RESUMO
PGC-1α is well established as a metazoan transcriptional coactivator of cellular adaptation in response to stress. However, the mechanisms by which PGC-1α activates gene transcription are incompletely understood. Here, we report that PGC-1α serves as a scaffold protein that physically and functionally connects the DNA-binding protein estrogen-related receptor α (ERRα), cap-binding protein 80 (CBP80), and Mediator to overcome promoter-proximal pausing of RNAPII and transcriptionally activate stress-response genes. We show that PGC-1α promotes pausing release in a two-arm mechanism (1) by recruiting the positive transcription elongation factor b (P-TEFb) and (2) by outcompeting the premature transcription termination complex Integrator. Using mice homozygous for five amino acid changes in the CBP80-binding motif (CBM) of PGC-1α that destroy CBM function, we show that efficient differentiation of primary myoblasts to myofibers and timely skeletal muscle regeneration after injury require PGC-1α binding to CBP80. Our findings reveal how PGC-1α activates stress-response gene transcription in a previously unanticipated pre-mRNA quality-control pathway.
Assuntos
Precursores de RNA , Fatores de Transcrição , Animais , Camundongos , Proteínas de Ligação a DNA/genética , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Regiões Promotoras Genéticas , Proteínas de Ligação ao Cap de RNA/genética , RNA Polimerase II/metabolismo , Precursores de RNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The functional role of Pax7-expressing satellite cells (SCs) in postnatal skeletal muscle development beyond weaning remains obscure. Therefore, the relevance of SCs during prepubertal growth, a period after weaning but prior to the onset of puberty, has not been examined. Here, we have characterized mouse skeletal muscle growth during prepuberty and found significant increases in myofiber cross-sectional area that correlated with SC-derived myonuclear number. Remarkably, genome-wide RNA-sequencing analysis established that post-weaning juvenile and early adolescent skeletal muscle have markedly different gene expression signatures. These distinctions are consistent with extensive skeletal muscle maturation during this essential, albeit brief, developmental phase. Indelible labeling of SCs with Pax7CreERT2/+ ; Rosa26nTnG/+ mice demonstrated SC-derived myonuclear contribution during prepuberty, with a substantial reduction at puberty onset. Prepubertal depletion of SCs in Pax7CreERT2/+ ; Rosa26DTA/+ mice reduced myofiber size and myonuclear number, and caused force generation deficits to a similar extent in both fast and slow-contracting muscles. Collectively, these data demonstrate SC-derived myonuclear accretion as a cellular mechanism that contributes to prepubertal hypertrophic skeletal muscle growth.
Assuntos
Desenvolvimento Muscular , Fator de Transcrição PAX7/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Maturidade Sexual , Animais , Animais Recém-Nascidos , Fenômenos Biomecânicos , Regulação da Expressão Gênica no Desenvolvimento , Hipertrofia , Camundongos Endogâmicos C57BL , Contração Muscular , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologiaRESUMO
The niche is a conserved regulator of stem cell quiescence and function. During ageing, stem cell function declines. To what extent and by what means age-related changes within the niche contribute to this phenomenon are unknown. Here we demonstrate that the aged muscle stem cell niche, the muscle fibre, expresses Fgf2 under homeostatic conditions, driving a subset of satellite cells to break quiescence and lose their self-renewing capacity. We show in mice that relatively dormant aged satellite cells robustly express sprouty 1 (Spry1), an inhibitor of fibroblast growth factor (FGF) signalling. Increasing FGF signalling in aged satellite cells under homeostatic conditions by removing Spry1 results in the loss of quiescence, satellite cell depletion and diminished regenerative capacity. Conversely, reducing niche-derived FGF activity through inhibition of Fgfr1 signalling or overexpression of Spry1 in satellite cells prevents their depletion. These experiments identify an age-dependent change in the stem cell niche that directly influences stem cell quiescence and function.
