RESUMO
PURPOSE OF REVIEW: Nuclear factor-kappaB (NF-κB) activation is associated with a wide range of muscle-related diseases. Here, we review the evidence implicating specific NF-κB components in different disease pathologies and discuss therapies designed to target aberrant NF-κB signaling for the treatment of those pathologies. RECENT FINDINGS: Many components of the NF-κB signaling pathway contribute to muscle pathologies, presumably through activation of the transcription factor. In addition, an increasing number of upstream factors have been connected to disease progression. Genetic models and therapeutic approaches affecting these upstream targets associate with ameliorating disease progression. SUMMARY: Dissecting the crosstalk between NF-κB, its upstream mediators, and other signaling pathways is vital to our understanding of how activation of this signaling pathway is mediated in various diseases. The strides made in therapeutically inhibiting the NF-κB pathway provide some promise for the treatment of these diseases.
Assuntos
Músculo Esquelético/patologia , Doenças Musculares/patologia , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Animais , Caquexia/genética , Caquexia/patologia , Modelos Animais de Doenças , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Modelos Genéticos , Músculo Esquelético/metabolismo , Doenças Musculares/genética , Doenças Musculares/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Fator de Transcrição RelA/genética , Fatores de Transcrição , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Mitochondrial DNA (mtDNA) depletion/deletions syndromes (MDDS) encompass a clinically and etiologically heterogenous group of mitochondrial disorders caused by impaired mtDNA maintenance. Among the most frequent causes of MDDS are defects in nucleoside/nucleotide metabolism, which is critical for synthesis and homeostasis of the deoxynucleoside triphosphate (dNTP) substrates of mtDNA replication. A central enzyme for generating dNTPs is ribonucleotide reductase, a critical mediator of de novo nucleotide synthesis composed of catalytic RRM1 subunits in complex with RRM2 or p53R2. Here, we report 5 probands from 4 families who presented with ptosis and ophthalmoplegia as well as other clinical manifestations and multiple mtDNA deletions in muscle. We identified 3 RRM1 loss-of-function variants, including a dominant catalytic site variant (NP_001024.1: p.N427K) and 2 homozygous recessive variants at p.R381, which has evolutionarily conserved interactions with the specificity site. Atomistic molecular dynamics simulations indicate mechanisms by which RRM1 variants affect protein structure. Cultured primary skin fibroblasts of probands manifested mtDNA depletion under cycling conditions, indicating impaired de novo nucleotide synthesis. Fibroblasts also exhibited aberrant nucleoside diphosphate and dNTP pools and mtDNA ribonucleotide incorporation. Our data reveal that primary RRM1 deficiency and, by extension, impaired de novo nucleotide synthesis are causes of MDDS.
Assuntos
Doenças Mitocondriais , Ribonucleotídeo Redutases , Replicação do DNA , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Humanos , Doenças Mitocondriais/genética , Nucleosídeos , Nucleotídeos/genética , Ribonucleosídeo Difosfato Redutase/genética , Ribonucleosídeo Difosfato Redutase/metabolismo , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismoRESUMO
BACKGROUND: The activation of p38 mitogen-activated protein kinases (MAPK) is implicated in cold ischemia-reperfusion injury of donor organs. The islet isolation process, from pancreas procurement through islet collection, may activate p38MAPK leading to cytokine release and islet damage. This damage may be prevented by treating pancreata with a p38MAPK inhibitor (p38IH) before cold preservation. METHODS: Pancreata removed from Beagle dogs were infused with University of Wisconsin solution containing the p38IH, SB203580, and Pefabloc (n=6) or vehicle (dimethyl sulfoxide and Pefabloc) alone (n=7), through the pancreatic duct and preserved using the two-layer method. After 20 to 22 hr, islets were isolated and 3000 IEQ/kg were autotransplanted into the corresponding dog to monitor glucose metabolism. RESULTS: p38IH-treated pancreata yielded significantly more islets than control pancreata (IEQ/g: 2134+/-297 vs. 1477+/-145 IEQ/g or 65,012+/-9385 vs. 45,700+/-5103 IEQ/pancreas; P<0.05). Apoptotic beta-cell percentages assessed by laser scanning cytometry were lower in p38IH-treated than the controls (44%+/-9.4% vs. 61.6%+/-4.8%, P<0.05). Tumor necrosis factor-alpha expression assessed by real-time reverse transcription polymerase chain reaction was significantly lower in the p38IH-treated group than controls. All dogs (3000 IEQ/kg) transplanted with p38IH-treated islets (n=5) became euglycemic versus four of five dogs that received untreated islets. Plasma C-peptide levels after glucagon challenge were higher in animals receiving p38IH-treated islets (n=5) versus untreated islets (n=4) (0.40+/-0.78 vs. 0.21+/-0.05 ng/mL, P<0.05). CONCLUSIONS: Infusion of pancreata with University of Wisconsin solution containing p38IH through the duct before preservation suppresses cytokine release, prevents beta-cell apoptosis, and improves islet yield significantly with no adverse effect on islet function after transplantation. p38IH treatment of human pancreata may improve islet yield for use in clinical transplantation.
