyopathy. A myriad of in vitro membrane repair studies suggest that dysferlin plays an integral role in the membrane repair complex in skeletal muscle. In comparison, less is known about dysferlin in the heart, but mounting evidence suggests that dysferlin's role is similar in both muscle types. Recent findings have shown that dysferlin regulates Ca2+ handling in striated muscle via multiple mechanisms and that this becomes more important in conditions of stress. Maintenance of the transverse (t)-tubule network and the tight coordination of excitation-contraction coupling are essential for muscle contractility. Dysferlin regulates the maintenance and repair of t-tubules, and it is suspected that dysferlin regulates t-tubules and sarcolemmal repair through a similar mechanism. This review focuses on the emerging complexity of dysferlin's activity in striated muscle. Such insights will progress our understanding of the proteins and pathways that regulate basic heart and skeletal muscle function and help guide research into striated muscle pathology, especially that which arises due to dysferlin dysfunction.

Keywords:  Ca2+; EC coupling; cardiac muscle and skeletal muscle; dysferlin; membrane repair; t‐tubule

DOI:  https://doi.org/10.1113/JP285103
Acta Neuropathol. 2024 Apr 18. 147(1): 72

Characterization of NEB pathogenic variants in patients reveals novel nemaline myopathy disease mechanisms and omecamtiv mecarbil force effects.

Esmat Karimi, Jochen Gohlke, Mila van der Borgh, Johan Lindqvist, Zaynab Hourani, Justin Kolb, Stacy Cossette, Michael W Lawlor, Coen Ottenheijm, Henk Granzier.

  Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Pathogenic variants in the nebulin gene (NEB) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking curative therapies. In this study, we examined a cohort of ten NEM2 patients, each with unique pathogenic variants, aiming to understand their impact on mRNA, protein, and functional levels. Results show that pathogenic truncation variants affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with pathogenic splicing variants that are expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with either relatively normal or markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, or reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a pathogenic duplication variant in nebulin that resulted in a four-copy gain in the triplicate region of NEB and a much larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type 1 muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87 to 318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from pathogenic splicing variants. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.

Keywords:  Cyptic splice-site; Nebulin; Nemaline myopathy; Omecamtiv mecarbil

DOI:  https://doi.org/10.1007/s00401-024-02726-w
J Cachexia Sarcopenia Muscle. 2024 Apr 17.

Hepatic signal transducer and activator of transcription-3 signalling drives early-stage pancreatic cancer cachexia via suppressed ketogenesis.

Paige C Arneson-Wissink, Heike Mendez, Katherine Pelz, Jessica Dickie, Alexandra Q Bartlett, Beth L Worley, Stephanie M Krasnow, Robert Eil, Aaron J Grossberg.

  BACKGROUND: Patients with pancreatic ductal adenocarcinoma (PDAC) often suffer from cachexia, a wasting syndrome that significantly reduces both quality of life and survival. Although advanced cachexia is associated with inflammatory signalling and elevated muscle catabolism, the early events driving wasting are poorly defined. During periods of nutritional scarcity, the body relies on hepatic ketogenesis to generate ketone bodies, and lipid metabolism via ketogenesis is thought to protect muscle from catabolizing during nutritional scarcity.METHODS: We developed an orthotopic mouse model of early PDAC cachexia in 12-week-old C57BL/6J mice. Murine pancreatic cancer cells (KPC) were orthotopically implanted into the pancreas of wild-type, IL-6-/-, and hepatocyte STAT3-/- male and female mice. Mice were subject to fasting, 50% food restriction, ad libitum feeding or ketogenic diet interventions. We measured longitudinal body composition by EchoMRI, body mass and food intake. At the endpoint, we measured tissue mass, tissue gene expression by quantitative real-time polymerase chain reaction, whole-body calorimetry, circulating hormone levels, faecal protein and lipid content, hepatic lipid content and ketogenic response to medium-chain fatty acid bolus. We assessed muscle atrophy in vivo and C2C12 myotube atrophy in vitro.
RESULTS: Pre-cachectic PDAC mice did not preserve gastrocnemius muscle mass during 3-day food restriction (-13.1 ± 7.7% relative to food-restricted sham, P = 0.0117) and displayed impaired fatty acid oxidation during fasting, resulting in a hypoketotic state (ketogenic response to octanoate bolus, -83.0 ± 17.3%, P = 0.0328; Hmgcs2 expression, -28.3 ± 7.6%, P = 0.0004). PDAC human patients display impaired fasting ketones (-46.9 ± 7.1%, P < 0.0001) and elevated circulating interleukin-6 (IL-6) (12.4 ± 16.5-fold increase, P = 0.0001). IL-6-/- PDAC mice had improved muscle mass (+35.0 ± 3.9%, P = 0.0031) and ketogenic response (+129.4 ± 44.4%, P = 0.0033) relative to wild-type PDAC mice. Hepatocyte-specific signal transducer and activator of transcription 3 (STAT3) deletion prevented muscle loss (+9.3 ± 4.0%, P = 0.009) and improved fasting ketone levels (+52.0 ± 43.3%, P = 0.018) in PDAC mice. Without affecting tumour growth, a carbohydrate-free diet improved tibialis anterior myofibre diameter (+16.5 ± 3.5%, P = 0.0089), circulating ketone bodies (+333.0 ± 117.6%, P < 0.0001) and Hmgcs2 expression (+106.5 ± 36.1%, P < 0.0001) in PDAC mice. Ketone supplementation protected muscle against PDAC-induced atrophy in vitro (+111.0 ± 17.6%, P < 0.0001 myofibre diameter).
CONCLUSIONS: In early PDAC cachexia, muscle vulnerability to wasting is dependent on inflammation-driven metabolic reprogramming in the liver. PDAC suppresses lipid β-oxidation and impairs ketogenesis in the liver, which is reversed in genetically modified mouse models deficient in IL-6/STAT3 signalling or through ketogenic diet supplementation. This work establishes a direct link between skeletal muscle homeostasis and hepatic metabolism. Dietary and anti-inflammatory interventions that restore ketogenesis may be a viable preventative approach for pre-cachectic patients with pancreatic cancer.

