bims-moremu 2024-10-13 papers

bims-moremu

Biomed News

on Molecular regulators of muscle mass

Issue of 2024–10–13
thirty-two papers selected by
Anna Vainshtein, Craft Science Inc.

Electrical stimulation of biofidelic engineered muscle enhances myotube size, force, fatigue resistance, and induces a fast-to-slow-phenotype shift.
Overexpression of CPT1A disrupts the maintenance and regenerative function of muscle stem cells.
Inducible and reversible SOD2 knockdown in mouse skeletal muscle drives impaired pyruvate oxidation and reduced metabolic flexibility.
Effects of age on human skeletal muscle: A systematic review and meta-analysis of myosin heavy chain isoform protein expression, fiber size and distribution.
A partial loss-of-function variant (Ile191Val) of the TAS1R2 glucose receptor is associated with enhanced responses to exercise training in older adults with obesity: A translational study.
SRSF1 Is Crucial for Maintaining Satellite Cell Homeostasis During Skeletal Muscle Growth and Regeneration.
Inhibition of CILP2 Improves Glucose Metabolism and Mitochondrial Dysfunction in Sarcopenia via the Wnt Signalling Pathway.
Laminin-α2 chain deficiency in skeletal muscle causes dysregulation of multiple cellular mechanisms.
The proliferation and differentiation of skeletal muscle stem cells are enhanced in a bioreactor.
Advanced glycation end products promote ROS production via PKC/p47 phox axis in skeletal muscle cells.
ATF4-dependent and independent mitokine secretion from OPA1 deficient skeletal muscle in mice is sexually dimorphic.
Bioavailable testosterone and androgen receptor activation, but not total testosterone, are associated with muscle mass and strength in females.
Decreased mitochondrial creatine kinase 2 impairs skeletal muscle mitochondrial function independently of insulin in type 2 diabetes.
Alternative splicing dysregulation across tissue and therapeutic approaches in a mouse model of myotonic dystrophy type 1.
Activity and phosphatidylcholine transfer protein interactions of skeletal muscle thioesterase Them2 enable hepatic steatosis and insulin resistance.
Lysosomal LRRC8 complex regulates lysosomal pH, morphology and systemic glucose metabolism.
Spatial multi-omics in whole skeletal muscle reveals complex tissue architecture.
Effects of Exercise Training on Mitochondrial and Capillary Growth in Human Skeletal Muscle: A Systematic Review and Meta-Regression.
Functional role of myosin-binding protein H in thick filaments of developing vertebrate fast-twitch skeletal muscle.
Tail-anchored membrane protein SLMAP3 is essential for targeting centrosomal proteins to the nuclear envelope in skeletal myogenesis.
Muscle Atrophy and mRNA-miRNA Network Analysis of Vascular Endothelial Growth Factor (VEGF) in a Mouse Model of Denervation-Induced Disuse.
DELE1 maintains muscle proteostasis to promote growth and survival in mitochondrial myopathy.
Spatial analysis of transcript and protein levels in skeletal muscle.
Repeated passive heat treatment increases muscle tissue capillarization, but does not affect postprandial muscle protein synthesis rates in healthy older adults.
Acetylcholine receptor-β inhibition by interleukin-6 in skeletal muscles contributes to modulating neuromuscular junction during aging.
Sorafenib induces cachexia by impeding transcriptional signaling of the SET1/MLL complex on muscle-specific genes.
High-intensity exercise alongside insulin alleviates muscle atrophy in type 1 diabetes mellitus concomitant with modulation of mitophagy-related proteins in skeletal muscle.
Mechanisms of the NAD+ salvage pathway in enhancing skeletal muscle function.
Isolation of small extracellular vesicles from regenerating muscle tissue using tangential flow filtration and size exclusion chromatography.
Identification and analysis of differentially expressed lncRNAs and their ceRNA networks in DMD/mdx primary myoblasts.
A combination of major histocompatibility complex (MHC) I overexpression and type I interferon induce mitochondrial dysfunction in human skeletal myoblasts.
Dynamics of cardio-muscular networks in exercise and fatigue.

Physiol Rep. 2024 Oct;12(19): e70051

Electrical stimulation of biofidelic engineered muscle enhances myotube size, force, fatigue resistance, and induces a fast-to-slow-phenotype shift.

Isabella Pallotta, Michael J Stec, Brian Schriver, David R Golann, Kevin Considine, Qi Su, Victor Barahona, Julia E Napolitano, Sarah Stanley, Meghan Garcia, Nicole T Feric, Krista M Durney, Roozbeh Aschar-Sobbi, Nathan Bays, Tea Shavlakadze, Michael P Graziano.

  Therapeutic development for skeletal muscle diseases is challenged by a lack of ex vivo models that recapitulate human muscle physiology. Here, we engineered 3D human skeletal muscle tissue in the Biowire II platform that could be maintained and electrically stimulated long-term. Increasing differentiation time enhanced myotube formation, modulated myogenic gene expression, and increased twitch and tetanic forces. When we mimicked exercise training by applying chronic electrical stimulation, the "exercised" skeletal muscle tissues showed increased myotube size and a contractility profile, fatigue resistance, and gene expression changes comparable to in vivo models of exercise training. Additionally, tissues also responded with expected physiological changes to known pharmacological treatment. To our knowledge, this is the first evidence of a human engineered 3D skeletal muscle tissue that recapitulates in vivo models of exercise. By recapitulating key features of human skeletal muscle, we demonstrated that the Biowire II platform may be used by the pharmaceutical industry as a model for identifying and optimizing therapeutic drug candidates that modulate skeletal muscle function.

Keywords:  dexamethasone; electrical stimulation; engineered skeletal muscle; skeletal muscle contractility

DOI:  https://doi.org/10.14814/phy2.70051
FASEB J. 2024 Oct 15. 38(19): e70071

Overexpression of CPT1A disrupts the maintenance and regenerative function of muscle stem cells.

Jiamin Qiu, Feng Yue, Kun Ho Kim, Xiyue Chen, Mennatallah A Khedr, Jingjuan Chen, Lijie Gu, Junxiao Ren, Christina R Ferreira, Jessica Ellis, Shihuan Kuang.

  The skeletal muscle satellite cells (SCs) mediate regeneration of myofibers upon injury. As they switch from maintenance (quiescence) to regeneration, their relative reliance on glucose and fatty acid metabolism alters. To explore the contribution of mitochondrial fatty acid oxidation (FAO) pathway to SCs and myogenesis, we examined the role of carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme of FAO. CPT1A is highly expressed in quiescent SCs (QSCs) compared with activated and proliferating SCs, and its expression level decreases during myogenic differentiation. Myod1Cre-driven overexpression (OE) of Cpt1a in embryonic myoblasts (Cpt1aMTG) reduces muscle weight, grip strength, and contractile force without affecting treadmill endurance of adult mice. Adult Cpt1aMTG mice have reduced number of SC, impairing muscle regeneration and promoting lipid infiltration. Similarly, Pax7CreER-driven, tamoxifen-inducible Cpt1a-OE in QSCs of adult muscles (Cpt1aPTG) leads to depletion of SCs and compromises muscle regeneration. The reduced proliferation of Cpt1a-OE SCs is associated with elevated level of acyl-carnitine, and acyl-carnitine treatment impedes proliferation of wildtype SCs. These findings indicate that aberrant level of CPT1A elevates acyl-carnitine to impair the maintenance, proliferation and regenerative function of SCs.

Keywords:  CPT1A; acyl‐carnitine; fatty acid oxidation; muscle regeneration; satellite cell

DOI:  https://doi.org/10.1096/fj.202400947R
bioRxiv. 2024 Sep 25. pii: 2024.09.23.614547. [Epub ahead of print]

Inducible and reversible SOD2 knockdown in mouse skeletal muscle drives impaired pyruvate oxidation and reduced metabolic flexibility.

Ethan L Ostrom, Rudy Stuppard, Aurora Mattson-Hughes, David J Marcinek.

Abstract Figure:
Highlights: SOD2 knockdown and recovery is achieved in skeletal muscle by using a shRNA targeted to SOD2 mRNA controlled by a tetracycline Response Element and reverse tetracycline transactivator proteinSOD2 KD is induced by administering doxycycline in the drinking waterMitochondrial functional decline and recovery follows the time course of SOD2 protein decline and recoverySustained SOD2 KD precipitates reduced metabolic flexibility in skeletal muscle mitochondria characterized by impaired pyruvate respiration in the presence of other substrates.
Introduction: Skeletal muscle mitochondrial dysfunction is a key characteristic of aging muscle and contributes to age related diseases such as sarcopenia, frailty, and type 2 diabetes. Mitochondrial oxidative distress has been implicated as a driving factor in these age-related diseases, however whether it is a cause, or a consequence of mitochondrial dysfunction remains to be determined. The development of more flexible genetic models is an important tool to test the mechanistic role of mitochondrial oxidative stress on skeletal muscle metabolic dysfunction. We characterize a new model of inducible and reversible mitochondrial redox stress using a tetracycline controlled skeletal muscle specific short hairpin RNA targeted to superoxide dismutase 2 (iSOD2).
Methods: iSOD2 KD and control (CON) animals were administered doxycycline for 3-or 12-weeks and followed for up to 24 weeks and mitochondrial respiration and muscle contraction were measured to define the time course of SOD2 KD and muscle functional changes and recovery.
Results: Maximum knockdown of SOD2 protein occurred by 6 weeks and recovered by 24 weeks after DOX treatment. Mitochondrial aconitase activity and maximum mitochondrial respiration declined in KD muscle by 12 weeks and recovered by 24 weeks. There were minimal changes in gene expression between KD and CON muscle. Twelve-week KD showed a small, but significant decrease in muscle fatigue resistance. The primary phenotype was reduced metabolic flexibility characterized by impaired pyruvate driven respiration when other substrates are present. The pyruvate dehydrogenase kinase inhibitor dichloroacetate partially restored pyruvate driven respiration, while the thiol reductant DTT did not.
Conclusion: We use a model of inducible and reversible skeletal muscle SOD2 knockdown to demonstrate that elevated matrix superoxide reversibly impairs mitochondrial substrate flexibility characterized by impaired pyruvate oxidation. Despite the bioenergetic effect, the limited change in gene expression suggests that the elevated redox stress in this model is confined to the mitochondrial matrix.

