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Reduced skeletal muscle mass and oxidative capacity coexist in patients with pulmonary emphysema and are independently associated with higher mortality. If reduced cellular respiration contributes to muscle atrophy in that setting remains unknown. Using a mouse with genetically induced pulmonary emphysema that recapitulates muscle dysfunction, we found that reduced activity of succinate dehydrogenase (SDH) is a hallmark of its myopathic changes. We generated an inducible, muscle-specific SDH knockout mouse that demonstrates lower mitochondrial oxygen consumption, myofiber contractility, and exercise endurance. Respirometry analyses show that in vitro complex I respiration is unaffected by loss of SDH subunit C in muscle mitochondria, which is consistent with the pulmonary emphysema animal data. SDH knockout initially causes succinate accumulation associated with a down-regulated transcriptome but modest proteome effects. Muscle mass, myofiber type composition, and overall body mass constituents remain unaltered in the transgenic mice. Thus, while SDH regulates myofiber respiration in experimental pulmonary emphysema, it does not control muscle mass or other body constituents.
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Respiração Celular , Camundongos Knockout , Contração Muscular , Músculo Esquelético , Enfisema Pulmonar , Succinato Desidrogenase , Animais , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/genética , Enfisema Pulmonar/patologia , Enfisema Pulmonar/etiologia , Succinato Desidrogenase/metabolismo , Succinato Desidrogenase/genética , Camundongos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Complexo II de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/genética , Modelos Animais de Doenças , Camundongos Transgênicos , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/patologia , Consumo de OxigênioRESUMO
PURPOSE OF THE REVIEW: Cancer-associated cachexia is a wasting syndrome entailing loss in body mass and a shortened life expectancy. There is currently no effective treatment to abrogate this syndrome, which leads to 20-30% of deaths in patients with cancer. While there have been advancements in defining signaling factors/pathways in cancer-induced muscle wasting, targeting the same in the clinic has not been as successful. Krüppel-like factor 10 (KLF10), a transcription factor implicated in muscle regulation, is regulated by the transforming growth factor-beta signaling pathway. This review proposes KLF10 as a potential convergence point of diverse signaling pathways involved in muscle wasting. RECENT FINDINGS: KLF10 was discovered as a target of transforming growth factor-beta decades ago but more recently it has been shown that deletion of KLF10 rescues cancer-induced muscle wasting. Moreover, KLF10 has also been shown to bind key atrophy genes associated with muscle atrophy in vitro . SUMMARY: There is an elevated need to explore targets in cachexia, which will successfully translate into the clinic. Investigating a convergence point downstream of multiple signaling pathways might hold promise in developing effective therapies for cachexia.
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Caquexia , Fatores de Transcrição de Resposta de Crescimento Precoce , Fatores de Transcrição Kruppel-Like , Neoplasias , Transdução de Sinais , Caquexia/etiologia , Caquexia/genética , Caquexia/fisiopatologia , Humanos , Fatores de Transcrição Kruppel-Like/genética , Neoplasias/complicações , Fatores de Transcrição de Resposta de Crescimento Precoce/genética , Atrofia Muscular/genética , Fator de Crescimento Transformador beta/metabolismo , Músculo Esquelético/metabolismoRESUMO
Cancer cachexia, and its associated complications, represent a large and currently untreatable roadblock to effective cancer management. Many potential therapies have been proposed and tested-including appetite stimulants, targeted cytokine blockers, and nutritional supplementation-yet highly effective therapies are lacking. Innovative approaches to treating cancer cachexia are needed. Members of the Kruppel-like factor (KLF) family play wide-ranging and important roles in the development, maintenance, and metabolism of skeletal muscle. Within the KLF family, we identified KLF10 upregulation in a multitude of wasting contexts-including in pancreatic, lung, and colon cancer mouse models as well as in human patients. We subsequently interrogated loss-of-function of KLF10 as a potential strategy to mitigate cancer associated muscle wasting. In vivo studies leveraging orthotopic implantation of pancreas cancer cells into wild-type and KLF10 KO mice revealed significant preservation of lean mass and robust suppression of pro-atrophy muscle-specific ubiquitin ligases Trim63 and Fbxo32, as well as other factors implicated in atrophy, calcium signaling, and autophagy. Bioinformatics analyses identified Transforming growth factor beta (TGF-ß), a known inducer of KLF10 and cachexia promoting factor, as a key upstream regulator of KLF10. We provide direct in vivo evidence that KLF10 KO mice are resistant to the atrophic effects of TGF-ß. ChIP-based binding studies demonstrated direct binding to Trim63, a known wasting-associated atrogene. Taken together, we report a critical role for the TGF-ß/KLF10 axis in the etiology of pancreatic cancer-associated muscle wasting and highlight the utility of targeting KLF10 as a strategy to prevent muscle wasting and limit cancer-associated cachexia.
