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1.
PLoS One ; 16(10): e0258419, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34644361

RESUMO

Kinetin or N6-furfuryladenine (K) belongs to a class of plant hormones called cytokinins, which are biologically active molecules modulating many aspects of plant growth and development. However, biological activities of cytokinins are not only limited to plants; their effects on animals have been widely reported in the literature. Here, we found that Kinetin is a potent small molecule that efficiently stimulates differentiation of C2C12 myoblasts into myotubes in vitro. The highest efficacy was achieved at 1µM and 10µM Kinetin concentrations, in both mitogen-poor and rich media. More importantly, Kinetin was able to strongly stimulate the MyoD-dependent conversion of fibroblasts into myotubes. Kinetin alone did not give rise to fibroblast conversion and required MyoD; this demonstrates that Kinetin augments the molecular repertoire of necessary key regulatory factors to facilitate MyoD-mediated myogenic differentiation. This novel Kinetin pro-myogenic function may be explained by its ability to alter intracellular calcium levels and by its potential to impact on Reactive Oxygen Species (ROS) signalling. Taken together, our findings unravel the effects of a new class of small molecules with potent pro-myogenic activities. This opens up new therapeutic avenues with potential for treating skeletal muscle diseases related to muscle aging and wasting.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Cinetina/farmacologia , Desenvolvimento Muscular/efeitos dos fármacos , Animais , Linhagem Celular , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Proteína MyoD/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Espécies Reativas de Oxigênio/metabolismo
2.
Cells ; 10(10)2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34685705

RESUMO

Skeletal myogenesis is required to maintain muscle mass and integrity, and impaired myogenesis is causally linked to the etiology of muscle wasting. Recently, it was shown that excessive uptake of saturated fatty acids (SFA) plays a significant role in the pathogenesis of muscle wasting. Although microRNA (miRNA) is implicated in the regulation of myogenesis, the molecular mechanism whereby SFA-induced miRNAs impair myogenic differentiation remains largely unknown. Here, we investigated the regulatory roles of miR-325-3p on CFL2 expression and myogenic differentiation in C2C12 myoblasts. PA impeded myogenic differentiation, concomitantly suppressed CFL2 and induced miR-325-3p. Dual-luciferase analysis revealed that miR-325-3p directly targets the 3'UTR of CFL2, thereby suppressing the expression of CFL2, a crucial factor for actin dynamics. Transfection with miR-325-3p mimic resulted in the accumulation of actin filaments (F-actin) and nuclear Yes-associated protein (YAP) in myoblasts and promoted myoblast proliferation and cell cycle progression. Consequently, miR-325-3p mimic significantly attenuated the expressions of myogenic factors and thereby impaired the myogenic differentiation of myoblasts. The roles of miR-325-3p on CFL2 expression, F-actin modulation, and myogenic differentiation suggest a novel miRNA-mediated regulatory mechanism of myogenesis and PA-inducible miR-325-3p may be a critical mediator between obesity and muscle wasting.


Assuntos
Diferenciação Celular/genética , Cofilina 2/genética , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Desenvolvimento Muscular/genética , Mioblastos/citologia , Mioblastos/metabolismo , Regiões 3' não Traduzidas/genética , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sequência de Bases , Ciclo Celular/genética , Núcleo Celular/metabolismo , Proliferação de Células/genética , Cofilina 2/metabolismo , Camundongos , MicroRNAs/genética
3.
Nat Commun ; 12(1): 6264, 2021 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-34716330

RESUMO

Lack of dystrophin causes muscle degeneration, which is exacerbated by chronic inflammation and reduced regenerative capacity of muscle stem cells in Duchenne Muscular Dystrophy (DMD). To date, glucocorticoids remain the gold standard for the treatment of DMD. These drugs are able to slow down the progression of the disease and increase lifespan by dampening the chronic and excessive inflammatory process; however, they also have numerous harmful side effects that hamper their therapeutic potential. Here, we investigated Resolvin-D2 as a new therapeutic alternative having the potential to target multiple key features contributing to the disease progression. Our in vitro findings showed that Resolvin-D2 promotes the switch of macrophages toward their anti-inflammatory phenotype and increases their secretion of pro-myogenic factors. Moreover, Resolvin-D2 directly targets myogenic cells and promotes their differentiation and the expansion of the pool of myogenic progenitor cells leading to increased myogenesis. These effects are ablated when the receptor Gpr18 is knocked-out, knocked-down, or blocked by the pharmacological antagonist O-1918. Using different mouse models of DMD, we showed that Resolvin-D2 targets both inflammation and myogenesis leading to enhanced muscle function compared to glucocorticoids. Overall, this preclinical study has identified a new therapeutic approach that is more potent than the gold-standard treatment for DMD.


Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Desenvolvimento Muscular/efeitos dos fármacos , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/fisiopatologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Glucocorticoides/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Camundongos Endogâmicos mdx , Camundongos Knockout , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Desenvolvimento Muscular/fisiologia , Mioblastos/efeitos dos fármacos , Utrofina/genética
4.
Molecules ; 26(19)2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34641349

RESUMO

High-throughput, pillar-strip-based assays have been proposed as a drug-safety screening tool for developmental toxicity. In the assay described here, muscle cell culture and differentiation were allowed to occur at the end of a pillar strip (eight pillars) compatible with commercially available 96-well plates. Previous approaches to characterize cellular differentiation with immunostaining required a burdensome number of washing steps; these multiple washes also resulted in a high proportion of cellular loss resulting in poor yield. To overcome these limitations, the approach described here utilizes cell growth by easily moving the pillars for washing and immunostaining without significant loss of cells. Thus, the present pillar-strip approach is deemed suitable for monitoring high-throughput myogenic differentiation. Using this experimental high-throughput approach, eight drugs (including two well-known myogenic inhibitory drugs) were tested at six doses in triplicate, which allows for the generation of dose-response curves of nuclei and myotubes in a 96-well platform. As a result of comparing these F-actin (an actin-cytoskeleton protein), nucleus, and myotube data, two proposed differentiation indices-curve-area-based differentiation index (CA-DI) and maximum-point-based differentiation index (MP-DI) were generated. Both indices successfully allowed for screening of high-myogenic inhibitory drugs, and the maximum-point-based differentiation index (MP-DI) experimentally demonstrated sensitivity for quantifying drugs that inhibited myogenic differentiation.


Assuntos
Bioensaio/métodos , Diferenciação Celular , Fibras Musculares Esqueléticas/citologia , Mioblastos/citologia , Preparações Farmacêuticas/administração & dosagem , Animais , Proliferação de Células , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Mioblastos/efeitos dos fármacos
5.
Phytomedicine ; 93: 153791, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34666284

RESUMO

BACKGROUND: Recent advancements in understanding ß-escin action provide basis for new therapeutic claims for the drug. ß-escin-evoked attenuation of NF-κB-dependent signaling, increase in MMP-14 and decrease in COUP-TFII content and a rise in cholesterol biosynthesis could be beneficial in alleviating muscle-damaging processes. PURPOSE: The aim of this study was to investigate the effect of ß-escin on skeletal muscle regeneration. METHODS: Rat model of cardiotoxin-induced injury of fast-twich extensor digitorum longus (EDL) and slow-twich soleus (SOL) muscles and C2C12 myoblast cells were used in the study. We evaluated muscles obtained on day 3 and 14 post-injury by histological analyses of muscle fibers, connective tissue, and mononuclear infiltrate, by immunolocalization of macrophages and by qPCR to quantify the expression of muscle regeneration-related genes. Mechanism of drug action was investigated in vitro by assessing cell viability, NF-κB activation, MMP-2 and MMP-9 secretion, and ALDH activity. RESULTS: In rat model, ß-escin rescues regenerating muscles from atrophy. The drug reduces inflammatory infiltration, increases the number of muscle fibers and decreases fibrosis. ß-escin reduces macrophage infiltration into injured muscles and promotes their M2 polarization. It also alters transcription of muscle regeneration-related genes: Myf5, Myh2, Myh3, Myh8, Myod1, Pax3 and Pax7, and Pcna. In C2C12 myoblasts in vitro, ß-escin inhibits TNF-α-induced activation of NF-κB, reduces secretion of MMP-9 and increases ALDH activity. CONCLUSIONS: The data reveal beneficial role of ß-escin in muscle regeneration, particularly in poorly regenerating slow-twitch muscles. The findings provide rationale for further studies on ß-escin repositioning into conditions associated with muscle damage such as strenuous exercise, drug-induced myotoxicity or age-related disuse atrophy.


