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1.
Int J Mol Sci ; 22(18)2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34575979

RESUMO

MicroRNAs (miRNAs) play an essential role in the regulation of a number of physiological functions. miR-133a and other muscular miRs (myomiRs) play a key role in muscle cell growth and in some type of cancers. Here, we show that miR133a is upregulated in individuals that undertake physical exercise. We used a skeletal muscle differentiation model to dissect miR-133a's role and to identify new targets, identifying Tropomyosin-4 (TPM4). This protein is expressed during muscle differentiation, but importantly it is an essential component of microfilament cytoskeleton and stress fibres formation. The microfilament scaffold remodelling is an essential step in cell transformation and tumour progression. Using the muscle system, we obtained valuable information about the microfilament proteins, and the knowledge on these molecular players can be transferred to the cytoskeleton rearrangement observed in cancer cells. Further investigations showed a role of TPM4 in cancer physiology, specifically, we found that miR-133a downregulation leads to TPM4 upregulation in colon carcinoma (CRC), and this correlates with a lower patient survival. At molecular level, we demonstrated in myocyte differentiation that TPM4 is positively regulated by the TA isoform of the p63 transcription factor. In muscles, miR-133a generates a myogenic stimulus, reducing the differentiation by downregulating TPM4. In this system, miR-133a counteracts the differentiative TAp63 activity. Interestingly, in CRC cell lines and in patient biopsies, miR-133a is able to regulate TPM4 activity, while TAp63 is not active. The downregulation of the miR leads to TPM4 overexpression, this modifies the architecture of the cell cytoskeleton contributing to increase the invasiveness of the tumour and associating with a poor prognosis. These results add data to the interesting question about the link between physical activity, muscle physiology and protection against colorectal cancer. The two phenomena have in common the cytoskeleton remodelling, due to the TPM4 activity, that is involved in stress fibres formation.


Assuntos
Diferenciação Celular/genética , Neoplasias do Colo/genética , MicroRNAs/genética , Fatores de Transcrição/genética , Tropomiosina/genética , Proteínas Supressoras de Tumor/genética , Citoesqueleto de Actina/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias do Colo/patologia , Citoesqueleto/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células Musculares/citologia , Desenvolvimento Muscular/genética , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Fibras de Estresse/genética
2.
Int J Mol Sci ; 22(17)2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34502539

RESUMO

Muscular dystrophies are a heterogeneous group of inherited diseases characterized by the progressive degeneration and weakness of skeletal muscles, leading to disability and, often, premature death. To date, no effective therapies are available to halt or reverse the pathogenic process, and meaningful treatments are urgently needed. From this perspective, it is particularly important to establish reliable in vitro models of human muscle that allow the recapitulation of disease features as well as the screening of genetic and pharmacological therapies. We herein review and discuss advances in the development of in vitro muscle models obtained from human induced pluripotent stem cells, which appear to be capable of reproducing the lack of myofiber proteins as well as other specific pathological hallmarks, such as inflammation, fibrosis, and reduced muscle regenerative potential. In addition, these platforms have been used to assess genetic correction strategies such as gene silencing, gene transfer and genome editing with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), as well as to evaluate novel small molecules aimed at ameliorating muscle degeneration. Furthermore, we discuss the challenges related to in vitro drug testing and provide a critical view of potential therapeutic developments to foster the future clinical translation of preclinical muscular dystrophy studies.


Assuntos
Diferenciação Celular/fisiologia , Descoberta de Drogas/métodos , Terapia Genética/métodos , Células-Tronco Pluripotentes Induzidas/fisiologia , Células Musculares/fisiologia , Distrofias Musculares/terapia , Animais , Distrofina/genética , Distrofina/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células Musculares/citologia , Distrofias Musculares/genética , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/terapia
3.
Nat Commun ; 12(1): 4744, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362895

RESUMO

Human induced pluripotent stem cells (iPSC) hold promise for modeling diseases in individual human genetic backgrounds and thus for developing precision medicine. Here, we generate sensorimotor organoids containing physiologically functional neuromuscular junctions (NMJs) and apply the model to different subgroups of amyotrophic lateral sclerosis (ALS). Using a range of molecular, genomic, and physiological techniques, we identify and characterize motor neurons and skeletal muscle, along with sensory neurons, astrocytes, microglia, and vasculature. Organoid cultures derived from multiple human iPSC lines generated from individuals with ALS and isogenic lines edited to harbor familial ALS mutations show impairment at the level of the NMJ, as detected by both contraction and immunocytochemical measurements. The physiological resolution of the human NMJ synapse, combined with the generation of major cellular cohorts exerting autonomous and non-cell autonomous effects in motor and sensory diseases, may prove valuable to understand the pathophysiological mechanisms of ALS.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Junção Neuromuscular/metabolismo , Organoides/fisiologia , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Astrócitos , Edição de Genes , Humanos , Células-Tronco Pluripotentes Induzidas , Neurônios Motores , Células Musculares , Músculo Esquelético , Mutação , Organoides/patologia , Células-Tronco
4.
Mol Biol (Mosk) ; 55(4): 683-696, 2021.
Artigo em Russo | MEDLINE | ID: mdl-34432786

