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1.
Ultrasonics ; 110: 106243, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32961400

RESUMO

Skeletal muscle is an important secretory organ in mammals, producing myriad chemical mediators ("myokines") with distinct biological action in different tissues, including anti-inflammatory activity. Extracellular vesicles (EVs) have recently been identified as a mode of myokine transport from muscle, facilitating such anti-inflammatory activity. In this report, we have demonstrated that high-intensity ultrasound (US) strongly induces EV secretion from cultured myotubes without a reduction in cell viability. High-intensity US of 3.0 W/cm2 with 20% duty cycle increased the number of EVs by 2-fold compared to control at 6 h. This effect was specific to EVs in the 100-150 nm size range. Thus, high-intensity US is a novel modality for inducing myocellular EV release and may hold therapeutic value.


Assuntos
Vesículas Extracelulares/metabolismo , Tratamento por Ondas de Choque Extracorpóreas/métodos , Fibras Musculares Esqueléticas/metabolismo , Animais , Células Cultivadas , Camundongos
2.
PLoS One ; 15(9): e0239047, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32941492

RESUMO

Muscle aging is accompanied by blunted muscle regeneration in response to injury and disuse. Oxidative stress likely underlies this diminished response, but muscle redox sensors that act in regeneration have not yet been characterized. Calmodulin contains multiple redox sensitive methionines whose oxidation alters the regulation of numerous cellular targets. We have used the CRISPR-Cas9 system to introduce a single amino acid substitution M109Q that mimics oxidation of methionine to methionine sulfoxide in one or both alleles of the CALM1 gene, one of three genes encoding the muscle regulatory protein calmodulin, in C2C12 mouse myoblasts. When signaled to undergo myogenesis, mutated myoblasts failed to differentiate into myotubes. Although early myogenic regulatory factors were present, cells with the CALM1 M109Q mutation in one or both alleles were unable to withdraw from the cell cycle and failed to express late myogenic factors. We have shown that a single oxidative modification to a redox-sensitive muscle regulatory protein can halt myogenesis, suggesting a molecular target for mitigating the impact of oxidative stress in age-related muscle degeneration.


Assuntos
Calmodulina/metabolismo , Desenvolvimento Muscular/fisiologia , Animais , Calmodulina/genética , Calmodulina/fisiologia , Diferenciação Celular/fisiologia , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Mioblastos/metabolismo , Oxirredução , Estresse Oxidativo/fisiologia
3.
PLoS One ; 15(9): e0238441, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32881965

RESUMO

Limb-girdle muscular dystrophy type 2B (LGMD2B) is caused by mutations in the dysferlin gene, resulting in non-functional dysferlin, a key protein found in muscle membrane. Treatment options available for patients are chiefly palliative in nature and focus on maintaining ambulation. Our hypothesis is that galectin-1 (Gal-1), a soluble carbohydrate binding protein, increases membrane repair capacity and myogenic potential of dysferlin-deficient muscle cells and muscle fibers. To test this hypothesis, we used recombinant human galectin-1 (rHsGal-1) to treat dysferlin-deficient models. We show that rHsGal-1 treatments of 48 h-72 h promotes myogenic maturation as indicated through improvements in size, myotube alignment, myoblast migration, and membrane repair capacity in dysferlin-deficient myotubes and myofibers. Furthermore, increased membrane repair capacity of dysferlin-deficient myotubes, independent of increased myogenic maturation is apparent and co-localizes on the membrane of myotubes after a brief 10min treatment with labeled rHsGal-1. We show the carbohydrate recognition domain of Gal-1 is necessary for observed membrane repair. Improvements in membrane repair after only a 10 min rHsGal-1treatment suggest mechanical stabilization of the membrane due to interaction with glycosylated membrane bound, ECM or yet to be identified ligands through the CDR domain of Gal-1. rHsGal-1 shows calcium-independent membrane repair in dysferlin-deficient and wild-type myotubes and myofibers. Together our novel results reveal Gal-1 mediates disease pathologies through both changes in integral myogenic protein expression and mechanical membrane stabilization.