DOI: https://doi.org/10.1101/2024.09.23.614547
Am J Physiol Cell Physiol. 2024 Oct 07.

Effects of age on human skeletal muscle: A systematic review and meta-analysis of myosin heavy chain isoform protein expression, fiber size and distribution.

Christopher Lee, Philip C Woods, Amanda E Paluch, Mark S Miller.

  Human studies examining the cellular mechanisms behind sarcopenia, or age-related loss of skeletal muscle mass and function, have produced inconsistent results. A systematic review and meta-analysis were performed to determine the aging effects on protein expression, size and distribution of fibers with various myosin heavy chain (MyHC) isoforms. Study eligibility included MyHC comparisons between young (18-49 years) and older (≥ 60 years) adults, with 27 studies identified. Relative protein expression was higher with age for the slow-contracting MyHC I fibers, with correspondingly lower fast-contracting MyHC II and IIA values. Fiber sizes were similar with age for MyHC I, while smaller for MyHC II and IIA. Fiber distributions were similar with age. When separated by sex, the few studies that examined females showed atrophy of MyHC II and IIA fibers with age, but no change in MyHC protein expression. Additional analyses by measurement technique, physical activity, and muscle biopsied provided important insights. In summary, age-related atrophy in fast-contracting fibers lead to more of the slow-contracting, lower force-producing isoform in older male muscles, which helps explain their age-related loss in whole muscle force, velocity, and power. Exercise or pharmacological interventions that shift MyHC expression towards faster isoforms and/or increase fast-contracting fiber size should decrease the prevalence of sarcopenia. Our findings also indicate that future studies need to include or focus solely on females, measure MyHC IIA and IIX isoforms separately, examine fiber type distribution, sample additional muscles to the vastus lateralis, and incorporate an objective measurement of physical activity.

Keywords:  MHC; aging; physical activity; sarcopenia; sex

DOI:  https://doi.org/10.1152/ajpcell.00347.2024
Metabolism. 2024 Oct 09. pii: S0026-0495(24)00273-7. [Epub ahead of print] 156045

A partial loss-of-function variant (Ile191Val) of the TAS1R2 glucose receptor is associated with enhanced responses to exercise training in older adults with obesity: A translational study.

Joan Serrano, Saki Kondo, Grace M Link, Ian S Brown, Richard E Pratley, Kedryn K Baskin, Bret H Goodpaster, Paul M Coen, George A Kyriazis.

   BACKGROUND: The TAS1R2 receptor, known for its role in taste perception, has also emerged as a key regulator of muscle physiology. Previous studies have shown that genetic ablation of TAS1R2 in mice enhances muscle fitness mimicking responses to endurance exercise training. However, the translational relevance of these findings to humans remains uncertain.
METHODS: We explored responses to endurance exercise training in mice and humans with genetic deficiency of TAS1R2. First, we assessed the effects of muscle-specific deletion of TAS1R2 in mice (mKO) or wild type controls (mWT) following 4 weeks of voluntary wheel running (VWR). Next, we investigated the effects of the TAS1R2-Ile191Val (rs35874116) partial loss-of-function variant on responses to a 6-month diet-induced weight loss with exercise training (WLEX), weight loss alone (WL), or education control (CON) interventions in older individuals with obesity. Participants were retrospectively genotyped for the TAS1R2-Ile191Val polymorphism and classified as conventional function (Ile/Ile) or partial loss-of-function (Val carriers: Ile/Val and Val/Val). Body composition, cardiorespiratory fitness, and skeletal muscle mitochondrial function were assessed before and after the intervention.
RESULTS: In response to VWR, mKO mice demonstrated enhanced running endurance and mitochondrial protein content. Similarly, TAS1R2 Val carriers exhibited distinctive improvements in body composition, including increased muscle mass, along with enhanced cardiorespiratory fitness and mitochondrial function in skeletal muscle following the WLEX intervention compared to Ile/Ile counterparts. Notably, every Val carrier demonstrated substantial responses to exercise training and weight loss, surpassing all Ile/Ile participants in overall performance metrics.
CONCLUSIONS: Our findings suggest that TAS1R2 partial loss-of-function confers beneficial effects on muscle function and metabolism in humans in response to exercise training, akin to observations in TAS1R2 muscle-deficient mice. Targeting TAS1R2 may help enhancing exercise training adaptations in individuals with compromised exercise tolerance or metabolic disorders, presenting a potential avenue for personalized exercise interventions.

Keywords:  Aging; Exercise; Genetics; HbA1c; Mitochondria; Muscle mass; Muscle metabolism; NAD; Obesity; PDE; Polymorphism; Running; Sweet taste receptor; TAS1R2; Weight loss; rs35874116

DOI:  https://doi.org/10.1016/j.metabol.2024.156045
J Cachexia Sarcopenia Muscle. 2024 Oct 09.

SRSF1 Is Crucial for Maintaining Satellite Cell Homeostasis During Skeletal Muscle Growth and Regeneration.

Zhenzhen Wang, Qian Peng, Zhige Zhang, Xue You, Huimin Duan, Rula Sha, Ningyang Yuan, Zhigang Li, Zhiqin Xie, Jun Han, Ying Feng.

   BACKGROUND: The splicing factor SRSF1 emerges as a mater regulator of cell proliferation, displaying high expression in actively proliferative satellite cells (SCs). In SRSF1 knockout mice (KO) generated via MyoD-Cre, early mortality and muscle atrophy are observed during postnatal muscle growth. Despite these findings, the precise mechanisms through which SRSF1 loss influences SCs' functions and its role in muscle regeneration remain to be elucidated.
METHODS: To unravel the exact mechanisms underlying the impact of SRSF1 deficiency SC functions, we employed single-cell RNA sequencing (scRNA-seq) on a mononuclear cell suspension isolated from the newborn diaphragm of KO and control mice. Concurrently, we subjected diaphragm muscles to RNA-seq analysis to identify dysregulated splicing events associated with SRSF1 deletion. For the analysis of the effect of SRSF1 deletion on muscle regeneration, we generated mice with inducible SC-specific Srsf1 ablation through Pax7-CreER. SRSF1 ablation was induced by intraperitoneal injection of tamoxifen. Using cardiotoxin-induced muscle injury, we examined the consequences of SRSF1 depletion on SC function through HE staining, immunostaining and EdU incorporation assay. C2C12 myoblasts and isolated myoblasts were employed to assess stem cell function and senescence.
RESULTS: Utilizing scRNA-seq analysis, we observed a noteworthy increase in activated and proliferating myoblasts when SRSF1 was absent. This increase was substantial, with the proportion rising from 28.68% in the control group to 77.06% in the knockout group. However, these myoblasts experienced mitotic abnormalities in the absence of SRSF1, resulting in cell cycle arrest and the onset of cellular senescence. In the knockout mice, the proportion of Pax7+ cells within improper niche positioning increased significantly to 25% compared to 12% in the control cells (n ≥ 10, p < 0.001). Furthermore, there was an observation of persistent cell cycle exit specifically in the Pax7+ cells deficient in SRSF1 (n = 6, p < 0.001). SRSF1 plays a pivotal role in regulating the splicing of Fgfr1op2, favouring the full-length isoform crucial for mitotic spindle organization. Disrupting SRSF1 in C2C12 and primary myoblasts results in multipolar spindle formation (p < 0.001) and dysregulated splicing of Fgfr1op2 and triggers cellular senescence. Consequently, adult SCs lacking SRSF1 initially activate upon injury but face substantial challenge in proliferation (n = 4, p < 0.001), leading to a failure in muscle regeneration.
CONCLUSIONS: SRSF1 plays a critical role in SCs by ensuring proper splicing, maintaining mitotic progression and preventing premature senescence. These findings underscore the significant role of SRSF1 in controlling SC proliferation during skeletal muscle growth and regeneration.

Keywords:  SRSF1; cellular senescence; dysregulated splicing; muscle regeneration; satellite cells; scRNA‐seq

DOI:  https://doi.org/10.1002/jcsm.13607
J Cachexia Sarcopenia Muscle. 2024 Oct 10.

Inhibition of CILP2 Improves Glucose Metabolism and Mitochondrial Dysfunction in Sarcopenia via the Wnt Signalling Pathway.