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Neoplasias Pancreáticas , Fator de Crescimento Transformador beta , Humanos , Camundongos , Animais , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Caquexia/genética , Atrofia Muscular/genética , Neoplasias Pancreáticas/complicações , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Músculo Esquelético/metabolismo , Fatores de Transcrição de Resposta de Crescimento Precoce/genética , Fatores de Transcrição de Resposta de Crescimento Precoce/metabolismoRESUMO
BACKGROUND: Over the past decade, advances in sepsis identification and management have resulted in decreased sepsis mortality. This increase in survivorship has highlighted a new clinical obstacle: chronic critical illness (CCI), for which there are no effective treatment options. Up to half of sepsis survivors suffer from CCI, which can include multi-organ dysfunction, chronic inflammation, muscle wasting, physical and mental disabilities, and enhanced frailty. These symptoms prevent survivors from returning to regular day-to-day activities and are directly associated with poor quality of life. METHODS: Mice were subjected to cecal ligation and puncture (CLP) with daily chronic stress (DCS) as an in vivo model to study sepsis late-effects/sequelae on skeletal muscle components. Longitudinal monitoring was performed via magnetic resonance imaging, skeletal muscle and/or muscle stem cell (MuSCs) assays (e.g., post-necropsy wet muscle weights, minimum Feret diameter measurements, in vitro MuSC proliferation and differentiation, number of regenerating myofibres and numbers of Pax7-positive nuclei per myofibre), post-sepsis whole muscle metabolomics and MuSC isolation and high-content transcriptional profiling. RESULTS: We report several findings supporting the hypothesis that MuSCs/muscle regeneration are critically involved in post-sepsis muscle recovery. First, we show that genetic ablation of muscle stem cells (MuSCs) impairs post-sepsis muscle recovery (maintenance of 5-8% average lean mass loss compared with controls). Second, we observe impaired MuSCs expansion capacity and morphological defects at 26 days post-sepsis compared with control MuSCs (P < 0.001). Third, when subjected to an experimental muscle injury, sepsis-recovered mice exhibited evidence of impaired muscle regeneration compared with non-septic mice receiving the same muscle injury (CLP/DCS injured mean minimum Feret is 92.1% of control injured, P < 0.01). Fourth, we performed a longitudinal RNA sequencing study on MuSCs isolated from post-sepsis mice and found clear transcriptional differences in all post-sepsis samples compared with controls. At Day 28, CLP/DCS mice satellite cells have multiple altered metabolic pathways, such as oxidative phosphorylation, mitochondrial dysfunction, sirtuin signalling and oestrogen receptor signalling, compared with controls (P < 0.001). CONCLUSIONS: Our data show that MuSCs and muscle regeneration are required for effective post-sepsis muscle recovery and that sepsis triggers morphological, functional, and transcriptional changes in MuSCs. Moving forward, we strive to leverage a more complete understanding of post-sepsis MuSC/regenerative defects to identify and test novel therapies that promote muscle recovery and improve quality of life in sepsis survivors.