Assuntos
Escina , Músculo Esquelético , Animais , Metaloproteinase 2 da Matriz , Mioblastos , Ratos , Regeneração
6.
Curr Protoc ; 1(10): e263, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34612611

RESUMO

Skeletal muscle stem cells (MuSCs) reside in a complex niche composed of the muscle fiber plasma membrane and the laminin-rich basal lamina surrounded by the microvasculature, as well as different supportive cell types such as fibro-adipogenic progenitors residing in the interstitial extracellular matrix. Within the first few hours after tissue damage, MuSCs undergo cytoskeletal rearrangements and transcriptional changes that prime the cells for activation. Due to their time-consuming nature, enzymatic methods for liberation of single muscle fibers with fully quiescent MuSCs are challenging. Moreover, during enzymatic digestion, important niche components including the microvasculature and the collagenous interstitial matrix are destroyed. Here, we provide a method for the visualization of MuSCs on muscle fibers in their intact niche. Our method relies on mechanical teasing of fiber bundles from fixed skeletal muscles. We demonstrate that teased muscle fiber bundles allow the investigator to capture a representative snapshot of the MuSC niche in skeletal muscle, and outline how stem cell morphology and different microenvironmental components can be visualized. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of fiber bundles Basic Protocol 2: Immunofluorescence staining of MuSCs on fiber bundles Support Protocol: Preparation of Sylgard dishes.


Assuntos
Músculo Esquelético , Nicho de Células-Tronco , Adipogenia , Mioblastos , Células-Tronco
7.
Ecotoxicol Environ Saf ; 226: 112850, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34607188

RESUMO

As a common mycotoxin, deoxynivalenol (DON) contaminates cereal grains and feed in field or during processing and storage. DON elicits a spectrum of adverse effects in animals including anorexia and growth retardation. Especially, the presence of DON has also been detected in muscle, suggesting that DON may has the potential to affect the development of muscle. However, the relevant research is very rare and the molecular mechanism remains unclear. Myoblasts differentiation into multinucleated myotubes is one of the crucial steps of skeletal muscle development. In the present study, we investigated the effects of DON on differentiation of myoblasts using murine C2C12 cells model. The results indicated that DON dose-dependent inhibited the formation of myotubes in C2C12 cells. After performing omics techniques, a total of 149 differentially expressed genes were identified. The expression of cytoskeleton proteins and extracellular matrix (ECM) proteins were downregulated by DON. Furthermore, DON significantly downregulated the expression of integrin αv and integrin ß5, leading to inhibition of the ECM-integrin receptor interaction. The focal adhesion kinase (FAK) and phosphorylated forms, ras-related C3 botulinum toxin substrate (RAC) and p21-activated kinases 1 (PAK1) were also downregulated by DON. Taken together, our findings suggest that DON has the potent to affect the differentiation of myoblasts via downregulating of cytoskeleton and ECM-integrin-FAK-RAC-PAK signaling pathway.


Assuntos
Micotoxinas , Animais , Diferenciação Celular , Citoesqueleto/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Integrinas/genética , Camundongos , Mioblastos/metabolismo , Transdução de Sinais , Tricotecenos
8.
Nat Commun ; 12(1): 5977, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34645816

RESUMO

Muscle diseases and aging are associated with impaired myogenic stem cell self-renewal and fewer proliferating progenitors (MPs). Importantly, distinct metabolic states induced by glycolysis or oxidative phosphorylation have been connected to MP proliferation and differentiation. However, how these energy-provisioning mechanisms cooperate remain obscure. Herein, we describe a mechanism by which mitochondrial-localized transcriptional co-repressor p107 regulates MP proliferation. We show p107 directly interacts with the mitochondrial DNA, repressing mitochondrial-encoded gene transcription. This reduces ATP production by limiting electron transport chain complex formation. ATP output, controlled by the mitochondrial function of p107, is directly associated with the cell cycle rate. Sirt1 activity, dependent on the cytoplasmic glycolysis product NAD+, directly interacts with p107, impeding its mitochondrial localization. The metabolic control of MP proliferation, driven by p107 mitochondrial function, establishes a cell cycle paradigm that might extend to other dividing cell types.