RESUMO

Hydrophobic molecules may be toxic when present in excess. When dissolved in membranes, hydrophobic molecules disrupt membrane function. Studies on the effects of free fatty acids (FFA) on cultured cells contradict each other. Here we describe the effects of FFA on various human cells in culture. The addition of long-chain FFA (oleic, palmitic, linoleic, linolenic, etc.) to cultured cells led to lipid accumulation in hepatocytes and muscle cells, initiation of autophagy, and uncoupling of oxidative phosphorylation. Although treated cells increase their oxygen consumption, metabolic shifts in favor of glycolysis were observed. All these effects were expressed to varying degrees in different cells and with the addition of different FFAs. The mechanisms of these FFA effects are discussed, as well their practical implications.


Assuntos
Ácidos Graxos não Esterificados , Lisossomos , Glicólise , Hepatócitos , Humanos , Células Musculares
5.
Biomolecules ; 11(8)2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34439754

RESUMO

Increasing energy expenditure through activation of brown fat thermogenesis is a promising therapeutic strategy for the treatment of obesity. Epigenetic regulation has emerged as a key player in regulating brown fat development and thermogenic program. Here, we aimed to study the role of DNA methyltransferase 3b (Dnmt3b), a DNA methyltransferase involved in de novo DNA methylation, in the regulation of brown fat function and energy homeostasis. We generated a genetic model with Dnmt3b deletion in brown fat-skeletal lineage precursor cells (3bKO mice) by crossing Dnmt3b-floxed (fl/fl) mice with Myf5-Cre mice. Female 3bKO mice are prone to diet-induced obesity, which is associated with decreased energy expenditure. Dnmt3b deficiency also impairs cold-induced thermogenic program in brown fat. Surprisingly, further RNA-seq analysis reveals a profound up-regulation of myogenic markers in brown fat of 3bKO mice, suggesting a myocyte-like remodeling in brown fat. Further motif enrichment and pyrosequencing analysis suggests myocyte enhancer factor 2C (Mef2c) as a mediator for the myogenic alteration in Dnmt3b-deficient brown fat, as indicated by decreased methylation at its promoter. Our data demonstrate that brown fat Dnmt3b is a key regulator of brown fat development, energy metabolism and obesity in female mice.


Assuntos
Tecido Adiposo Marrom/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Fator Regulador Miogênico 5/metabolismo , Obesidade/metabolismo , Adipócitos Marrons/metabolismo , Tecido Adiposo Branco/metabolismo , Alelos , Animais , Peso Corporal , Cruzamentos Genéticos , DNA (Citosina-5-)-Metiltransferases/genética , Modelos Animais de Doenças , Metabolismo Energético , Epigênese Genética , Feminino , Resistência à Insulina , Camundongos , Camundongos Knockout , Células Musculares/metabolismo , RNA-Seq , Termogênese
6.
J Gen Physiol ; 153(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34264306

RESUMO

The intercalated disk (ID) is a specialized subcellular region that provides electrical and mechanical connections between myocytes in the heart. The ID has a clearly defined passive role in cardiac tissue, transmitting mechanical forces and electrical currents between cells. Recent studies have shown that Na+ channels, the primary current responsible for cardiac excitation, are preferentially localized at the ID, particularly within nanodomains such as the gap junction-adjacent perinexus and mechanical junction-associated adhesion-excitability nodes, and that perturbations of ID structure alter cardiac conduction. This suggests that the ID may play an important, active role in regulating conduction. However, the structures of the ID and intercellular cleft are not well characterized and, to date, no models have incorporated the influence of ID structure on conduction in cardiac tissue. In this study, we developed an approach to generate realistic finite element model (FEM) meshes replicating nanoscale of the ID structure, based on experimental measurements from transmission electron microscopy images. We then integrated measurements of the intercellular cleft electrical conductivity, derived from the FEM meshes, into a novel cardiac tissue model formulation. FEM-based calculations predict that the distribution of cleft conductances is sensitive to regional changes in ID structure, specifically the intermembrane separation and gap junction distribution. Tissue-scale simulations predict that ID structural heterogeneity leads to significant spatial variation in electrical polarization within the intercellular cleft. Importantly, we found that this heterogeneous cleft polarization regulates conduction by desynchronizing the activation of postjunctional Na+ currents. Additionally, these heterogeneities lead to a weaker dependence of conduction velocity on gap junctional coupling, compared with prior modeling formulations that neglect or simplify ID structure. Further, we found that disruption of local ID nanodomains can either slow or enhance conduction, depending on gap junctional coupling strength. Our study therefore suggests that ID nanoscale structure can play a significant role in regulating cardiac conduction.


