bims-moremu Biomed News
on Molecular regulators of muscle mass
Issue of 2023‒03‒26
thirty-two papers selected by
Anna Vainshtein
Craft Science Inc.

  1. Aging (Albany NY). 2023 Mar 20. 15
      Aging coincides with the accumulation of senescent cells within skeletal muscle that produce inflammatory products, known as the senescence-associated secretory phenotype, but the relationship of senescent cells to muscle atrophy is unclear. Previously, we found that a metformin + leucine (MET+LEU) treatment had synergistic effects in aged mice to improve skeletal muscle structure and function during disuse atrophy. Therefore, the study's purpose was to determine the mechanisms by which MET+LEU exhibits muscle atrophy protection in vitro and if this occurs through cellular senescence. C2C12 myoblasts differentiated into myotubes were used to determine MET+LEU mechanisms during atrophy. Additionally, aged mouse single myofibers and older human donor primary myoblasts were individually isolated to determine the translational potential of MET+LEU on muscle cells. MET+LEU (25 + 125 μM) treatment increased myotube differentiation and prevented myotube atrophy. Low concentration (0.1 + 0.5 μM) MET+LEU had unique effects to prevent muscle atrophy and increase transcripts related to protein synthesis and decrease transcripts related to protein breakdown. Myotube atrophy resulted in dysregulated proteostasis that was reversed with MET+LEU and individually with proteasome inhibition (MG-132). Inflammatory and cellular senescence transcriptional pathways and respective transcripts were increased following myotube atrophy yet reversed with MET+LEU treatment. Dasatinib + quercetin (D+Q) senolytic prevented myotube atrophy similar to MET+LEU. Finally, MET+LEU prevented loss in myotube size in alternate in vitro models of muscle atrophy as well as in aged myofibers while, in human primary myotubes, MET+LEU prevented reductions in myonuclei fusion. These data support that MET+LEU has skeletal muscle cell-autonomous properties to prevent atrophy by reversing senescence and improving proteostasis.
    Keywords:  AMPK; inflammation; protein breakdown; senolytic; skeletal muscle atrophy
  2. Geroscience. 2023 Mar 23.
      Progressive muscle atrophy and loss of muscle strength associated with old age have been well documented. Although age-associated impairments in skeletal muscle regeneration following injury have been demonstrated, less is known about whether aging impacts the regenerative response of neuromuscular junctions (NMJ) following contraction-induced injury. Reduced ability of NMJs to regenerate could lead to increased numbers of denervated muscle fibers and therefore play a contributing role to age-related sarcopenia. To investigate the relationship between age and NMJ regeneration following injury, extensor digitorum longus (EDL) muscles of middle-aged (18-19 months) and old mice (27-28 months) were subjected to a protocol of lengthening contractions (LC) that resulted in an acute force deficit of ~55% as well as functional and histological evidence of a similar magnitude of injury 3 days post LCs that was not different between age groups. After 28 days, the architecture and innervation of the NMJs were evaluated. The numbers of fragmented endplates increased and of fully innervated NMJs decreased post-injury for the muscle of both middle-aged and old mice and for contralateral uninjured muscles of old compared with uninjured muscles of middle-aged controls. Thus, the diminished ability of the skeletal muscle of old mice to recover following injury may be due in part to an age-related decrease in the ability to regenerate NMJs in injured muscles. The impaired ability to regenerate NMJs may be a triggering factor for degenerative changes at the NMJ contributing to muscle fiber weakness and loss in old age.
    Keywords:  Aging; Injury; Innervation; Lengthening contractions; Muscle; Neuromuscular junction; Regeneration; Sarcopenia
  3. Int J Sports Med. 2023 Mar 21.
      Resistance training is employed for pursuing muscle strength characterized by activation of mammalian target of rapamycin (mTOR)-mediated hypertrophic signaling for protein production. Endurance training elevates peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) signaling of mitochondrial adaptations for oxidative phosphorylation. Now, emerging evidence suggests that, like endurance training, resistance training also elicits profound effects on mitochondrial adaptations in skeletal muscle, which means that resistance training yields both strength and endurance phenotypes in myofibers, which has treatment value for the muscle loss and poor aerobic capacity in humans. Our review outlines a brief overview of muscle hypertrophic signals with resistance training and focused on the effects of resistance training on mitochondrial biogenesis and respiration in skeletal muscle, providing novel insights into the therapeutic strategy of resistance training for the metabolically dysfunctional individuals with declined mitochondrial function.
  4. Stem Cells. 2023 Mar 21. pii: sxad023. [Epub ahead of print]
      Skeletal muscle is mainly composed of multinucleated cells called myofibers, and has excellent regenerative and adaptive abilities. These abilities are granted by muscle satellite cells (MuSCs), which are anatomically defined cells located between myofibers and basal lamina. In addition to myofibers and MuSCs, skeletal muscle contains several types of cells located in interstitial areas, such as mesenchymal progenitors. These cells are positive for platelet-derived growth factor receptor alpha and are called fibro/adipogenic progenitors (FAPs) or mesenchymal stromal cells. Although mesenchymal progenitors were originally identified as the causative cells of ectopic fat accumulation in skeletal muscles, recent studies have shed light on their beneficial roles in homeostasis, regeneration, and hypertrophy. Furthermore, the heterogeneity of mesenchymal progenitors is of great interest in understanding skeletal muscle development, homeostasis, regeneration, aging, and diseases. In this concise review, we summarize recent findings on the physiological roles of mesenchymal progenitors and their heterogeneity and discuss the remaining critical concerns.