Zhibo Deng, Chao Song, Long Chen, Rongsheng Zhang, Linhai Yang, Peng Zhang, Yu Xiu, Yibin Su, Fenqi Luo, Jun Luo, Hanhao Dai, Jie Xu.

   BACKGROUND: Skeletal muscle is the primary organ involved in insulin-mediated glucose metabolism. Elevated levels of CILP2 are a significant indicator of impaired glucose tolerance and are predominantly expressed in skeletal muscle. It remains unclear whether CILP2 contributes to age-related muscle atrophy through regulating the glucose homeostasis and insulin sensitivity.
METHODS: Initially, the expression levels of CILP2 were assessed in elderly mice and patients with sarcopenia. Lentiviral vectors were used to induce either silencing or overexpression of CILP2 in C2C12 myoblast cells. The effects of CILP2 on proliferation, myogenic differentiation, insulin sensitivity and glucose uptake were evaluated using immunofluorescence, western blotting, real-time quantitative polymerase chain reaction, RNA sequencing, glucose uptake experiments, dual-luciferase reporter assays and co-immunoprecipitation (CO-IP). An adeno-associated virus-9 containing a muscle-specific promoter was injected into SAMP8 senile mice to observe the efficacy of CILP2 knockout.
RESULTS: We found that there was more CLIP2 expressed in the skeletal muscle of ageing mice (+1.1-fold, p < 0.01) and in patients with sarcopenia (+2.5-fold, p < 0.01) compared to the control group. Following the overexpression of CILP2, Ki67 (-65%, p < 0.01), PCNA (-32%, p < 0.05), MyoD1 (-89%, p < 0.001), MyoG (-31%, p < 0.05) and MyHC (-85%, p < 0.001), which indicate proliferation and differentiation potential, were significantly reduced. In contrast, MuRF-1 (+59%, p < 0.05), atrogin-1 (+43%, p < 0.05) and myostatin (+31%, p < 0.05), the markers of muscular atrophy, were significantly increased. Overexpression of CILP2 decreased insulin sensitivity, glucose uptake (-18%, p < 0.001), GLUT4 translocation to the membrane and the maximum respiratory capacity of mitochondria. Canonical Wnt signalling was identified through RNA sequencing as a potential pathway for CILP2 regulation in C2C12, and Wnt3a was confirmed as an interacting protein of CILP2 in the CO-IP assay. The addition of recombinant Wnt3a protein reversed the inhibitory effects on myogenesis and glucose metabolism caused by CILP2 overexpression. Conversely, CILP2 knockdown promoted myogenesis and glucose metabolism. CILP2 knockdown improved muscle atrophy in mice, characterized by significant increases in time to exhaustion (+42%, p < 0.001), grip strength (+19%, p < 0.01), muscle mass (+15%, p < 0.001) and mean muscle cross-sectional area (+37%, p < 0.01). CILP2 knockdown enhanced glycogen synthesis (+83%, p < 0.001) and the regeneration of oxidative and glycolytic muscle fibres in SAMP8 ageing mice via the Wnt/β-catenin signalling pathway.
CONCLUSIONS: Our results indicate that CILP2 interacts with Wnt3a to suppress the Wnt/β-catenin signalling pathway and its downstream cascade, leading to impaired insulin sensitivity and glucose metabolism in skeletal muscle. Targeting CILP2 inhibition could offer potential therapeutic benefits for sarcopenia.

Keywords:  CILP2; Wnt/β‐catenin pathway; glucose metabolism; insulin resistance; sarcopenia

DOI:  https://doi.org/10.1002/jcsm.13597
Life Sci Alliance. 2024 Dec;pii: e202402829. [Epub ahead of print]7(12):

Laminin-α2 chain deficiency in skeletal muscle causes dysregulation of multiple cellular mechanisms.

Susana G Martins, Vanessa Ribeiro, Catarina Melo, Cláudia Paulino-Cavaco, Dario Antonini, Sharadha Dayalan Naidu, Fernanda Murtinheira, Inês Fonseca, Bérénice Saget, Mafalda Pita, Diogo R Fernandes, Pedro Gameiro Dos Santos, Gabriela Rodrigues, Rita Zilhão, Federico Herrera, Albena T Dinkova-Kostova, Ana Rita Carlos, Sólveig Thorsteinsdóttir.

LAMA2, coding for the laminin-α2 chain, is a crucial ECM component, particularly abundant in skeletal muscle. Mutations in LAMA2 trigger the often-lethal LAMA2-congenital muscular dystrophy (LAMA2-CMD). Various phenotypes have been linked to LAMA2-CMD; nevertheless, the precise mechanisms that malfunction during disease onset in utero remain unknown. We generated Lama2-deficient C2C12 cells and found that Lama2-deficient myoblasts display proliferation, differentiation, and fusion defects, DNA damage, oxidative stress, and mitochondrial dysfunction. Moreover, fetal myoblasts isolated from the dy W mouse model of LAMA2-CMD display impaired differentiation and fusion in vitro. We also showed that disease onset during fetal development is characterized by a significant down-regulation of gene expression in muscle fibers, causing pronounced effects on cytoskeletal organization, muscle differentiation, and altered DNA repair and oxidative stress responses. Together, our findings provide unique insights into the critical importance of the laminin-α2 chain for muscle differentiation and muscle cell homeostasis.

DOI: https://doi.org/10.26508/lsa.202402829
Biotechnol Bioeng. 2024 Oct 06.

The proliferation and differentiation of skeletal muscle stem cells are enhanced in a bioreactor.

Wei-Hsuan Lin, Chung-Yuh Tzeng, Fan-Che Kao, Chia-Wen Tsao, Ning Li, Chuan-Che Wu, Sheng-Huei Lee, Kai-Fan Huang, Wei-Wen Hu, Shen-Liang Chen.

  Skeletal muscle (SKM) is the largest organ in mammalian body and it can repair damages by using the residential myogenic stem cells (MuSC), but this repairing capacity reduces with age and in some genetic muscular dystrophy. Under these circumstances, artificial amplification of autologous MuSC in vitro might be necessary to repair the damaged SKM. The amplification of MuSC is highly dependent on myogenic signals, such as sonic hedgehog (Shh), Wnt3a, and fibroblast growth factors, so formulating an optimum myogenic kit composed of specific myogenic signals might increase the proliferation and differentiation of MuSC efficiently. In this study, various myogenic signals have been tested on C2C12 myoblasts and primary MuSC, and a myogenic kit consists of insulin, lithium chloride, T3, and retinoic acid has been formulated, and we found it significantly increased the fusion index and MHC expression level of both C2C12 and MuSC myotubes. A novel bioreactor providing cyclic stretching (CS) and electrical stimulation (ES) has been fabricated to enhance the myogenic differentiation of both C2C12 and MuSC. We further found that coating the bioreactor substratum with collagen gave the best effect on proliferation and differentiation of MuSC. Furthermore, combining the collagen coating and physical stimuli (CS + ES) in the bioreactor can generate more proliferative primary MuSC cells. Our results have demonstrated that the combination of myogenic kit and bioreactor can provide environment for efficient MuSC proliferation and differentiation. These MuSC and mature myotubes amplified in the bioreactor might be useful for clinical grafting into damaged SKM in the future.

Keywords:  bioreactor; differentiation; muscle; myogenesis; proliferation; stem cell

DOI:  https://doi.org/10.1002/bit.28857
J Physiol Sci. 2024 Oct 05. 74(1): 51

Advanced glycation end products promote ROS production via PKC/p47 phox axis in skeletal muscle cells.

Shinichiro Suzuki, Tatsuya Hayashi, Tatsuro Egawa.

  Advanced glycation end products (AGEs) are risk factors for various diseases, including sarcopenia. One of the deleterious effects of AGEs is the induction of abnormal reactive oxygen species (ROS) production in skeletal muscle. However, the underlying mechanism remains poorly understood. Therefore, the aim of this study was to elucidate how AGEs induce ROS production in skeletal muscle cells. This study demonstrated that AGEs treatment promoted ROS production in myoblasts and myotubes while PKC inhibitor abolished ROS production by AGEs stimulation. Phosphorylation of p47 phox by kinases such as PKCα is required to form the Nox2 complex, which induces ROS production. In this study, AGEs treatment promoted the phosphorylation of PKCα and p47 phox in myoblasts and myotubes. Our findings suggest that AGEs promote ROS production through the phosphorylation of PKCα and p47 phox in skeletal muscle cells.

Keywords:  AGEs; Myoblast; Myotube; Nox2; ROS

DOI:  https://doi.org/10.1186/s12576-024-00944-1
Front Endocrinol (Lausanne). 2024 ;15 1325286

ATF4-dependent and independent mitokine secretion from OPA1 deficient skeletal muscle in mice is sexually dimorphic.

Jennifer Streeter, Luis Persaud, Jason Gao, Deeraj Manika, Will Fairman, Luis Miguel García-Peña, Alex Marti, Chethan Manika, Shreya Gaddi, Brandon Schickling, Renata O Pereira, E Dale Abel.