Assuntos
Apoptose , Inibidores Enzimáticos/farmacologia , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/patologia , Preservação de Órgãos/métodos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adenosina/uso terapêutico , Alopurinol/uso terapêutico , Animais , Citocinas/metabolismo , Cães , Ativação Enzimática , Glutationa/uso terapêutico , Insulina/uso terapêutico , Células Secretoras de Insulina/patologia , Masculino , Soluções para Preservação de Órgãos/uso terapêutico , Rafinose/uso terapêutico , Traumatismo por ReperfusãoRESUMO
Duchenne muscular dystrophy (DMD) is a neuromuscular disorder causing progressive muscle degeneration. Although cardiomyopathy is a leading mortality cause in DMD patients, the mechanisms underlying heart failure are not well understood. Previously, we showed that NF-κB exacerbates DMD skeletal muscle pathology by promoting inflammation and impairing new muscle growth. Here, we show that NF-κB is activated in murine dystrophic (mdx) hearts, and that cardiomyocyte ablation of NF-κB rescues cardiac function. This physiological improvement is associated with a signature of upregulated calcium genes, coinciding with global enrichment of permissive H3K27 acetylation chromatin marks and depletion of the transcriptional repressors CCCTC-binding factor, SIN3 transcription regulator family member A, and histone deacetylase 1. In this respect, in DMD hearts, NF-κB acts differently from its established role as a transcriptional activator, instead promoting global changes in the chromatin landscape to regulate calcium genes and cardiac function.
Assuntos
Distrofia Muscular de Duchenne/metabolismo , Miócitos Cardíacos/metabolismo , NF-kappa B/metabolismo , Animais , Fator de Ligação a CCCTC/metabolismo , Cálcio/metabolismo , Células Cultivadas , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/fisiologia , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Complexo Correpressor Histona Desacetilase e Sin3 , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismoRESUMO
Mitochondrial function is a key component of skeletal muscle health, and its dysfunction has been associated with a wide variety of diseases. Microplate-based respirometry measures aerobic respiration of live cells through extracellular changes in oxygen concentration. Here, we describe a methodology to measure aerobic respiration of intact murine skeletal muscle tissue. The tissues are not cultured, permeabilized, or enzymatically dissociated to single fibers, so there is minimal experimental manipulation affecting the samples prior to acquiring measurements.