Assuntos
Lactato Desidrogenase 5/genética , Mitocôndrias/genética , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Proteína p107 Retinoblastoma-Like/genética , Células-Tronco/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Ciclo Celular/genética , Linhagem Celular , Proliferação de Células , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica , Glicólise , Humanos , Lactato Desidrogenase 5/antagonistas & inibidores , Lactato Desidrogenase 5/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Músculo Esquelético/citologia , Mioblastos/citologia , Fosforilação Oxidativa , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína p107 Retinoblastoma-Like/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Células-Tronco/citologia , Transcrição Genética
9.
In Vivo ; 35(6): 3181-3191, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34697149

RESUMO

BACKGROUND/AIM: At present, there are no effective drugs for the treatment of insulin resistance. MTH-3, a curcumin derivative, exerts potent anti-cancer effects. The aim of the present study was to explore whether MTH-3 is capable of regulating palmitic acid (PA)-induced insulin resistance in C2C12 cells. MATERIALS AND METHODS: Cell viability was examined using the MTT assay. C2C12 cells were treated with PA and evaluated for the production of oil droplets using an Oil Red O assay. Glucose uptake was analysed by the 2-NBDG assay. RESULTS: Treatment of cells with up to 500 µM PA for 24 h or with 5 or 10 µM MTH-3 had no effect on cell viability. PA induced production of oil droplets in C2C12 cells. After adding MTH-3, the quantity of oil droplets decreased significantly and glucose uptake recovered. CONCLUSION: MTH-3 may become an efficient drug for the treatment of insulin resistance and associated diseases.


Assuntos
Curcumina , Resistência à Insulina , Animais , Linhagem Celular , Curcumina/farmacologia , Insulina , Camundongos , Mioblastos , Palmitatos
10.
Molecules ; 26(18)2021 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-34577156

RESUMO

DPY19L3 has been identified as a C-mannosyltransferase for thrombospondin type-1 repeat domain-containing proteins. In this study, we focused on the role of DPY19L3 in the myogenic differentiation of C2C12 mouse myoblast cells. We carried out DPY19L3 gene depletion using the CRISPR/Cas9 system. The result showed that these DPY19L3-knockout cells could not be induced for differentiation. Moreover, the phosphorylation levels of MEK/ERK and p70S6K were suppressed in the DPY19L3-knockout cells compared with that of parent cells, suggesting that the protein(s) that is(are) DPY19L3-mediated C-mannosylated and regulate(s) MEK/ERK or p70S6K signaling is(are) required for the differentiation.


Assuntos
Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Manosiltransferases/genética , Manosiltransferases/fisiologia , Mioblastos/fisiologia , Animais , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Glicosilação , Manosiltransferases/metabolismo , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mioblastos/citologia , Fosforilação/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/genética , Regulação para Cima/genética
11.
PLoS One ; 16(9): e0249438, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34473703

RESUMO

Muscle derived stem cells (MDSCs) and myoblast play an important role in myotube regeneration when muscle tissue is injured. However, these cells can be induced to differentiate into adipocytes once exposed to PPARγ activator like EPA and DHA that are highly suggested during pregnancy. The objective of this study aims at determining the identity of trans-differentiated cells by exploring the effect of EPA and DHA on C2C12 undergoing differentiation into brown and white adipocytes. DHA but not EPA committed C2C12 cells reprograming into white like adipocyte phenotype. Also, DHA promoted the expression of lipolysis regulating genes but had no effect on genes regulating ß-oxidation referring to its implication in lipid re-esterification. Furthermore, DHA impaired C2C12 cells differentiation into brown adipocytes through reducing the thermogenic capacity and mitochondrial biogenesis of derived cells independent of UCP1. Accordingly, DHA treated groups showed an increased accumulation of lipid droplets and suppressed mitochondrial maximal respiration and spare respiratory capacity. EPA, on the other hand, reduced myogenesis regulating genes, but no significant differences were observed in the expression of adipogenesis key genes. Likewise, EPA suppressed the expression of WAT signature genes indicating that EPA and DHA have an independent role on white adipogensis. Unlike DHA treatment, EPA supplementation had no effect on the differential of C2C12 cells into brown adipocytes. In conclusion, DHA is a potent adipogenic and lipogenic factor that can change the metabolic profile of muscle cells by increasing myocellular fat.