Assuntos
Envelhecimento/fisiologia , Ciclo Celular , Células Musculares/citologia , Células Satélites de Músculo Esquelético/citologia , Nicho de Células-Tronco/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Contagem de Células , Diferenciação Celular , Senescência Celular , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Citometria de Fluxo , Homeostase , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/citologia , Fator de Transcrição PAX7/metabolismo , Fosfoproteínas/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/transplante , Transdução de Sinais , Fatores de TempoRESUMO
Across different niches, subsets of highly functional stem cells are maintained in a relatively dormant rather than proliferative state. Our understanding of proliferative dynamics in tissue-specific stem cells during conditions of increased tissue turnover remains limited. Using a TetO-H2B-GFP reporter of proliferative history, we identify skeletal muscle stem cell, or satellite cells, that retain (LRC) or lose (nonLRC) the H2B-GFP label. We show in mice that LRCs and nonLRCs are formed at birth and persist during postnatal growth and adult muscle repair. Functionally, LRCs and nonLRCs are born equivalent and transition during postnatal maturation into distinct and hierarchically organized subsets. Adult LRCs give rise to LRCs and nonLRCs; the former are able to self-renew, whereas the latter are restricted to differentiation. Expression analysis revealed the CIP/KIP family members p21(cip1) (Cdkn1a) and p27(kip1) (Cdkn1b) to be expressed at higher levels in LRCs. In accordance with a crucial role in LRC fate, loss of p27(kip1) promoted proliferation and differentiation of LRCs in vitro and impaired satellite cell self-renewal after muscle injury. By contrast, loss of p21(cip1) only affected nonLRCs, in which myogenic commitment was inhibited. Our results provide evidence that restriction of self-renewal potential to LRCs is established early in life and is maintained during increased tissue turnover through the cell cycle inhibitor p27(kip1). They also reveal the differential role of CIP/KIP family members at discrete steps within the stem cell hierarchy.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Músculo Esquelético/citologia , Coloração e Rotulagem , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Progressão da Doença , Proteínas de Fluorescência Verde/metabolismo , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Distrofia Muscular Animal/patologia , FenótipoRESUMO
Embryonic skeletal muscle growth is contingent upon a population of somite derived satellite cells, however, the contribution of these cells to early postnatal skeletal muscle growth remains relatively high. As prepubertal postnatal development proceeds, the activity and contribution of satellite cells to skeletal muscle growth diminishes. Eventually, at around puberty, a population of satellite cells escapes terminal commitment, continues to express the paired box transcription factor Pax7, and reside in a quiescent state orbiting the myofiber periphery adjacent to the basal lamina. After adolescence, some satellite cell contributions to muscle maintenance and adaptation occur, however, their necessity is reduced relative to embryonic, early postnatal, and prepubertal growth.
Assuntos
Desenvolvimento Muscular , Músculo Esquelético , Células Satélites de Músculo Esquelético , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/fisiologia , Animais , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Humanos , Fator de Transcrição PAX7/metabolismo , Fator de Transcrição PAX7/genética , Diferenciação CelularRESUMO
Aging is associated with a decline in stem cell functionality and number across the organism. In this study, we aimed to further unravel Muscle Stem Cells (MuSCs) aging by assessing how systemic factors influence MuSC fate decisions through long-term epigenetic landscape remodelling. As aging is intricately linked to a pro-inflammatory shift, we studied the epigenetic effects of inflammatory signals in MuSCs and measured decreased H4K20me1 levels. This loss disrupts MuSC quiescence, largely through epigenetic silencing of Notch target genes. In the setting of inflammatory signals or aging, the lack of Kmt5a and the subsequent absence of de novoH4K20me1 culminate in cell death by ferroptosis. Aged MuSCs manifest abnormal iron metabolism and reduced Gpx4 levels, resulting in the accumulation of intracellular iron, increased reactive oxygen species, genomic instability, and lipid peroxidation. We showed that ferroptosis is the predominant mode of cell death in aged MuSCs, with remarkably high levels of lipid peroxidation; a phenomenon we also observed in aged hematopoietic stem cells. Implementing preventative strategies to inhibit systemic inflammation prevented aged MuSC ferroptosis, preserving their numbers and regenerative capabilities. This intervention significantly enhanced aged muscle regeneration and strength recovery and extended both lifespan and healthspan in mice. This study delineates a previously underappreciated fate trajectory for stem cell aging, and offers meaningful insights into the treatment of age-related disorders.