Assuntos
Respiração Celular/fisiologia , Músculo Esquelético/fisiologia , Animais , Metabolismo Energético , Camundongos , Mitocôndrias Musculares/metabolismo , Consumo de OxigênioRESUMO
Skeletal muscle growth immediately following birth is critical for proper body posture and locomotion. However, compared with embryogenesis and adulthood, the processes regulating the maturation of neonatal muscles is considerably less clear. Studies in the 1960s predicted that neonatal muscle growth results from nuclear accretion of myoblasts preferentially at the tips of myofibers. Remarkably, little information has been added since then to resolve how myoblasts migrate to the ends of fibers. Here, we provide insight into this process by revealing a unique NF-κB-dependent communication between NG2(+) interstitial cells and myoblasts. NF-κB in NG2(+) cells promotes myoblast migration to the tips of myofibers through cell-cell contact. This occurs through expression of ephrinA5 from NG2(+) cells, which we further deduce is an NF-κB target gene. Together, these results suggest that NF-κB plays an important role in the development of newborn muscles to ensure proper myoblast migration for fiber growth.
Assuntos
Antígenos/metabolismo , Diferenciação Celular/fisiologia , Efrina-A5/metabolismo , Desenvolvimento Muscular/fisiologia , Mioblastos/metabolismo , NF-kappa B/metabolismo , Proteoglicanas/metabolismo , Animais , Animais Recém-Nascidos , Movimento Celular/fisiologia , Masculino , Camundongos Knockout , Camundongos Transgênicos , Músculo Esquelético/metabolismoRESUMO
MyoD is a key regulator of skeletal myogenesis that directs contractile protein synthesis, but whether this transcription factor also regulates skeletal muscle metabolism has not been explored. In a genome-wide ChIP-seq analysis of skeletal muscle cells, we unexpectedly observed that MyoD directly binds to numerous metabolic genes, including those associated with mitochondrial biogenesis, fatty acid oxidation, and the electron transport chain. Results in cultured cells and adult skeletal muscle confirmed that MyoD regulates oxidative metabolism through multiple transcriptional targets, including PGC-1ß, a master regulator of mitochondrial biogenesis. We find that PGC-1ß expression is cooperatively regulated by MyoD and the alternative NF-κB signaling pathway. Bioinformatics evidence suggests that this cooperativity between MyoD and NF-κB extends to other metabolic genes as well. Together, these data identify MyoD as a regulator of the metabolic capacity of mature skeletal muscle to ensure that sufficient energy is available to support muscle contraction.
Assuntos
Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Proteína MyoD/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Animais , Camundongos , Mitocôndrias/genética , Contração Muscular/genética , Desenvolvimento Muscular/genética , Proteína MyoD/metabolismo , Mioblastos/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Oxirredução , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Ligação Proteica , Transdução de Sinais , Fator de Transcrição RelB/genética , Fator de Transcrição RelB/metabolismoRESUMO
OBJECTIVES: Allogeneic mesenchymal stem cells (MSCs) and bone marrow cells (BMCs) were cotransplanted in nonobese diabetic mice after none myeloablative preconditioning and the development of chimerism, insulitis, diabetes, and graft-versus-host disease (GVHD) were monitored. METHODS: Eight-week-old female nonobese diabetic mice were injected intravenously with 2 × 10 BMCs and 5 × 10 MSCs from C57BL/6 mice after treatment with 2 intraperitoneal injections of anti-CD3 antibody (days -7 and -4) and 3-Gy total body irradiation (day -1). Thereafter, blood glucose and chimerism were monitored on peripheral blood samples. RESULTS: Stable mixed chimerism (3->90% of donor phenotype) was induced in 63.2% of BMCs-MSCs recipients (n = 19) and 45.0% of BMCs-alone recipients (n = 20, P = 0.256). Insulitis was prevented, and euglycemia persisted for more than 18 weeks in 89.5% of BMCs-MSCs recipients including those with less than 3% chimerism and 55% of BM-alone recipients (P < 0.05). In controls, 9.1% of mice receiving preconditioning treatment alone (n = 11) and 16.7% of preconditioned mice receiving only MSCs (n = 12) were nondiabetic. Graft-versus-host disease was not detected in all mice. CONCLUSIONS: Coinjection of MSCs and BMCs increased the success rate in inducing chimerism and preventing insulitis and overt diabetes with no incidence of GVHD. Results also indicated that even microchimerism with less than 3% donor cells is sufficient for blocking autoimmunity.