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Células Satélites de Músculo Esquelético , Sepse , Camundongos , Animais , Qualidade de Vida , Músculo Esquelético/patologia , Células Satélites de Músculo Esquelético/metabolismo , Diferenciação Celular , Sepse/metabolismoRESUMO
Acquired sporadic late onset nemaline myopathy (SLONM) and inherited nemaline myopathy (iNM) both feature accumulation of nemaline rods in muscle fibers. Unlike iNM, SLONM is amenable to therapy. The distinction between these disorders is therefore crucial when the diagnosis remains ambiguous after initial investigations. We sought to identify biomarkers facilitating this distinction and to investigate the pathophysiology of nemaline rod formation in these different disorders. Twenty-two muscle samples from patients affected by SLONM or iNM underwent quantitative histological analysis, laser capture microdissection for proteomic analysis of nemaline rod areas and rod-free areas, and transcriptomic analysis. In all iNM samples, nemaline rods were found in subsarcolemmal or central aggregates, whereas they were diffusely distributed within muscle fibers in most SLONM samples. In SLONM, muscle fibers harboring nemaline rods were smaller than those without rods. Necrotic fibers, increased endomysial connective tissue, and atrophic fibers filled with nemaline rods were more common in SLONM. Proteomic analysis detected differentially expressed proteins between nemaline rod areas and rod-free areas, as well as between SLONM and iNM. These differentially expressed proteins implicated immune, structural, metabolic, and cellular processes in disease pathophysiology. Notably, immunoglobulin overexpression with accumulation in nemaline rod areas was detected in SLONM. Transcriptomic analysis corroborated proteomic findings and further revealed substantial gene expression differences between SLONM and iNM. Overall, we identified unique pathological and molecular signatures associated with SLONM and iNM, suggesting distinct underlying pathophysiological mechanisms. These findings represent a step towards enhanced diagnostic tools and towards development of treatments for SLONM.
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Miopatias da Nemalina , Humanos , Miopatias da Nemalina/genética , Miopatias da Nemalina/patologia , Proteômica , Fibras Musculares Esqueléticas/patologia , Miocárdio/patologia , Músculo Esquelético/patologiaRESUMO
BACKGROUNDA patient-derived organoid (PDO) platform may serve as a promising tool for translational cancer research. In this study, we evaluated PDO's ability to predict clinical response to gastrointestinal (GI) cancers.METHODSWe generated PDOs from primary and metastatic lesions of patients with GI cancers, including pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and cholangiocarcinoma. We compared PDO response with the observed clinical response for donor patients to the same treatments.RESULTSWe report an approximately 80% concordance rate between PDO and donor tumor response. Importantly, we found a profound influence of culture media on PDO phenotype, where we showed a significant difference in response to standard-of-care chemotherapies, distinct morphologies, and transcriptomes between media within the same PDO cultures.CONCLUSIONWhile we demonstrate a high concordance rate between donor tumor and PDO, these studies also showed the important role of culture media when using PDOs to inform treatment selection and predict response across a spectrum of GI cancers.TRIAL REGISTRATIONNot applicable.FUNDINGThe Joan F. & Richard A. Abdoo Family Fund in Colorectal Cancer Research, GI Cancer program of the Mayo Clinic Cancer Center, Mayo Clinic SPORE in Pancreatic Cancer, Center of Individualized Medicine (Mayo Clinic), Department of Laboratory Medicine and Pathology (Mayo Clinic), Incyte Pharmaceuticals and Mayo Clinic Hepatobiliary SPORE, University of Minnesota-Mayo Clinic Partnership, and the Early Therapeutic program (Department of Oncology, Mayo Clinic).