Keywords:  STAT3; cachexia; interleukin‐6; ketogenesis; pancreatic cancer

DOI:  https://doi.org/10.1002/jcsm.13466
Biochem Biophys Res Commun. 2024 Apr 16. pii: S0006-291X(24)00480-7. [Epub ahead of print]712-713 149944

Effect of 2-aminoethoxydiphenyl borate on the functions of mouse skeletal muscle mitochondria.

Mikhail V Dubinin, Alexander V Chulkov, Anastasia D Igoshkina, Alena A Cherepanova, Natalia V Mikina.

  This work examined the effect of 2-aminoethoxydiphenyl borate (2-APB) on the functioning of isolated mouse skeletal muscle mitochondria and modeled its putative interaction with mitochondrial proteins. We have shown that 2-APB is able to dose-dependently suppress mitochondrial respiration in state 3 and 3UDNP driven by substrates of complex I and II. This effect of 2-APB was accompanied by a slight dose-dependent decrease in mitochondrial membrane potential and appears to be due to inhibition of complex I and complex III of the electron transport chain (ETC) with IC50 values of 200 and 120 μM, respectively. The results of molecular docking identified putative 2-APB interaction sites in these ETC complexes. 2-APB was shown to dose-dependently inhibit both mitochondrial Ca2+ uptake and Ca2+ efflux, which seems to be caused by a decrease in the membrane potential of the organelles. We have found that 2-APB has no significant effect on mitochondrial calcium retention capacity. On the other hand, 2-APB exhibited antioxidant effect by reducing mitochondrial hydrogen peroxide production but without affecting superoxide generation. It is concluded that the effect of 2-APB on mitochondrial targets should be taken into account when interpreting the results of cell and in vivo experiments.

Keywords:  2-Aminoethoxydiphenyl borate; Calcium; Electron transport chain; MPT-Pore; Skeletal muscle mitochondria

DOI:  https://doi.org/10.1016/j.bbrc.2024.149944
Sci Rep. 2024 04 17. 14(1): 8871

HOIL-1L deficiency induces cell cycle alteration which causes immaturity of skeletal muscle and cardiomyocytes.

Kentaro Akagi, Shiro Baba, Hiroaki Fujita, Yasuhiro Fuseya, Daisuke Yoshinaga, Hirohito Kubota, Eitaro Kume, Fumiaki Fukumura, Koichi Matsuda, Takayuki Tanaka, Takuya Hirata, Megumu K Saito, Kazuhiro Iwai, Junko Takita.

HOIL-1L deficiency was recently reported to be one of the causes of myopathy and dilated cardiomyopathy (DCM). However, the mechanisms by which myopathy and DCM develop have not been clearly elucidated. Here, we sought to elucidate these mechanisms using the murine myoblast cell line C2C12 and disease-specific human induced pluripotent stem cells (hiPSCs). Myotubes differentiated from HOIL-1L-KO C2C12 cells exhibited deteriorated differentiation and mitotic cell accumulation. CMs differentiated from patient-derived hiPSCs had an abnormal morphology with a larger size and were excessively multinucleated compared with CMs differentiated from control hiPSCs. Further analysis of hiPSC-derived CMs showed that HOIL-1L deficiency caused cell cycle alteration and mitotic cell accumulation. These results demonstrate that abnormal cell maturation possibly contribute to the development of myopathy and DCM. In conclusion, HOIL-1L is an important intrinsic regulator of cell cycle-related myotube and CM maturation and cell proliferation.

