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1.
Exp Cell Res ; 411(2): 112990, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-34973262

RESUMO

Human pluripotent stem cells (hPSCs) provide a human model for developmental myogenesis, disease modeling and development of therapeutics. Differentiation of hPSCs into muscle stem cells has the potential to provide a cell-based therapy for many skeletal muscle wasting diseases. This review describes the current state of hPSCs towards recapitulating human myogenesis ex vivo, considerations of stem cell and progenitor cell state as well as function for future use of hPSC-derived muscle cells in regenerative medicine.


Assuntos
Desenvolvimento Muscular/fisiologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Diferenciação Celular/fisiologia , Humanos , Modelos Biológicos , Desenvolvimento Muscular/genética , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/fisiologia , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/fisiologia
2.
Sci Rep ; 12(1): 1377, 2022 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-35082348

RESUMO

Muscle wasting is a major problem leading to reduced quality of life and higher risks of mortality and various diseases. Muscle atrophy is caused by multiple conditions in which protein degradation exceeds its synthesis, including disuse, malnutrition, and microgravity. While Vitamin D receptor (VDR) is well known to regulate calcium and phosphate metabolism to maintain bone, recent studies have shown that VDR also plays roles in skeletal muscle development and homeostasis. Moreover, its expression is upregulated in muscle undergoing atrophy as well as after muscle injury. Here we show that VDR regulates simulated microgravity-induced atrophy in C2C12 myotubes in vitro. After 8 h of microgravity simulated using 3D-clinorotation, the VDR-binding motif was associated with chromatin regions closed by the simulated microgravity and enhancer regions inactivated by it, which suggests VDR mediates repression of enhancers. In addition, VDR was induced and translocated into the nuclei in response to simulated microgravity. VDR-deficient C2C12 myotubes showed resistance to simulated microgravity-induced atrophy and reduced induction of FBXO32, an atrophy-associated ubiquitin ligase. These results demonstrate that VDR contributes to the regulation of simulated microgravity-induced atrophy at least in part by controlling expression of atrophy-related genes.


Assuntos
Fibras Musculares Esqueléticas/metabolismo , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , Mioblastos Esqueléticos/metabolismo , Receptores de Calcitriol/metabolismo , Transdução de Sinais/genética , Simulação de Ausência de Peso/efeitos adversos , Animais , Linhagem Celular , Técnicas de Inativação de Genes/métodos , Homeostase/genética , Camundongos , Desenvolvimento Muscular/genética , Atrofia Muscular/genética , Receptores de Calcitriol/genética , Transfecção
3.
Exp Cell Res ; 411(2): 112991, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-34958765

RESUMO

The processes of myogenesis during both development and regeneration share a number of similarities across both amniotes and teleosts. In amniotes, the process of muscle formation is considered largely biphasic, with developmental myogenesis occurring through hyperplastic fibre deposition and postnatal muscle growth driven through hypertrophy of existing fibres. In contrast, teleosts continue generating new muscle fibres during adult myogenesis through a process of eternal hyperplasia using a dedicated stem cell system termed the external cell layer. During developmental and regenerative myogenesis alike, muscle progenitors interact with their niche to receive cues guiding their transition into myoblasts and ultimately mature myofibres. During development, muscle precursors receive input from neighbouring embryological tissues; however, during repair, this role is fulfilled by other injury resident cell types, such as those of the innate immune response. Recent work has focused on the role of macrophages as a pro-regenerative cell type which provides input to muscle satellite cells during regenerative myogenesis. As zebrafish harbour a satellite cell system analogous to that of mammals, the processes of regeneration can be interrogated in vivo with the imaging intensive approaches afforded in the zebrafish system. This review discusses the strengths of zebrafish with a focus on both the similarities and differences to amniote myogenesis during both development and repair.


Assuntos
Desenvolvimento Muscular/fisiologia , Regeneração/fisiologia , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/fisiologia , Animais , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Macrófagos/fisiologia , Modelos Biológicos , Desenvolvimento Muscular/genética , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/fisiologia , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/metabolismo , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Regeneração/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
4.
Biomed Pharmacother ; 146: 112584, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34968921

RESUMO

Heart muscle injury and an elevated troponin level signify myocardial infarction (MI), which may result in defective and uncoordinated segments, reduced cardiac output, and ultimately, death. Physicians apply thrombolytic therapy, coronary artery bypass graft (CABG) surgery, or percutaneous coronary intervention (PCI) to recanalize and restore blood flow to the coronary arteries, albeit they were not convincingly able to solve the heart problems. Thus, researchers aim to introduce novel substitutional therapies for regenerating and functionalizing damaged cardiac tissue based on engineering concepts. Cell-based engineering approaches, utilizing biomaterials, gene, drug, growth factor delivery systems, and tissue engineering are the most leading studies in the field of heart regeneration. Also, understanding the primary cause of MI and thus selecting the most efficient treatment method can be enhanced by preparing microdevices so-called heart-on-a-chip. In this regard, microfluidic approaches can be used as diagnostic platforms or drug screening in cardiac disease treatment. Additionally, bioprinting technique with whole organ 3D printing of human heart with major vessels, cardiomyocytes and endothelial cells can be an ideal goal for cardiac tissue engineering and remarkable achievement in near future. Consequently, this review discusses the different aspects, advancements, and challenges of the mentioned methods with presenting the advantages and disadvantages, chronological indications, and application prospects of various novel therapeutic approaches.