RESUMO
Mammalian limb and trunk skeletal muscles are composed of muscle fibers that differ in contractile and molecular properties. They are commonly divided into four categories according to the myosin heavy chain that they express: I, IIA, IIX and IIB, ranging from slowest to fastest. Individual motor axons innervate tens of muscle fibers, nearly all of which are of the same type. The mechanisms accounting for this striking specificity, termed motor unit homogeneity, remain incompletely understood, in part because there have been no markers for motoneuron types. Here we show in mice that the synaptic vesicle protein SV2A is selectively localized in motor nerve terminals on slow (type I and small type IIA) muscle fibers; its close relatives, SV2B and SV2C, are present in all motor nerve terminals. SV2A is broadly expressed at birth; fast motoneurons downregulate its expression during the first postnatal week. An inducible transgene incorporating regulatory elements from the Sv2a gene permits selective labeling of slow motor units and reveals their composition. Overexpression of the transcriptional co-regulator PGC1α in muscle fibers, which converts them to a slow phenotype, leads to an increased frequency of SV2A-positive motor nerve terminals, indicating a fiber type-specific retrograde influence of muscle fibers on their innervation. This retrograde influence must be integrated with known anterograde influences in order to understand how motor units become homogeneous.
Assuntos
Marcadores Genéticos/genética , Glicoproteínas de Membrana/metabolismo , Neurônios Motores/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/inervação , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Animais , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Fibras Musculares de Contração Lenta/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Transativadores/metabolismo , Fatores de Transcrição , Transgenes/genéticaRESUMO
Skeletal muscle fibers vary in contractile and metabolic properties. Four main fiber types are present in mammalian trunk and limb muscles; they are called I, IIA, IIX, and IIB, ranging from slowest- to fastest-contracting. Individual muscles contain stereotyped proportions of two or more fiber types. Fiber type is determined by a combination of nerve-dependent and -independent influences, leading to formation of "homogeneous motor units" in which all branches of a single motor neuron form synapses on fibers of a single type. Fiber type composition of muscles can be altered in adulthood by multiple factors including exercise, denervation, hormones, and aging. To facilitate analysis of muscle development, plasticity, and innervation, we generated transgenic mouse lines in which Type I, Type IIA, and Type IIX+B fibers can be selectively labeled with distinguishable fluorophores. We demonstrate their use for motor unit reconstruction and live imaging of nerve-dependent alterations in fiber type.
Assuntos
Fibras Musculares Esqueléticas/classificação , Fibras Musculares Esqueléticas/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Animais , Processamento de Imagem Assistida por Computador , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Contração Muscular/fisiologiaRESUMO
During development, resident stem cell populations contribute to the growth and maturation of tissue and organs. In skeletal muscle, muscle stem cells, or satellite cells (SCs), are responsible for the maturation of postnatal myofibers. However, the role SCs play in later stages of postnatal growth, and thus, when they enter a mature quiescent state is controversial. Here, we discuss the current literature regarding the role SCs play in all stages of postnatal growth, from birth to puberty onset to young adulthood. We additionally highlight the implications of SC loss or dysfunction during developmental stages, both in the context of experimental paradigms and disease settings.