Assuntos
Disferlina/genética , Galectina 1/farmacologia , Distrofia Muscular do Cíngulo dos Membros/terapia , Animais , Linhagem Celular , Modelos Animais de Doenças , Disferlina/metabolismo , Galectina 1/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Membranas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Miofibrilas/metabolismo
4.
PLoS Negl Trop Dis ; 14(8): e0008282, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32817655

RESUMO

Muscle cells are potential targets of many arboviruses, such as Ross River, Dengue, Sindbis, and chikungunya viruses, that may be involved in the physiopathological course of the infection. During the recent outbreak of Zika virus (ZIKV), myalgia was one of the most frequently reported symptoms. We investigated the susceptibility of human muscle cells to ZIKV infection. Using an in vitro model of human primary myoblasts that can be differentiated into myotubes, we found that myoblasts can be productively infected by ZIKV. In contrast, myotubes were shown to be resistant to ZIKV infection, suggesting a differentiation-dependent susceptibility. Infection was accompanied by a caspase-independent cytopathic effect, associated with paraptosis-like cytoplasmic vacuolization. Proteomic profiling was performed 24h and 48h post-infection in cells infected with two different isolates. Proteome changes indicate that ZIKV infection induces an upregulation of proteins involved in the activation of the Interferon type I pathway, and a downregulation of protein synthesis. This work constitutes the first observation of primary human muscle cells susceptibility to ZIKV infection, and differentiation-dependent restriction of infection from myoblasts to myotubes. Since myoblasts constitute the reservoir of stem cells involved in reparation/regeneration in muscle tissue, the infection of muscle cells and the viral-induced alterations observed here could have consequences in ZIKV infection pathogenesis.


Assuntos
Diferenciação Celular , Células Musculares/metabolismo , Células Musculares/virologia , Proteômica , Infecção por Zika virus , Morte Celular , Linhagem Celular , Efeito Citopatogênico Viral , Suscetibilidade a Doenças , Interações Hospedeiro-Patógeno , Humanos , Interferon Tipo I/metabolismo , Células Musculares/patologia , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/virologia , Mioblastos/metabolismo , Mioblastos/virologia , Proteínas/metabolismo , Células-Tronco , Replicação Viral , Zika virus/patogenicidade , Infecção por Zika virus/patologia , Infecção por Zika virus/virologia
5.
PLoS One ; 15(8): e0237095, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32756599

RESUMO

Regular exercise is an effective strategy that is used to prevent and treat obesity as well as type 2 diabetes. Exercise-induced myokine secretion is considered a mechanism that coordinates communication between muscles and other organs. In order to examine the possibility of novel communications from muscle to adipose tissue mediated by myokines, we treated 3T3-L1 adipocytes with C2C12 myotube electrical pulse stimulation-conditioned media (EPS-CM), using a C2C12 myotube contraction system stimulated by an electrical pulse. Continuous treatment with myotube EPS-CM promoted adipogenesis of 3T3-L1 pre-adipocytes via the upregulation of the peroxisome proliferator-activated receptor-gamma (PPARγ) 2 and PPARγ-regulated gene expression. Furthermore, our results revealed that myotube EPS-CM induces lipolysis and secretion of adiponectin in mature adipocytes. EPS-CM obtained from a C2C12 myoblast culture did not induce such changes in these genes, suggesting that contraction-induced myokine(s) secretion occurs particularly in differentiated myotubes. Thus, contraction-induced secretion of myokine(s) promotes adipogenesis and lipid metabolism in 3T3-L1 adipocytes. These findings suggest the possibility that skeletal muscle communicates to adipose tissues during exercise, probably by the intermediary of unidentified myokines.


Assuntos
Adipócitos/citologia , Diferenciação Celular , Lipólise , Fibras Musculares Esqueléticas/metabolismo , Células 3T3 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia , Adiponectina/metabolismo , Animais , Comunicação Celular , Meios de Cultivo Condicionados/farmacologia , Camundongos , PPAR gama/metabolismo
6.
Nucleic Acids Res ; 48(16): 8927-8942, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32692361

RESUMO

The differentiation and regeneration of skeletal muscle from myoblasts to myotubes involves myogenic transcription factors, such as myocardin-related transcription factor A (MRTF-A) and serum response factor (SRF). In addition, post-transcriptional regulation by miRNAs is required during myogenesis. Here, we provide evidence for novel mechanisms regulating MRTF-A during myogenic differentiation. Endogenous MRTF-A protein abundance and activity decreased during C2C12 differentiation, which was attributable to miRNA-directed inhibition. Conversely, overexpression of MRTF-A impaired differentiation and myosin expression. Applying miRNA trapping by RNA affinity purification (miTRAP), we identified miRNAs which directly regulate MRTF-A via its 3'UTR, including miR-1a-3p, miR-206-3p, miR-24-3p and miR-486-5p. These miRNAs were upregulated during differentiation and specifically recruited to the 3'UTR of MRTF-A. Concomitantly, Ago2 recruitment to the MRTF-A 3'UTR was considerably increased, whereas Dicer1 depletion or 3'UTR deletion elevated MRTF-A and inhibited differentiation. MRTF-A protein expression was inhibited by ectopic miRNA expression in murine C2C12 and primary human myoblasts. 3'UTR reporter activity diminished upon differentiation or miRNA expression, whereas deletion of the predicted binding sites reversed these effects. Furthermore, TGF-ß abolished MRTF-A reduction and decreased miR-486-5p expression. Our findings implicate miR-24-3p and miR-486-5p in the repression of MRTF-A and suggest a complex network of transcriptional and post-transcriptional mechanisms regulating myogenesis.