  5. JCI Insight. 2023 Mar 21. pii: e151933. [Epub ahead of print]
      Phosphoinositides (PI) are membrane lipids that regulate signal transduction and vesicular trafficking. X-linked centronuclear myopathy (XLCNM), also called myotubular myopathy, results from loss-of-function mutations in the Mtm1 gene, which encodes the myotubularin phosphatidylinositol 3-phosphate (PtdIns3P) lipid phosphatase. No therapy for this disease is currently available. Previous studies showed that loss of expression of the class II phosphoinositide 3-kinase (PI3K) PI3K-C2β protein improved the phenotypes of a XLCNM mouse model. PI3Ks are well known to have extensive scaffolding functions and the importance of the catalytic activity of this PI3K for rescue remains unclear. Here, using PI3K-C2β kinase-dead mice, we show that the selective inactivation of PI3K-C2β kinase activity is sufficient to fully prevent muscle atrophy and weakness, histopathology, and sarcomere and triad disorganization in Mtm1 knockout mice. This rescue correlates with normalization of PtdIns3P level and mTORC1 activity, a key regulator of protein synthesis and autophagy. Conversely, lack of PI3K-C2β kinase activity did not rescue the histopathology of the BIN1 autosomal centronuclear myopathy mouse model. Overall, these findings support the development of specific PI3K-C2β kinase inhibitors to cure myotubular myopathy.
    Keywords:  Inositol phosphates; Muscle Biology; Protein kinases; Skeletal muscle
  6. FASEB J. 2023 Apr;37(4): e22851
      Sarcopenia is a geriatric syndrome characterized by an age-related decline in skeletal muscle mass and strength. Here, we show that suppression of mitochondrial calcium uniporter (MCU)-mediated Ca2+ influx into mitochondria in the body wall muscles of the nematode Caenorhabditis elegans improved the sarcopenic phenotypes, blunting movement and mitochondrial structural and functional decline with age. We found that normally aged muscle cells exhibited elevated resting mitochondrial Ca2+ levels and increased mitophagy to eliminate damaged mitochondria. Similar to aging muscle, we found that suppressing MCU function in muscular dystrophy improved movement via reducing elevated resting mitochondrial Ca2+ levels. Taken together, our results reveal that elevated resting mitochondrial Ca2+ levels contribute to muscle decline with age and muscular dystrophy. Further, modulation of MCU activity may act as a potential pharmacological target in various conditions involving muscle loss.
    Keywords:  MCU; aging; calcium; dystrophy; mitophagy; sarcopenia
  7. Am J Physiol Endocrinol Metab. 2023 Mar 22.
      The quality of skeletal muscle is maintained by a balance between protein biosynthesis and degradation. Disruption in this balance results in sarcopenia. However, its underlying mechanisms remain under-investigated. Selenoprotein P (SeP; encoded by Selenop in mice) is a hepatokine that is upregulated in type 2 diabetes and aging and causes signal resistances via reductive stress. We created immobilized muscle atrophy (IMM) model in Selenop knockout (KO) mice. IMM significantly reduced cross-sectional areas and the size of skeletal muscle fibers, which were ameliorated in KO mice. IMM upregulated the genes encoding E3 ubiquitin ligases and their upstream FoxO1, FoxO3, and KLF15 transcription factors in the skeletal muscle, which were suppressed in KO mice. These findings suggest a possible involvement of SeP-mediated reductive stress in physical inactivity-mediated sarcopenia, which may be a therapeutic target against sarcopenia.
    Keywords:  E3 ligases; Immobilization; Muscle atrophy; Reductive stress; Selenoprotein P
  8. Front Cardiovasc Med. 2023 ;10 1118738
      Skeletal muscle injury in peripheral artery disease (PAD) has been attributed to vascular insufficiency, however evidence has demonstrated that muscle cell responses play a role in determining outcomes in limb ischemia. Here, we demonstrate that genetic ablation of Pax7+ muscle progenitor cells (MPCs) in a model of hindlimb ischemia (HLI) inhibited muscle regeneration following ischemic injury, despite a lack of morphological or physiological changes in resting muscle. Compared to control mice (Pax7WT), the ischemic limb of Pax7-deficient mice (Pax7Δ) was unable to generate significant force 7 or 28 days after HLI. A significant increase in adipose was observed in the ischemic limb 28 days after HLI in Pax7Δ mice, which replaced functional muscle. Adipogenesis in Pax7Δ mice corresponded with a significant increase in PDGFRα+ fibro/adipogenic progenitors (FAPs). Inhibition of FAPs with batimastat decreased muscle adipose but increased fibrosis. In vitro, Pax7Δ MPCs failed to form myotubes but displayed increased adipogenesis. Skeletal muscle from patients with critical limb threatening ischemia displayed increased adipose in more ischemic regions of muscle, which corresponded with fewer satellite cells. Collectively, these data demonstrate that Pax7+ MPCs are required for muscle regeneration after ischemia and suggest that muscle regeneration may be an important therapeutic target in PAD.
    Keywords:  adipogenesis; critical limb threatening ischemia; fibro/adipogenic progenitor cells; hind limb ischemia; muscle progenitor cells; peripheral artery disease; skeletal muscle regeneration
  9. Elife. 2023 Mar 23. pii: e85289. [Epub ahead of print]12
      Reactive oxygen species (ROS) accumulation is a cardinal feature of skeletal muscle atrophy. ROS refers to a collection of radical molecules whose cellular signals are vast, and it is unclear which downstream consequences of ROS are responsible for the loss of muscle mass and strength. Here we show that lipid hydroperoxides (LOOH) are increased with age and disuse, and the accumulation of LOOH by deletion of glutathione peroxidase 4 (GPx4) is sufficient to augment muscle atrophy. LOOH promoted atrophy in a lysosomal-dependent, proteasomal-independent manner. In young and old mice, genetic and pharmacologic neutralization of LOOH or their secondary reactive lipid aldehydes robustly prevented muscle atrophy and weakness, indicating that LOOH-derived carbonyl stress mediates age- and disuse-induced muscle dysfunction. Our findings provide novel insights for the role of LOOH in sarcopenia including a therapeutic implication by pharmacologic suppression.