Assuntos
Infarto do Miocárdio/fisiopatologia , Regeneração/fisiologia , Materiais Biocompatíveis/metabolismo , Engenharia Celular/métodos , Sistemas de Liberação de Medicamentos/métodos , Vesículas Extracelulares/metabolismo , Fibroblastos/metabolismo , Terapia Genética/métodos , Insuficiência Cardíaca/fisiopatologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células-Tronco Mesenquimais/metabolismo , Microfluídica/métodos , Mioblastos Esqueléticos/metabolismo , Isquemia Miocárdica/fisiopatologia , Células-Tronco/metabolismo , Engenharia Tecidual/métodos
5.
Front Endocrinol (Lausanne) ; 12: 785242, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34917036

RESUMO

Intrauterine growth restricted (IUGR) fetuses are born with lower skeletal muscle mass, fewer proliferating myoblasts, and fewer myofibers compared to normally growing fetuses. Plasma concentrations of insulin, a myogenic growth factor, are lower in IUGR fetuses. We hypothesized that a two-week insulin infusion at 75% gestation would increase myoblast proliferation and fiber number in IUGR fetal sheep. Catheterized control fetuses received saline (CON-S, n=6), and the IUGR fetuses received either saline (IUGR-S, n=7) or insulin (IUGR-I, 0.014 ± 0.001 units/kg/hr, n=11) for 14 days. Fetal arterial blood gases and plasma amino acid levels were measured. Fetal skeletal muscles (biceps femoris, BF; and flexor digitorum superficialis, FDS) and pancreases were collected at necropsy (126 ± 2 dGA) for immunochemistry analysis, real-time qPCR, or flow cytometry. Insulin concentrations in IUGR-I and IUGR-S were lower vs. CON-S (P ≤ 0.05, group). Fetal arterial PaO2, O2 content, and glucose concentrations were lower in IUGR-I vs. CON-S (P ≤ 0.01) throughout the infusion period. IGF-1 concentrations tended to be higher in IUGR-I vs. IUGR-S (P=0.06), but both were lower vs. CON-S (P ≤ 0.0001, group). More myoblasts were in S/G2 cell cycle stage in IUGR-I vs. both IUGR-S and CON-S (145% and 113%, respectively, P ≤ 0.01). IUGR-I FDS muscle weighed 40% less and had 40% lower fiber number vs. CON-S (P ≤ 0.05) but were not different from IUGR-S. Myonuclear number per fiber and the mRNA expression levels of muscle regulatory factors were not different between groups. While the pancreatic ß-cell mass was lower in both IUGR-I and IUGR-S compared to CON-S, the IUGR groups were not different from each other indicating that feedback inhibition by endogenous insulin did not reduce ß-cell mass. A two-week insulin infusion at 75% gestation promoted myoblast proliferation in the IUGR fetus but did not increase fiber or myonuclear number. Myoblasts in the IUGR fetus retain the capacity to proliferate in response to mitogenic stimuli, but intrinsic defects in the fetal myoblast by 75% gestation may limit the capacity to restore fiber number.


Assuntos
Desenvolvimento Fetal/efeitos dos fármacos , Retardo do Crescimento Fetal/tratamento farmacológico , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Fibras Musculares Esqueléticas/efeitos dos fármacos , Mioblastos Esqueléticos/efeitos dos fármacos , Animais , Esquema de Medicação , Feminino , Desenvolvimento Fetal/fisiologia , Retardo do Crescimento Fetal/patologia , Infusões Intravenosas , Desenvolvimento Muscular/efeitos dos fármacos , Desenvolvimento Muscular/fisiologia , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Mioblastos Esqueléticos/patologia , Mioblastos Esqueléticos/fisiologia , Gravidez , Ovinos
6.
Molecules ; 26(24)2021 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-34946743

RESUMO

Steric blocking antisense oligonucleotides (ASO) are promising tools for splice modulation such as exon-skipping, although their therapeutic effect may be compromised by insufficient delivery. To address this issue, we investigated the synthesis of a 20-mer 2'-OMe PS oligonucleotide conjugated at 3'-end with ursodeoxycholic acid (UDCA) involved in the targeting of human DMD exon 51, by exploiting both a pre-synthetic and a solution phase approach. The two approaches have been compared. Both strategies successfully provided the desired ASO 51 3'-UDC in good yield and purity. It should be pointed out that the pre-synthetic approach insured better yields and proved to be more cost-effective. The exon skipping efficiency of the conjugated oligonucleotide was evaluated in myogenic cell lines and compared to that of unconjugated one: a better performance was determined for ASO 51 3'-UDC with an average 9.5-fold increase with respect to ASO 51.