Assuntos
MicroRNAs/metabolismo , Desenvolvimento Muscular , Mioblastos Esqueléticos/citologia , Transativadores/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Chlorocebus aethiops , Humanos , Camundongos , Fibras Musculares Esqueléticas/metabolismo
7.
Am J Pathol ; 190(10): 2039-2055, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32650005

RESUMO

This study investigated intercellular adhesion molecule-1 (ICAM-1), a membrane protein that mediates cell-to-cell adhesion and communication, as a mechanism through which the inflammatory response facilitates muscle regeneration after injury. Toxin-induced muscle injury to tibialis anterior muscles of wild-type mice caused ICAM-1 to be expressed by a population of satellite cells/myoblasts and myofibers. Myogenic cell expression of ICAM-1 contributed to the restoration of muscle structure after injury, as regenerating myofibers were more abundant and myofiber size was larger for wild-type compared with Icam1-/- mice during 28 days of recovery. Contrastingly, restoration of muscle function after injury was similar between the genotypes. ICAM-1 facilitated the restoration of muscle structure after injury through mechanisms involving the regulation of myofiber branching, protein synthesis, and the organization of nuclei within myofibers after myogenic cell fusion. These findings provide support for a paradigm in which ICAM-1 expressed by myogenic cells after muscle injury augments their adhesive and fusogenic properties, which, in turn, facilitates regenerative and hypertrophic processes that restore structure to injured muscle.


Assuntos
Adesão Celular/fisiologia , Molécula 1 de Adesão Intercelular/metabolismo , Desenvolvimento Muscular/fisiologia , Células Satélites de Músculo Esquelético/metabolismo , Animais , Comunicação Celular/fisiologia , Feminino , Hipertrofia/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Regeneração/genética
8.
Am J Physiol Cell Physiol ; 319(2): C432-C440, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32608991

RESUMO

microRNAs (miRNAs) are important regulators of cellular homeostasis and exert their effect by directly controlling protein expression. We have previously reported an age-dependent negative association between microRNA-99b (miR-99b-5p) expression and muscle protein synthesis in human muscle in vivo. Here we investigated the role of miR-99b-5p as a potential negative regulator of protein synthesis via inhibition of mammalian target for rapamycin (MTOR) signaling in human primary myocytes. Overexpressing miR-99b-5p in human primary myotubes from young and old subjects significantly decreased protein synthesis with no effect of donor age. A binding interaction between miR-99b-5p and its putative binding site within the MTOR 3'-untranslated region (UTR) was confirmed in C2C12 myoblasts. The observed decline in protein synthesis was, however, not associated with a suppression of the MTOR protein but of its regulatory associated protein of mTOR complex 1 (RPTOR). These results demonstrate that modulating the expression levels of a miRNA can regulate protein synthesis in human muscle cells and provide a potential mechanism for muscle wasting in vivo.


Assuntos
MicroRNAs/genética , Fibras Musculares Esqueléticas/metabolismo , Biossíntese de Proteínas/genética , Serina-Treonina Quinases TOR/genética , Regiões 3' não Traduzidas/genética , Animais , Proliferação de Células/genética , Regulação da Expressão Gênica/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Mioblastos/metabolismo , Transdução de Sinais/genética
9.
Am J Physiol Cell Physiol ; 319(3): C541-C551, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32697599