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Neoplasias Gastrointestinais , Neoplasias Pancreáticas , Humanos , Meios de Cultura , Organoides/patologia , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/patologia , Neoplasias Pancreáticas/patologia , Neoplasias PancreáticasRESUMO
Hereditary muscle diseases are disabling disorders lacking effective treatments. UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) myopathy (GNEM) is an autosomal recessive distal myopathy with rimmed vacuoles typically manifesting in late adolescence/early adulthood. GNE encodes the rate-limiting enzyme in sialic acid biosynthesis, which is necessary for the proper function of numerous biological processes. Outside of the causative gene, very little is known about the mechanisms contributing to the development of GNE myopathy. In the present study, we aimed to address this knowledge gap by querying the underlying mechanisms of GNE myopathy using a patient-derived induced pluripotent stem-cell (iPSC) model. Control and patient-specific iPSCs were differentiated down a skeletal muscle lineage, whereby patient-derived GNEM iPSC clones were able to recapitulate key characteristics of the human pathology and further demonstrated defects in myogenic progression. Single-cell RNA sequencing time course studies revealed clear differences between control and GNEM iPSC-derived muscle precursor cells (iMPCs), while pathway studies implicated altered stress and autophagy signaling in GNEM iMPCs. Treatment of GNEM patient-derived iMPCs with an autophagy activator improved myogenic differentiation. In summary, we report an in vitro, iPSC-based model of GNE myopathy and implicate defective myogenesis as a contributing mechanism to the etiology of GNE myopathy.
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ABSTRACT: Sepsis is a highly prevalent cause of death in intensive care units. Characterized by severe immune cell derangements, sepsis is often associated with multiorgan dysfunction. For many sepsis survivors, these deficits can persist long after clinical resolution of the underlying infection. Although many studies report on the impact of sepsis on individual immune cell subtypes, a comprehensive analysis of sepsis-induced alterations within and across the immune cell landscape is lacking. In this study, we used single-cell RNA sequencing to assess sepsis-associated transcriptional changes in immune cells isolated from bone marrow at single-cell resolution. We used a high-survival fecal-induced peritonitis sepsis model using Friend leukemia virus B mice. Single-cell RNA sequencing classified 3402 single cells from control subjects into 14 clusters representing long-term hematopoietic stem cell (HSC), short-term HSC, basophil, dendritic cell, eosinophil, erythroblast, erythrocyte, macrophage, neutrophil, natural killer cell, plasma cell, plasmacytoid dendritic cell, pre-B cell, and T memory cell lineages. One day following experimentally induced sepsis, cell type compositions shifted significantly and included notable decreases in HSC and myeloid cell abundance. In addition to proportional cell composition changes, acute sepsis induced significant transcriptional alterations in most immune cell types analyzed-changes that failed to completely resolve 1 month after sepsis. Taken together, we report widespread and persistent transcriptional changes in diverse immune cells in response to polymicrobial infection. This study will serve as a valuable resource for future work investigating acute and/or long-term sepsis-associated immune cell derangements.
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Coinfecção , Peritonite , Sepse , Animais , Medula Óssea , Células da Medula Óssea , Células-Tronco Hematopoéticas , Humanos , Camundongos , Peritonite/complicaçõesRESUMO
Culturing primary muscle stem cells ex vivo is a useful method for studying this cell population in controlled environments. Primary muscle stem cells respond to external stimuli differently than immortalized myoblasts (C2C12 cells), making ex vivo culture of muscle stem cells an important tool in understanding cell responses to stimuli. Primary muscle stem cells cultured ex vivo retain a majority of the characteristics they possess in vivo such as the abilities to differentiate into multinucleated structures, and self-renew a stem cell-like population. In this chapter, we describe methods for isolating primary muscle stem cells, controlled differentiation into myotubes, and quantification of differentiation using IncuCyte live cell imaging and analysis software.
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Desenvolvimento Muscular , Músculo Esquelético , Diferenciação Celular , Células Cultivadas , Desenvolvimento Muscular/fisiologia , Fibras Musculares Esqueléticas , MioblastosRESUMO
Approximately 80% of pancreatic cancer patients suffer from cachexia, and one-third die due to cachexia-related complications such as respiratory failure and cardiac arrest. Although there has been considerable research into cachexia mechanisms and interventions, there are, to date, no FDA-approved therapies. A major contributing factor for the lack of therapy options could be the failure of animal models to accurately recapitulate the human condition. In this study, we generated an aged model of pancreatic cancer cachexia to compare cachexia progression in young versus aged tumor-bearing mice. Comparative skeletal muscle transcriptome analyses identified 3-methyladenine (3-MA) as a candidate antiwasting compound. In vitro analyses confirmed antiwasting capacity, while in vivo analysis revealed potent antitumor effects. Transcriptome analyses of 3-MA-treated tumor cells implicated Perp as a 3-MA target gene. We subsequently (a) observed significantly higher expression of Perp in cancer cell lines compared with control cells, (b) noted a survival disadvantage associated with elevated Perp, and (c) found that 3-MA-associated Perp reduction inhibited tumor cell growth. Finally, we have provided in vivo evidence that survival benefits conferred by 3-MA administration are independent of its effect on tumor progression. Taken together, we report a mechanism linking 3-MA to Perp inhibition, and we further implicate Perp as a tumor-promoting factor in pancreatic cancer.