DOI: https://doi.org/10.1038/s41598-024-57504-1
Biochemistry (Mosc). 2024 Feb;89(2): 299-312

Age-Dependent Changes in the Production of Mitochondrial Reactive Oxygen Species in Human Skeletal Muscle.

Mikhail Yu Vyssokikh, Maksim A Vigovskiy, Vladislav V Philippov, Yakov R Boroday, Mariya V Marey, Olga A Grigorieva, Tatiana F Vepkhvadze, Nadezhda S Kurochkina, Ludmila A Manukhova, Anastasiya Yu Efimenko, Daniil V Popov, Vladimir P Skulachev.

  A decrease in muscle mass and its functionality (strength, endurance, and insulin sensitivity) is one of the integral signs of aging. One of the triggers of aging is an increase in the production of mitochondrial reactive oxygen species. Our study was the first to examine age-dependent changes in the production of mitochondrial reactive oxygen species related to a decrease in the proportion of mitochondria-associated hexokinase-2 in human skeletal muscle. For this purpose, a biopsy was taken from m. vastus lateralis in 10 young healthy volunteers and 70 patients (26-85 years old) with long-term primary arthrosis of the knee/hip joint. It turned out that aging (comparing different groups of patients), in contrast to inactivity/chronic inflammation (comparing young healthy people and young patients), causes a pronounced increase in peroxide production by isolated mitochondria. This correlated with the age-dependent distribution of hexokinase-2 between mitochondrial and cytosolic fractions, a decrease in the rate of coupled respiration of isolated mitochondria and respiration when stimulated with glucose (a hexokinase substrate). It is discussed that these changes may be caused by an age-dependent decrease in the content of cardiolipin, a potential regulator of the mitochondrial microcompartment containing hexokinase. The results obtained contribute to a deeper understanding of age-related pathogenetic processes in skeletal muscles and open prospects for the search for pharmacological/physiological approaches to the correction of these pathologies.

Keywords:  aging; hexokinase; mitochondria; mitochondrial reactive oxygen species; skeletal muscle

DOI:  https://doi.org/10.1134/S0006297924020093
J Appl Physiol (1985). 2024 Apr 18.

Molecular Transducers of Physical Activity Consortium (MoTrPAC): Human Studies Design and Protocol.

MoTrPAC Study Group.

  Physical activity, including structured exercise, is associated with favorable health-related chronic disease outcomes. While there is evidence of various molecular pathways that affect these responses, a comprehensive molecular map of these molecular responses to exercise has not been developed. The Molecular Transducers of Physical Activity Consortium (MoTrPAC) is a multi-center study designed to isolate the effects of structured exercise training on the molecular mechanisms underlying the health benefits of exercise and physical activity. MoTrPAC contains both a pre-clinical and human component. The details of the human studies component of MoTrPAC that include the design and methods are presented here. The human studies contain both an adult and pediatric component. In the adult component, sedentary participants are randomized to 12 weeks of Control, Endurance Exercise Training, or Resistance Exercise Training with outcomes measures completed before and following the 12 weeks. The adult component also includes recruitment of highly active endurance trained or resistance trained participants who only complete measures once. A similar design is used for the pediatric component; however, only endurance exercise is examined. Phenotyping measures include weight, body composition, vital signs, cardiorespiratory fitness, muscular strength, physical activity and diet, and other questionnaires. Participants also complete an acute rest period (adults only) or exercise session (adults, pediatrics) with collection of biospecimens (blood only for pediatrics) to allow for examination of the molecular responses. The design and methods of MoTrPAC may inform other studies. Moreover, MoTrPAC will provide a repository of data that can be used broadly across the scientific community.

Keywords:  adipose tissue; exercise; molecular transducers; physical activity; skeletal muscle

DOI:  https://doi.org/10.1152/japplphysiol.00102.2024
Am J Pathol. 2024 May;pii: S0002-9440(24)00034-8. [Epub ahead of print]194(5): 759-771

Uremia Impedes Skeletal Myocyte Myomixer Expression and Fusogenic Activity: Implication for Uremic Sarcopenia.

Takaaki Higashihara, Motoki Odawara, Hiroshi Nishi, Takehito Sugasawa, Yumika Suzuki, Satoshi Kametaka, Reiko Inagi, Masaomi Nangaku.