Assuntos
Células Satélites de Músculo Esquelético , Diferenciação Celular , Desenvolvimento Muscular , Músculo Esquelético , Regeneração/fisiologia , Células-TroncoRESUMO
BACKGROUND: As paediatric cancer survivors are living into adulthood, they suffer from the age-related, accelerated decline of functional skeletal muscle tissue, termed sarcopenia. With ionizing radiation (radiotherapy) at the core of paediatric cancer therapies, its direct and indirect effects can have lifelong negative impacts on paediatric growth and maintenance of skeletal muscle. Utilizing our recently developed preclinical rhabdomyosarcoma mouse model, we investigated the late effects of paediatric radiation treatment on skeletal muscles from late adolescent (8 weeks old) and middle-aged (16 months old) mice. METHODS: Paediatric C57BL/6J male mice (3 weeks old) were injected with rhabdomyosarcoma cells into their right hindlimbs, and then fractionated irradiation (3 × 8.2 Gy) was administered to those limbs at 4 weeks old to eliminate the tumours. Radiation-alone and tumour-irradiated mice were assessed at either 8 weeks (3 weeks post-irradiation) or 16 months (14 months post-irradiation) of age for muscle physiology, myofibre characteristics, cell loss, histopathology, fibrosis, inflammatory gene expression, and fibrotic gene expression. RESULTS: Mice that received only paediatric radiation demonstrated reduced muscle mass (-17%, P < 0.001), muscle physiological function (-25%, P < 0.01), muscle contractile kinetics (-25%, P < 0.05), satellite cell number (-45%, P < 0.05), myofibre cross-sectional area (-30%, P < 0.0001), and myonuclear number (-17%, P < 0.001). Paediatric radiation increased inflammatory gene expression, increased fibrotic gene expression, and induced extracellular matrix protein deposition (fibrosis) with tumour elimination exacerbating some phenotypes. Paediatric tumour-eliminated mice demonstrated exacerbated deficits to function (-20%, P < 0.05) and myofibre size (-17%, P < 0.001) in some muscles as well as further increases to inflammatory and fibrotic gene expression. Examining the age-related effects of paediatric radiotherapy in middle-aged mice, we found persistent myofibre atrophy (-20%, P < 0.01), myonuclear loss (-18%, P < 0.001), up-regulated inflammatory and fibrotic signalling, and lifelong fibrosis. CONCLUSIONS: The results from this paediatric radiotherapy model are consistent and recapitulate the clinical and molecular features of accelerated sarcopenia, musculoskeletal frailty, and radiation-induced fibrosis experienced by paediatric cancer survivors. We believe that this preclinical mouse model is well poised for future mechanistic insights and therapeutic interventions that improve the quality of life for paediatric cancer survivors.
Assuntos
Neoplasias , Qualidade de Vida , Adolescente , Adulto , Animais , Fibrose , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Neoplasias/patologiaRESUMO
BACKGROUND: Radiotherapy is commonly used to treat childhood cancers and can have adverse effects on muscle function, but the underlying mechanisms have yet to be fully elucidated. We hypothesized that endurance exercise following radiation treatment would improve skeletal muscle function. METHODS: We utilized the Small Animal Radiation Research Platform (SARRP) to irradiate juvenile male mice with a clinically relevant fractionated dose of 3× (every other day over 5 days) 8.2 Gy X-ray irradiation locally from the knee to footpad region of the right hindlimb. Mice were then singly housed for 1 month in cages equipped with either locked or free-spinning voluntary running wheels. Ex vivo muscle contractile function, RT-qPCR analyses, resting cytosolic and sarcoplasmic reticulum (SR) store Ca2+ levels, mitochondrial reactive oxygen species levels (MitoSOX), and immunohistochemical and biochemical analyses of muscle samples were conducted to assess the muscle pathology and the relative therapeutic impact of voluntary wheel running (VWR). RESULTS: Irradiation reduced fast-twitch extensor digitorum longus (EDL) muscle-specific force by 27% compared to that of non-irradiated mice, while VWR post-irradiation improved muscle-specific force by 37%. Radiation treatment similarly reduced slow-twitch soleus muscle-specific force by 14% compared to that of non-irradiated mice, while VWR post-irradiation improved specific force by 18%. We assessed intracellular Ca2+ regulation, oxidative stress, and mitochondrial homeostasis as potential mechanisms of radiation-induced pathology and exercise-mediated rescue. We found a significant reduction in resting cytosolic Ca2+ concentration following irradiation in sedentary mice. Intriguingly, however, SR Ca2+ store content was increased in myofibers from irradiated mice post-VWR compared to mice that remained sedentary. We observed a 73% elevation in the overall protein oxidization in muscle post-irradiation, while VWR reduced protein nitrosylation by 35% and mitochondrial reactive oxygen species (ROS) production by 50%. Finally, we found that VWR significantly increased the expression of PGC1α at both the transcript and protein levels, consistent with an exercise-dependent increase in mitochondrial biogenesis. CONCLUSIONS: Juvenile irradiation stunted muscle development, disrupted proper Ca2+ handling, damaged mitochondria, and increased oxidative and nitrosative stress, paralleling significant deficits in muscle force production. Exercise mitigated aberrant Ca2+ handling, mitochondrial homeostasis, and increased oxidative and nitrosative stress in a manner that correlated with improved skeletal muscle function after radiation.