Assuntos
Éxons , Distrofia Muscular de Duchenne , Mioblastos Esqueléticos/metabolismo , Oligonucleotídeos Antissenso , Precursores de RNA , Ácido Ursodesoxicólico , Linhagem Celular Transformada , Humanos , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos Antissenso/farmacologia , Precursores de RNA/genética , Precursores de RNA/metabolismo , Ácido Ursodesoxicólico/química , Ácido Ursodesoxicólico/farmacocinética , Ácido Ursodesoxicólico/farmacologia
7.
Nutrients ; 13(12)2021 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-34959937

RESUMO

Sarcopenia, also known as skeletal muscle atrophy, is characterized by significant loss of muscle mass and strength. Oyster (Crassostrea gigas) hydrolysates have anti-cancer, antioxidant, and anti-inflammation properties. However, the anti-sarcopenic effect of oyster hydrolysates remains uninvestigated. Therefore, we prepared two different oyster hydrolysates, namely TGPN and PNY. This study aimed to determine the anti-muscle atrophy efficacy and molecular mechanisms of TGPN and PNY on both C2C12 cell lines and mice. In vitro, the TGPN and PNY recovered the dexamethasone-induced reduction in the myotube diameters. In vivo, TGPN and PNY administration not only improved grip strength and exercise endurance, but also attenuated the loss of muscle mass and muscle fiber cross-sectional area. Mechanistically, TGPN and PNY increased the expression of protein synthesis-related protein levels via phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of the rapamycin pathway, and reduced the expression of protein degradation-related protein levels via the PI3K/Akt/forkhead box O pathway. Also, TGPN and PNY stimulated NAD-dependent deacetylase sirtuin-1(SIRT1), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), nuclear respiratory factor 1,2, mitochondrial transcription factor A, along with mitochondrial DNA content via SIRT1/PGC-1α signaling. These findings suggest oyster hydrolysates could be used as a valuable natural material that inhibits skeletal muscle atrophy via regulating protein turnover and mitochondrial biogenesis.


Assuntos
Mitocôndrias/efeitos dos fármacos , Proteínas Musculares/metabolismo , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/metabolismo , Biogênese de Organelas , Ostreidae/química , Hidrolisados de Proteína/farmacologia , Hidrolisados de Proteína/uso terapêutico , Sarcopenia/tratamento farmacológico , Sarcopenia/metabolismo , Animais , Células Cultivadas , Tolerância ao Exercício/efeitos dos fármacos , Força da Mão , Camundongos , Atrofia Muscular/etiologia , Atrofia Muscular/fisiopatologia , Mioblastos Esqueléticos , Hidrolisados de Proteína/isolamento & purificação , Sarcopenia/etiologia , Sarcopenia/fisiopatologia
8.
Development ; 148(23)2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34738614

RESUMO

Autosomal dominant PDGFRß gain-of-function mutations in mice and humans cause a spectrum of wasting and overgrowth disorders afflicting the skeleton and other connective tissues, but the cellular origin of these disorders remains unknown. We demonstrate that skeletal stem cells (SSCs) isolated from mice with a gain-of-function D849V point mutation in PDGFRß exhibit colony formation defects that parallel the wasting or overgrowth phenotypes of the mice. Single-cell RNA transcriptomics with SSC-derived polyclonal colonies demonstrates alterations in osteogenic and chondrogenic precursors caused by PDGFRßD849V. Mutant cells undergo poor osteogenesis in vitro with increased expression of Sox9 and other chondrogenic markers. Mice with PDGFRßD849V exhibit osteopenia. Increased STAT5 phosphorylation and overexpression of Igf1 and Socs2 in PDGFRßD849V cells suggests that overgrowth in mice involves PDGFRßD849V activating the STAT5-IGF1 axis locally in the skeleton. Our study establishes that PDGFRßD849V causes osteopenic skeletal phenotypes that are associated with intrinsic changes in SSCs, promoting chondrogenesis over osteogenesis.