In patients with chronic kidney disease (CKD), skeletal muscle mass and function are known to occasionally decline. However, the muscle regeneration and differentiation process in uremia has not been extensively studied. In mice with CKD induced by adenine-containing diet, the tibialis anterior muscle injured using a barium chloride injection method recovered poorly as compared to control mice. In the cultured murine skeletal myocytes, stimulation with indoxyl sulfate (IS), a representative uremic toxin, morphologically jeopardized the differentiation, which was counteracted by L-ascorbic acid (L-AsA) treatment. Transcriptome analysis of cultured myocytes identified a set of genes whose expression was down-regulated by IS stimulation but up-regulated by L-AsA treatment. Gene silencing of myomixer, one of the genes in the set, impaired myocyte fusion during differentiation. By contrast, lentiviral overexpression of myomixer compensated for a hypomorphic phenotype caused by IS treatment. The split-luciferase technique demonstrated that IS stimulation negatively affected early myofusion activity that was rescued by L-AsA treatment. Lastly, in mice with CKD compared with control mice, myomixer expression in the muscle tissue in addition to the muscle weight after the injury was reduced, both of which were restored with L-AsA treatment. Collectively, data showed that the uremic milieu impairs the expression of myomixer and impedes the myofusion process. Considering frequent musculoskeletal injuries in uremic patients, defective myocyte fusion followed by delayed muscle damage recovery could underlie their muscle loss and weakness.

DOI: https://doi.org/10.1016/j.ajpath.2024.01.005
J Cachexia Sarcopenia Muscle. 2024 Apr 20.

DNA methylation of skeletal muscle function-related secretary factors identifies FGF2 as a potential biomarker for sarcopenia.

Jia-Wen Li, Zheng-Kai Shen, Yu-Shuang Lin, Zhi-Yue Wang, Mei-Lin Li, Hui-Xian Sun, Quan Wang, Can Zhao, Jin-Shui Xu, Xiang Lu, Wei Gao.

  BACKGROUND: Sarcopenia is characterized by progressive loss of muscle mass and function due to aging. DNA methylation has been identified to play important roles in the dysfunction of skeletal muscle. The aim of our present study was to explore the whole blood sample-based methylation changes of skeletal muscle function-related factors in patients with sarcopenia.METHODS: The overall DNA methylation levels were analysed by using MethlTarget™ DNA Methylation Analysis platform in a discovery set consistent of 50 sarcopenic older adults (aged ≥65 years) and 50 age- and sex-matched non-sarcopenic individuals. The candidate differentially methylated regions (DMRs) were further validated by Methylation-specific PCR (MSP) in another two independent larger sets and confirmed by pyrosequencing. Receiver operating characteristic (ROC) curve analysis was used to determine the optimum cut-off levels of fibroblast growth factor 2 (FGF2)_30 methylation best predicting sarcopenia and area under the ROC curve (AUC) was measured. The correlation between candidate DMRs and the risk of sarcopenia was investigated by univariate analysis and multivariate logistic regression analysis.
RESULTS: Among 1149 cytosine-phosphate-guanine (CpG) sites of 27 skeletal muscle function-related secretary factors, 17 differentially methylated CpG sites and 7 differentially methylated regions (DMRs) were detected between patients with sarcopenia and control subjects in the discovery set. Further methylation-specific PCR identified that methylation of fibroblast growth factor 2 (FGF2)_30 was lower in patients with sarcopenia and the level was decreased as the severity of sarcopenia increased, which was confirmed by pyrosequencing. Correlation analysis demonstrated that the methylation level of FGF2_30 was positively correlated to ASMI (r = 0.372, P < 0.001), grip strength (r = 0.334, P < 0.001), and gait speed (r = 0.411, P < 0.001). ROC curve analysis indicated that the optimal cut-off value of FGF2_30 methylation level that predicted sarcopenia was 0.15 with a sensitivity of 84.6% and a specificity of 70.1% (AUC = 0.807, 95% CI = 0.756-0.858, P < 0.001). Multivariate logistic regression analyses showed that lower FGF2_30 methylation level (<0.15) was significantly associated with increased risk of sarcopenia even after adjustment for potential confounders including age, sex, and BMI (adjusted OR = 9.223, 95% CI: 6.614-12.861, P < 0.001).
CONCLUSIONS: Our results suggest that lower FGF2_30 methylation is correlated with the risk and severity of sarcopenia in the older adults, indicating that FGF2 methylation serve as a surrogate biomarker for the screening and evaluation of sarcopenia.

Keywords:  DNA methylation; FGF2; Older adults; Sarcopenia

DOI:  https://doi.org/10.1002/jcsm.13472