RESUMO
Tissue regeneration declines with ageing but little is known about whether this arises from changes in stem-cell heterogeneity. Here, in homeostatic skeletal muscle, we identify two quiescent stem-cell states distinguished by relative CD34 expression: CD34High, with stemness properties (genuine state), and CD34Low, committed to myogenic differentiation (primed state). The genuine-quiescent state is unexpectedly preserved into later life, succumbing only in extreme old age due to the acquisition of primed-state traits. Niche-derived IGF1-dependent Akt activation debilitates the genuine stem-cell state by imposing primed-state features via FoxO inhibition. Interventions to neutralize Akt and promote FoxO activity drive a primed-to-genuine state conversion, whereas FoxO inactivation deteriorates the genuine state at a young age, causing regenerative failure of muscle, as occurs in geriatric mice. These findings reveal transcriptional determinants of stem-cell heterogeneity that resist ageing more than previously anticipated and are only lost in extreme old age, with implications for the repair of geriatric muscle.
Assuntos
Antígenos CD34/metabolismo , Proliferação de Células , Autorrenovação Celular , Senescência Celular , Fatores de Transcrição Forkhead/metabolismo , Músculo Esquelético/metabolismo , Regeneração , Células Satélites de Músculo Esquelético/metabolismo , Fatores Etários , Animais , Cardiotoxinas/toxicidade , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Autorrenovação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Músculo Esquelético/transplante , Fenótipo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regeneração/efeitos dos fármacos , Regeneração/genética , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Células Satélites de Músculo Esquelético/patologia , Células Satélites de Músculo Esquelético/transplante , Transdução de Sinais , Nicho de Células-TroncoRESUMO
Normal homeostatic functions of adult stem cells have rhythmic daily oscillations that are believed to become arrhythmic during aging. Unexpectedly, we find that aged mice remain behaviorally circadian and that their epidermal and muscle stem cells retain a robustly rhythmic core circadian machinery. However, the oscillating transcriptome is extensively reprogrammed in aged stem cells, switching from genes involved in homeostasis to those involved in tissue-specific stresses, such as DNA damage or inefficient autophagy. Importantly, deletion of circadian clock components did not reproduce the hallmarks of this reprogramming, underscoring that rewiring, rather than arrhythmia, is associated with physiological aging. While age-associated rewiring of the oscillatory diurnal transcriptome is not recapitulated by a high-fat diet in young adult mice, it is significantly prevented by long-term caloric restriction in aged mice. Thus, stem cells rewire their diurnal timed functions to adapt to metabolic cues and to tissue-specific age-related traits.
Assuntos
Células-Tronco Adultas/patologia , Senescência Celular , Ritmo Circadiano , Epiderme/patologia , Músculo Esquelético/patologia , Células-Tronco Adultas/fisiologia , Animais , Autofagia , Restrição Calórica , Relógios Circadianos , Dano ao DNA , Dieta Hiperlipídica , Homeostase , Camundongos , Estresse Fisiológico , TranscriptomaRESUMO
Mitochondrial dysfunction and accumulation of damaged mitochondria are considered major contributors to aging. However, the molecular mechanisms responsible for these mitochondrial alterations remain unknown. Here, we demonstrate that mitofusin 2 (Mfn2) plays a key role in the control of muscle mitochondrial damage. We show that aging is characterized by a progressive reduction in Mfn2 in mouse skeletal muscle and that skeletal muscle Mfn2 ablation in mice generates a gene signature linked to aging. Furthermore, analysis of muscle Mfn2-deficient mice revealed that aging-induced Mfn2 decrease underlies the age-related alterations in metabolic homeostasis and sarcopenia. Mfn2 deficiency reduced autophagy and impaired mitochondrial quality, which contributed to an exacerbated age-related mitochondrial dysfunction. Interestingly, aging-induced Mfn2 deficiency triggers a ROS-dependent adaptive signaling pathway through induction of HIF1α transcription factor and BNIP3. This pathway compensates for the loss of mitochondrial autophagy and minimizes mitochondrial damage. Our findings reveal that Mfn2 repression in muscle during aging is a determinant for the inhibition of mitophagy and accumulation of damaged mitochondria and triggers the induction of a mitochondrial quality control pathway.