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Adenina/análogos & derivados , Caquexia , Proteínas de Membrana , Músculo Esquelético , Neoplasias Pancreáticas , Adenina/metabolismo , Adenina/farmacologia , Fatores Etários , Animais , Autofagia/efeitos dos fármacos , Caquexia/etiologia , Caquexia/metabolismo , Caquexia/terapia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Neoplasias Pancreáticas/complicações , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/terapiaRESUMO
Skeletal muscle wasting drives negative clinical outcomes and is associated with a spectrum of pathologies including cancer. Cancer cachexia is a multi-factorial syndrome that encompasses skeletal muscle wasting and remains understudied, despite being a frequent and serious co-morbidity. Deviation from the homeostatic balance between breakdown and regeneration leads to muscle wasting disorders, such as cancer cachexia. Muscle stem cells (MuSCs) are the cellular compartment responsible for muscle regeneration, which makes MuSCs an intriguing target in the context of wasting muscle. Molecular studies investigating MuSCs and skeletal muscle wasting largely focus on transcriptional changes, but our group and others propose that metabolic changes are another layer of cellular regulation underlying MuSC dysfunction in cancer cachexia. In the present study, we combined gene expression and non-targeted metabolomic profiling of myoblasts exposed to wasting conditions (cancer cell conditioned media, CC-CM) to derive a more complete picture of the myoblast response to wasting factors. After mapping these features to annotated pathways, we found that more than half of the mapped pathways were amino acid-related, linking global amino acid metabolic disruption to conditioned media-induced myoblast defects. Notably, arginine metabolism was a highly enriched pathway in combined metabolomic and transcriptomic data. Arginine catabolism generates nitric oxide (NO), an important signaling molecule known to have negative effects on mature muscle. We hypothesize that tumor-derived disruptions in Nitric Oxide Synthase (NOS)2-regulated arginine catabolism impair differentiation of MuSCs. The work presented here further investigates the effect of NOS2 overactivity on myoblast proliferation and differentiation. We show that NOS2 inhibition is sufficient to rescue wasting phenotypes associated with inflammatory cytokines. Ultimately, this work provides new insights into MuSC biology and opens up potential therapeutic avenues for addressing disrupted MuSC dynamics in cancer cachexia.
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Caquexia/metabolismo , Citocinas/metabolismo , Mioblastos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Caquexia/complicações , Caquexia/patologia , Diferenciação Celular/fisiologia , Humanos , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/metabolismo , Atrofia Muscular/patologia , RegeneraçãoRESUMO
Many chronic disease patients experience a concurrent loss of lean muscle mass. Skeletal muscle is a dynamic tissue maintained by continuous protein turnover and progenitor cell activity. Muscle stem cells, or satellite cells, differentiate (by a process called myogenesis) and fuse to repair and regenerate muscle. During myogenesis, satellite cells undergo extensive metabolic alterations; therefore, pathologies characterized by metabolic derangements have the potential to impair myogenesis, and consequently exacerbate skeletal muscle wasting. How disease-associated metabolic disruptions in satellite cells might be contributing to wasting is an important question that is largely neglected. With this review we highlight the impact of various metabolic disruptions in disease on myogenesis and skeletal muscle regeneration. We also discuss metabolic therapies with the potential to improve myogenesis, skeletal muscle regeneration, and ultimately muscle mass.