    Keywords:  cell biology; mouse
  10. Front Physiol. 2023 ;14 1114595
      Methods of isolating mitochondria commonly utilise mechanical force and shear stress to homogenize tissue followed by purification by multiple rounds of ultracentrifugation. Existing protocols can be time-consuming with some physically impairing integrity of the sensitive mitochondrial double membrane. Here, we describe a method for the recovery of intact, respiring mitochondria from murine skeletal muscle tissue and cell lines using nitrogen cavitation. This protocol results in high-yield, pure and respiring mitochondria without the need for purification gradients or ultracentrifugation. The protocol takes under an hour and requires limited specialised equipment. Our methodology is successful in extracting mitochondria of both cell extracts and skeletal muscle tissue. This represents an improved yield in comparison to many of the existing methods. Western blotting and electron microscopy demonstrate the enrichment of mitochondria with their ultrastructure well-preserved and an absence of contamination from cytoplasmic or nuclear fractions. Using respirometry analysis we show that mitochondria extracted from murine skeletal muscle cell lines (C2C12) and tibialis anterior tissue have an appropriate respiratory control ratio. These measures are indicative of healthy coupled mitochondria. Our method successfully demonstrates the rapid isolation of functional mitochondria and will benefit researchers studying mitochondrial bioenergetics as well as providing greater throughput and application for time-sensitive assays.
    Keywords:  cavitation; methodolody; mitochondria; protocol; skeletal muscle
  11. Toxicology. 2023 Mar 16. pii: S0300-483X(23)00075-6. [Epub ahead of print]489 153489
      Ripretinib is a multikinase inhibitor drug approved in 2020 by the FDA and in 2021 by EMA for use in the treatment of advanced gastrointestinal stromal tumors (GIST) which have not adequately responded to previous treatments with kinase inhibitors. The most common side effects of the drug are myalgia and fatigue, which likely causes interruption of the treatment or reduction of the dose. Skeletal muscle cells highly depend on ATP to perform their functions and mitochondrial damage may play a role in skeletal muscle toxicity induced by kinase inhibitors. However, the molecular mechanism has not been clearly identified in the literature yet. In this study, it has been aimed to elucidate the role of mitochondria in the toxic effect of ripretinib on skeletal muscle using the mouse C2C12 myoblast-derived myotubes. The myotubes were exposed to ripretinib at the range of 1-20 μM concentrations for 24 h. To determine the potential role of mitochondrial impairment in ripretinib-induced skeletal muscle toxicity, intracellular ATP level, mitochondrial membrane potential (MMP), mitochondrial ROS production (mtROS), mitochondrial DNA (mtDNA) copy number, and mitochondrial mass were examined after ripretinib treatment. Furthermore, changes in PGC 1α/NRF 1/NRF 2 expression levels that play a role in mitochondrial biogenesis and mitophagy were investigated. Additionally, the mitochondrial electron transport chain (ETC) enzyme activities were evaluated. Lastly, a molecular docking study was done to see ripretinib's possible interaction with DNA polymerase gamma (POLG) which is important for DNA replication in the mitochondria. According to the findings, ripretinib decreases the ATP level and mtDNA copy number, induces loss of MMP, and reduces mitochondrial mass. The activities of the ETC complexes were inhibited with ripretinib exposure which is in line with the observed ATP depletion and MMP loss. The molecular docking study revealed that ripretinib has inhibitory potential against POLG which supports the observed inhibition of mtDNA. The expression of PGC 1α was reduced in the nuclear fraction indicating that PGC-1α was not activated since the NRF 1 expression was reduced and NRF 2 level did not show significant change. Consequently, mtROS production increased in all treatment groups and mitophagy-related gene expressions and Parkin protein expression level were up-regulated at high doses. In conclusion, mitochondrial damage/loss can be one of the underlying causes of ripretinib-induced skeletal muscle toxicity. However, further studies are needed to confirm the results in vivo.
    Keywords:  C2C12 myotubes; Kinase inhibitors; Mitochondrial damage; Myotoxicity; Ripretinib
  12. Sci Robot. 2023 Mar 22. 8(76): eadd9369
      Robot-actuated mechanical loading (ML)-based therapies ("mechanotherapies") can promote regeneration after severe skeletal muscle injury, but the effectiveness of such approaches during aging is unknown and may be influenced by age-associated decline in the healing capacity of skeletal muscle. To address this knowledge gap, this work used a noninvasive, load-controlled robotic device to impose highly defined tissue stresses to evaluate the age dependence of ML on muscle repair after injury. The response of injured muscle to robot-actuated cyclic compressive loading was found to be age sensitive, revealing not only a lack of reparative benefit of ML on injured aged muscles but also exacerbation of tissue inflammation. ML alone also disrupted the normal regenerative processes of aged muscle stem cells. However, these negative effects could be reversed by introducing anti-inflammatory therapy alongside ML application, leading to enhanced skeletal muscle regeneration even in aged mice.
  13. Cell Rep. 2023 Mar 22. pii: S2211-1247(23)00300-5. [Epub ahead of print]42(4): 112289
      Myofibers are broadly characterized as fatigue-resistant slow-twitch (type I) fibers and rapidly fatiguing fast-twitch (type IIa/IIx/IIb) fibers. However, the molecular regulation of myofiber type is not entirely understood; particularly, information on regulators of fast-twitch muscle is scarce. Here, we demonstrate that the large Maf transcription factor family dictates fast type IIb myofiber specification in mice. Remarkably, the ablation of three large Mafs leads to the drastic loss of type IIb myofibers, resulting in enhanced endurance capacity and the reduction of muscle force. Conversely, the overexpression of each large Maf in the type I soleus muscle induces type IIb myofibers. Mechanistically, a large Maf directly binds to the Maf recognition element on the promoter of myosin heavy chain 4, which encodes the type IIb myosin heavy chain, driving its expression. This work identifies the large Maf transcription factor family as a major regulator for fast type IIb muscle determination.