Assuntos
Mutação com Ganho de Função , Mioblastos Esqueléticos/metabolismo , Mutação Puntual , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Substituição de Aminoácidos , Animais , Condrogênese/genética , Regulação da Expressão Gênica , Camundongos , Camundongos Transgênicos , Mioblastos Esqueléticos/patologia , Osteogênese/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais/genética
9.
Int J Mol Sci ; 22(19)2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34639225

RESUMO

Current treatment protocols for myocardial infarction improve the outcome of disease to some extent but do not provide the clue for full regeneration of the heart tissues. An increasing body of evidence has shown that transplantation of cells may lead to some organ recovery. However, the optimal stem cell population has not been yet identified. We would like to propose a novel pro-regenerative treatment for post-infarction heart based on the combination of human skeletal myoblasts (huSkM) and mesenchymal stem cells (MSCs). huSkM native or overexpressing gene coding for Cx43 (huSKMCx43) alone or combined with MSCs were delivered in four cellular therapeutic variants into the healthy and post-infarction heart of mice while using molecular reporter probes. Single-Photon Emission Computed Tomography/Computed Tomography (SPECT/CT) performed right after cell delivery and 24 h later revealed a trend towards an increase in the isotopic uptake in the post-infarction group of animals treated by a combination of huSkMCx43 with MSC. Bioluminescent imaging (BLI) showed the highest increase in firefly luciferase (fluc) signal intensity in post-infarction heart treated with combination of huSkM and MSCs vs. huSkM alone (p < 0.0001). In healthy myocardium, however, nanoluciferase signal (nanoluc) intensity varied markedly between animals treated with stem cell populations either alone or in combinations with the tendency to be simply decreased. Therefore, our observations seem to show that MSCs supported viability, engraftment, and even proliferation of huSkM in the post-infarction heart.


Assuntos
Células-Tronco Mesenquimais/citologia , Imagem Molecular/métodos , Mioblastos Esqueléticos/citologia , Infarto do Miocárdio/patologia , Miocárdio/patologia , Animais , Modelos Animais de Doenças , Genes Reporter , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mioblastos Esqueléticos/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo
10.
Biochem Biophys Res Commun ; 578: 115-121, 2021 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-34562651

RESUMO

Earth's gravity is essential for maintaining skeletal muscle mass and function in the body. The role of gravity in the myogenic mechanism has been studied with animal experiments in the International Space Station. Recently, gravity-control devices allow to study the effects of gravity on cultured cells on the ground. This study demonstrated that simulated microgravity accelerated aging of human skeletal muscle myoblasts in an in-vitro culture. The microgravity culture induced a significant decrease in cell proliferation and an enlargement of the cytoskeleton and nucleus of cells. Similar changes are often observed in aged myoblasts following several passages. In fact, by the microgravity culture the expression of senescence associated ß-Gal was significantly enhanced, and some muscle-specific proteins decreased in the enlarged cells. Importantly, these microgravity effects remained with the cells even after a return to normal gravity conditions. Consequently, the microgravity-affected myoblasts demonstrated a reduced capability of differentiation into myotubes. In the body, it is difficult to interpret the disability of microgravity-affected myoblasts, since muscle regeneration is linked to the supply of new myogenic cells. Therefore, our in-vitro cell culture study will be advantageous to better understand the role of each type of myogenic cell in human muscle without gravitational stress at the single cell level.


Assuntos
Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/patologia , Mioblastos Esqueléticos/patologia , Análise de Célula Única/métodos , Simulação de Ausência de Peso/métodos , Envelhecimento/fisiologia , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Citoesqueleto/metabolismo , Humanos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/metabolismo
11.
Biomed Pharmacother ; 143: 112188, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34563947

RESUMO

An extract from Artemisia dracunculus L. (termed PMI-5011) improves glucose homeostasis by enhancing insulin action and reducing ectopic lipid accumulation, while increasing fat oxidation in skeletal muscle tissue in obese insulin resistant male mice. A chalcone, DMC-2, in PMI-5011 is the major bioactive that enhances insulin signaling and activation of AKT. However, the mechanism by which PMI-5011 improves lipid metabolism is unknown. AMPK is the cellular energy and metabolic sensor and a key regulator of lipid metabolism in muscle. This study examined PMI-5011 activation of AMPK signaling using murine C2C12 muscle cell culture and skeletal muscle tissue. Findings show that PMI-5011 increases Thr172-phosphorylation of AMPK in muscle cells and skeletal muscle tissue, while hepatic AMPK activation by PMI-5011 was not observed. Increased AMPK activity by PMI-5011 affects downstream signaling of AMPK, resulting in inhibition of ACC and increased SIRT1 protein levels. Selective deletion of DMC-2 from PMI-5011 demonstrates that compounds other than DMC-2 in a "DMC-2 knock out extract" (KOE) are responsible for AMPK activation and its downstream effects. Compared to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and metformin, the phytochemical mixture characterizing the KOE appears to more efficiently activate AMPK in muscle cells. KOE-mediated AMPK activation was LKB-1 independent, suggesting KOE does not activate AMPK via LKB-1 stimulation. Through AMPK activation, compounds in PMI-5011 may regulate lipid metabolism in skeletal muscle. Thus, the AMPK-activating potential of the KOE adds therapeutic value to PMI-5011 and its constituents in treating insulin resistance or type 2 diabetes.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Artemisia , Ativadores de Enzimas/farmacologia , Hipoglicemiantes/farmacologia , Resistência à Insulina , Músculo Esquelético/efeitos dos fármacos , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/farmacologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Artemisia/química , Linhagem Celular , Dieta Hiperlipídica , Modelos Animais de Doenças , Ativação Enzimática , Ativadores de Enzimas/isolamento & purificação , Hipoglicemiantes/isolamento & purificação , Masculino , Metformina/farmacologia , Camundongos Endogâmicos C57BL , Músculo Esquelético/enzimologia , Mioblastos Esqueléticos/efeitos dos fármacos , Mioblastos Esqueléticos/enzimologia , Fosforilação , Compostos Fitoquímicos/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Ribonucleotídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos
12.
Am J Physiol Regul Integr Comp Physiol ; 321(5): R781-R790, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34585616