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Tactics to increase the number of underrepresented (UR) students in biomedical research PhD training programs have not yet translated to UR faculty numbers that reflect the diversity of the United States. Continued interventions are required to build skills beyond those that result in placement into a PhD program. We hypothesize that successful interventions must build skills that give UR students foundations for confident self-efficacy in leadership. We seek interventions that allow UR students to envision themselves as successful faculty. We posit that development of such skills is difficult in the classroom or laboratory alone. Therefore, novel interventions are required. As part of the NIH-funded Post-baccalaureate Research Education Program (PREP) and Initiative for Maximizing Student Development (IMSD) at the Mayo Clinic Graduate School of Biomedical Sciences, we designed and implemented a unique intervention to support development of student leadership skills: a biannual student-organized and student-led national research conference titled "Scientific Innovation Through Diverse Perspectives" (SITDP). This initiative is based on the concept that students who actively live out realistic roles as scientific leaders will be encouraged to persist to scientific leadership as faculty. Here we describe the motivation for, design of, and outcomes from, the first three pilot conferences of this series. We further discuss approaches needed to rigorously evaluate the effectiveness of such interventions in the future.
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Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies - including single-cell RNA-sequencing and single-cell proteomics - is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.
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Tecido Adiposo/citologia , Técnicas de Cultura de Células/métodos , Epididimo/citologia , Células-Tronco/citologia , Adipócitos/citologia , Animais , Anticorpos/metabolismo , Sobrevivência Celular , Citometria de Fluxo , Masculino , CamundongosRESUMO
Background: Muscle wasting is a debilitating co-morbidity affecting most advanced cancer patients. Alongside enhanced muscle catabolism, defects in muscle repair/regeneration contribute to cancer-associated wasting. Among the factors implicated in suppression of muscle regeneration are cytokines that interfere with myogenic signal transduction pathways. Less understood is how other cancer/wasting-associated cues, such as metabolites, contribute to muscle dysfunction. This study investigates how the metabolite succinate affects myogenesis and muscle regeneration. Methods: We leveraged an established ectopic metabolite treatment (cell permeable dimethyl-succinate) strategy to evaluate the ability of intracellular succinate elevation to 1) affect myoblast homeostasis (proliferation, apoptosis), 2) disrupt protein dynamics and induce wasting-associated atrophy, and 3) modulate in vitro myogenesis. In vivo succinate supplementation experiments (2% succinate, 1% sucrose vehicle) were used to corroborate and extend in vitro observations. Metabolic profiling and functional metabolic studies were then performed to investigate the impact of succinate elevation on mitochondria function. Results: We found that in vitro succinate supplementation elevated intracellular succinate about 2-fold, and did not have an impact on proliferation or apoptosis of C2C12 myoblasts. Elevated succinate had minor effects on protein homeostasis (~25% decrease in protein synthesis assessed by OPP staining), and no significant effect on myotube atrophy. Succinate elevation interfered with in vitro myoblast differentiation, characterized by significant decreases in late markers of myogenesis and fewer nuclei per myosin heavy chain positive structure (assessed by immunofluorescence staining). While mice orally administered succinate did not exhibit changes in overall body composition or whole muscle weights, these mice displayed smaller muscle myofiber diameters (~6% decrease in the mean of non-linear regression curves fit to the histograms of minimum feret diameter distribution), which was exacerbated when muscle regeneration was induced with barium chloride injury. Significant decreases in the mean of non-linear regression curves fit to the histograms of minimum feret diameter distributions were observed 7 days and 28 days post injury. Elevated numbers of myogenin positive cells (3-fold increase) supportive of the differentiation defects observed in vitro were observed 28 days post injury. Metabolic profiling and functional metabolic assessment of myoblasts revealed that succinate elevation caused both widespread metabolic changes and significantly lowered maximal cellular respiration (~35% decrease). Conclusions: This study broadens the repertoire of wasting-associated factors that can directly modulate muscle progenitor cell function and strengthens the hypothesis that metabolic derangements are significant contributors to impaired muscle regeneration, an important aspect of cancer-associated muscle wasting.