    Keywords:  CP: Developmental biology; large Maf transcription factor; myofiber type; myosin heavy chain; skeletal muscle
  14. Curr Opin Clin Nutr Metab Care. 2023 Feb 10.
      PURPOSE OF REVIEW: Systemic cancer therapy-associated skeletal muscle wasting is emerging as a powerful impetus to the overall loss of skeletal muscle experienced by patients with cancer. This review explores the clinical magnitude and biological mechanisms of muscle wasting during systemic cancer therapy to illuminate this adverse effect. Emerging strategies for mitigation are also discussed.RECENT FINDINGS: Clinical findings include precise, specific measures of muscle loss over the course of chemotherapy, targeted therapy and immunotherapy. All these therapeutic classes associate with quantitatively important muscle loss, independent of tumor response. Parallel experimental studies provide understanding of the specific molecular basis of wasting, which can include inhibition of protein synthesis, proliferation and differentiation, and activation of inflammation, reactive oxygen species, autophagy, mitophagy, apoptosis, protein catabolism, fibrosis and steatosis in muscle. Strategies to mitigate these muscle-specific adverse effects of cancer therapy remain in the earliest stages of development.
    SUMMARY: The adverse side effect of cancer therapy on skeletal muscle has been largely ignored in the development of cancer therapeutics. Given the extent to which loss of muscle mass and function can bear on patients' function and quality of life, protection/mitigation of these side effects is a research priority.
  15. J Tissue Eng. 2023 Jan-Dec;14:14 20417314221139794
      Skeletal muscle-derived cells (SMDC) hold tremendous potential for replenishing dysfunctional muscle lost due to disease or trauma. Current therapeutic usage of SMDC relies on harvesting autologous cells from muscle biopsies that are subsequently expanded in vitro before re-implantation into the patient. Heterogeneity can arise from multiple factors including quality of the starting biopsy, age and comorbidity affecting the processed SMDC. Quality attributes intended for clinical use often focus on minimum levels of myogenic cell marker expression. Such approaches do not evaluate the likelihood of SMDC to differentiate and form myofibres when implanted in vivo, which ultimately determines the likelihood of muscle regeneration. Predicting the therapeutic potency of SMDC in vitro prior to implantation is key to developing successful therapeutics in regenerative medicine and reducing implementation costs. Here, we report on the development of a novel SMDC profiling tool to examine populations of cells in vitro derived from different donors. We developed an image-based pipeline to quantify morphological features and extracted cell shape descriptors. We investigated whether these could predict heterogeneity in the formation of myotubes and correlate with the myogenic fusion index. Several of the early cell shape characteristics were found to negatively correlate with the fusion index. These included total area occupied by cells, area shape, bounding box area, compactness, equivalent diameter, minimum ferret diameter, minor axis length and perimeter of SMDC at 24 h after initiating culture. The information extracted with our approach indicates live cell imaging can detect a range of cell phenotypes based on cell-shape alone and preserving cell integrity could be used to predict propensity to form myotubes in vitro and functional tissue in vivo.
    Keywords:  Cell therapy; fusion index; imaging pipeline; potency; skeletal muscle
  16. Front Physiol. 2023 ;14 1169923
    Keywords:  cellular signaling; energy metabolism; exercise; fatty acid oxidation; glucose uptake; metabolic homeostasis; molecular regulation; skeletal muscle
  17. FASEB J. 2023 Apr;37(4): e22891
      Respiratory complex IV (CIV, cytochrome c oxidase) is the terminal enzyme of the mitochondrial electron transport chain. Some CIV subunits have two or more isoforms, which are ubiquitously expressed or are expressed in specific tissues like the lung, muscle, and testis. Among the tissue-specific CIV isoforms, the muscle-specific isoforms are expressed in adult cardiac and skeletal muscles. To date, the physiological and biochemical association between the muscle-specific CIV isoforms and aerobic respiration in muscles remains unclear. In this study, we profiled the CIV organization and expression pattern of muscle-specific CIV isoforms in different mouse muscle tissues. We found extensive CIV-containing supramolecular organization in murine musculature at advanced developmental stages, while a switch in the expression from ubiquitous to muscle-specific isoforms of CIV was also detected. Such a switch was confirmed during the in vitro differentiation of mouse C2C12 myoblasts. Unexpectedly, a CIV expression decrease was observed during C2C12 differentiation, which was probably due to a small increase in the expression of muscle-specific isoforms coupled with a dramatic decrease in the ubiquitous isoforms. We also found that the enzymatic activity of CIV containing the muscle-specific isoform COX6A2 was higher than that with COX6A1 in engineered HEK293T cells. Overall, our results indicate that switching the expression from ubiquitous to muscle-specific CIV isoforms is indispensable for optimized oxidative phosphorylation in mature skeletal muscles. We also note that the in vitro C2C12 differentiation model is not suitable for the study of muscular aerobic respiration due to insufficient expression of muscle-specific CIV isoforms.