RESUMO

At-risk alcohol use is prevalent and increases dysglycemia among people living with human immunodeficiency virus (PLWH). Skeletal muscle (SKM) bioenergetic dysregulation is implicated in dysglycemia and type 2 diabetes. The objective of this study was to determine the relationship between at-risk alcohol, glucose tolerance, and SKM bioenergetic function in PLWH. Thirty-five PLWH (11 females, 24 males, age: 53 ± 9 yr, body mass index: 29.0 ± 6.6 kg/m2) with elevated fasting glucose enrolled in the ALIVE-Ex study provided medical history and alcohol use information [Alcohol Use Disorders Identification Test (AUDIT)], then underwent an oral glucose tolerance test (OGTT) and SKM biopsy. Bioenergetic health and function and mitochondrial volume were measured in isolated myoblasts. Mitochondrial gene expression was measured in SKM. Linear regression adjusting for age, sex, and smoking was performed to examine the relationship between glucose tolerance (2-h glucose post-OGTT), AUDIT, and their interaction with each outcome measure. Negative indicators of bioenergetic health were significantly (P < 0.05) greater with higher 2-h glucose (proton leak) and AUDIT (proton leak, nonmitochondrial oxygen consumption, and bioenergetic health index). Mitochondrial volume was increased with the interaction of higher 2-h glucose and AUDIT. Mitochondrial gene expression decreased with higher 2-h glucose (TFAM, PGC1B, PPARG, MFN1), AUDIT (MFN1, DRP1, MFF), and their interaction (PPARG, PPARD, MFF). Decreased expression of mitochondrial genes were coupled with increased mitochondrial volume and decreased bioenergetic health in SKM of PLWH with higher AUDIT and 2-h glucose. We hypothesize these mechanisms reflect poorer mitochondrial health and may precede overt SKM bioenergetic dysregulation observed in type 2 diabetes.


Assuntos
Consumo de Bebidas Alcoólicas/efeitos adversos , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/sangue , Metabolismo Energético , Infecções por HIV/metabolismo , Sobreviventes de Longo Prazo ao HIV , Mitocôndrias Musculares/metabolismo , Mioblastos Esqueléticos/metabolismo , Músculo Quadríceps/metabolismo , Adulto , Consumo de Bebidas Alcoólicas/sangue , Consumo de Bebidas Alcoólicas/epidemiologia , Consumo de Bebidas Alcoólicas/fisiopatologia , Biomarcadores/sangue , Células Cultivadas , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Infecções por HIV/epidemiologia , Infecções por HIV/fisiopatologia , Humanos , Resistência à Insulina , Louisiana/epidemiologia , Masculino , Pessoa de Meia-Idade , Consumo de Oxigênio , Músculo Quadríceps/fisiopatologia , Medição de Risco , Fatores de Risco , Adulto Jovem
13.
Int J Mol Sci ; 22(17)2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34502418

RESUMO

As the largest tissue in the body, skeletal muscle has multiple functions in movement and energy metabolism. Skeletal myogenesis is controlled by a transcriptional cascade including a set of muscle regulatory factors (MRFs) that includes Myogenic Differentiation 1 (MYOD1), Myocyte Enhancer Factor 2 (MEF2), and Myogenin (MYOG), which direct the fusion of myogenic myoblasts into multinucleated myotubes. Neddylation is a posttranslational modification that covalently conjugates ubiquitin-like NEDD8 (neural precursor cell expressed, developmentally downregulated 8) to protein targets. Inhibition of neddylation impairs muscle differentiation; however, the underlying molecular mechanisms remain less explored. Here, we report that neddylation is temporally regulated during myoblast differentiation. Inhibition of neddylation through pharmacological blockade using MLN4924 (Pevonedistat) or genetic deletion of NEDD8 Activating Enzyme E1 Subunit 1 (NAE1), a subunit of the E1 neddylation-activating enzyme, blocks terminal myoblast differentiation partially through repressing MYOG expression. Mechanistically, we found that neddylation deficiency enhances the mRNA and protein expressions of class IIa histone deacetylases 4 and 5 (HDAC4 and 5) and prevents the downregulation and nuclear export of class III HDAC (NAD-Dependent Protein Deacetylase Sirtuin-1, SIRT1), all of which have been shown to repress MYOD1-mediated MYOG transcriptional activation. Together, our findings for the first time identify the crucial role of neddylation in mediating class IIa and III HDAC co-repressors to control myogenic program and provide new insights into the mechanisms of muscle disease and regeneration.