   Introduction: Reducing Optic Atrophy 1 (OPA1) expression in skeletal muscle in male mice induces Activation Transcription Factor 4 (ATF4) and the integrated stress response (ISR). Additionally, skeletal muscle secretion of Fibroblast Growth Factor 21 (FGF21) is increased, which mediates metabolic adaptations including resistance to diet-induced obesity (DIO) and glucose intolerance in these mice. Although FGF21 induction in this model can be reversed with pharmacological attenuation of ER stress, it remains to be determined if ATF4 is responsible for FGF21 induction and its metabolic benefits in this model.
Methods: We generated mice with homozygous floxed Opa1 and Atf4 alleles and a tamoxifen-inducible Cre transgene controlled by the human skeletal actin promoter to enable simultaneous depletion of OPA1 and ATF4 in skeletal muscle (mAO DKO). Mice were fed high fat (HFD) or control diet and evaluated for ISR activation, body mass, fat mass, glucose tolerance, insulin tolerance and circulating concentrations of FGF21 and growth differentiation factor 15 (GDF15).
Results: In mAO DKO mice, ATF4 induction is absent. Other indices of ISR activation, including XBP1s, ATF6, and CHOP were induced in mAO DKO males, but not in mOPA1 or mAO DKO females. Resistance to diet-induced obesity was not reversed in mAO DKO mice of both sexes. Circulating FGF21 and GDF15 illustrated sexually dimorphic patterns. Loss of OPA1 in skeletal muscle increases circulating FGF21 in mOPA1 males, but not in mOPA1 females. Additional loss of ATF4 decreased circulating FGF21 in mAO DKO male mice, but increased circulating FGF21 in female mAO DKO mice. Conversely, circulating GDF15 was increased in mAO DKO males and mOPA1 females, but not in mAO DKO females.
Conclusion: Sex differences exist in the transcriptional outputs of the ISR following OPA deletion in skeletal muscle. Deletion of ATF4 in male and female OPA1 KO mice does not reverse the resistance to DIO. Induction of circulating FGF21 is ATF4 dependent in males, whereas induction of circulating GDF15 is ATF4 dependent in females. Elevated GDF15 in males and FGF21 in females could reflect activation by other transcriptional outputs of the ISR, that maintain mitokine-dependent metabolic protection in an ATF4-independent manner.

Keywords:  FGF21; insulin resistance; integrated stress response; mitochondria; obesity

DOI:  https://doi.org/10.3389/fendo.2024.1325286
J Physiol. 2024 Oct 11.

Bioavailable testosterone and androgen receptor activation, but not total testosterone, are associated with muscle mass and strength in females.

Sarah E Alexander, Briana Gatto, Olivia E Knowles, Ross M Williams, Kinga N Fiebig, Paul Jansons, Paul A Della Gatta, Andrew Garnham, Nir Eynon, Glenn D Wadley, Brad Aisbett, Danielle Hiam, Séverine Lamon.

  Testosterone, the major androgen, influences the reproductive and non-reproductive systems in males and females via binding to the androgen receptor (AR). Both circulating endogenous testosterone and muscle AR protein content are positively associated with muscle mass and strength in males, but there is no such evidence in females. Here, we tested whether circulating testosterone levels were associated with muscle mass, function, or the muscle anabolic response to resistance training in pre-menopausal females. Twenty-seven pre-menopausal, untrained females (aged 23.5 ± 4.8 years) underwent a 12-week resistance training programme. Muscle strength, size, power, and plasma and urine androgen hormone levels were measured. Skeletal muscle biopsies were collected before and after the training programme to quantify the effect of resistance training on AR content and nuclear localisation. Primary muscle cell lines were cultured from a subset (n = 6) of the participants' biopsies and treated with testosterone to investigate its effect on myotube diameter, markers of muscle protein synthesis and AR cellular localisation. Physiological levels of total testosterone were not associated with muscle mass or strength at baseline or with the changes in muscle mass and strength that occurred in response to resistance training in our cohort of pre-menopausal females. In contrast, bioavailable testosterone and the proportion of nuclear-localised AR were positively associated with skeletal muscle mass and strength in pre-menopausal females. In vitro, supra-physiological doses of testosterone increased myocyte diameter, but this did not occur via the Akt/mTOR pathway as previously suggested. Instead, we show a marked increase in AR nuclear localisation with testosterone administration in vitro. KEY POINTS: Total circulating testosterone was not related to muscle mass or strength before or after resistance training in pre-menopausal females. Bioavailable testosterone was positively related to exercise-induced muscle hypertrophy in pre-menopausal females. In vivo nuclear localisation of the androgen receptor was positively related to muscle mass in pre-menopausal females at baseline, but not to resistance training-induced hypertrophy. Testosterone treatment induced androgen receptor nuclear translocation but did not induce mTOR signalling in primary skeletal myocytes cultured from pre-menopausal female muscle.

Keywords:  androgens; exercise; female; muscle; sex hormones; women

DOI:  https://doi.org/10.1113/JP286803
Sci Transl Med. 2024 Oct 09. 16(768): eado3022

Decreased mitochondrial creatine kinase 2 impairs skeletal muscle mitochondrial function independently of insulin in type 2 diabetes.

David Rizo-Roca, Dimitrius Santiago P S F Guimarães, Logan A Pendergrast, Nicolas Di Leo, Alexander V Chibalin, Salwan Maqdasy, Mikael Rydén, Erik Näslund, Juleen R Zierath, Anna Krook.

Increased plasma creatine concentrations are associated with the risk of type 2 diabetes, but whether this alteration is associated with or causal for impairments in metabolism remains unexplored. Because skeletal muscle is the main disposal site of both creatine and glucose, we investigated the role of intramuscular creatine metabolism in the pathophysiology of insulin resistance in type 2 diabetes. In men with type 2 diabetes, plasma creatine concentrations were increased, and intramuscular phosphocreatine content was reduced. These alterations were coupled to reduced expression of sarcomeric mitochondrial creatine kinase 2 (CKMT2). In C57BL/6 mice fed a high-fat diet, neither supplementation with creatine for 2 weeks nor treatment with the creatine analog β-GPA for 1 week induced changes in glucose tolerance, suggesting that increased circulating creatine was associated with insulin resistance rather than causing it. In C2C12 myotubes, silencing Ckmt2 using small interfering RNA reduced mitochondrial respiration, membrane potential, and glucose oxidation. Electroporation-mediated overexpression of Ckmt2 in skeletal muscle of high-fat diet-fed male mice increased mitochondrial respiration, independent of creatine availability. Given that overexpression of Ckmt2 improved mitochondrial function, we explored whether exercise regulates CKMT2 expression. Analysis of public data revealed that CKMT2 content was up-regulated by exercise training in both humans and mice. We reveal a previously underappreciated role of CKMT2 in mitochondrial homeostasis beyond its function for creatine phosphorylation, independent of insulin action. Collectively, our data provide functional evidence for how CKMT2 mediates mitochondrial dysfunction associated with type 2 diabetes.

DOI: https://doi.org/10.1126/scitranslmed.ado3022
Mol Ther Nucleic Acids. 2024 Dec 10. 35(4): 102338

Alternative splicing dysregulation across tissue and therapeutic approaches in a mouse model of myotonic dystrophy type 1.

Sawyer M Hicks, Jesus A Frias, Subodh K Mishra, Marina Scotti, Derek R Muscato, M Carmen Valero, Leanne M Adams, John D Cleary, Masayuki Nakamori, Eric Wang, J Andrew Berglund.

  Myotonic dystrophy type 1 (DM1), the leading cause of adult-onset muscular dystrophy, is caused by a CTG repeat expansion. Expression of the repeat causes widespread alternative splicing (AS) defects and downstream pathogenesis, including significant skeletal muscle impacts. The HSA LR mouse model plays a significant role in therapeutic development. This mouse model features a transgene composed of approximately 220 interrupted CTG repeats, which results in skeletal muscle pathology that mirrors DM1. To better understand this model and the growing number of therapeutic approaches developed with it, we performed a meta-analysis of publicly available RNA sequencing data for AS changes across three widely examined skeletal muscles: quadriceps, gastrocnemius, and tibialis anterior. Our analysis demonstrated that transgene expression correlated with the extent of splicing dysregulation across these muscles from gastrocnemius (highest), quadriceps (medium), to tibialis anterior (lowest). We identified 95 splicing events consistently dysregulated across all examined datasets. Comparison of splicing rescue across seven therapeutic approaches showed a range of rescue across the 95 splicing events from the three muscle groups. This analysis contributes to our understanding of the HSA LR model and the growing number of therapeutic approaches currently in preclinical development for DM1.

Keywords:  DM1; HSALR; MT: Bioinformatics; RNA sequencing; RNA-seq; alternative splicing; therapeutics

DOI:  https://doi.org/10.1016/j.omtn.2024.102338
J Biol Chem. 2024 Oct 04. pii: S0021-9258(24)02357-3. [Epub ahead of print] 107855

Activity and phosphatidylcholine transfer protein interactions of skeletal muscle thioesterase Them2 enable hepatic steatosis and insulin resistance.

Yang Xie, Xu Liu, Wenpeng Liu, Logan R Carr, Luke P Lee, Norihiro Imai, Eric A Ortlund, David E Cohen.