RESUMO

Lin28a/miRNA let-7b-5p pathway has emerged as a key regulators of energy homeostasis in the skeletal muscle. However, the mechanism through which this pathway is regulated in the skeletal muscle has remained unclear. We have found that 8 wk of aerobic training (Tr) markedly decreased let-7b-5p expression in murine skeletal muscle, whereas high-fat diet (Hfd) increased its expression. Conversely, Lin28a expression, a well-known inhibitor of let-7b-5p, was induced by Tr and decreased by Hfd. Similarly, in human muscle biopsies, Tr increased LIN28 expression and decreased let-7b-5p expression. Bioinformatics analysis of LIN28a DNA sequence revealed that its enrichment in peroxisome proliferator-activated receptor delta (PPARδ) binding sites, which is a well-known metabolic regulator of exercise. Treatment of primary mouse skeletal muscle cells or C2C12 cells with PPARδ activators GW501516 and AICAR increased Lin28a expression. Lin28a and let-7b-5p expression was also regulated by PPARδ coregulators. While PPARγ coactivator-1α (PGC1α) increased Lin28a expression, corepressor NCoR1 decreased its expression. Furthermore, PGC1α markedly reduced the let-7b-5p expression. PGC1α-mediated induction of Lin28a expression was blocked by the PPARδ inhibitor GSK0660. In agreement, Lin28a expression was downregulated in PPARδ knocked-down cells leading to increased let-7b-5p expression. Finally, we show that modulation of the Lin28a-let-7b-5p pathway in muscle cells leads to changes in mitochondrial metabolism in PGC1α dependent fashion. In summary, we demonstrate that Lin28a-let-7b-5p is a direct target of PPARδ in the skeletal muscle, where it impacts mitochondrial respiration.


Assuntos
Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , PPAR delta/metabolismo , Proteínas de Ligação a RNA/genética , Animais , Linhagem Celular , Regulação para Baixo , Camundongos , Fibras Musculares Esqueléticas/metabolismo , PPAR delta/genética
10.
Anim Sci J ; 91(1): e13425, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32691493

RESUMO

The difference of muscle fiber type composition affects several parameters related to meat quality; however, the relationship between muscle fiber types and meat taste is unclear. To elucidate this relationship, we determined the taste of various beef samples using a taste sensor (INSENT SA402B) and analyzed its correlation with different muscle fiber type composition. We used 22 kinds of beef samples and measured nine tastes, including the relative and change of membrane potential caused by adsorption (CPA) values, using six sensors (GL1, CT0, CA0, AAE, C00, and AE1). The taste sensor analysis indicated positive value outputs for the relative C00, AAE, and GL1 values as well as for the CPA value of AAE, which corresponded to bitterness, umami, sweetness, and richness, respectively. We found significant positive correlations of the myosin heavy chain 1 (MyHC1) composition with umami taste, and with richness. This result suggests that high levels of slow MyHC1 can induce strong umami taste and richness in beef. We expect that our results will contribute to the elucidation of the relationship between muscle fiber types and meat palatability.


Assuntos
Análise de Alimentos/instrumentação , Qualidade dos Alimentos , Fibras Musculares Esqueléticas/classificação , Cadeias Pesadas de Miosina/análise , Carne Vermelha/análise , Paladar , Animais , Bovinos , Potenciais da Membrana , Fibras Musculares Esqueléticas/metabolismo
11.
Nat Commun ; 11(1): 2695, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483258

RESUMO

Obesity and type 2 diabetes (T2D) are metabolic disorders influenced by lifestyle and genetic factors that are characterized by insulin resistance in skeletal muscle, a prominent site of glucose disposal. Numerous genetic variants have been associated with obesity and T2D, of which the majority are located in non-coding DNA regions. This suggests that most variants mediate their effect by altering the activity of gene-regulatory elements, including enhancers. Here, we map skeletal muscle genomic enhancer elements that are dynamically regulated after exposure to the free fatty acid palmitate or the inflammatory cytokine TNFα. By overlapping enhancer positions with the location of disease-associated genetic variants, and resolving long-range chromatin interactions between enhancers and gene promoters, we identify target genes involved in metabolic dysfunction in skeletal muscle. The majority of these genes also associate with altered whole-body metabolic phenotypes in the murine BXD genetic reference population. Thus, our combined genomic investigations identified genes that are involved in skeletal muscle metabolism.


Assuntos
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Elementos Facilitadores Genéticos , Resistência à Insulina/genética , Músculo Esquelético/metabolismo , Obesidade/genética , Obesidade/metabolismo , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Diabetes Mellitus Tipo 2/patologia , Feminino , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Masculino , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Obesidade/patologia , Ácido Palmítico/farmacologia , Fatores de Iniciação de Peptídeos/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Fator de Necrose Tumoral alfa/farmacologia
12.
PLoS Biol ; 18(6): e3000731, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32479501