Assuntos
Diferenciação Celular , Histona Desacetilases/metabolismo , Mioblastos Esqueléticos/metabolismo , Proteína NEDD8/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/metabolismo , Sirtuína 1/metabolismo , Linhagem Celular , Histona Desacetilases/genética , Humanos , Proteína MyoD/genética , Proteína MyoD/metabolismo , Miogenina/genética , Miogenina/metabolismo , Proteína NEDD8/genética , Proteínas Repressoras/genética , Sirtuína 1/genética , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo
14.
Elife ; 102021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34519272

RESUMO

Skeletal muscle fibers are multinucleated cellular giants formed by the fusion of mononuclear myoblasts. Several molecules involved in myoblast fusion have been discovered, and finger-like projections coincident with myoblast fusion have also been implicated in the fusion process. The role of these cellular projections in muscle cell fusion was investigated herein. We demonstrate that these projections are filopodia generated by class X myosin (Myo10), an unconventional myosin motor protein specialized for filopodia. We further show that Myo10 is highly expressed by differentiating myoblasts, and Myo10 ablation inhibits both filopodia formation and myoblast fusion in vitro. In vivo, Myo10 labels regenerating muscle fibers associated with Duchenne muscular dystrophy and acute muscle injury. In mice, conditional loss of Myo10 from muscle-resident stem cells, known as satellite cells, severely impairs postnatal muscle regeneration. Furthermore, the muscle fusion proteins Myomaker and Myomixer are detected in myoblast filopodia. These data demonstrate that Myo10-driven filopodia facilitate multinucleated mammalian muscle formation.


Assuntos
Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Mioblastos Esqueléticos/metabolismo , Miosinas/metabolismo , Pseudópodes/metabolismo , Animais , Diferenciação Celular , Fusão Celular , Linhagem Celular , Proliferação de Células , Modelos Animais de Doenças , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Knockout , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Mioblastos Esqueléticos/patologia , Miosinas/genética , Pseudópodes/genética , Regeneração , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/patologia , Fatores de Tempo
15.
Life Sci Alliance ; 4(10)2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34389686

RESUMO

Absence of dystrophin, an essential sarcolemmal protein required for muscle contraction, leads to the devastating muscle-wasting disease Duchenne muscular dystrophy. Dystrophin has an actin-binding domain, which binds and stabilises filamentous-(F)-actin, an integral component of the RhoA-actin-serum-response-factor-(SRF) pathway. This pathway plays a crucial role in circadian signalling, whereby the suprachiasmatic nucleus (SCN) transmits cues to peripheral tissues, activating SRF and transcription of clock-target genes. Given dystrophin binds F-actin and disturbed SRF-signalling disrupts clock entrainment, we hypothesised dystrophin loss causes circadian deficits. We show for the first time alterations in the RhoA-actin-SRF-signalling pathway, in dystrophin-deficient myotubes and dystrophic mouse models. Specifically, we demonstrate reduced F/G-actin ratios, altered MRTF levels, dysregulated core-clock and downstream target-genes, and down-regulation of key circadian genes in muscle biopsies from Duchenne patients harbouring an array of mutations. Furthermore, we show dystrophin is absent in the SCN of dystrophic mice which display disrupted circadian locomotor behaviour, indicative of disrupted SCN signalling. Therefore, dystrophin is an important component of the RhoA-actin-SRF pathway and novel mediator of circadian signalling in peripheral tissues, loss of which leads to circadian dysregulation.