  Thioesterase superfamily member 2 (Them2), a long-chain fatty acyl-CoA thioesterase that is highly expressed in oxidative tissues, interacts with phosphatidylcholine transfer protein (PC-TP) to regulate hepatic lipid and glucose metabolism and to suppress insulin signaling. High-fat diet (HFD)-fed mice lacking Them2 globally or specifically in skeletal muscle, but not liver, exhibit reduced hepatic steatosis and insulin resistance. Here, we report that the capacity of Them2 in skeletal muscle to promote hepatic steatosis and insulin resistance depends on both its catalytic activity and interaction with PC-TP. Two residues of Them2 catalytic site were mutated (N50A/D65A) to produce the inactive enzyme while maintaining its homotetrameric structure and interaction with PC-TP. Restoration of skeletal muscle expression in Them2-/- mice using recombinant adeno-associated virus revealed that wild-type (WT), but not N50A/D65A Them2, promoted HFD-induced weight gain and hepatic steatosis. This was accompanied by greater impairment of insulin sensitivity in WT compared with N50A/D65A Them2. Pharmacological inhibition or genetic ablation of PC-TP attenuated these effects. In reductionist experiments, conditioned medium collected from WT primary cultured myotubes promoted excess lipid accumulation in oleic acid-treated primary cultured hepatocytes relative to Them2-/- myotubes, which was attributable to secreted extracellular vesicles (EV). Reconstitution of Them2 expression in Them2-/- myotubes affirmed the requirements for catalytic activity and PC-TP interactions for EV to promote lipid accumulation in hepatocytes. These studies provide valuable mechanistic insights whereby Them2 in skeletal muscle promotes hepatic steatosis and establish both Them2 and PC-TP as represent attractive targets for managing metabolic dysfunction-associated steatotic liver disease.

Keywords:  fatty acid metabolism; glucose metabolism; hepatocyte; insulin resistance; lipid binding protein; skeletal muscle metabolism

DOI:  https://doi.org/10.1016/j.jbc.2024.107855
bioRxiv. 2024 Sep 23. pii: 2024.09.22.614256. [Epub ahead of print]

Lysosomal LRRC8 complex regulates lysosomal pH, morphology and systemic glucose metabolism.

Ashutosh Kumar, Yonghui Zhao, Litao Xie, Rahul Chadda, Nihil Abraham, Juan Hong, Ethan Feng, John D Tranter, David Rawnsley, Haiyan Liu, Kyla M Henry, Gretchen Meyer, Meiqin Hu, Haoxing Xu, Antentor Hinton, Chad E Grueter, E Dale Abel, Andrew W Norris, Abhinav Diwan, Rajan Sah.

The lysosome integrates anabolic signalling and nutrient-sensing to regulate intracellular growth pathways. The leucine-rich repeat containing 8 (LRRC8) channel complex forms a lysosomal anion channel and regulates PI3K-AKT-mTOR signalling, skeletal muscle differentiation, growth, and systemic glucose metabolism. Here, we define the endogenous LRRC8 subunits localized to a subset of lysosomes in differentiated myotubes. We show LRRC8A regulates leucine-stimulated mTOR, lysosome size, number, pH, and expression of lysosomal proteins LAMP2, P62, LC3B, suggesting impaired autophagic flux. Mutating a LRRC8A lysosomal targeting dileucine motif sequence (LRRC8A-L706A;L707A) in myotubes recapitulates the abnormal AKT signalling and altered lysosomal morphology and pH observed in LRRC8A KO cells. In vivo , LRRC8A-L706A;L707A KI mice exhibit increased adiposity, impaired glucose tolerance and insulin resistance characterized by reduced skeletal muscle glucose-uptake, and impaired incorporation of glucose into glycogen. These data reveal a lysosomal LRRC8 mediated metabolic signalling function that regulates lysosomal activity, systemic glucose homeostasis and insulin-sensitivity.

DOI: https://doi.org/10.1101/2024.09.22.614256
Commun Biol. 2024 Oct 05. 7(1): 1272

Spatial multi-omics in whole skeletal muscle reveals complex tissue architecture.

Clara Martínez Mir, Paola Pisterzi, Isabel De Poorter, Maria Rilou, Melissa van Kranenburg, Bram Heijs, Anna Alemany, Fanny Sage, Niels Geijsen.

Myofibers are large multinucleated cells that have long thought to have a rather simple organization. Single-nucleus transcriptomics, spatial transcriptomics and spatial metabolomics analysis have revealed distinct transcription profiles in myonuclei related to myofiber type. However, the use of local tissue collection or dissociation methods have obscured the spatial organization. To elucidate the full tissue architecture, we combine two spatial omics, RNA tomography and mass spectrometry imaging. This enables us to map the spatial transcriptomic, metabolomic and lipidomic organization of the whole murine tibialis anterior muscle. Our findings on heterogeneity in fiber type proportions are validated with multiplexed immunofluorescence staining in tibialis anterior, extensor digitorum longus and soleus. Our results demonstrate unexpectedly strong regionalization of gene expression, metabolic differences and variable myofiber type proportion along the proximal-distal axis. These new insights in whole-tissue level organization reconcile sometimes conflicting results coming from previous studies relying on local sampling methods.

DOI: https://doi.org/10.1038/s42003-024-06949-1
Sports Med. 2024 Oct 10.

Effects of Exercise Training on Mitochondrial and Capillary Growth in Human Skeletal Muscle: A Systematic Review and Meta-Regression.

Knut Sindre Mølmen, Nicki Winfield Almquist, Øyvind Skattebo.

BACKGROUND: Skeletal muscle mitochondria and capillaries are crucial for aerobic fitness, and suppressed levels are associated with chronic and age-related diseases. Currently, evidence-based exercise training recommendations to enhance these characteristics are limited. It is essential to explore how factors, such as fitness level, age, sex, and disease affect mitochondrial and capillary adaptations to different exercise stimuli.
OBJECTIVES: The main aim of this study was to compare the effects of low- or moderate intensity continuous endurance training (ET), high-intensity interval or continuous training (HIT), and sprint interval training (SIT) on changes in skeletal muscle mitochondrial content and capillarization. Secondarily, the effects on maximal oxygen consumption (VO2max), muscle fiber cross-sectional area, and fiber type proportion were investigated.
METHODS: A systematic literature search was conducted in PubMed, Web of Science, and SPORTDiscus databases, with no data restrictions, up to 2 February 2022. Exercise training intervention studies of ET, HIT, and SIT were included if they had baseline and follow-up measures of at least one marker of mitochondrial content or capillarization. In total, data from 5973 participants in 353 and 131 research articles were included for the mitochondrial and capillary quantitative synthesis of this review, respectively. Additionally, measures of VO2max, muscle fiber cross-sectional area, and fiber type proportion were extracted from these studies.
RESULTS: After adjusting for relevant covariates, such as training frequency, number of intervention weeks, and initial fitness level, percentage increases in mitochondrial content in response to exercise training increased to a similar extent with ET (23 ± 5%), HIT (27 ± 5%), and SIT (27 ± 7%) (P > 0.138), and were not influenced by age, sex, menopause, disease, or the amount of muscle mass engaged. Higher training frequencies (6 > 4 > 2 sessions/week) were associated with larger increases in mitochondrial content. Per total hour of exercise, SIT was ~ 2.3 times more efficient in increasing mitochondrial content than HIT and ~ 3.9 times more efficient than ET, while HIT was ~ 1.7 times more efficient than ET. Capillaries per fiber increased similarly with ET (15 ± 3%), HIT (13 ± 4%) and SIT (10 ± 11%) (P = 0.556) after adjustments for number of intervention weeks and initial fitness level. Capillaries per mm2 only increased after ET (13 ± 3%) and HIT (7 ± 4%), with increases being larger after ET compared with HIT and SIT (P < 0.05). This difference coincided with increases in fiber cross-sectional area after ET (6.5 ± 3.5%), HIT (8.9 ± 4.9%), and SIT (11.9 ± 15.1%). Gains in capillarization occurred primarily in the early stages of training (< 4 weeks) and were only observed in untrained to moderately trained participants. The proportion of type I muscle fibers remained unaltered by exercise training (P > 0.116), but ET and SIT exhibited opposing effects (P = 0.041). VO2max increased similarly with ET, HIT, and SIT, although HIT showed a tendency for greater improvement compared with both ET and SIT (P = 0.082), while SIT displayed the largest increase per hour of exercise. Higher training frequencies (6 > 4 > 2 sessions/week) were associated with larger increases in VO2max. Women displayed greater percentage gains in VO2max compared with men (P = 0.008). Generally, lower initial fitness levels were associated with greater percentage improvements in mitochondrial content, capillarization, and VO2max. SIT was particularly effective in improving mitochondrial content and VO2max in the early stages of training, while ET and HIT showed slower but steady improvements over a greater number of training weeks.
CONCLUSIONS: The magnitude of change in mitochondrial content, capillarization, and VO2max to exercise training is largely determined by the initial fitness level, with greater changes observed in individuals with lower initial fitness. The ability to adapt to exercise training is maintained throughout life, irrespective of sex and presence of disease. While training load (volume × intensity) is a suitable predictor of changes in mitochondrial content and VO2max, this relationship is less clear for capillary adaptations.

DOI: https://doi.org/10.1007/s40279-024-02120-2
J Gen Physiol. 2024 Dec 02. pii: e202413604. [Epub ahead of print]156(12):

Functional role of myosin-binding protein H in thick filaments of developing vertebrate fast-twitch skeletal muscle.