    Keywords:  electron transport complex IV; myoblasts; oxidative phosphorylation; protein isoforms; skeletal muscle
  18. Physiol Genomics. 2023 Mar 20.
      Acute exercise elicits dynamic transcriptional changes that, when repeated, form the fundamental basis of health, resilience, and performance adaptations. While moderate-intensity endurance training combined with conventional resistance training (traditional, TRAD) is often prescribed and recommended by public health guidance, high-intensity training combining maximal-effort intervals with intensive, limited-rest resistance training is a time-efficient alternative that may be used tactically (HITT) to confer similar benefits. Mechanisms of action of these distinct stimuli are incompletely characterized and have not been directly compared. We assessed transcriptome-wide responses in skeletal muscle and circulating extracellular vehicles (EVs) to a single exercise bout in young adults randomized to TRAD (n=21, 12M/9F, 22±3y) or HITT (n=19, 11M/8F, 22±2y). Next-generation sequencing captured small, long, and circular RNA in muscle and EVs. Analysis identified differentially expressed transcripts (|log2FC|>1, FDR≤0.05) immediately (h0, EVs only), h3, and h24 post-exercise within and between exercise protocols. Additionally, all apparently responsive transcripts (FDR<0.2) underwent singular value decomposition to summarize data structures into latent variables (LVs) to deconvolve molecular expression circuits and inter-regulatory relationships. LVs were compared across time and exercise protocol. TRAD, a longer but more sustained stimulus, generally elicited a stronger transcriptional response than HITT, but considerable overlap and key differences existed. Findings reveal shared and unique molecular responses to the exercise stimuli and lay groundwork toward establishing relationships between protein-coding genes and lesser-understood transcripts that serve regulatory roles following exercise. Future work should advance the understanding of these circuits and whether they repeat in other populations or following other types of exercise/stress.
    Keywords:  exercise physiology; extracellular vesicles; gene expression; skeletal muscle; transcriptomics
  19. Mol Cell Endocrinol. 2023 Mar 21. pii: S0303-7207(23)00065-5. [Epub ahead of print] 111914
      OBJECTIVE: To determine whether alcohol consumed within the meal influences the feeding induced increase in mTORC1 signaling.METHODS: Alcohol provided in the liquid diet was consumed by alcohol naïve, fasted, C57BL/6Hsd female mice and gastrocnemius was collected 1hr after the refeeding. Subsequent experiments determined the extent to which changes in mTORC1 signaling persisted across the day.
    RESULTS: Compared with control mice, protein synthesis, mTORC1 (Ser2448), 4EBP1 (Ser65), S6K1 (Thr389), rpS6 (Ser240/244), Akt (Thr308), and ULK1 (Ser757) were lower in EtOH. Similar suppressive patterns were observed in the hours following consumption of alcohol containing food throughout the dark cycle. Higher peak blood alcohol concentrations induced by intraperitoneal injection of alcohol extended the time and magnitude of mTORC1 pathway suppression.
    CONCLUSION: Alcohol administered as part of the meal results in lower skeletal muscle mTORC1 signaling while subsequent models show that alcohol may influence this pathway across the day.
    Keywords:  Alcohol; Anabolic response; Ethanol; Skeletal muscle
  20. Front Immunol. 2023 ;14 1099799
      Introduction: Macrophages play an important role in the innate immunity. While macrophage inflammation is necessary for biological defense, it must be appropriately controlled. Extracellular vesicles (EVs) are small vesicles released from all types of cells and play a central role in intercellular communication. Skeletal muscle has been suggested to release anti-inflammatory factors, but the effect of myotube-derived EVs on macrophages is unknown. As an anti-inflammatory mechanism of macrophages, the immune responsive gene 1 (IRG1)-itaconate pathway is essential. In this study, we show that skeletal muscle-derived EVs suppress macrophage inflammatory responses, upregulating the IRG1-itaconate pathway.Methods: C2C12 myoblasts were differentiated into myotubes and EVs were extracted by ultracentrifugation. Skeletal myotube-derived EVs were administered to mouse bone marrow-derived macrophages, then lipopolysaccharide (LPS) stimulation was performed and inflammatory cytokine expression was measured by RT-qPCR. Metabolite abundance in macrophages after addition of EVs was measured by CE/MS, and IRG1 expression was measured by RT-PCR. Furthermore, RNA-seq analysis was performed on macrophages after EV treatment.
    Results: EVs attenuated the expression of LPS-induced pro-inflammatory factors in macrophages. Itaconate abundance and IRG1 expression were significantly increased in the EV-treated group. RNA-seq analysis revealed activation of the PI3K-Akt and JAK-STAT pathways in macrophages after EV treatment. The most abundant miRNA in myotube EVs was miR-206-3p, followed by miR-378a-3p, miR-30d-5p, and miR-21a-5p.
    Discussion: Skeletal myotube EVs are supposed to increase the production of itaconate via upregulation of IRG1 expression and exhibited an anti-inflammatory effect in macrophages. This anti-inflammatory effect was suggested to involve the PI3K-Akt and JAK-STAT pathways. The miRNA profiles within EVs implied that miR-206-3p, miR-378a-3p, miR-30d-5p, and miR-21a-5p may be responsible for the anti-inflammatory effects of the EVs. In summary, in this study we showed that myotube-derived EVs prevent macrophage inflammatory responses by activating the IRG1-itaconate pathway.