RESUMO

The nuclear lamina protein lamin A/C is a key component of the nuclear envelope. Mutations in the lamin A/C gene (LMNA) are identified in patients with various types of laminopathy-containing diseases, which have features of accelerated aging and osteoporosis. However, the underlying mechanisms for laminopathy-associated osteoporosis remain largely unclear. Here, we provide evidence that loss of lamin A/C in skeletal muscles, but not osteoblast (OB)-lineage cells, results in not only muscle aging-like deficit but also trabecular bone loss, a feature of osteoporosis. The latter is due in large part to elevated bone resorption. Further cellular studies show an increase of osteoclast (OC) differentiation in cocultures of bone marrow macrophages/monocytes (BMMs) and OBs after treatment with the conditioned medium (CM) from lamin A/C-deficient muscle cells. Antibody array screening analysis of the CM proteins identifies interleukin (IL)-6, whose expression is markedly increased in lamin A/C-deficient muscles. Inhibition of IL-6 by its blocking antibody in BMM-OB cocultures diminishes the increase of osteoclastogenesis. Knockout (KO) of IL-6 in muscle lamin A/C-KO mice diminishes the deficits in trabecular bone mass but not muscle. Further mechanistic studies reveal an elevation of cellular senescence marked by senescence-associated beta-galactosidase (SA-ß-gal), p16Ink4a, and p53 in lamin A/C-deficient muscles and C2C12 muscle cells, and the p16Ink4a may induce senescence-associated secretory phenotype (SASP) and IL-6 expression. Taken together, these results suggest a critical role for skeletal muscle lamin A/C to prevent cellular senescence, IL-6 expression, hyperosteoclastogenesis, and trabecular bone loss, uncovering a pathological mechanism underlying the link between muscle aging/senescence and osteoporosis.


Assuntos
Envelhecimento/patologia , Lamina Tipo A/deficiência , Músculo Esquelético/patologia , Osteoporose/patologia , Animais , Anticorpos Bloqueadores/farmacologia , Fenômenos Biomecânicos , Reabsorção Óssea/complicações , Reabsorção Óssea/patologia , Osso Esponjoso/efeitos dos fármacos , Osso Esponjoso/patologia , Diferenciação Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Interleucina-6/metabolismo , Camundongos Knockout , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Osteogênese/efeitos dos fármacos , Osteoporose/sangue , Fenótipo
13.
Gene ; 752: 144765, 2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32413480

RESUMO

The natural flight response in shrimp is powered by rapid contractions of the abdominal muscle fibres to propel themselves backwards away from perceived danger. This muscle contraction is dependent on repetitive depolarization of muscle plasma membrane, triggering tightly spaced cytoplasmic [Ca2+] transients and rapidly rising tetanic force responses. To achieve such high amplitude and high frequency of Ca2+ transients requires a high abundance of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) to rapidly clear cytoplasmic Ca2+ between each transient and an efficient Ca2+ release system consisting of the Ryanodine Receptor (RyR), and voltage gated Ca2+ channels (CaVs). With the aim to expand our knowledge of muscle gene function and identify orthologous genes regulating muscle excitation-contraction (EC) coupling, this study assembled nine Penaeid shrimp muscle transcriptomes. On average, the nine transcriptomes contained 27,000 contigs, with an annotation rate of 36% and a BUSCO completeness of 70%. Despite maintaining their function, the crustacean RyR and CaV proteins showed evidence of significant diversification from mammalian orthologs, while SERCA remained more conserved. Several key components of protein interaction were conserved, while others showed distinct crustacean specific evolutionary adaptations. Lastly, this study revealed approximately 1,000 orthologous genes involved in muscle specific processes present across all nine species.


Assuntos
Acoplamento Excitação-Contração/genética , Penaeidae/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Animais , Evolução Biológica , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/fisiologia , Citosol/metabolismo , Evolução Molecular , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Especificidade da Espécie , Transcriptoma/genética
14.
Am J Physiol Endocrinol Metab ; 319(1): E187-E195, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32396388

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic fat accumulation and impaired insulin sensitivity. Reduced hepatic ketogenesis may promote these pathologies, but data are inconclusive in humans and the link between NAFLD and reduced insulin sensitivity remains obscure. We investigated individuals with obesity-related NAFLD and hypothesized that ß-hydroxybutyrate (ßOHB; the predominant ketone species) would be reduced and related to hepatic fat accumulation and insulin sensitivity. Furthermore, we hypothesized that ketones would impact skeletal muscle mitochondrial respiration in vitro. Hepatic fat was assessed by 1H-MRS in 22 participants in a parallel design, case control study [Control: n = 7, age 50 ± 6 yr, body mass index (BMI) 30 ± 1 kg/m2; NAFLD: n = 15, age 57 ± 3 yr, BMI 35 ± 1 kg/m2]. Plasma assessments were conducted in the fasted state. Whole body insulin sensitivity was determined by the gold-standard hyperinsulinemic-euglycemic clamp. The effect of ketone dose (0.5-5.0 mM) on mitochondrial respiration was conducted in human skeletal muscle cell culture. Fasting ßOHB, a surrogate measure of hepatic ketogenesis, was reduced in NAFLD (-15.6%, P < 0.01) and correlated negatively with liver fat (r2 = 0.21, P = 0.03) and positively with insulin sensitivity (r2 = 0.30, P = 0.01). Skeletal muscle mitochondrial oxygen consumption increased with low-dose ketones, attributable to increases in basal respiration (135%, P < 0.05) and ATP-linked oxygen consumption (136%, P < 0.05). NAFLD pathophysiology includes impaired hepatic ketogenesis, which is associated with hepatic fat accumulation and impaired insulin sensitivity. This reduced capacity to produce ketones may be a potential link between NAFLD and NAFLD-associated reductions in whole body insulin sensitivity, whereby ketone concentrations impact skeletal muscle mitochondrial respiration.