Andrew F Mead, Neil B Wood, Shane R Nelson, Bradley M Palmer, Lin Yang, Samantha Beck Previs, Angela Ploysangngam, Guy G Kennedy, Jennifer F McAdow, Sarah M Tremble, Marcus A Zimmermann, Marilyn J Cipolla, Alicia M Ebert, Aaron N Johnson, Christina A Gurnett, Michael J Previs, David M Warshaw.

Myosin-binding protein H (MyBP-H) is a component of the vertebrate skeletal muscle sarcomere with sequence and domain homology to myosin-binding protein C (MyBP-C). Whereas skeletal muscle isoforms of MyBP-C (fMyBP-C, sMyBP-C) modulate muscle contractility via interactions with actin thin filaments and myosin motors within the muscle sarcomere "C-zone," MyBP-H has no known function. This is in part due to MyBP-H having limited expression in adult fast-twitch muscle and no known involvement in muscle disease. Quantitative proteomics reported here reveal that MyBP-H is highly expressed in prenatal rat fast-twitch muscles and larval zebrafish, suggesting a conserved role in muscle development and prompting studies to define its function. We take advantage of the genetic control of the zebrafish model and a combination of structural, functional, and biophysical techniques to interrogate the role of MyBP-H. Transgenic, FLAG-tagged MyBP-H or fMyBP-C both localize to the C-zones in larval myofibers, whereas genetic depletion of endogenous MyBP-H or fMyBP-C leads to increased accumulation of the other, suggesting competition for C-zone binding sites. Does MyBP-H modulate contractility in the C-zone? Globular domains critical to MyBP-C's modulatory functions are absent from MyBP-H, suggesting that MyBP-H may be functionally silent. However, our results suggest an active role. In vitro motility experiments indicate MyBP-H shares MyBP-C's capacity as a molecular "brake." These results provide new insights and raise questions about the role of the C-zone during muscle development.

DOI: https://doi.org/10.1085/jgp.202413604
Open Biol. 2024 Oct;14(10): 240094

Tail-anchored membrane protein SLMAP3 is essential for targeting centrosomal proteins to the nuclear envelope in skeletal myogenesis.

Ana Paula Dias, Taha Rehmani, Maysoon Salih, Balwant Tuana.

  The positioning and communication between the nucleus and centrosomes are essential in cell division, differentiation and tissue formation. During skeletal myogenesis, the nuclei become evenly spaced with the switch of the microtubule-organizing centre (MTOC) from the centrosome to the nuclear envelope (NE). We report that the tail-anchored sarcolemmal membrane associated protein 3 (SLMAP3), a component of the MTOC and NE, is crucial for myogenesis because its deletion in mice leads to a reduction in the NE-MTOC formation, mislocalization of the nuclei, dysregulation of the myogenic programme and abnormal embryonic myofibres. SLMAP3-/- myoblasts also displayed a similar disorganized distribution of nuclei with an aberrant NE-MTOC and defective myofibre formation and differentiation programming. We identified novel interactors of SLMAP3, including pericentrin, PCM1 (pericentriolar material 1), AKAP9 (A-kinase anchoring protein 9), kinesin-1 members Kif5B (kinesin family member 5B), KCL1 (kinesin light chain 1), KLC2 (kinesin light chain 2) and nuclear lamins, and observed that the distribution of centrosomal proteins at the NE together with Nesprin-1 was significantly altered by the loss of SLMAP3 in differentiating myoblasts. SLMAP3 is believed to negatively regulate Hippo signalling, but its loss was without impact on this pathway in developing muscle. These results reveal that SLMAP3 is essential for skeletal myogenesis through unique mechanisms involving the positioning of nuclei, NE-MTOC dynamics and gene programming.

Keywords:  Golgi; LINC complex; SLMAP3; non-centrosomal microtubule-organizing centre; nuclear envelope; skeletal myogenesis

DOI:  https://doi.org/10.1098/rsob.240094
Cureus. 2024 Sep;16(9): e68974

Muscle Atrophy and mRNA-miRNA Network Analysis of Vascular Endothelial Growth Factor (VEGF) in a Mouse Model of Denervation-Induced Disuse.

Gaku Oguri, Ryo Ikegami, Haruka Ugawa, Manami Katoh, Syotaro Obi, Masashi Sakuma, Norihiko Takeda, Yutaka Kano, Shigeru Toyoda, Toshiaki Nakajima.

   BACKGROUND: Skeletal muscle atrophy is frequently caused by the disuse of muscles. It impacts quality of life, especially in aging populations and those with chronic diseases. Understanding the molecular mechanisms underlying muscle atrophy is crucial for developing effective therapies.
OBJECTIVE: To investigate the roles of vascular endothelial growth factor (VEGF) and various microRNAs (miRNAs) in muscle atrophy using a mouse model of denervation (DEN)-induced disuse, and to elucidate their interactions and regulatory functions through comprehensive network analysis.
METHODS: The right sciatic nerve of C57BL/6J mice (n=6) was excised to simulate DEN, with the left serving as a sham surgery control (Sham). Following a two-week period, wet muscle weight was measured. Total RNA was extracted from the tibialis anterior muscle for microarray analysis. Significant expression changes were analyzed via Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and miRNet for miRNAs.
RESULTS: Denervated limbs showed a significant reduction in muscle weight. Over 1,000 genes displayed increased expression, while 527 showed reductions to less than half of control levels. VEGF, along with specific miRNAs such as miR-106a-5p, miR-mir20a-5p, mir93-5p and mir17-5p, occupied central regulatory nodes within the gene network. Functional analysis revealed that these molecules are involved in key biological processes including regulation of cell migration, vasculature development, and regulation of endothelial cell proliferation. The increased miRNAs were subjected to further network analysis that revealed significant regulatory interactions with target mRNAs.
CONCLUSION: VEGF and miRNAs play crucial roles in the progression of skeletal muscle atrophy, offering potential targets for therapeutic interventions aimed at reducing atrophy and enhancing muscle regeneration.

Keywords:  denervation; gene expression; microrna; muscle atrophy; vascular endothelial growth factor (vegf)

DOI:  https://doi.org/10.7759/cureus.68974
EMBO J. 2024 Oct 08.

DELE1 maintains muscle proteostasis to promote growth and survival in mitochondrial myopathy.

Hsin-Pin Lin, Jennifer D Petersen, Alexandra J Gilsrud, Angelo Madruga, Theresa M D'Silva, Xiaoping Huang, Mario K Shammas, Nicholas P Randolph, Kory R Johnson, Yan Li, Drew R Jones, Michael E Pacold, Derek P Narendra.

  Mitochondrial dysfunction causes devastating disorders, including mitochondrial myopathy, but how muscle senses and adapts to mitochondrial dysfunction is not well understood. Here, we used diverse mouse models of mitochondrial myopathy to show that the signal for mitochondrial dysfunction originates within mitochondria. The mitochondrial proteins OMA1 and DELE1 sensed disruption of the inner mitochondrial membrane and, in response, activated the mitochondrial integrated stress response (mt-ISR) to increase the building blocks for protein synthesis. In the absence of the mt-ISR, protein synthesis in muscle was dysregulated causing protein misfolding, and mice with early-onset mitochondrial myopathy failed to grow and survive. The mt-ISR was similar following disruptions in mtDNA maintenance (Tfam knockout) and mitochondrial protein misfolding (CHCHD10 G58R and S59L knockin) but heterogenous among mitochondria-rich tissues, with broad gene expression changes observed in heart and skeletal muscle and limited changes observed in liver and brown adipose tissue. Taken together, our findings identify that the DELE1 mt-ISR mediates a similar response to diverse forms of mitochondrial stress and is critical for maintaining growth and survival in early-onset mitochondrial myopathy.

Keywords:  Mitochondria Unfolded Protein Response (mt-UPR); Mitochondrial Disorders; Mitohormesis; Mitonuclear Communication; Mitophagy

DOI:  https://doi.org/10.1038/s44318-024-00242-x
STAR Protoc. 2024 Oct 09. pii: S2666-1667(24)00543-4. [Epub ahead of print]5(4): 103378

Spatial analysis of transcript and protein levels in skeletal muscle.

Paola Pisterzi, Clara Martinez Mir, Ouafa Dahri, Isabel de Poorter, Sandra Batlles Parera, Milica Dostanić, Massimo Mastrangeli, Christine Mummery, Niels Geijsen, Fanny Sage.

  Skeletal muscle spatial analyses have revealed unexpected regionalized gene expression patterns challenging the understanding of muscle as a homogeneous tissue. Here, we present a protocol for the spatial analysis of transcript and protein levels in murine skeletal muscle. We describe steps for tibialis anterior dissection, formaldehyde fixation, tissue chopper cutting, and hybridization chain reaction (HCR) detection and amplification. We then detail procedures for immunostaining, tissue clearing, and imaging. This protocol is easily adaptable to other tissues.

Keywords:  Gene Expression; In Situ Hybridization; Microscopy; Tissue Engineering

DOI:  https://doi.org/10.1016/j.xpro.2024.103378
J Physiol. 2024 Oct 07.

Repeated passive heat treatment increases muscle tissue capillarization, but does not affect postprandial muscle protein synthesis rates in healthy older adults.

Cas J Fuchs, Milan W Betz, Heather L Petrick, Jil Weber, Joan M Senden, Floris K Hendriks, Julia L M Bels, Luc J C van Loon, Tim Snijders.