Assuntos
Atividade Motora , Músculo Esquelético , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Atividade Motora/fisiologia , Músculo Esquelético/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
Mesenchymal progenitors of the lateral plate mesoderm give rise to various cell fates within limbs, including a heterogeneous group of muscle-resident mesenchymal cells. Often described as fibro-adipogenic progenitors, these cells are key players in muscle development, disease, and regeneration. To further define this cell population(s), we perform lineage/reporter analysis, flow cytometry, single-cell RNA sequencing, immunofluorescent staining, and differentiation assays on normal and injured murine muscles. Here we identify six distinct Pdgfra+ non-myogenic muscle-resident mesenchymal cell populations that fit within a bipartite differentiation trajectory from a common progenitor. One branch of the trajectory gives rise to two populations of immune-responsive mesenchymal cells with strong adipogenic potential and the capability to respond to acute and chronic muscle injury, whereas the alternative branch contains two cell populations with limited adipogenic capacity and inherent mineralizing capabilities; one of the populations displays a unique neuromuscular junction association and an ability to respond to nerve injury.
Assuntos
Desenvolvimento Muscular , Músculo Esquelético , Adipogenia , Animais , Diferenciação Celular , Camundongos , Fibras Musculares Esqueléticas , Músculo Esquelético/fisiologiaRESUMO
A therapeutic strategy to treat Duchenne muscular dystrophy (DMD) involves identifying compounds that can elevate utrophin A expression in muscle fibers of affected patients. The dystrophin homologue utrophin A can functionally substitute for dystrophin when its levels are enhanced in the mdx mouse model of DMD. Utrophin A expression in skeletal muscle is regulated by mechanisms that promote the slow myofiber program. Since activation of peroxisome proliferator-activated receptor (PPAR) beta/delta promotes the slow oxidative phenotype in skeletal muscle, we initiated studies to determine whether pharmacological activation of PPARbeta/delta provides functional benefits to the mdx mouse. GW501516, a PPARbeta/delta agonist, was found to stimulate utrophin A mRNA levels in C2C12 muscle cells through an element in the utrophin A promoter. Expression of PPARbeta/delta was greater in skeletal muscles of mdx versus wild-type mice. We treated 5-7-week-old mdx mice with GW501516 for 4 weeks. This treatment increased the percentage of muscle fibers expressing slower myosin heavy chain isoforms and stimulated utrophin A mRNA levels leading to its increased expression at the sarcolemma. Expression of alpha1-syntrophin and beta-dystroglycan was restored to the sarcolemma. Improvement of mdx sarcolemmal integrity was evidenced by decreased intracellular IgM staining and decreased in vivo Evans blue dye (EBD) uptake. GW501516 treatment also conferred protection against eccentric contraction (ECC)-induced damage of mdx skeletal muscles, as shown by a decreased contraction-induced force drop and reduction of dye uptake during ECC. These results demonstrate that pharmacological activation of PPARbeta/delta might provide functional benefits to DMD patients through enhancement of utrophin A expression.