Assuntos
Envelhecimento , Autofagia , GTP Fosfo-Hidrolases/metabolismo , Mitofagia , Músculo Esquelético/patologia , Sarcopenia/patologia , Animais , Camundongos , Camundongos KnockoutRESUMO
During ageing, muscle stem-cell regenerative function declines. At advanced geriatric age, this decline is maximal owing to transition from a normal quiescence into an irreversible senescence state. How satellite cells maintain quiescence and avoid senescence until advanced age remains unknown. Here we report that basal autophagy is essential to maintain the stem-cell quiescent state in mice. Failure of autophagy in physiologically aged satellite cells or genetic impairment of autophagy in young cells causes entry into senescence by loss of proteostasis, increased mitochondrial dysfunction and oxidative stress, resulting in a decline in the function and number of satellite cells. Re-establishment of autophagy reverses senescence and restores regenerative functions in geriatric satellite cells. As autophagy also declines in human geriatric satellite cells, our findings reveal autophagy to be a decisive stem-cell-fate regulator, with implications for fostering muscle regeneration in sarcopenia.
Assuntos
Autofagia/fisiologia , Senescência Celular , Células Satélites de Músculo Esquelético/citologia , Envelhecimento/patologia , Animais , Contagem de Células , Inibidor p16 de Quinase Dependente de Ciclina/genética , Epigênese Genética , Homeostase , Humanos , Masculino , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitofagia , Músculo Esquelético/citologia , Músculo Esquelético/patologia , Organelas/metabolismo , Estresse Oxidativo , Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regeneração , Sarcopenia/patologia , Sarcopenia/prevenção & controle , Células Satélites de Músculo Esquelético/patologiaRESUMO
Regeneration of skeletal muscle depends on a population of adult stem cells (satellite cells) that remain quiescent throughout life. Satellite cell regenerative functions decline with ageing. Here we report that geriatric satellite cells are incapable of maintaining their normal quiescent state in muscle homeostatic conditions, and that this irreversibly affects their intrinsic regenerative and self-renewal capacities. In geriatric mice, resting satellite cells lose reversible quiescence by switching to an irreversible pre-senescence state, caused by derepression of p16(INK4a) (also called Cdkn2a). On injury, these cells fail to activate and expand, undergoing accelerated entry into a full senescence state (geroconversion), even in a youthful environment. p16(INK4a) silencing in geriatric satellite cells restores quiescence and muscle regenerative functions. Our results demonstrate that maintenance of quiescence in adult life depends on the active repression of senescence pathways. As p16(INK4a) is dysregulated in human geriatric satellite cells, these findings provide the basis for stem-cell rejuvenation in sarcopenic muscles.
Assuntos
Envelhecimento/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Adulto , Animais , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Inibidor p16 de Quinase Dependente de Ciclina/genética , Fator de Transcrição E2F1/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Progéria/metabolismo , Progéria/patologia , Regeneração , Rejuvenescimento , Proteína do Retinoblastoma/metabolismo , Adulto JovemRESUMO
In Duchenne muscular dystrophy (DMD), a persistently altered and reorganizing extracellular matrix (ECM) within inflamed muscle promotes damage and dysfunction. However, the molecular determinants of the ECM that mediate inflammatory changes and faulty tissue reorganization remain poorly defined. Here, we show that fibrin deposition is a conspicuous consequence of muscle-vascular damage in dystrophic muscles of DMD patients and mdx mice and that elimination of fibrin(ogen) attenuated dystrophy progression in mdx mice. These benefits appear to be tied to: (i) a decrease in leukocyte integrin α(M)ß(2)-mediated proinflammatory programs, thereby attenuating counterproductive inflammation and muscle degeneration; and (ii) a release of satellite cells from persistent inhibitory signals, thereby promoting regeneration. Remarkably, Fib-gamma(390-396A) (Fibγ(390-396A)) mice expressing a mutant form of fibrinogen with normal clotting function, but lacking the α(M)ß(2) binding motif, ameliorated dystrophic pathology. Delivery of a fibrinogen/α(M)ß(2) blocking peptide was similarly beneficial. Conversely, intramuscular fibrinogen delivery sufficed to induce inflammation and degeneration in fibrinogen-null mice. Thus, local fibrin(ogen) deposition drives dystrophic muscle inflammation and dysfunction, and disruption of fibrin(ogen)-α(M)ß(2) interactions may provide a novel strategy for DMD treatment.