Assuntos
Ácido 3-Hidroxibutírico/metabolismo , Fígado/metabolismo , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/metabolismo , Adulto , Idoso , Estudos de Casos e Controles , Ácidos Graxos não Esterificados/metabolismo , Feminino , Técnica Clamp de Glucose , Humanos , Técnicas In Vitro , Resistência à Insulina , Corpos Cetônicos/metabolismo , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/etiologia , Obesidade/complicações , Espectroscopia de Prótons por Ressonância Magnética
15.
PLoS Genet ; 16(5): e1008754, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365093

RESUMO

FSHD is characterized by the misexpression of DUX4 in skeletal muscle. Although DUX4 upregulation is thought to be the pathogenic cause of FSHD, DUX4 is lowly expressed in patient samples, and analysis of the consequences of DUX4 expression has largely relied on artificial overexpression. To better understand the native expression profile of DUX4 and its targets, we performed bulk RNA-seq on a 6-day differentiation time-course in primary FSHD2 patient myoblasts. We identify a set of 54 genes upregulated in FSHD2 cells, termed FSHD-induced genes. Using single-cell and single-nucleus RNA-seq on myoblasts and differentiated myotubes, respectively, we captured, for the first time, DUX4 expressed at the single-nucleus level in a native state. We identified two populations of FSHD myotube nuclei based on low or high enrichment of DUX4 and FSHD-induced genes ("FSHD-Lo" and "FSHD Hi", respectively). FSHD-Hi myotube nuclei coexpress multiple DUX4 target genes including DUXA, LEUTX and ZSCAN4, and also upregulate cell cycle-related genes with significant enrichment of E2F target genes and p53 signaling activation. We found more FSHD-Hi nuclei than DUX4-positive nuclei, and confirmed with in situ RNA/protein detection that DUX4 transcribed in only one or two nuclei is sufficient for DUX4 protein to activate target genes across multiple nuclei within the same myotube. DUXA (the DUX4 paralog) is more widely expressed than DUX4, and depletion of DUXA suppressed the expression of LEUTX and ZSCAN4 in late, but not early, differentiation. The results suggest that the DUXA can take over the role of DUX4 to maintain target gene expression. These results provide a possible explanation as to why it is easier to detect DUX4 target genes than DUX4 itself in patient cells and raise the possibility of a self-sustaining network of gene dysregulation triggered by the limited DUX4 expression.


Assuntos
Núcleo Celular/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular Facioescapuloumeral , RNA-Seq/métodos , Análise de Célula Única/métodos , Estudos de Casos e Controles , Diferenciação Celular , Núcleo Celular/química , Núcleo Celular/classificação , Núcleo Celular/patologia , Células Cultivadas , Regulação da Expressão Gênica , Células HEK293 , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/fisiologia , Fibras Musculares Esqueléticas/ultraestrutura , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/metabolismo , Distrofia Muscular Facioescapuloumeral/patologia , Mioblastos/metabolismo , Mioblastos/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Sequenciamento Completo do Exoma
16.
Muscle Nerve ; 62(2): 284-288, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32367547

RESUMO

INTRODUCTION: The mechanism by which weakness develops in idiopathic inflammatory myopathies (IIMs) is still unclear. In this study we investigated the maximum force of single muscle fibers from patients with IIMs. METHODS: Permeabilized single muscle fibers from patients with IIMs and healthy controls were subjected to contractility measurements. Maximum force and specific force production (maximum force normalized to fiber size) and fiber type were determined for each isolated fiber. RESULTS: A total of 178 fibers were studied from five patients with IIMs and 95 fibers from four controls. Specific force production was significantly lower in the IIM group for all fiber types. DISCUSSION: The findings from this exploratory study suggest that weakness in IIMs may, in part, be caused by dysfunction of the contractile apparatus. These findings provide a basis for further studies into the mechanisms underlying weakness in IIMs.