Assuntos
Expressão Gênica/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/tratamento farmacológico , PPAR alfa/metabolismo , PPAR beta/metabolismo , Tiazóis/administração & dosagem , Utrofina/genética , Animais , Linhagem Celular , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Fibras Musculares Esqueléticas/efeitos dos fármacos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , PPAR alfa/agonistas , PPAR alfa/genética , PPAR beta/agonistas , PPAR beta/genética , Sarcolema/efeitos dos fármacos , Sarcolema/metabolismo , Utrofina/metabolismoRESUMO
BACKGROUND: Skeletal muscle wasting (SMW) in cancer patients is associated with increased morbidity, mortality, treatment intolerance and discontinuation, and poor quality of life. This is particularly true for patients with pancreatic ductal adenocarcinoma (PDAC), as over 85% experience SMW, which is responsible for ~30% of patient deaths. While the established paradigm to explain SMW posits that muscle catabolism from systemic inflammation and nutritional deficiencies, the cause of death, and the cellular and molecular mechanisms responsible remain to be elucidated. To address this, we investigated the relationship between tumour burden and survival in the KCKO murine PDAC model. METHODS: Female C57BL/6J mice 6-8 weeks of age underwent orthotopic injection with KCKO-luc tumour cells. Solid tumour was verified on Day 5, post-tumour inoculation. In vivo, longitudinal lean mass and tumour burden were assessed via dual-energy X-ray absorptiometry and IVIS imaging, respectively, and total body weight was assessed, weekly. Animals were sacrificed at a designated end point of 'failure to thrive'. After sacrifice, lower limb hind muscles were harvested for histology and RNA extraction. RESULTS: We found a strong correlation between primary tumour size and survival (r2 = 0.83, P < 0.0001). A significant decrease in lower limb lean mass was first detected at Day 38 post-implantation vs. no tumour controls (NTCs) (P < 0.0001). SMW was confirmed by histology, which demonstrated a 38%, 32.7%, and 39.9% decrease in fibre size of extensor digitorum longus, soleus, and tibialis anterior muscles, respectively, in PDAC mice vs. NTC (P < 0.002). Histology also revealed a 67.6% increase in haematopoietic cells within the muscle of PDAC mice when compared with NTC. Bulk RNAseq on muscles from PDAC mice vs. NTC revealed significant increases in c/ebpß/Δ, il-1, il-6, and tnf gene expression. Pathway analyses to identify potential upstream factors revealed increased adipogenic gene expression, including a four-fold increase in igfbp-3. Histomorphometry of Oil Red-O staining for fat content in tibialis anterior muscles demonstrated a 95.5% increase in positively stained fibres from PDAC mice vs. NTC. CONCLUSIONS: Together, these findings support a novel model of PDAC-associated SMW and mortality in which systemic inflammation leads to inflammatory cell infiltration into skeletal muscle with up-regulated myocellular lipids.
Assuntos
Caquexia , Neoplasias Pancreáticas , Animais , Caquexia/etiologia , Modelos Animais de Doenças , Feminino , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético , Neoplasias Pancreáticas/complicações , Qualidade de VidaRESUMO
During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume and function impacting mobility and quality of life. Muscle loss is often associated with denervation and a loss of resident muscle stem cells (satellite cells or MuSCs); however, the relationship between MuSCs and innervation has not been established. Herein, we administered severe neuromuscular trauma to a transgenic murine model that permits MuSC lineage tracing. We show that a subset of MuSCs specifically engraft in a position proximal to the neuromuscular junction (NMJ), the synapse between myofibers and motor neurons, in healthy young adult muscles. In aging and in a mouse model of neuromuscular degeneration (Cu/Zn superoxide dismutase knockout - Sod1-/-), this localized engraftment behavior was reduced. Genetic rescue of motor neurons in Sod1-/- mice reestablished integrity of the NMJ in a manner akin to young muscle and partially restored MuSC ability to engraft into positions proximal to the NMJ. Using single cell RNA-sequencing of MuSCs isolated from aged muscle, we demonstrate that a subset of MuSCs are molecularly distinguishable from MuSCs responding to myofiber injury and share similarity to synaptic myonuclei. Collectively, these data reveal unique features of MuSCs that respond to synaptic perturbations caused by aging and other stressors.