Assuntos
Fibrina/metabolismo , Antígeno de Macrófago 1/metabolismo , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/terapia , Animais , Matriz Extracelular/metabolismo , Fibrinogênio/antagonistas & inibidores , Fibrinogênio/genética , Fibrinogênio/metabolismo , Fibrinogênio/farmacologia , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Inflamação/terapia , Leucócitos/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos mdx , Camundongos Knockout , Camundongos Mutantes , Modelos Biológicos , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Regeneração/fisiologia , Células Satélites de Músculo Esquelético/patologia , Células Satélites de Músculo Esquelético/fisiologiaRESUMO
Repair of damaged tissue requires the coordinated action of inflammatory and tissue-specific cells to restore homeostasis, but the underlying regulatory mechanisms are poorly understood. In this paper, we report new roles for MKP-1 (mitogen-activated protein kinase [MAPK] phosphatase-1) in controlling macrophage phenotypic transitions necessary for appropriate muscle stem cell-dependent tissue repair. By restricting p38 MAPK activation, MKP-1 allows the early pro- to antiinflammatory macrophage transition and the later progression into a macrophage exhaustion-like state characterized by cytokine silencing, thereby permitting resolution of inflammation as tissue fully recovers. p38 hyperactivation in macrophages lacking MKP-1 induced the expression of microRNA-21 (miR-21), which in turn reduced PTEN (phosphatase and tensin homologue) levels, thereby extending AKT activation. In the absence of MKP-1, p38-induced AKT activity anticipated the acquisition of the antiinflammatory gene program and final cytokine silencing in macrophages, resulting in impaired tissue healing. Such defects were reversed by temporally controlled p38 inhibition. Conversely, miR-21-AKT interference altered homeostasis during tissue repair. This novel regulatory mechanism involving the appropriate balance of p38, MKP-1, miR-21, and AKT activities may have implications in chronic inflammatory degenerative diseases.
Assuntos
Fosfatase 1 de Especificidade Dupla/fisiologia , Inflamação , Macrófagos/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Cicatrização , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Animais , Citocinas , Regulação da Expressão Gênica , Camundongos , MicroRNAs , PTEN Fosfo-HidrolaseRESUMO
Adult skeletal muscle is a very stable tissue containing a small population of myofiber-associated quiescent satellite cells compared with late embryonic/neonatal skeletal muscle, which contains highly proliferating myoblasts and small actively growing myofibers, suggesting that specific regulatory pathways may control myogenesis at distinct developmental stages. The p38 MAPK signaling pathway is central for myogenesis, based on studies using immortalized and neonatal primary myoblasts in vitro. However, the contribution of this pathway to adult myogenesis has never been investigated. Four p38 isoforms (p38alpha, p38beta, p38gamma and p38delta) exist in mammalian cells, being p38alpha and p38gamma the most abundantly expressed isoforms in adult skeletal muscle. Given the embryonic/neonatal lethality of p38alpha-deficient mice, here we investigate the relative contribution of p38beta, p38gamma and p38delta to adult myogenesis. Regeneration and myofiber growth of adult muscle proceeds with similar efficiency in mice lacking p38beta, p38gamma and p38delta as in wild-type control mice. In agreement with this, there is no difference in adult primary myoblasts behavior in vitro among the different genotypes. Importantly, the pattern of p38 activation (ascribed to p38alpha) remains unperturbed during satellite cell-mediated myogenesis in vitro and adult muscle regeneration in wild type and p38beta-, p38gamma- and p38delta-deficient mice, rendering p38alpha as the essential p38 isoform sustaining adult myogenesis. This study constitutes the first analysis addressing the functionality of p38beta, p38gamma and p38delta in satellite cell-dependent adult muscle regeneration and growth.
Assuntos
Músculo Esquelético/enzimologia , Músculo Esquelético/fisiologia , Regeneração , Proteínas Quinases p38 Ativadas por Mitógeno/deficiência , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Diferenciação Celular , Fusão Celular , Proliferação de Células , Células Cultivadas , Camundongos , Proteína Quinase 11 Ativada por Mitógeno/deficiência , Proteína Quinase 12 Ativada por Mitógeno/deficiência , Proteína Quinase 13 Ativada por Mitógeno/deficiência , Desenvolvimento Muscular , Mioblastos/citologia , Mioblastos/enzimologia , FenótipoRESUMO
In the fatal degenerative Duchenne muscular dystrophy (DMD), skeletal muscle is progressively replaced by fibrotic tissue. Here, we show that fibrinogen accumulates in dystrophic muscles of DMD patients and mdx mice. Genetic loss or pharmacological depletion of fibrinogen in these mice reduced fibrosis and dystrophy progression. Our results demonstrate that fibrinogen-Mac-1 receptor binding, through induction of IL-1beta, drives the synthesis of transforming growth factor-beta (TGFbeta) by mdx macrophages, which in turn induces collagen production in mdx fibroblasts. Fibrinogen-produced TGFbeta further amplifies collagen accumulation through activation of profibrotic alternatively activated macrophages. Fibrinogen, by engaging its alphavbeta3 receptor on fibroblasts, also directly promotes collagen synthesis. These data unveil a profibrotic role of fibrinogen deposition in muscle dystrophy.