Assuntos
Adipócitos Brancos/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Adipócitos Marrons/efeitos dos fármacos , Adipócitos Brancos/citologia , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/efeitos dos fármacos , Animais , Linhagem Celular , Transdiferenciação Celular/efeitos dos fármacos , Transdiferenciação Celular/genética , DNA Mitocondrial , Regulação da Expressão Gênica/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Lipólise/efeitos dos fármacos , Camundongos , Mioblastos/citologia , Mioblastos/efeitos dos fármacos
12.
Life Sci ; 285: 119918, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34480939

RESUMO

AIMS: Insulin resistance is defined as the decreased sensitivity of tissues and organs to insulin and it is the main pathological basis of metabolic syndrome. PDCD5 is widely expressed in tissues including skeletal muscle and liver, but its exact function and the role in insulin resistance has not been studied. The present study is to explore the effect of PDCD5 on insulin resistance in skeletal muscle, the largest target organ of insulin, and its mechanism. MATERIALS AND METHODS: Mice were fed with high-fat diet to establish obesity model. C2C12 myoblasts differentiated into myotubes and then were treated with palmitate to induce insulin resistance. Gain-of-function and loss-of-function experiments were performed by infecting C2C12 with adenovirus containing PDCD5 cDNA or PDCD5 shRNA. KEY FINDINGS: PDCD5 protein was first increased and then decreased in the skeletal muscle from high-fat diet induced obese mice and consistently in palmitate induced insulin resistance C2C12 myotubes. Overexpression of PDCD5 in C2C12 cells did not affect the sensitivity to insulin but inhibited the palmitate induced insulin resistance, while knockdown of PDCD5 aggravated the insulin resistance. Mechanistically, PDCD5 interacted with ubiquitin ligase MDM2; overexpression of PDCD5 decreased MDM2 protein level, inhibited the increased interaction of MDM2 with IRS-1 and the degradation of IRS-1 by palmitate stimulation. SIGNIFICANCE: PDCD5 is upregulated during the early stage of insulin resistance in skeletal muscle. The increased PDCD5 inhibits IRS-1 ubiquitination, increases the stability of IRS-1 by interacting with and degrading MDM2, thus providing a protective effect on insulin resistance in skeletal muscle.


Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Proteínas Substratos do Receptor de Insulina/metabolismo , Resistência à Insulina , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Ubiquitinação , Animais , Proteínas Reguladoras de Apoptose/genética , Diferenciação Celular , Linhagem Celular , Dieta Hiperlipídica , Modelos Animais de Doenças , Estabilidade Enzimática , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/citologia , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Proteínas de Neoplasias/genética , Obesidade/genética , Obesidade/metabolismo , Palmitatos/farmacologia , Proteólise/efeitos dos fármacos
13.
Int J Mol Sci ; 22(17)2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34502296

RESUMO

Proper muscle function depends on the neuromuscular junctions (NMJs), which mature postnatally to complex "pretzel-like" structures, allowing for effective synaptic transmission. Postsynaptic acetylcholine receptors (AChRs) at NMJs are anchored in the actin cytoskeleton and clustered by the scaffold protein rapsyn, recruiting various actin-organizing proteins. Mechanisms driving the maturation of the postsynaptic machinery and regulating rapsyn interactions with the cytoskeleton are still poorly understood. Drebrin is an actin and microtubule cross-linker essential for the functioning of the synapses in the brain, but its role at NMJs remains elusive. We used immunohistochemistry, RNA interference, drebrin inhibitor 3,5-bis-trifluoromethyl pyrazole (BTP2) and co-immunopreciptation to explore the role of this protein at the postsynaptic machinery. We identify drebrin as a postsynaptic protein colocalizing with the AChRs both in vitro and in vivo. We also show that drebrin is enriched at synaptic podosomes. Downregulation of drebrin or blocking its interaction with actin in cultured myotubes impairs the organization of AChR clusters and the cluster-associated microtubule network. Finally, we demonstrate that drebrin interacts with rapsyn and a drebrin interactor, plus-end-tracking protein EB3. Our results reveal an interplay between drebrin and cluster-stabilizing machinery involving rapsyn, actin cytoskeleton, and microtubules.