Assuntos
Envelhecimento , Músculo Esquelético/lesões , Mioblastos Esqueléticos/fisiologia , Junção Neuromuscular/fisiologia , Superóxido Dismutase-1/deficiência , Animais , Feminino , Masculino , Camundongos KnockoutRESUMO
We examined the role of post-transcriptional mechanisms in controlling utrophin A mRNA expression in slow versus fast skeletal muscles. First, we determined that the half-life of utrophin A mRNA is significantly shorter in the presence of proteins isolated from fast muscles. Direct plasmid injection experiments using reporter constructs containing the full-length or truncated variants of the utrophin 3'UTR into slow soleus and fast extensor digitorum longus muscles revealed that a region of 265 nucleotides is sufficient to confer lower levels of reporter mRNA in fast muscles. Further analysis of this region uncovered a conserved AU-rich element (ARE) that suppresses expression of reporter mRNAs in cultured muscle cells. Moreover, stability of reporter mRNAs fused to the utrophin full-length 3'UTR was lower in the presence of fast muscle protein extracts. This destabilization effect seen in vivo was lost upon deletion of the conserved ARE. Finally, we observed that calcineurin signaling affects utrophin A mRNA stability through the conserved ARE. These results indicate that ARE-mediated mRNA decay is a key mechanism that regulates expression of utrophin A mRNA in slow muscle fibers. This is the first demonstration of ARE-mediated mRNA decay regulating the expression of a gene associated with the slow myogenic program.
Assuntos
Regiões 3' não Traduzidas/química , Calcineurina/metabolismo , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Estabilidade de RNA , Sequências Reguladoras de Ácido Ribonucleico , Utrofina/genética , Adenosina/análise , Animais , Sequência de Bases , Linhagem Celular , Sequência Conservada , Regulação da Expressão Gênica , Genes Reporter , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais , Uridina/análise , Utrofina/metabolismoRESUMO
During aging, muscle mass decreases, leading to sarcopenia, associated with low-level chronic inflammation (inflammaging), which induces sarcopenia by promoting proteolysis of muscle fibers and inhibiting their regeneration. Patients with a variety of pathologic conditions associated with sarcopenia, including rheumatoid arthritis (RA), have systemically elevated TNFα serum levels, and transgenic mice with TNFα overexpression (TNF-Tg mice, a model of RA) develop sarcopenia between adolescence and adulthood before they age. However, if and how TNFα contributes to the pathogenesis of sarcopenia during the normal aging process and in RA remains largely unknown. We report that TNFα levels are increased in skeletal muscles of aged WT mice, associated with muscle atrophy and decreased numbers of satellite cells and Type IIA myofibers, a phenotype that we also observed in adult TNF-Tg mice. Aged WT mice also have increased numbers of myeloid lineage cells in their skeletal muscles, including macrophages and granulocytes. These cells have increased TNFα expression, which impairs myogenic cell differentiation. Expression levels of TNF receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase, which mediates signaling by some TNF receptor (TNFR) family members, are elevated in skeletal muscles of both aged WT mice and adult TNF-Tg mice. TRAF6 binds to TNFR2 in C2C12 myoblasts and mediates TNFα-induced muscle atrophy through NF-κB-induced transcription of the muscle-specific E3 ligases, Atrogen1 and Murf1, which promote myosin heavy-chain degradation. Haplo-deficiency of TRAF6 prevents muscle atrophy and the decrease in numbers of satellite cells, Type IIA myofibers, and myogenic regeneration in TRAF6+/- ;TNF-Tg mice. Our findings suggest that pharmacologic inhibition of TRAF6 signaling in skeletal muscles during aging could treat/prevent age- and RA-related sarcopenia by preventing TNFα-induced proteolysis and inhibition of muscle fiber regeneration. © 2020 American Society for Bone and Mineral Research.