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BACKGROUND: Persistent loss of skeletal muscle mass and function as well as altered fat metabolism are frequently observed in severe sepsis survivors. Studies examining sepsis-associated tissue dysfunction from the perspective of the tissue microenvironment are scarce. In this study, we comprehensively assessed transcriptional changes in muscle and fat at single-cell resolution following experimental sepsis induction. METHODS: Skeletal muscle and visceral white adipose tissue from control mice or mice 1 day or 1 month following faecal slurry-induced sepsis were used. Single cells were mechanically and enzymatically prepared from whole tissue, and viable cells were further isolated by fluorescence activated cell sorting. Droplet-based single-cell RNA-sequencing (scRNA-seq; 10× Genomics) was used to generate single-cell gene expression profiles of thousands of muscle and fat-resident cells. Bioinformatics analyses were performed to identify and compare individual cell populations in both tissues. RESULTS: In skeletal muscle, scRNA-seq analysis classified 1438 single cells into myocytes, endothelial cells, fibroblasts, mesenchymal stem cells, macrophages, neutrophils, T-cells, B-cells, and dendritic cells. In adipose tissue, scRNA-seq analysis classified 2281 single cells into adipose stem cells, preadipocytes, endothelial cells, fibroblasts, macrophages, dendritic cells, B-cells, T-cells, NK cells, and gamma delta T-cells. One day post-sepsis, the proportion of most non-immune cell populations was decreased, while immune cell populations, particularly neutrophils and macrophages, were highly enriched. Proportional changes of endothelial cells, neutrophils, and macrophages were validated using faecal slurry and cecal ligation and puncture models. At 1 month post-sepsis, we observed persistent enrichment/depletion of cell populations and further uncovered a cell-type and tissue-specific ability to return to a baseline transcriptomic state. Differential gene expression analyses revealed key genes and pathways altered in post-sepsis muscle and fat and highlighted the engagement of infection/inflammation and tissue damage signalling. Finally, regulator analysis identified gonadotropin-releasing hormone and Bay 11-7082 as targets/compounds that we show can reduce sepsis-associated loss of lean or fat mass. CONCLUSIONS: These data demonstrate persistent post-sepsis muscle and adipose tissue disruption at the single-cell level and highlight opportunities to combat long-term post-sepsis tissue wasting using bioinformatics-guided therapeutic interventions.
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Tecido Adiposo , Músculo Esquelético , Sepse , Animais , Células Endoteliais , Feminino , Masculino , Camundongos , Sepse/etiologia , TranscriptomaRESUMO
BACKGROUND: Cancer-associated muscle wasting (CAW), a symptom of cancer cachexia, is associated with approximately 20% of lung cancer deaths and remains poorly characterized on a mechanistic level. Current animal models for lung cancer-associated cachexia are limited in that they (1) primarily employ flank transplantation methods, (2) have short survival times not reflective of the patient condition, and (3) are typically performed in young mice not representative of mean patient age. This study investigates a new model for lung cancer-associated cachexia that can address these issues and also implicates muscle regeneration as a contributor to CAW. METHODS: We used tail vein injection as a method to introduce tumor cells that seed primarily in the lungs of mice. Body composition of tumor-bearing mice was longitudinally tracked using NMR-based, echo magnetic resonance imaging (echoMRI). These data were combined with histological and molecular assessments of skeletal muscle to provide a complete analysis of muscle wasting. RESULTS: In this new lung CAW model, we observed (1) progressive loss in whole body weight, (2) progressive loss of lean and fat mass, (3) a circulating cytokine/inflammatory profile similar to that seen in other models of CAW, (4) histological changes associated with muscle wasting, and (5) molecular changes in muscle that implicate suppression of muscle repair/regeneration. Finally, we show that survival can be extended without lessening CAW by titrating injected cell number. CONCLUSIONS: Overall, this study describes a new model of CAW that could be useful for further studies of lung cancer-associated wasting and accompanying changes in the regenerative capacity of muscle. Additionally, this model addresses many recent concerns with existing models such as immunocompetence, tumor location, and survival time.