Assuntos
Acetilcolina/metabolismo , Microtúbulos/fisiologia , Mioblastos/fisiologia , Junção Neuromuscular/fisiologia , Neuropeptídeos/farmacologia , Receptores Colinérgicos/metabolismo , Sinapses/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Células Cultivadas , Camundongos , Microtúbulos/efeitos dos fármacos , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Receptores Colinérgicos/genética , Transmissão Sináptica
14.
Int J Mol Sci ; 22(17)2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34502309

RESUMO

Skeletal muscles represent 40% of body mass and its native regenerative capacity can be permanently lost after a traumatic injury, congenital diseases, or tumor ablation. The absence of physiological regeneration can hinder muscle repair preventing normal muscle tissue functions. To date, tissue engineering (TE) represents one promising option for treating muscle injuries and wasting. In particular, hydrogels derived from the decellularized extracellular matrix (dECM) are widely investigated in tissue engineering applications thanks to their essential role in guiding muscle regeneration. In this work, the myogenic potential of dECM substrate, obtained from decellularized bovine pericardium (Tissuegraft Srl), was evaluated in vitro using C2C12 murine muscle cells. To assess myotubes formation, the width, length, and fusion indexes were measured during the differentiation time course. Additionally, the ability of dECM to support myogenesis was assessed by measuring the expression of specific myogenic markers: α-smooth muscle actin (α-sma), myogenin, and myosin heavy chain (MHC). The results obtained suggest that the dECM niche was able to support and enhance the myogenic potential of C2C12 cells in comparison of those grown on a plastic standard surface. Thus, the use of extracellular matrix proteins, as biomaterial supports, could represent a promising therapeutic strategy for skeletal muscle tissue engineering.


Assuntos
Diferenciação Celular , Matriz Extracelular/fisiologia , Desenvolvimento Muscular , Mioblastos/citologia , Pericárdio/citologia , Engenharia Tecidual/métodos , Animais , Bovinos , Hidrogéis/química , Camundongos , Tecidos Suporte/química
15.
Biomaterials ; 277: 121097, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34481290

RESUMO

Skeletal muscle tissue is mechanically dynamic with changes in stiffness influencing function, maintenance, and regeneration. We modeled skeletal muscle mechanical changes in culture with dynamically stiffening hydrogels demonstrating that the chaperone protein BAG3 transduces matrix stiffness by redistributing YAP and TAZ subcellular localization in muscle progenitor cells. BAG3 depletion increases cytoplasmic retention of YAP and TAZ, desensitizing myoblasts to changes in hydrogel elastic moduli. Upon differentiation, muscle progenitors depleted of BAG3 formed enlarged, round myotubes lacking the typical cylindrical morphology. The aberrant morphology is dependent on YAP/TAZ signaling, which was sequestered in the cytoplasm in BAG3-depleted myotubes but predominately nuclear in cylindrical myotubes of control cells. Control progenitor cells induced to differentiate on soft (E' = 4 and 12 kPa) hydrogels formed circular myotubes similar to those observed in BAG3-depleted cells. Inhibition of the Hippo pathway partially restored myotube morphologies, permitting nuclear translocation of YAP and TAZ in BAG3-depleted myogenic progenitors. Thus, BAG3 is a critical mediator of dynamic stiffness changes in muscle tissue, coupling mechanical alterations to intracellular signals and inducing changes in gene expression that influence muscle progenitor cell morphology and differentiation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Mecanotransdução Celular , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo
16.
Int J Mol Sci ; 22(17)2021 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-34502512

RESUMO

Primary cilia are non-motile, cell cycle-associated organelles that can be found on most vertebrate cell types. Comprised of microtubule bundles organised into an axoneme and anchored by a mature centriole or basal body, primary cilia are dynamic signalling platforms that are intimately involved in cellular responses to their extracellular milieu. Defects in ciliogenesis or dysfunction in cilia signalling underlie a host of developmental disorders collectively referred to as ciliopathies, reinforcing important roles for cilia in human health. Whilst primary cilia have long been recognised to be present in striated muscle, their role in muscle is not well understood. However, recent studies indicate important contributions, particularly in skeletal muscle, that have to date remained underappreciated. Here, we explore recent revelations that the sensory and signalling functions of cilia on muscle progenitors regulate cell cycle progression, trigger differentiation and maintain a commitment to myogenesis. Cilia disassembly is initiated during myoblast fusion. However, the remnants of primary cilia persist in multi-nucleated myotubes, and we discuss their potential role in late-stage differentiation and myofiber formation. Reciprocal interactions between cilia and the extracellular matrix (ECM) microenvironment described for other tissues may also inform on parallel interactions in skeletal muscle. We also discuss emerging evidence that cilia on fibroblasts/fibro-adipogenic progenitors and myofibroblasts may influence cell fate in both a cell autonomous and non-autonomous manner with critical consequences for skeletal muscle ageing and repair in response to injury and disease. This review addresses the enigmatic but emerging role of primary cilia in satellite cells in myoblasts and myofibers during myogenesis, as well as the wider tissue microenvironment required for skeletal muscle formation and homeostasis.