Assuntos
Diabetes Mellitus Tipo 1/prevenção & controle , Transplante de Células-Tronco Mesenquimais , Quimeras de Transplante , Animais , Transplante de Medula Óssea/imunologia , Feminino , Doença Enxerto-Hospedeiro/prevenção & controle , Tolerância Imunológica , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Endogâmicos NOD , Quimeras de Transplante/imunologia , Condicionamento Pré-Transplante , Transplante HomólogoRESUMO
BACKGROUND: Bone marrow-derived mesenchymal stem cells (MSCs) are known to produce vascular endothelial growth factor. We hypothesize that co-transplantation of MSCs and islets promotes revascularization and improves islet graft function. METHODS: Lewis rat islets were infused into the liver of streptozotocin-diabetic syngeneic recipients or transplanted under the renal capsule of nonobese diabetic severe combined immunodeficiency (NOD SCID) mice with MSCs isolated from Lewis bone marrow and expanded in culture. RESULTS: Co-transplantation of 500 islets and 107 MSCs (islet-MSCs) reversed diabetes in all eight recipients, whereas islet-alone transplantation achieved euglycemia in 3 of 10 recipients. With 300 islets, five of nine islet-MSCs and 1 of 10 islets-alone recipients reversed diabetes. Results of intravenous glucose tolerance tests performed on day 56 were significantly better in islet-MSCs than islet-alone recipients. One week after transplantation, well-preserved islet structure and higher number of capillaries were found in the liver of islet-MSCs recipients, whereas islet-alone grafts were fragmented with very few capillaries. Islets showed a similar morphology when transplanted with MSCs in nonobese diabetic severe combined immunodeficiency mice with a significantly higher capillary per [beta]-cell ratio than that in islet-alone grafts (0.135+/-0.046 vs. 0.052+/-0.028 capillary segments per [beta]-cell, P<0.01). One week after transplantation, islets were surrounded by MSCs labeled with carboxyfluorescein succinimidyl ester or Qdot nanocrystals, and some labeled MSCs positively stained for vascular endothelial growth factor or von Willebrand factor. CONCLUSION: Our results demonstrate the improvement of islet graft morphology and function by co-transplantation with MSCs. This improvement is attributable, at least in part, to the promotion of graft revascularization mediated by MSCs.
Assuntos
Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/citologia , Células-Tronco Mesenquimais/citologia , Animais , Separação Celular , Citometria de Fluxo , Teste de Tolerância a Glucose , Sobrevivência de Enxerto , Imuno-Histoquímica/métodos , Camundongos , Camundongos SCID , Neovascularização Fisiológica , Ratos , Ratos Endogâmicos Lew , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator de von Willebrand/metabolismoRESUMO
OBJECTIVES: We investigated whether the recovery of cultured human islets is improved through the addition of a p38alpha-selective mitogen-activated protein kinase inhibitor, SD-282, to clinically used serum-free culture medium. METHODS: Immediately after isolation, islets were cultured for 24 hours in medium alone (control) or medium containing dimethyl sulfoxide, 0.1 microM SD-282, or 0.3 microM SD-282. Cytokine expression, apoptotic beta-cell percentage, and islet function were assessed postculture. RESULTS: Expression of p38 and phosphorylated p38 in islets increased during culture. Interleukin 6 mRNA expression in cultured islets, as well as IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor released into the medium, was significantly reduced by adding SD-282. The apoptotic beta-cell percentage was significantly lower in islets cultured with 0.1 microM SD-282, but not 0.3 microM, as compared with the control. Stimulation indices measured in vitro were higher but without significance (P = 0.06); the function of transplanted islets in diabetic NOD-scid mice was also better in 0.1-microM SD-282 group as compared with control. CONCLUSIONS: Better islet function was obtained by adding 0.1 microM SD-282 to the serum-free culture medium. This improvement was associated with suppression of cytokine production and prevention of beta-cell apoptosis. However, this beneficial effect was diminished at a higher concentration.