Assuntos
Sarcopenia , Fator 6 Associado a Receptor de TNF , Envelhecimento , Animais , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/patologia , Sarcopenia/patologia , Fator 6 Associado a Receptor de TNF/metabolismoRESUMO
Muscle regeneration depends on a robust albeit transient inflammatory response. Persistent inflammation is a feature of age-related regenerative deficits, yet the underlying mechanisms are poorly understood. Here, we find inflammatory-related CC-chemokine-receptor 2 (Ccr2) expression in non-hematopoietic myogenic progenitors (MPs) during regeneration. After injury, the expression of Ccr2 in MPs corresponds to the levels of its ligands, the chemokines Ccl2, 7, and 8. We find stimulation of Ccr2-activity inhibits MP fusion and contribution to myofibers. This occurs in association with increases in MAPKp38δ/γ signaling, MyoD phosphorylation, and repression of the terminal myogenic commitment factor Myogenin. High levels of Ccr2-chemokines are a feature of regenerating aged muscle. Correspondingly, deletion of Ccr2 in MPs is necessary for proper fusion into regenerating aged muscle. Finally, opportune Ccr2 inhibition after injury enhances aged regeneration and functional recovery. These results demonstrate that inflammatory-induced activation of Ccr2 signaling in myogenic cells contributes to aged muscle regenerative decline.
Assuntos
Mediadores da Inflamação/metabolismo , Músculo Esquelético/fisiopatologia , Receptores CCR2/metabolismo , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Fatores Etários , Animais , Transplante de Células/métodos , Quimiocina CCL2/metabolismo , Quimiocina CCL7/metabolismo , Quimiocina CCL8/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Desenvolvimento Muscular/genética , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Miogenina/genética , Miogenina/metabolismo , Receptores CCR2/genética , Regeneração/genética , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/transplante , Transdução de Sinais/genética , Ferimentos e Lesões/genética , Ferimentos e Lesões/fisiopatologia , Ferimentos e Lesões/terapiaRESUMO
Pediatric cancer treatment often involves chemotherapy and radiation, where off-target effects can include skeletal muscle decline. The effect of such treatments on juvenile skeletal muscle growth has yet to be investigated. We employed a small animal irradiator to administer fractionated hindlimb irradiation to juvenile mice bearing implanted rhabdomyosarcoma (RMS) tumors. Hindlimb-targeted irradiation (3 × 8.2 Gy) of 4-week-old mice successfully eliminated RMS tumors implanted one week prior. After establishment of this preclinical model, a cohort of tumor-bearing mice were injected with the chemotherapeutic drug, vincristine, alone or in combination with fractionated irradiation (5 × 4.8 Gy). Single myofiber analysis of fast-contracting extensor digitorum longus (EDL) and slow-contracting soleus (SOL) muscles was conducted 3 weeks post-treatment. Although a reduction in myofiber size was apparent, EDL and SOL myonuclear number were differentially affected by juvenile irradiation and/or vincristine treatment. In contrast, a decrease in myonuclear domain (myofiber volume/myonucleus) was observed regardless of muscle or treatment. Thus, inhibition of myofiber hypertrophic growth is a consistent feature of pediatric cancer treatment.
Assuntos
Quimiorradioterapia/efeitos adversos , Fibras Musculares Esqueléticas/patologia , Rabdomiossarcoma/terapia , Envelhecimento , Animais , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Fracionamento da Dose de Radiação , Membro Posterior/efeitos dos fármacos , Membro Posterior/patologia , Membro Posterior/efeitos da radiação , Hipertrofia , Masculino , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos da radiação , Teste de Desempenho do Rota-Rod , Transplante Isogênico , Vincristina/farmacologiaRESUMO
During prepubertal development, muscle stem cells (satellite cells, SCs) actively contribute to myofiber growth. Because some SCs are active during this time, they may be particularly susceptible to damage. Using a Small Animal Radiation Research Platform (SARRP), we investigated the effects of local fractionated radiation treatment on prepubertal SCs. Immediately after this regimen, there was a reduction in SC number. Although surviving SCs had deficiencies in function, some myogenic potential remained. Indeed, some muscle regenerative capacity persisted immediately after irradiation. Lastly, we assessed the long-term consequences of radiation-induced SC loss during prepuberty. We observed a reduction of myofiber size and corresponding loss of nuclei in both fast- and slow-contracting muscles 14 months post-irradiation. Notably, prepubertal SC depletion mimicked these lifelong deficits. This work highlights the susceptibility of prepubertal SCs to radiation exposure. We also reveal the importance of prepubertal SC contribution to the lifelong maintenance of skeletal muscle.