  Prolonged passive heat treatment (PHT) has been suggested to trigger skeletal muscle adaptations that may improve muscle maintenance in older individuals. To assess the effects of PHT on skeletal muscle tissue capillarization, perfusion capacity, protein synthesis rates, hypertrophy and leg strength, 14 older adults (9 males, 5 females; 73 ± 6 years) underwent 8 weeks of PHT (infrared sauna: 3× per week, 45 min at ∼60°C). Before and after PHT we collected muscle biopsies to assess skeletal muscle capillarization and fibre cross-sectional area (CSA). Basal and postprandial muscle tissue perfusion kinetics and protein synthesis rates were assessed using contrast-enhanced ultrasound and primed continuous l-[ring-13C6]phenylalanine infusions, respectively. One-repetition maximum (1RM) leg strength and vastus lateralis muscle CSA were assessed. Type I and type II muscle fibre capillarization strongly increased following PHT (capillary-to-fibre perimeter exchange index: +31 ± 18 and +33 ± 30%, respectively; P < 0.001). No changes were observed in basal (0.24 ± 0.27 vs. 0.18 ± 0.11 AU; P = 0.266) or postprandial (0.20 ± 0.12 vs. 0.18 ± 0.14 AU; P = 0.717) microvascular blood flow following PHT. Basal (0.048 ± 0.014 vs. 0.051 ± 0.019%/h; P = 0.630) and postprandial (0.041 ± 0.012 vs. 0.051 ± 0.024%/h; P = 0.199) muscle protein synthesis rates did not change in response to prolonged PHT. Furthermore, no changes in vastus lateralis muscle CSA (15.3 ± 4.6 vs. 15.2 ± 4.6 cm2; P = 0.768) or 1RM leg strength (46 ± 12 vs. 47 ± 12 kg; P = 0.087) were observed over time. In conclusion, prolonged PHT increases muscle tissue capillarization but this does not improve muscle microvascular blood flow or increase muscle protein synthesis rates in healthy, older adults. Prolonged PHT does not induce skeletal muscle hypertrophy or increase leg strength in healthy, older adults. KEY POINTS: Repeated exposure to heat has been suggested to trigger skeletal muscle adaptive responses. We investigated the effect of 8 weeks of whole-body passive heat treatment (PHT; infrared sauna: 3× per week for 45 min at ∼60°C) on skeletal muscle tissue capillarization, perfusion capacity, basal, and postprandial muscle protein synthesis rates, muscle (fibre) hypertrophy, and leg strength in healthy, older adults. Prolonged PHT increases muscle tissue capillarization, but this does not improve muscle microvascular blood flow or increase muscle protein synthesis rates. Despite increases in muscle tissue capillarization, prolonged PHT does not suffice to induce skeletal muscle hypertrophy or increase leg strength in healthy, older adults.

Keywords:  adaptation; ageing; blood pressure; glucose metabolism; heat stress; hypertrophy; mitochondrial content; mitochondrial function; stable isotope tracers; vascular conductance

DOI:  https://doi.org/10.1113/JP286986
Mol Med. 2024 Oct 10. 30(1): 171

Acetylcholine receptor-β inhibition by interleukin-6 in skeletal muscles contributes to modulating neuromuscular junction during aging.

Yanling Zhao, Han Yan, Ke Liu, Jiangping Ma, Wenlan Sun, Hejin Lai, Hongli Li, Jianbang Gu, He Huang.

   BACKGROUND: Aging-related strength decline contributes to physiological deterioration and is a good predictor of poor prognosis. However, the mechanisms underlying neuromuscular junction disorders affecting contraction in aging are not well described. We hypothesized that the autocrine effect of interleukin (IL)-6 secreted by skeletal muscle inhibits acetylcholine receptor (AChR) expression, potentially causing aging-related strength decline. Therefore, we investigated IL-6 and AChR β-subunit (AChR-β) expression in the muscles and sera of aging C57BL/6J mice and verified the effect of IL-6 on AChR-β expression.
METHODS: Animal experiments, in vitro studies, bioinformatics, gene manipulation, dual luciferase reporter gene assays, and chromatin immunoprecipitation experiments were used to explore the role of the transcription cofactor peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α) and its interacting transcription factors in the IL-6-mediated regulation of AChR-β expression.
RESULTS: IL-6 expression gradually increased during aging, inhibiting AChR-β expression, which was reversed by tocilizumab. Both tocilizumab and the PGC1α agonist reversed the inhibiting effect of IL-6 expression on AChR-β. Compared to inhibition of signal transducer and activator of transcription 3, extracellular signal-regulated kinases 1/2 (ERK1/2) inhibition suppressed the effects of IL-6 on AChR-β and PGC1α. In aging mouse muscles and myotubes, myocyte enhancer factor 2 C (MEF2C) was recruited by PGC1α, which directly binds to the AChR-β promoter to regulate its expression.
CONCLUSIONS: This study verifies AChR-β regulation by the IL-6/IL-6R-ERK1/2-PGC1α/MEF2C pathway. Hence, evaluating muscle secretion, myokines, and AChRs at an earlier stage to determine pathological progression is important. Moreover, developing intervention strategies for monitoring, maintaining, and improving muscle structure and function is necessary.

Keywords:  Acetylcholine receptor β; Aging; Interleukin-6; Myocyte enhancer factor 2 C; Neuromuscular junction; Peroxisome proliferator-activated receptor gamma coactivator 1α

DOI:  https://doi.org/10.1186/s10020-024-00943-3
iScience. 2024 Oct 18. 27(10): 110913

Sorafenib induces cachexia by impeding transcriptional signaling of the SET1/MLL complex on muscle-specific genes.

Bushra Khan, Chiara Lanzuolo, Valentina Rosti, Philina Santarelli, Andreas Pich, Theresia Kraft, Mamta Amrute-Nayak, Arnab Nayak.

  Chemotherapeutics used in cancer therapy are often linked to muscle wasting or cachexia. Insights into the molecular basis of chemotherapy-induced cachexia is essential to improve treatment strategies. Here, we demonstrated that Sorafenib-tyrosine kinase inhibitor (TKI) class of chemotherapeutic agents-induced cachexia. System-wide analyses revealed that Sorafenib alters the global transcriptional program and proteostasis in muscle cells. Mechanistically, Sorafenib treatment reduced active epigenetic mark H3K4 methylation on distinct muscle-specific genes by impeding chromatin association of SET1A-catalytic component of the SET1/MLL histone methyltransferase complex. This mechanism favored transcriptional disorientation that led to disrupted sarcomere assembly, calcium homeostasis and mitochondrial respiration. Consequently, the contractile ability of muscle cells was severely compromised. Interestingly, the other prominent TKIs Nilotinib and Imatinib did not exert similar effects on muscle cell physiology. Collectively, we identified an unanticipated transcriptional mechanism underlying Sorafenib-induced cachexia. Our findings hold the potential to strategize therapy regimens to minimize chemotherapy-induced cachexia.

Keywords:  Health sciences; Internal medicine; Medical specialty; Medicine; Oncology

DOI:  https://doi.org/10.1016/j.isci.2024.110913
Arch Physiol Biochem. 2024 Oct 09. 1-13

High-intensity exercise alongside insulin alleviates muscle atrophy in type 1 diabetes mellitus concomitant with modulation of mitophagy-related proteins in skeletal muscle.

Manal Moustafa Mahmoud, Nahed Qutb Abdel Hameed, Basant Adel Al Dreny Abd Al Latef, Samaa Samir Kamar, Laila Ahmed Rashed, Sarah Ali Abdelhameed Gouda.

  Background: Diabetes patients' quality of life can be severely impacted by diabetic muscle atrophy.Aim: This study aimed to explore the impact of high-intensity exercise (HIE) alongside insulin treatment on muscle atrophy in a rat model of type 1 diabetes mellitus (T1DM).Methodology: Fifty rats were allocated into five groups; Group 1, control sedentary (CS), T1DM was elicited in the rest of the groups by giving them Streptozotocin (STZ) (60 mg/kg), where group 2 (DS) remained sedentary, while groups 3,4,5 were treated with insulin after induction of diabetes. Group 4 (DI+MIE) and 5 (DI+ HIE) underwent moderate and high-intensity exercise, respectively.Results: HIE for 14 days combined with insulin treatment significantly restored muscle strength and mass with a significant modification in the mitophagy-related proteins and fibroblast growth factor 21 (FGF 21) compared to other treated groups.Conclusion: This study concluded that there is a therapeutic role for HIE with insulin against T1DM-induced muscle atrophy.

Keywords:  FGF 21; Type 1 DM; high-intensity exercise; mitophagy; muscle atrophy

DOI:  https://doi.org/10.1080/13813455.2024.2410791
Front Cell Dev Biol. 2024 ;12 1464815

Mechanisms of the NAD+ salvage pathway in enhancing skeletal muscle function.

Mengzhu Su, Fanghui Qiu, Yansong Li, Tongtong Che, Ningning Li, Shuangshuang Zhang.