Assuntos
Centrossomo/metabolismo , Cílios/fisiologia , Músculo Esquelético/fisiologia , Animais , Axonema , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Cílios/metabolismo , Citoesqueleto , Matriz Extracelular , Humanos , Desenvolvimento Muscular/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Organelas , Transdução de Sinais
17.
FASEB J ; 35(10): e21928, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34559924

RESUMO

Limb contractures are a debilitating and progressive consequence of a wide range of upper motor neuron injuries that affect skeletal muscle function. One type of perinatal brain injury causes cerebral palsy (CP), which affects a child's ability to move and is often painful. While several rehabilitation therapies are used to treat contractures, their long-term effectiveness is marginal since such therapies do not change muscle biological properties. Therefore, new therapies based on a biological understanding of contracture development are needed. Here, we show that myoblast progenitors from contractured muscle in children with CP are hyperproliferative. This phenotype is associated with DNA hypermethylation and specific gene expression patterns that favor cell proliferation over quiescence. Treatment of CP myoblasts with 5-azacytidine, a DNA hypomethylating agent, reduced this epigenetic imprint to TD levels, promoting exit from mitosis and molecular mechanisms of cellular quiescence. Together with previous studies demonstrating reduction in myoblast differentiation, this suggests a mechanism of contracture formation that is due to epigenetic modifications that alter the myogenic program of muscle-generating stem cells. We suggest that normalization of DNA methylation levels could rescue myogenesis and promote regulated muscle growth in muscle contracture and thus may represent a new nonsurgical approach to treating this devastating neuromuscular condition.


Assuntos
Lesões Encefálicas/genética , Lesões Encefálicas/patologia , Metilação de DNA , Perfilação da Expressão Gênica , Músculo Esquelético/patologia , Mioblastos/metabolismo , Mioblastos/patologia , Transcrição Genética , Adolescente , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Lesões Encefálicas/metabolismo , Proliferação de Células , Paralisia Cerebral/tratamento farmacológico , Paralisia Cerebral/patologia , Criança , Pré-Escolar , Metilação de DNA/efeitos dos fármacos , Feminino , Humanos , Masculino , Músculo Esquelético/metabolismo , Mioblastos/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
18.
Sci Rep ; 11(1): 16603, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400681

RESUMO

Vascular research is largely performed in rodents with the goal of developing treatments for human disease. Micro-computed tomography (micro-CT) provides non-destructive three-dimensional imaging that can be used to study the vasculature of rodents. However, to distinguish vasculature from other soft tissues, long-circulating contrast agents are required. In this study, we demonstrated that poly(ethylene glycol) (PEG)-coated gadolinium nanoparticles can be used as a vascular contrast agent in micro-CT. The coated particles could be lyophilized and then redispersed in an aqueous solution to achieve 100 mg/mL of gadolinium. After an intravenous injection of the contrast agent into mice, micro-CT scans showed blood pool contrast enhancements of at least 200 HU for 30 min. Imaging and quantitative analysis of gadolinium in tissues showed the presence of contrast agent in clearance organs including the liver and spleen and very low amounts in other organs. In vitro cell culture experiments, subcutaneous injections, and analysis of mouse body weight suggested that the agents exhibited low toxicity. Histological analysis of tissues 5 days after injection of the contrast agent showed cytotoxicity in the spleen, but no abnormalities were observed in the liver, lungs, kidneys, and bladder.