    Keywords:  IRG1; extracellular vesicle; itaconate; macrophage; skeletal muscle
  21. Physiol Rep. 2023 Mar;11(6): e15629
      Tyrosine kinase inhibitors (TKIs) including ponatinib are commonly used to treat cancer patients. Unfortunately, TKIs induce cardiac as well as skeletal muscle dysfunction as a side effect. Therefore, detailed mechanistic studies are required to understand its pathogenesis and to develop a therapeutic treatment. The current study was undertaken to examine whether ponatinib induces apoptosis and apoptotic mechanisms both in vitro and in vivo models and furthermore to test the potential of bone morphogenetic protein 7 (BMP-7) as a possible treatment option for its attenuation. Sol8 cells, a mouse myogenic cell line was exposed to ponatinib to generate an apoptotic cell culture model and were subsequently treated with BMP-7 to understand its protective effects. For the in vivo model, C57BL/6J mice were administered with ponatinib to understand apoptosis, cell signaling apoptotic mechanisms, and adverse muscle remodeling and its attenuation with BMP-7. TUNEL staining, immunohistochemistry (IHC), and real-time polymerase chain reaction (RT-PCR) methods were used. Our data show significantly (p < 0.05) increased TUNEL staining, caspase-3, BAX/Bcl2 ratio in the in vitro model. Furthermore, our in vivo muscle data show ponatinib-induced muscle myopathy, and loss in muscle function. The observed muscle myopathy was associated with increased apoptosis, caspase-3 staining, and BAX/Bcl-2 ratio as confirmed with IHC and RT-PCR. Furthermore, our data show a significant (p < 0.05) increase in the involvement of cell signaling apoptotic regulator protein PTEN and a decrease in cell survival protein AKT. These results suggest that increased apoptosis following ponatinib treatment showed an increase in skeletal muscle remodeling, sarcopenia, and fibrosis. Furthermore, BMP-7 treatment significantly (p < 0.05) attenuated ponatinib-induced apoptosis, BAX/Bcl2 ratio, decreased PTEN, and increased cell survival protein AKT, decreased adverse muscle remodeling, and improved muscle function. Overall, we provide evidence that ponatinib-induces apoptosis leading to sarcopenia and muscle myopathy with decreased function which was attenuated by BMP-7.
    Keywords:  apoptosis; atrophy; cell death; skeletal muscle myopathy; tyrosine kinase inhibitor
  22. bioRxiv. 2023 Mar 11. pii: 2023.03.10.532020. [Epub ahead of print]
      Insulin resistance (IR) is a complex metabolic disorder that underlies several human diseases, including type 2 diabetes and cardiovascular disease. Despite extensive research, the precise mechanisms underlying IR development remain poorly understood. Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, deficiency of coenzyme Q (CoQ), mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells results in CoQ depletion and loss of mitochondrial respiratory chain components, leading to mitochondrial dysfunction and IR. Further, decreasing mitochondrial ceramide levels in vitro and in animal models increased CoQ levels and was protective against IR. CoQ supplementation also rescued ceramide-associated IR. Examination of the mitochondrial proteome from human muscle biopsies revealed a strong correlation between the respirasome system and mitochondrial ceramide as key determinants of insulin sensitivity. Our findings highlight the mitochondrial Ceramide-CoQ-respiratory chain nexus as a potential foundation of an IR pathway that may also play a critical role in other conditions associated with ceramide accumulation and mitochondrial dysfunction, such as heart failure, cancer, and aging. These insights may have important clinical implications for the development of novel therapeutic strategies for the treatment of IR and related metabolic disorders.
  23. J Exp Biol. 2023 Apr 25. pii: jeb245158. [Epub ahead of print]226(Suppl_1):
      Skeletal muscle powers animal movement, making it an important determinant of fitness. The classic excitation-contraction coupling, sliding-filament and crossbridge theories are thought to describe the processes of muscle activation and the generation of force, work and power. Here, we review how the comparative, realistic muscle physiology typified by Journal of Experimental Biology over the last 100 years has supported and refuted these theories. We examine variation in the contraction rates and force-length and force-velocity relationships predicted by these theories across diverse muscles, and explore what has been learnt from the use of workloop and force-controlled techniques that attempt to replicate aspects of in vivo muscle function. We suggest inclusion of features of muscle contraction not explained by classic theories in our routine characterization of muscles, and the use of phylogenetic comparative methods to allow exploration of the effects of factors such as evolutionary history, ecology, behavior and size on muscle physiology and mechanics. We hope that these future directions will improve our understanding of the mechanisms of muscle contraction, allow us to better characterize the variation in muscle performance possible, and enable us to infer adaptation.
    Keywords:  Eccentric; Excitation–contraction coupling; Force–length; Force–velocity; History dependence; Phylogenetic comparative methods
  24. Biol Res. 2023 Mar 25. 56(1): 14
      The endocannabinoid system (ECS) regulates energy metabolism, has been implicated in the pathogenesis of metabolic diseases and exerts its actions mainly through the type 1 cannabinoid receptor (CB1). Likewise, autophagy is involved in several cellular processes. It is required for the normal development of muscle mass and metabolism, and its deregulation is associated with diseases. It is known that the CB1 regulates signaling pathways that control autophagy, however, it is currently unknown whether the ECS could regulate autophagy in the skeletal muscle of obese mice. This study aimed to investigate the role of the CB1 in regulating autophagy in skeletal muscle. We found concomitant deregulation in the ECS and autophagy markers in high-fat diet-induced obesity. In obese CB1-KO mice, the autophagy-associated protein LC3 II does not accumulate when mTOR and AMPK phosphorylation levels do not change. Acute inhibition of the CB1 with JD-5037 decreased LC3 II protein accumulation and autophagic flux. Our results suggest that the CB1 regulates autophagy in the tibialis anterior skeletal muscle in both lean and obese mice.