  Nicotinamide adenine dinucleotide (NAD+) is crucial for cellular energy production, serving as a coenzyme in oxidation-reduction reactions. It also supports enzymes involved in processes such as DNA repair, aging, and immune responses. Lower NAD+ levels have been associated with various diseases, highlighting the importance of replenishing NAD+. Nicotinamide phosphoribosyltransferase (NAMPT) plays a critical role in the NAD+ salvage pathway, which helps sustain NAD+ levels, particularly in high-energy tissues like skeletal muscle.This review explores how the NAMPT-driven NAD+ salvage pathway influences skeletal muscle health and functionality in aging, type 2 diabetes mellitus (T2DM), and skeletal muscle injury. The review offers insights into enhancing the salvage pathway through exercise and NAD+ boosters as strategies to improve muscle performance. The findings suggest significant potential for using this pathway in the diagnosis, monitoring, and treatment of skeletal muscle conditions.

Keywords:  NAD+; NAMPT; T2DM; aging; skelelal muscle

DOI:  https://doi.org/10.3389/fcell.2024.1464815
Skelet Muscle. 2024 Oct 11. 14(1): 22

Isolation of small extracellular vesicles from regenerating muscle tissue using tangential flow filtration and size exclusion chromatography.

Uxia Gurriaran-Rodriguez, Yves De Repentigny, Rashmi Kothary, Michael A Rudnicki.

  We have recently made the strikingly discovery that upon a muscle injury, Wnt7a is upregulated and secreted from new regenerating myofibers on the surface of exosomes to elicit its myogenerative response distally. Despite recent advances in extracellular vesicle (EVs) isolation from diverse tissues, there is still a lack of specific methodology to purify EVs from muscle tissue. To eliminate contamination with non-EV secreted proteins and cytoplasmic fragments, which are typically found when using classical methodology, such as ultracentrifugation, we adapted a protocol combining Tangential Flow Filtration (TFF) and Size Exclusion Chromatography (SEC). We found that this approach allows simultaneous purification of Wnt7a, bound to EVs (retentate fraction) and free non-EV Wnt7a (permeate fraction). Here we described this optimized protocol designed to specifically isolate EVs from hind limb muscle explants, without cross-contamination with other sources of non-EV bounded proteins. The first step of the protocol is to remove large EVs with sequential centrifugation. Extracellular vesicles are then concentrated and washed in exchange buffer by TFF. Lastly, SEC is performed to remove any soluble protein traces remaining after TFF. Overall, this procedure can be used to isolate EVs from conditioned media or biofluid that contains EVs derived from any cell type or tissue, improving reproducibility, efficiency, and purity of EVs preparations. Our purification protocol results in high purity EVs that maintain structural integrity and thus fully compatible with in vitro and in vivo bioactivity and analytic assays.

Keywords:  Myoregeneration; Skeletal muscle; Tangential flow filtration; Tissue derived extracellular vesicles

DOI:  https://doi.org/10.1186/s13395-024-00355-1
Sci Rep. 2024 10 10. 14(1): 23691

Identification and analysis of differentially expressed lncRNAs and their ceRNA networks in DMD/mdx primary myoblasts.

Abdolvahab Ebrahimpour Gorji, Kasra Ahmadian, Zahra Roudbari, Tomasz Sadkowski.

This study explored the significance of long non-coding RNAs (lncRNAs), particularly their role in maintaining dystrophin protein stability and regulating myocyte proliferation and differentiation. The investigation focused on DMD/mdx mouse skeletal muscle primary myoblasts, aiming to identify lncRNAs potential as biomarkers and therapeutic targets for Duchenne muscular dystrophy (DMD). Utilizing CLC Genomics Workbench software, 554 differentially expressed lncRNAs were identified in DMD/mdx mice compared to wild-type (WT) control. Among them, 373 were upregulated, and 181 were downregulated. The study highlighted specific lncRNAs (e.g., 5930430L01Rik, Gm10143, LncRNA1490, LncRNA580) and their potential regulatory roles in DMD key genes like IGF1, FN1, TNNI1, and MYOD1. By predicting miRNA and their connections with lncRNA and mRNA (ceRNA network) using tools such as miRNet, miRSYSTEM and miRCARTA, the study revealed potential indirect regulation of Dystrophin, IGF1R and UTRN genes by identified lncRNAs (e.g. 2310001H17Rik-203, C130073E24Rik-202, LncRNA2767, 5930430L01Rik and LncRNA580). These findings suggest that the identified lncRNAs may play crucial roles in the development and progression of DMD through their regulatory influence on key gene expression, providing valuable insights for potential therapeutic interventions.

DOI: https://doi.org/10.1038/s41598-024-75221-7
J Cell Physiol. 2024 Oct 09. e31458

A combination of major histocompatibility complex (MHC) I overexpression and type I interferon induce mitochondrial dysfunction in human skeletal myoblasts.

Anastasia Thoma, Razan Alomosh, Holly L Bond, Tania Akter-Miah, Nasser Al-Shanti, Hans Degens, Vanja Pekovic-Vaughan, Adam P Lightfoot.

  The overexpression of major histocompatibility complex (MHC) I on the surface of muscle fibers is a characteristic hallmark of the idiopathic inflammatory myopathies (IIMs), collectively termed myositis. Alongside MHC-I overexpression, subtypes of myositis, display a distinct type I interferon (IFN) signature. This study examined the combinational effects of elevated MHC-I and type I IFNs (IFNα/β) on mitochondrial function, as mitochondrial dysfunction is often seen in IIMs. Human skeletal muscle myoblasts were transfected with an MHC-I isoform using the mammalian HLA-A2/Kb vector. Mitochondrial respiration, mitochondrial membrane potential, and reactive oxygen/nitrogen species generation were assessed with or without IFNα and IFNβ. We show that MHC-I overexpression in human skeletal muscle myoblasts led to decreased basal glycolysis and mitochondrial respiration, cellular spare respiratory capacity, adenosine triphosphate-linked respiration, and an increased proton leak, which were all exaggerated by type I IFNs. Mitochondrial membrane depolarization was induced by MHC-I overexpression both in absence and presence of type I IFNs. Human myoblasts overexpressing MHC-I showed elevated nitric oxide generation that was abolished when combined with IFN. MHC-I on its own did not result in an increased reactive oxygen species (ROS) production, but IFN on their own, or combined with MHC-I overexpression did induce elevated ROS generation. Surprisingly, we observed no gross changes in mitochondrial reticular structure or markers of mitochondrial dynamics. We present new evidence that MHC-I overexpression and type I IFNs aggravate the effects each has on mitochondrial function in human skeletal muscle cells, providing novel insights into their mechanisms of action and suggesting important implications in the further study of myositis pathogenesis.

Keywords:  idiopathic inflammatory myopathies; major histocompatibility complex I; mitochondria; myositis; reactive and nitric oxygen species; type I interferon

DOI:  https://doi.org/10.1002/jcp.31458
J Physiol. 2024 Oct 11.

Dynamics of cardio-muscular networks in exercise and fatigue.

Sergi Garcia-Retortillo, Plamen Ch Ivanov.

  A fundamental question in cardiovascular and muscle physiology is how the heart operates in synchrony with distinct muscles to regulate homeostasis, enable movement and adapt to exercise demands and fatigue. Here we investigate how autonomic regulation of cardiac function synchronizes and integrates as a network with the activity of distinct muscles during exercise. Further, we establish how the network of cardio-muscular interactions reorganizes with fatigue. Thirty healthy young adults performed two body weight squat tests until exhaustion. Simultaneous recordings were taken of a 3-lead electrocardiogram (EKG) along with electromyography (EMG) signals from the left and right vastus lateralis, and left and right erector spinae. We first obtained instantaneous heart rate (HR) derived from the EKG signal and decomposed the EMG recordings in 10 frequency bands (F1-F10). We next quantified pair-wise coupling (cross-correlation) between the time series for HR and all EMG spectral power frequency bands in each leg and back muscle. We uncovered the first profiles of cardio-muscular network interactions, which depend on the role muscles play during exercise and muscle fibre histochemical characteristics. Additionally, we observed a significant decline in the degree of cardio-muscular coupling with fatigue, characterized by complex transitions from synchronous to asynchronous behaviour across a range of timescales. The network approach we utilized introduces new avenues for the development of novel network-based markers, with the potential to characterize multilevel cardio-muscular interactions to assess global health, levels of fatigue, fitness status or the effectiveness of cardiovascular and muscle injury rehabilitation programmes. KEY POINTS: The heart operates in synchrony with muscles to regulate homeostasis, enable movement, and adapt to exercise demands and fatigue. However, the precise mechanisms regulating cardio-muscular coupling remain unknown. This study introduces a pioneering approach to assess cardio-muscular network interactions by examining the synchronization of cardiac function with muscle activity during exercise and fatigue. We uncover the first profiles of cardio-muscular interactions characterized by specific hierarchical organization of link strength. We observe a significant decline in the degree of cardio-muscular coupling with fatigue, marked by complex transitions from synchronous to asynchronous behaviour. This network approach offers new network-based markers to characterize multilevel cardio-muscular interactions to assess global health, levels of fatigue, fitness status or the effectiveness of cardiovascular and muscle injury rehabilitation programmes.

Keywords:  cardiac electrophysiology; cardio‐muscular coordination; complex systems; dynamic networks; electromyography; fatigue; heart rate variability; muscle fibres; network physiology; synchronization

DOI:  https://doi.org/10.1113/JP286963