Assuntos
Distrofina/metabolismo , Fator de Resposta Sérica/metabolismo , Transdução de Sinais , Actinas/metabolismo , Animais , Linhagem Celular , Distrofina/genética , Camundongos , Mioblastos Esqueléticos/metabolismo , Utrofina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
16.
Elife ; 102021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34448452

RESUMO

Skeletal muscles are composed of hundreds of multinucleated muscle fibers (myofibers) whose myonuclei are regularly positioned all along the myofiber's periphery except the few ones clustered underneath the neuromuscular junction (NMJ) at the synaptic zone. This precise myonuclei organization is altered in different types of muscle disease, including centronuclear myopathies (CNMs). However, the molecular machinery regulating myonuclei position and organization in mature myofibers remains largely unknown. Conversely, it is also unclear how peripheral myonuclei positioning is lost in the related muscle diseases. Here, we describe the microtubule-associated protein, MACF1, as an essential and evolutionary conserved regulator of myonuclei positioning and maintenance, in cultured mammalian myotubes, in Drosophila muscle, and in adult mammalian muscle using a conditional muscle-specific knockout mouse model. In vitro, we show that MACF1 controls microtubules dynamics and contributes to microtubule stabilization during myofiber's maturation. In addition, we demonstrate that MACF1 regulates the microtubules density specifically around myonuclei, and, as a consequence, governs myonuclei motion. Our in vivo studies show that MACF1 deficiency is associated with alteration of extra-synaptic myonuclei positioning and microtubules network organization, both preceding NMJ fragmentation. Accordingly, MACF1 deficiency results in reduced muscle excitability and disorganized triads, leaving voltage-activated sarcoplasmic reticulum Ca2+ release and maximal muscle force unchanged. Finally, adult MACF1-KO mice present an improved resistance to fatigue correlated with a strong increase in mitochondria biogenesis.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/metabolismo , Junção Neuromuscular/metabolismo , Biogênese de Organelas , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestrutura , Acoplamento Excitação-Contração , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Microtúbulos/genética , Microtúbulos/ultraestrutura , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/ultraestrutura , Fadiga Muscular , Fibras Musculares Esqueléticas/ultraestrutura , Força Muscular , Mioblastos Esqueléticos/ultraestrutura , Junção Neuromuscular/genética , Junção Neuromuscular/ultraestrutura , Fatores de Tempo
17.
Cells ; 10(8)2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34440772

RESUMO

The Hedgehog (Hh) receptor PTCH1 and the integral membrane protein 2A (ITM2A) inhibit autophagy by reducing autolysosome formation. In this study, we demonstrate that ITM2A physically interacts with PTCH1; however, the two proteins inhibit autophagic flux independently, since silencing of ITM2A did not prevent the accumulation of LC3BII and p62 in PTCH1-overexpressing cells, suggesting that they provide alternative modes to limit autophagy. Knockdown of ITM2A potentiated PTCH1-induced autophagic flux blockade and increased PTCH1 expression, while ITM2A overexpression reduced PTCH1 protein levels, indicating that it is a negative regulator of PTCH1 non-canonical signalling. Our study also revealed that endogenous ITM2A is necessary for timely induction of myogenic differentiation markers in C2C12 cells since partial knockdown delays the timing of differentiation. We also found that basal autophagic flux decreases during myogenic differentiation at the same time that ITM2A expression increases. Given that canonical Hh signalling prevents myogenic differentiation, we investigated the effect of ITM2A on canonical Hh signalling using GLI-luciferase assays. Our findings demonstrate that ITM2A is a strong negative regulator of GLI transcriptional activity and of GLI1 stability. In summary, ITM2A negatively regulates canonical and non-canonical Hh signalling.


Assuntos
Autofagia , Diferenciação Celular , Proteínas de Membrana/metabolismo , Desenvolvimento Muscular , Mioblastos Esqueléticos/metabolismo , Receptor Patched-1/metabolismo , Transdução de Sinais , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/genética , Camundongos , Células NIH 3T3 , Receptor Patched-1/genética , Ligação Proteica , Mapas de Interação de Proteínas , Proteína GLI1 em Dedos de Zinco/metabolismo
18.
Carbohydr Polym ; 272: 118444, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34420709

RESUMO

In this study, a fully aligned microfibrous structure fabricated using fibrin-assisted alginate bioink and electrohydrodynamic direct-printing was proposed for skeletal muscle tissue engineering. To safely construct the aligned alginate/fibrin microfibrous structure laden with myoblasts or endothelial cells, various printing conditions, such as an applied electric field, distance between the nozzle and target, and nozzle moving speed, were selected appropriately. Furthermore, to accelerate the formation of myotubes more efficiently, the alginate/fibrin bioink with vascular endothelial cells was co-printed into a spatially patterned structure within a myoblast-laden structure. The myoblast-laden structure co-cultured with endothelial cells presented fully aligned myotube formation and significantly greater myogenic differentiation compared to the myoblast-laden structure without the endothelial cells owing to the more abundant secretion of angiogenic cytokines. Also, when adipose stem cell- and endothelial cell-laden fibrous structure was implanted in a mouse volumetric muscle loss model, accelerated volumetric muscle repair was observed compared to the defect model. Based on the results, this study demonstrates an alginate-based bioink and new bio-fabricating method to obtain microfibrous cell-laden alginate/fibrin structures with mechanically stable and topographical cues. The proposed method can provide a myoblast/endothelial cell-laden fibrous alginate structure to efficiently induce engineering of skeletal muscle tissue, which could be used in muscle-on-a-chip or recovering structures of volumetric muscle defects.