Assuntos
Contração Muscular/fisiologia , Fibras Musculares de Contração Rápida/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Fibras Musculares de Contração Lenta/fisiologia , Força Muscular/fisiologia , Miosite/fisiopatologia , Adulto , Biópsia , Estudos de Casos e Controles , Tamanho Celular , Dermatomiosite/metabolismo , Dermatomiosite/patologia , Dermatomiosite/fisiopatologia , Feminino , Humanos , Pessoa de Meia-Idade , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Rápida/patologia , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Fibras Musculares de Contração Lenta/metabolismo , Fibras Musculares de Contração Lenta/patologia , Cadeias Pesadas de Miosina/metabolismo , Miosite/metabolismo , Miosite/patologia , Polimiosite/metabolismo , Polimiosite/patologia , Polimiosite/fisiopatologia , Adulto Jovem
17.
Am J Physiol Endocrinol Metab ; 318(6): E848-E855, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32369416

RESUMO

Disturbances in skeletal muscle lipid oxidation might induce ectopic fat deposition and lipotoxicity. Nevertheless, the cellular mechanisms that regulate skeletal muscle lipid oxidation have not been fully determined. We aimed to determine whether there was an association between relative whole body lipid oxidation and mitochondrial size or mitochondria-sarcoplasmic reticulum interactions in the skeletal muscle. Twelve healthy men were included [mean (standard deviation), 24.7 (1.5) yr old, 24.4 (2.6) kg/m2]. The respiratory quotient (RQ) was used to estimate relative lipid oxidation at rest and during exercise (50% maximal oxygen consumption, 600 kcal expended). A skeletal muscle biopsy was obtained from the vastus lateralis at rest. Transmission electron microscopy was used to determine mitochondrial size and mitochondria-sarcoplasmic reticulum interactions (≤50 nm of distance between organelles). Protein levels of fusion/fission regulators were measured in skeletal muscle by Western blot. Resting RQ and exercise RQ associated inversely with intermyofibrillar mitochondrial size (r = -0.66 and r = -0.60, respectively, P < 0.05). Resting RQ also associated inversely with the percentage of intermyofibrillar mitochondria-sarcoplasmic reticulum interactions (r = -0.62, P = 0.03). Finally, intermyofibrillar mitochondrial size associated inversely with lipid droplet density (r = -0.66, P = 0.01) but directly with mitochondria fusion-to-fission ratio (r = 0.61, P = 0.03). Our results show that whole body lipid oxidation is associated with skeletal muscle intermyofibrillar mitochondrial size, fusion phenotype, and mitochondria-sarcoplasmic-reticulum interactions in nondiabetic humans.


Assuntos
Exercício Físico/fisiologia , Metabolismo dos Lipídeos , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Quadríceps/ultraestrutura , Retículo Sarcoplasmático/ultraestrutura , Adolescente , Adulto , Humanos , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Tamanho Mitocondrial , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Oxirredução , Consumo de Oxigênio , Músculo Quadríceps/metabolismo , Adulto Jovem
18.
Metabolism ; 108: 154257, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32370945

RESUMO

BACKGROUND: Protein degradation is an energy-dependent process, requiring ATP at multiple steps. However, reports conflict as to the relationship between intracellular energetics and the rate of proteasome-mediated protein degradation. METHODS: To determine whether the concentration of the adenine nucleotide pool (ATP + ADP + AMP) affects protein degradation in muscle cells, we overexpressed an AMP degrading enzyme, AMP deaminase 3 (AMPD3), via adenovirus in C2C12 myotubes. RESULTS: Overexpression of AMPD3 resulted in a dose- and time-dependent reduction of total adenine nucleotides (ATP, ADP and AMP) without increasing the ADP/ATP or AMP/ATP ratios. In agreement, the reduction of total adenine nucleotide concentration did not result in increased Thr172 phosphorylation of AMP-activated protein kinase (AMPK), a common indicator of intracellular energetic state. Furthermore, LC3 protein accumulation and ULK1 (Ser 555) phosphorylation were not induced. However, overall protein degradation and ubiquitin-dependent proteolysis were slowed by overexpression of AMPD3, despite unchanged content of several proteasome subunit proteins and proteasome activity in vitro under standard conditions. CONCLUSIONS: Altogether, these findings indicate that a physiologically relevant decrease in ATP content, without a concomitant increase in ADP or AMP, is sufficient to decrease the rate of protein degradation and activity of the ubiquitin-proteasome system in muscle cells. This suggests that adenine nucleotide degrading enzymes, such as AMPD3, may be a viable target to control muscle protein degradation and perhaps muscle mass.