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Caquexia/patologia , Neoplasias Pulmonares/patologia , Músculo Esquelético/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Caquexia/etiologia , Linhagem Celular Tumoral , Neoplasias Pulmonares/complicações , Masculino , Camundongos , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/metabolismoRESUMO
Obesity is a serious health concern and is associated with a reduced quality of life and a number of chronic diseases, including diabetes, heart disease, stroke, and cancer. With obesity rates on the rise worldwide, adipose tissue biology has become a top biomedical research priority. Despite steady growth in obesity-related research, more investigation into the basic biology of adipose tissue is needed to drive innovative solutions aiming to curtail the obesity epidemic. Adipose progenitor cells (APCs) play a central role in adipose tissue homeostasis and coordinate adipose tissue expansion and remodeling. Although APCs are well studied, defining and characterizing APC subsets remains ambiguous because of ill-defined cellular heterogeneity within this cellular compartment. In this study, we used single-cell RNA sequencing to create a cellular atlas of APC heterogeneity in mouse visceral adipose tissue. Our analysis identified two distinct populations of adipose tissue-derived stem cells (ASCs) and three distinct populations of preadipocytes (PAs). We identified novel cell surface markers that, when used in combination with traditional ASC and preadipocyte markers, could discriminate between these APC subpopulations by flow cytometry. Prospective isolation and molecular characterization of these APC subpopulations confirmed single-cell RNA sequencing gene expression signatures, and ex vivo culture revealed differential expansion/differentiation capabilities. Obese visceral adipose tissue featured relative expansion of less mature ASC and PA subpopulations, and expression analyses revealed major obesity-associated signaling alterations within each APC subpopulation. Taken together, our study highlights cellular and transcriptional heterogeneity within the APC pool, provides new tools to prospectively isolate and study these novel subpopulations, and underscores the importance of considering APC diversity when studying the etiology of obesity.
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Adipócitos/citologia , Obesidade/genética , Adipócitos/metabolismo , Adipócitos/patologia , Adipogenia/genética , Tecido Adiposo/citologia , Tecido Adiposo/patologia , Animais , Diferenciação Celular/fisiologia , Citometria de Fluxo/métodos , Perfilação da Expressão Gênica/métodos , Gordura Intra-Abdominal/citologia , Gordura Intra-Abdominal/patologia , Masculino , Camundongos , Camundongos Endogâmicos , Obesidade/metabolismo , Obesidade/patologia , Análise de Célula Única/métodos , Células-Tronco/citologia , Gordura Subcutânea/citologia , Gordura Subcutânea/patologia , Análise Serial de Tecidos/métodos , TranscriptomaRESUMO
Muscle wasting, a cardinal feature of cancer-associated cachexia (CAC), is a major clinical problem with few therapeutic options. In this Forum article we discuss cellular mechanisms of CAC, focusing on impaired muscle regeneration. We highlight muscle progenitor cell dysfunction and metabolism as two variables contributing to impaired regeneration in CAC.
Assuntos
Caquexia/etiologia , Caquexia/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Neoplasias/complicações , Caquexia/fisiopatologia , Diferenciação Celular , Proliferação de Células , Metabolismo Energético , Humanos , Músculo Esquelético/fisiopatologia , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Atrofia Muscular/fisiopatologia , Mioblastos/citologia , Mioblastos/metabolismo , Regeneração , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismoRESUMO
Tissue-specific stem cells contribute to adult tissue maintenance, repair, and regeneration. In skeletal muscle, many different mononuclear cell types are capable of giving rise to differentiated muscle. Of these tissue stem-like cells, satellite cells (SCs) are the most studied muscle stem cell population and are widely considered the main cellular source driving muscle repair and regeneration in adult tissue. Within the satellite cell pool, many distinct subpopulations exist, each exhibiting differential abilities to exit quiescence, expand, differentiate, and self-renew. In this chapter, we discuss the different stem cell types that can give rise to skeletal muscle tissue and then focus on satellite cell heterogeneity during the process of myogenesis/muscle regeneration. Finally, we highlight emerging opportunities to better characterize muscle stem cell heterogeneity, which will ultimately deepen our appreciation of stem cells in muscle development, repair/regeneration, aging, and disease.