Assuntos
Meios de Contraste , Gadolínio , Nanopartículas , Microtomografia por Raio-X/métodos , Animais , Coloides , Meios de Contraste/farmacocinética , Meios de Contraste/toxicidade , Gadolínio/farmacocinética , Gadolínio/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Nanopartículas/toxicidade , Polietilenoglicóis , Distribuição Tecidual , Imagem Corporal Total
19.
Cells ; 10(8)2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34440852

RESUMO

The objective of this study was to investigate fibromodulin (FMOD) and myostatin (MSTN) gene expressions during skeletal muscle aging and to understand their involvements in this process. The expressions of genes related to muscle aging (Atrogin 1 and Glb1), diabetes (RAGE and CD163), and lipid accumulation (CD36 and PPARγ) and those of FMOD and MSTN were examined in CTX-injected, aged, MSTN-/-, and high-fat diet (HFD) mice and in C2C12 myoblasts treated with ceramide or grown under adipogenic conditions. Results from CTX-injected mice and gene knockdown experiments in C2C12 cells suggested the involvement of FMOD during muscle regeneration and myoblast proliferation and differentiation. Downregulation of the FMOD gene in MSTN-/- mice, and MSTN upregulation and FMOD downregulation in FMOD and MSTN knockdown C2C12 cells, respectively, during their differentiation, suggested FMOD negatively regulates MSTN gene expression, and MSTN positively regulates FMOD gene expression. The results of our in vivo and in vitro experiments indicate FMOD inhibits muscle aging by negatively regulating MSTN gene expression or by suppressing the action of MSTN protein, and that MSTN promotes muscle aging by positively regulating the expressions of Atrogin1, CD36, and PPARγ genes in muscle.


Assuntos
Fibromodulina/metabolismo , Músculo Esquelético/metabolismo , Miostatina/metabolismo , Animais , Antígenos CD36/genética , Antígenos CD36/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ceramidas/farmacologia , Dieta Hiperlipídica , Fibromodulina/antagonistas & inibidores , Fibromodulina/genética , Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/patologia , Mioblastos/citologia , Mioblastos/metabolismo , Miostatina/antagonistas & inibidores , Miostatina/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor para Produtos Finais de Glicação Avançada/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Sarcopenia/metabolismo , Sarcopenia/patologia , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
20.
Exp Cell Res ; 407(1): 112779, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34428455

RESUMO

Skeletal muscle wasting drives negative clinical outcomes and is associated with a spectrum of pathologies including cancer. Cancer cachexia is a multi-factorial syndrome that encompasses skeletal muscle wasting and remains understudied, despite being a frequent and serious co-morbidity. Deviation from the homeostatic balance between breakdown and regeneration leads to muscle wasting disorders, such as cancer cachexia. Muscle stem cells (MuSCs) are the cellular compartment responsible for muscle regeneration, which makes MuSCs an intriguing target in the context of wasting muscle. Molecular studies investigating MuSCs and skeletal muscle wasting largely focus on transcriptional changes, but our group and others propose that metabolic changes are another layer of cellular regulation underlying MuSC dysfunction in cancer cachexia. In the present study, we combined gene expression and non-targeted metabolomic profiling of myoblasts exposed to wasting conditions (cancer cell conditioned media, CC-CM) to derive a more complete picture of the myoblast response to wasting factors. After mapping these features to annotated pathways, we found that more than half of the mapped pathways were amino acid-related, linking global amino acid metabolic disruption to conditioned media-induced myoblast defects. Notably, arginine metabolism was a highly enriched pathway in combined metabolomic and transcriptomic data. Arginine catabolism generates nitric oxide (NO), an important signaling molecule known to have negative effects on mature muscle. We hypothesize that tumor-derived disruptions in Nitric Oxide Synthase (NOS)2-regulated arginine catabolism impair differentiation of MuSCs. The work presented here further investigates the effect of NOS2 overactivity on myoblast proliferation and differentiation. We show that NOS2 inhibition is sufficient to rescue wasting phenotypes associated with inflammatory cytokines. Ultimately, this work provides new insights into MuSC biology and opens up potential therapeutic avenues for addressing disrupted MuSC dynamics in cancer cachexia.


Assuntos
Caquexia/metabolismo , Citocinas/metabolismo , Mioblastos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Caquexia/complicações , Caquexia/patologia , Diferenciação Celular/fisiologia , Humanos , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/metabolismo , Atrofia Muscular/patologia , Regeneração
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