Assuntos
AMP Desaminase/metabolismo , Trifosfato de Adenosina/metabolismo , Músculo Esquelético/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Fosforilação/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Proteólise , Ubiquitina/metabolismo
19.
Neurology ; 94(23): e2441-e2447, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32467133

RESUMO

OBJECTIVE: Facioscapulohumeral muscular dystrophy (FSHD) is a heterogenetic disorder predominantly characterized by progressive facial and scapular muscle weakness. Patients with FSHD either have a contraction of the D4Z4 repeat on chromosome 4q35 or mutations in D4Z4 chromatin modifiers SMCHD1 and DNMT3B, both causing D4Z4 chromatin relaxation and inappropriate expression of the D4Z4-encoded DUX4 gene in skeletal muscle. In this study, we tested the hypothesis whether LRIF1, a known SMCHD1 protein interactor, is a disease gene for idiopathic FSHD2. METHODS: Clinical examination of a patient with idiopathic FSHD2 was combined with pathologic muscle biopsy examination and with genetic, epigenetic, and molecular studies. RESULTS: A homozygous LRIF1 mutation was identified in a patient with a clinical phenotype consistent with FSHD. This mutation resulted in the absence of the long isoform of LRIF1 protein, D4Z4 chromatin relaxation, and DUX4 and DUX4 target gene expression in myonuclei, all molecular and epigenetic hallmarks of FSHD. In concordance, LRIF1 was shown to bind to the D4Z4 repeat, and knockdown of the LRIF1 long isoform in muscle cells results in DUX4 and DUX4 target gene expression. CONCLUSION: LRIF1 is a bona fide disease gene for FSHD2. This study further reinforces the unifying genetic mechanism, which postulates that FSHD is caused by D4Z4 chromatin relaxation, resulting in inappropriate DUX4 expression in skeletal muscle.


Assuntos
Proteínas de Ciclo Celular/genética , Distrofia Muscular Facioescapuloumeral/genética , Biópsia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Cromatina/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Humanos Par 4/genética , Códon sem Sentido , Consanguinidade , Fibroblastos , Mutação da Fase de Leitura , Duplicação Gênica , Regulação da Expressão Gênica , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/patologia , Linhagem , Isoformas de Proteínas/genética , Sequências Repetitivas de Ácido Nucleico
20.
Am J Physiol Cell Physiol ; 319(2): C277-C287, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32432932

RESUMO

Severe burn injury induces a myriad of deleterious effects to skeletal muscle, resulting in impaired function and delayed recovery. Following burn, catabolic signaling and myofiber atrophy are key fiber-intrinsic determinants of weakness; less well understood are alterations in the interstitial environment surrounding myofibers. Muscle quality, specifically alterations in the extracellular matrix (ECM), modulates force transmission and strength. We sought to determine the impact of severe thermal injury on adaptation to the muscle ECM and quantify muscle fibrotic burden. After a 30% total body surface area dorsal burn, spinotrapezius muscle was harvested from mice at 7 (7d, n = 5), 14 (14d, n = 4), and 21 days (21d, n = 4), and a sham control group was also examined (Sham, n = 4). Expression of transforming growth factor-ß (TGFß), myostatin, and downstream effectors and proteases involved in fibrosis and collagen remodeling were measured by immunoblotting, and immunohistochemical and biochemical analyses assessed fibrogenic cell abundance and collagen deposition. Myostatin signaling increased progressively through 21 days postburn alongside fibrogenic/adipogenic progenitor cell expansion, with abundance peaking at 14 days postburn. Postburn, elevated expression of tissue inhibitor of matrix metalloproteinase 1 supported collagen remodeling resulting in a net accumulation of muscle collagen content. Collagen accumulation peaked at 14 days postburn but remained elevated through 21 days postburn, demonstrating minimal resolution of burn-induced fibrosis. These findings highlight a progressive upregulation of fibrogenic processes following burn injury, eliciting a fibrotic muscle phenotype that hinders regenerative capacity and is not resolved with 21 days of recovery.


Assuntos
Queimaduras/genética , Fibrose/genética , Músculo Esquelético/metabolismo , Miostatina/genética , Fator de Crescimento Transformador beta/genética , Animais , Queimaduras/metabolismo , Queimaduras/patologia , Proliferação de Células/genética , Colágeno/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/metabolismo , Fibrose/patologia , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/lesões , Músculo Esquelético/patologia , Transdução de Sinais/genética
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