    Keywords:  Autophagy; Endocannabinoid receptor; High-fat diet; Skeletal muscle
  25. J Cachexia Sarcopenia Muscle. 2023 Mar 21.
      BACKGROUND: Ghrelin is a potential therapy for cachexia due to its orexigenic properties and anabolic effects on muscle and fat. However, its clinical use is limited by the short half-life of active (acylated) ghrelin (~11 min in humans). EXT418 is a novel long-acting, constitutively active ghrelin analog created by covalently linking it to a vitamin D derivative. Here, we evaluated the effects and mechanisms of action of EXT418 on Lewis lung carcinoma (LLC)-induced cachexia in mice.METHODS: Male C57BL/6J mice (5- to 7-month-old) were implanted with 1 × 106 heat-killed (HK) or live LLC cells. When the tumour was palpable, mice were injected with vehicle (T + V) or EXT418 daily (T + 418 Daily, 0.25 mg/kg/day) or every other day (T + 418 EOD, 0.5 mg/kg/EOD) for up to 14 days, whereas HK-treated mice were given vehicle (HK + V). Subsets of T + 418 Daily or EOD-treated mice were pair-fed to the T + V group. Body composition and grip strength were evaluated before tumour implantation and at the end of the experiment. Molecular markers were probed in muscles upon termination.
    RESULTS: In tumour-bearing mice, administration of EXT418 daily or EOD partially prevented weight loss (T + V vs. T + 418 Daily, P = 0.030; and vs. T + 418 EOD, P = 0.020). Similar effects were observed in whole body fat and lean body mass. Grip strength in tumour-bearing mice was improved by EXT418 daily (P = 0.010) or EOD (P = 0.008) administration compared with vehicle-treated mice. These effects of EXT418 on weight and grip strength were partially independent of food intake. EXT418 daily administration also improved type IIA (P = 0.015), IIB (P = 0.037) and IIX (P = 0.050) fibre cross-sectional area (CSA) in tibialis anterior (TA) and EXT418 EOD improved CSA of IIB fibres in red gastrocnemius (GAS; P = 0.005). In skeletal muscles, tumour-induced increases in atrogenes Fbxo32 and Trim63 were ameliorated by EXT418 treatments (TA and GAS/plantaris, PL), which were independent of food intake. EXT418 administration decreased expression of the mitophagy marker Bnip3 (GAS/PL; P ≤ 0.010). Similar effects of EXT418 EOD were observed in p62 (GAS/PL; P = 0.039). In addition, EXT418 treatments ameliorated the tumour-induced elevation in muscle Il6 transcript levels (TA and GAS/PL), independently of food intake. Il-6 transcript levels in adipose tissue and circulating IL-10 were elevated in response to the tumour but these increases were not significant with EXT418 administration. Tumour mass was not altered by EXT418.
    CONCLUSIONS: EXT418 mitigates LLC-induced cachexia by attenuating skeletal muscle inflammation, proteolysis, and mitophagy, without affecting tumour mass and partially independent of food intake.
    Keywords:  Cachexia; Ghrelin; Inflammation; Mitophagy; Wasting
  26. Sci Rep. 2023 Mar 23. 13(1): 4746
      Cell-based therapy is a major focus for treatment of stress urinary incontinence (SUI). However, derivation of primary cells requires tissue biopsies, which often have adverse effects on patients. A recent study used human induced pluripotent stem cells (iPSC)-derived smooth muscle myocytes for urethral sphincter regeneration in rats. Here, we establish a workflow using iPSC-derived fibroblasts and skeletal myocytes for urethral tissue regeneration: (1) Cells from voided urine of women were reprogrammed into iPSC. (2) The iPSC line U1 and hESC line H9 (control) were differentiated into fibroblasts expressing FSP1, TE7, vinculin, vimentin, αSMA, fibronectin and paxillin. (3) Myogenic differentiation of U1 and H9 was induced by small molecule CHIR99021 and confirmed by protein expression of myogenic factors PAX7, MYOD, MYOG, and MF20. Striated muscle cells enriched by FACS expressed NCAM1, TITIN, DESMIN, TNNT3. (4) Human iPSC-derived fibroblasts and myocytes were engrafted into the periurethral region of RNU rats. Injected cells were labelled with ferric nanoparticles and traced by Prussian Blue stain, human-specific nuclear protein KU80, and human anti-mitochondria antibody. This workflow allows the scalable derivation, culture, and in vivo tracing of patient-specific fibroblasts and myocytes, which can be assessed in rat SUI models to regenerate urethral damages and restore continence.
  27. Front Physiol. 2023 ;14 1126111
      Mechanosignaling describes processes by which biomechanical stimuli are transduced into cellular responses. External biophysical forces can be transmitted via structural protein networks that span from the cellular membrane to the cytoskeleton and the nucleus, where they can regulate gene expression through a series of biomechanical and/or biochemical mechanosensitive mechanisms, including chromatin remodeling, translocation of transcriptional regulators, and epigenetic factors. Striated muscle cells, including cardiac and skeletal muscle myocytes, utilize these nuclear mechanosignaling mechanisms to respond to changes in their intracellular and extracellular mechanical environment and mediate gene expression and cell remodeling. In this brief review, we highlight and discuss recent experimental work focused on the pathway of biomechanical stimulus propagation at the nucleus-cytoskeleton interface of striated muscles, and the mechanisms by which these pathways regulate gene regulation, muscle structure, and function. Furthermore, we discuss nuclear protein mutations that affect mechanosignaling function in human and animal models of cardiomyopathy. Furthermore, current open questions and future challenges in investigating striated muscle nuclear mechanosignaling are further discussed.