Assuntos
Alginatos/química , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/metabolismo , Impressão Tridimensional , Engenharia Tecidual/métodos , Tecidos Suporte/química , Tecido Adiposo/metabolismo , Animais , Bioimpressão/métodos , Diferenciação Celular , Técnicas de Cocultura/métodos , Células Endoteliais/metabolismo , Feminino , Fibrina/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Tinta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/química , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/química , Células-Tronco/metabolismo
19.
Am J Physiol Regul Integr Comp Physiol ; 321(4): R572-R587, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34431403

RESUMO

Hyperoxic conditions are known to accelerate skeletal muscle regeneration after injuries. In the early phase of regeneration, macrophages invade the injured area and subsequently secrete various growth factors, which regulate myoblast proliferation and differentiation. Although hyperoxic conditions accelerate muscle regeneration, it is unknown whether this effect is indirectly mediated by macrophages. Here, using C2C12 cells, we show that not only hyperoxia but also hypoxia enhance myoblast proliferation directly, without accelerating differentiation into myotubes. Under hyperoxic conditions (95% O2 + 5% CO2), the cell membrane was damaged because of lipid oxidization, and a disrupted cytoskeletal structure, resulting in suppressed cell proliferation. However, a culture medium containing vitamin C (VC), an antioxidant, prevented this lipid oxidization and cytoskeletal disruption, resulting in enhanced proliferation in response to hyperoxia exposure of ≤4 h/day. In contrast, exposure to hypoxic conditions (95% N2 + 5% CO2) for ≤8 h/day enhanced cell proliferation. Hyperoxia did not promote cell differentiation into myotubes, regardless of whether the culture medium contained VC. Similarly, hypoxia did not accelerate cell differentiation. These results suggest that regardless of hyperoxia or hypoxia, changes in oxygen tension can enhance cell proliferation directly, but do not influence differentiation efficiency in C2C12 cells. Moreover, excess oxidative stress abrogated the enhancement of myoblast proliferation induced by hyperoxia. This research will contribute to basic data for applying the effects of hyperoxia or hypoxia to muscle regeneration therapy.


Assuntos
Diferenciação Celular , Proliferação de Células , Desenvolvimento Muscular , Mioblastos Esqueléticos/metabolismo , Estresse Oxidativo , Oxigênio/metabolismo , Regeneração , Animais , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Cinética , Metabolismo dos Lipídeos , Camundongos , Desenvolvimento Muscular/efeitos dos fármacos , Mioblastos Esqueléticos/efeitos dos fármacos , Mioblastos Esqueléticos/patologia , Estresse Oxidativo/efeitos dos fármacos , Oxigênio/toxicidade , Regeneração/efeitos dos fármacos
20.
Biomolecules ; 11(8)2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34439731

RESUMO

Stromal interaction molecule 1 (STIM1) is the main protein that, along with Orai1, mediates store-operated Ca2+ entry (SOCE) in skeletal muscle. Abnormal SOCE due to mutations in STIM1 is one of the causes of human skeletal muscle diseases. STIM1-R304Q (a constitutively active form of STIM1) has been found in human patients with skeletal muscle phenotypes such as muscle weakness, myalgia, muscle stiffness, and contracture. However, the pathological mechanism(s) of STIM1-R304Q in skeletal muscle have not been well studied. To examine the pathological mechanism(s) of STIM1-R304Q in skeletal muscle, STIM1-R304Q was expressed in mouse primary skeletal myotubes, and the properties of the skeletal myotubes were examined using single-myotube Ca2+ imaging, transmission electron microscopy (TEM), and biochemical approaches. STIM1-R304Q did not interfere with the terminal differentiation of skeletal myoblasts to myotubes and retained the ability of STIM1 to attenuate dihydropyridine receptor (DHPR) activity. STIM1-R304Q induced hyper-SOCE (that exceeded the SOCE by wild-type STIM1) by affecting both the amplitude and the onset rate of SOCE. Unlike that by wild-type STIM1, hyper-SOCE by STIM1-R304Q contributed to a disturbance in Ca2+ distribution between the cytosol and the sarcoplasmic reticulum (SR) (high Ca2+ in the cytosol and low Ca2+ in the SR). Moreover, the hyper-SOCE and the high cytosolic Ca2+ level induced by STIM1-R304Q involve changes in mitochondrial shape. Therefore, a series of these cellular defects induced by STIM1-R304Q could induce deleterious skeletal muscle phenotypes in human patients carrying STIM1-R304Q.


Assuntos
Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/metabolismo , Proteínas de Neoplasias/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Humanos , Camundongos , Fibras Musculares Esqueléticas/citologia , Mioblastos Esqueléticos/citologia
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