Assuntos
Fibrinogênio/fisiologia , Ativação de Macrófagos/fisiologia , Distrofia Muscular de Duchenne/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Células Cultivadas , Criança , Pré-Escolar , Colágeno/metabolismo , Fibroblastos/metabolismo , Fibrose , Humanos , Integrina alfaVbeta3/metabolismo , Interleucina-1beta/metabolismo , Antígeno de Macrófago 1/metabolismo , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Animal/imunologia , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/imunologia , Distrofia Muscular de Duchenne/patologia , Ligação ProteicaRESUMO
The regulation of skeletal muscle formation (myogenesis) is essential for normal development as well as in pathological conditions such as muscular dystrophies and inflammatory myopathies. Findings published over the past years have established a key role for the p38 MAP kinase signaling pathway in the control of muscle gene expression and myotube formation. However, the relative contribution of the four p38 MAP kinases (p38alpha, p38beta, p38gamma and p38delta) to this process was unknown. We have recently demonstrated that myoblasts lacking p38alpha, but not those lacking p38beta or p38delta, were unable to differentiate and form multinucleated myotubes, while p38gamma-deficient myoblasts exhibited an attenuated fusion capacity. Defective myogenesis in the absence of p38alpha was attributed to delayed cell cycle exit and continuous proliferation in differentiation-promoting conditions, caused by enhanced activation of the JNK/cJun pathway. We discuss these findings in the context of the emerging crosstalk of p38 and JNK signaling pathways in controlling cell growth and differentiation.
Assuntos
Ciclo Celular/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Proteína Quinase 14 Ativada por Mitógeno/fisiologia , Mioblastos/citologia , Animais , Animais Recém-Nascidos , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Fusão Celular , Células Cultivadas/citologia , Ativação Enzimática , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Proteína Quinase 11 Ativada por Mitógeno/fisiologia , Proteína Quinase 12 Ativada por Mitógeno/fisiologia , Proteína Quinase 13 Ativada por Mitógeno/fisiologia , Proteína Quinase 14 Ativada por Mitógeno/deficiência , Proteína Quinase 14 Ativada por Mitógeno/genética , Fibras Musculares Esqueléticas/citologia , Proteínas Musculares/biossíntese , Proteínas Musculares/genética , Proteínas de Neoplasias/metabolismo , Especificidade de Órgãos , Fosforilação , Processamento de Proteína Pós-Traducional , Rabdomiossarcoma/patologiaRESUMO
The p38 mitogen-activated protein kinase (MAPK) pathway plays a critical role in skeletal muscle differentiation. However, the relative contribution of the four p38 MAPKs (p38alpha, p38beta, p38gamma and p38delta) to this process is unknown. Here we show that myoblasts lacking p38alpha, but not those lacking p38beta or p38delta, are unable to differentiate and form multinucleated myotubes, whereas p38gamma-deficient myoblasts exhibit an attenuated fusion capacity. The defective myogenesis in the absence of p38alpha is caused by delayed cell-cycle exit and continuous proliferation in differentiation-promoting conditions. Indeed, activation of JNK/cJun was enhanced in p38alpha-deficient myoblasts leading to increased cyclin D1 transcription, whereas inhibition of JNK activity rescued the proliferation phenotype. Thus, p38alpha controls myogenesis by antagonizing the activation of the JNK proliferation-promoting pathway, before its direct effect on muscle differentiation-specific gene transcription. More importantly, in agreement with the defective myogenesis of cultured p38alpha(Delta/Delta) myoblasts, neonatal muscle deficient in p38alpha shows cellular hyperproliferation and delayed maturation. This study provides novel evidence of a fundamental role of p38alpha in muscle formation in vitro and in vivo.