    Keywords:  LINC complex; cardiomyopathy; laminopathy; mechanosignaling; myocytes; nuclear morphology; nucleoskeleton; nucleus
  28. Protein J. 2023 Mar 23.
      Muscle weakness as a secondary feature of attenuated neuronal input often leads to disability and sometimes death in patients with neurogenic neuromuscular diseases. These impaired muscle function has been observed in several diseases including amyotrophic lateral sclerosis, Charcot-Marie-Tooth, spinal muscular atrophy and Myasthenia gravis. This has spurred the search for small molecules which could activate fast skeletal muscle troponin complex as a means to increase muscle strength. Discovered small molecules have however been punctuated by off-target and side effects leading to the development of the second-generation small molecule, Reldesemtiv. In this study, we investigated the impact of Reldesemtiv binding to the fast skeletal troponin complex and the molecular determinants that condition the therapeutic prowess of Redesemtiv through computational techniques. It was revealed that Reldesemtiv binding possibly potentiates troponin C compacting characterized by reduced exposure to solvent molecules which could favor the slow release of calcium ions and the resultant sensitization of the subunit to calcium. These conformational changes were underscored by conventional and carbon hydrogen bonds, pi-alkyl, pi-sulfur and halogen interactions between Reldesemtiv the binding site residues. Arg113 (-3.96 kcal/mol), Met116 (-2.23 kcal/mol), Val114 (-1.28 kcal/mol) and Met121 (-0.63 kcal/mol) of the switch region of the inhibitory subunit were among the residues that contributed the most to the total free binding energy of Reldesemtiv highlighting their importance. These findings present useful insights which could lay the foundation for the development of fast skeletal muscle small molecule activators with high specificity and potency.
    Keywords:  Fast skeletal troponin; Molecular dynamics simulation; Reldesemtiv; Troponin C; Troponin I
  29. Nat Metab. 2023 Mar 20.
      Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases, represents an important goal in improving human health. Here, we show that the lipid synthesis enzyme phosphatidylethanolamine cytidyltransferase (PCYT2/ECT) is critical to muscle health. Human deficiency in PCYT2 causes a severe disease with failure to thrive and progressive weakness. pcyt2-mutant zebrafish and muscle-specific Pcyt2-knockout mice recapitulate the participant phenotypes, with failure to thrive, progressive muscle weakness and accelerated ageing. Mechanistically, muscle Pcyt2 deficiency affects cellular bioenergetics and membrane lipid bilayer structure and stability. PCYT2 activity declines in ageing muscles of mice and humans, and adeno-associated virus-based delivery of PCYT2 ameliorates muscle weakness in Pcyt2-knockout and old mice, offering a therapy for individuals with a rare disease and muscle ageing. Thus, PCYT2 plays a fundamental and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2-synthesized lipids to severe muscle dystrophy and ageing.
  30. Sci Rep. 2023 Mar 22. 13(1): 4660
      Humans can inherently adapt their gait pattern in a way that minimizes the metabolic cost of transport, or walking economy, within a few steps, which is faster than any known direct physiological sensor of metabolic energy. Instead, walking economy may be indirectly sensed through mechanoreceptors that correlate with the metabolic cost per step to make such gait adaptations. We tested whether velocity feedback from tibialis anterior (TA) muscle fascicles during the early stance phase of walking could potentially act to indirectly sense walking economy. As participants walked within a range of steady-state speeds and step frequencies, we observed that TA fascicles lengthen on almost every step. Moreover, the average peak fascicle velocity experienced during lengthening reflected the metabolic cost of transport of the given walking condition. We observed that the peak TA muscle activation occurred earlier than could be explained by a short latency reflex response. The activation of the TA muscle just prior to heel strike may serve as a prediction of the magnitude of the ground collision and the associated energy exchange. In this scenario, any unexpected length change experienced by the TA fascicle would serve as an error signal to the nervous system and provide additional information about energy lost per step. Our work helps provide a biomechanical framework to understand the possible neural mechanisms underlying the rapid optimization of walking economy.
  31. bioRxiv. 2023 Mar 09. pii: 2023.03.09.531795. [Epub ahead of print]
      Mitochondrial diseases are a group of disorders defined by defects in oxidative phosphorylation caused by nuclear- or mitochondrial-encoded gene mutations. A main cellular phenotype of mitochondrial disease mutations are redox imbalances and inflammatory signaling underlying pathogenic signatures of these patients. Depending on the type of mitochondrial mutation, certain mechanisms can efficiently rescue cell death vulnerability. One method is the inhibition of mitochondrial translation elongation using tetracyclines, potent suppressors of cell death in mitochondrial disease mutant cells. However, the mechanisms whereby tetracyclines promote cell survival are unknown. Here, we show that in mitochondrial mutant disease cells, tetracycline-mediated inhibition of mitoribosome elongation promotes survival through suppression of the ER stress IRE1α protein. Tetracyclines increased levels of the splitting factor MALSU1 (Mitochondrial Assembly of Ribosomal Large Subunit 1) at the mitochondria with recruitment to the mitochondrial ribosome (mitoribosome) large subunit. MALSU1, but not other quality control factors, was required for tetracycline-induced cell survival in mitochondrial disease mutant cells during glucose starvation. In these cells, nutrient stress induced cell death through IRE1α activation associated with a strong protein loading in the ER lumen. Notably, tetracyclines rescued cell death through suppression of IRE1α oligomerization and activity. Consistent with MALSU1 requirement, MALSU1 deficient mitochondrial mutant cells were sensitive to glucose-deprivation and exhibited increased ER stress and activation of IRE1α that was not reversed by tetracyclines. These studies show that inhibition of mitoribosome elongation signals to the ER to promote survival, establishing a new interorganelle communication between the mitoribosome and ER with implications in basic mechanisms of cell survival and treatment of mitochondrial diseases.Significance Statement: Mitochondrial diseases are a rare and heterogenous class of diseases that result from mutations in mitochondrial genes. Currently, there are no curative therapies due to a lack of mechanistic insights into pathological transformation and signaling. Our lab has discovered that the class of mitochondrial ribosome targeting antibiotics, tetracyclines, promote survival and fitness in models of mitochondrial disease, establishing a new paradigm of cell survival under nutrient stress conditions. In the current study, we present mechanistic insights into tetracyclines ability to rescue mitochondrial disease cells, detailing an interorganelle communication between mitochondrial protein translation and the unfolded protein response during endoplasmic reticulum stress.