Assuntos
Junções Comunicantes , Miocárdio , Coração , Células Musculares , Miócitos Cardíacos , Sódio
7.
Am J Physiol Cell Physiol ; 321(3): C559-C568, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34319830

RESUMO

In organisms from flies to mammals, the initial formation of a functional tendon is completely dependent on chemical signals from muscles (myokines). However, how myokines affect the maturation, maintenance, and regeneration of tendons as a function of age is completely unstudied. Here we discuss the role of four myokines-fibroblast growth factors (FGF), myostatin, the secreted protein acidic and rich in cysteine (SPARC) miR-29-in tendon development and hypothesize a role for these factors in the progressive changes in tendon structure and function as a result of muscle wasting (disuse, aging, and disease). Because of the close relationship between mechanical loading and muscle and tendon regulation, disentangling muscle-tendon cross talk from simple mechanical loading is experimentally quite difficult. Therefore, we propose an experimental framework that hopefully will be useful in demonstrating muscle-tendon cross talk in vivo. Though understudied, the promise of a better understanding of muscle-tendon cross talk is the development of new interventions that will improve tendon development, regeneration, and function throughout the lifespan.


Assuntos
Envelhecimento/genética , Exossomos/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Tendões/metabolismo , Envelhecimento/metabolismo , Animais , Fenômenos Biomecânicos , Exossomos/genética , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Células Musculares/metabolismo , Células Musculares/patologia , Músculo Esquelético/patologia , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Miostatina/genética , Miostatina/metabolismo , Osteonectina/genética , Osteonectina/metabolismo , Transdução de Sinais , Tendões/patologia
8.
Methods Mol Biol ; 2319: 137-141, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331251

RESUMO

Lymphatic muscle cells (LMCs), with unique characteristics resembling a combination of both cardiac and smooth muscle cells, play an essential role in the spontaneous contraction of the lymphatic vessels to pump fluid forward. However, our understanding of the more detailed molecular phenotypes of LMCs is limited. Here, we described a method to isolate the LMCs from rat mesentery and then culture the cells in vitro, which will serve a lot more molecular biology study of LMCs and significantly improve our knowledge about the unique characteristics of LMCs.


Assuntos
Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Dissecação/métodos , Mesentério/citologia , Células Musculares/citologia , Animais , Imunofluorescência , Células Musculares/metabolismo , Ratos
9.
Molecules ; 26(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206320

RESUMO

Type 2 diabetes (T2D) is a chronic metabolic disease, which could affect the daily life of patients and increase their risk of developing other diseases. Synthetic anti-diabetic drugs usually show severe side effects. In the last few decades, plant-derived drugs have been intensively studied, particularly because of a rapid development of the instruments used in analytical chemistry. We tested the efficacy of Gundelia tournefortii L. (GT) in increasing the translocation of glucose transporter-4 (GLUT4) to the myocyte plasma membrane (PM), as a main strategy to manage T2D. In this study, GT methanol extract was sub-fractionated into 10 samples using flash chromatography. The toxicity of the fractions on L6 muscle cells, stably expressing GLUTmyc, was evaluated using the MTT assay. The efficacy with which GLUT4 was attached to the L6 PM was evaluated at non-toxic concentrations. Fraction 6 was the most effective, as it stimulated GLUT4 translocation in the absence and presence of insulin, 3.5 and 5.2 times (at 250 µg/mL), respectively. Fraction 1 and 3 showed no significant effects on GLUT4 translocation, while other fractions increased GLUT4 translocation up to 2.0 times. Gas chromatography-mass spectrometry of silylated fractions revealed 98 distinct compounds. Among those compounds, 25 were considered anti-diabetic and glucose disposal agents. These findings suggest that GT methanol sub-fractions exert an anti-diabetic effect by modulating GLUT4 translocation in L6 muscle cells, and indicate the potential of GT extracts as novel therapeutic agents for T2D.


Assuntos
Asteraceae/química , Diabetes Mellitus Tipo 2/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Hipoglicemiantes , Células Musculares/metabolismo , Animais , Linhagem Celular , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Transportador de Glucose Tipo 4/genética , Hipoglicemiantes/química , Hipoglicemiantes/isolamento & purificação , Hipoglicemiantes/farmacologia , Transporte Proteico/efeitos dos fármacos , Ratos
10.
Int J Mol Sci ; 22(13)2021 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-34281225

RESUMO

Thyroid hormones (THs) are key regulators of different biological processes. Their action involves genomic and non-genomic mechanisms, which together mediate the final effects of TH in target tissues. However, the proportion of the two processes and their contribution to the TH-mediated effects are still poorly understood. Skeletal muscle is a classical target tissue for TH, which regulates muscle strength and contraction, as well as energetic metabolism of myofibers. Here we address the different contribution of genomic and non-genomic action of TH in skeletal muscle cells by specifically silencing the deiodinase Dio2 or the ß3-Integrin expression via CRISPR/Cas9 technology. We found that myoblast proliferation is inversely regulated by integrin signal and the D2-dependent TH activation. Similarly, inhibition of the nuclear receptor action reduced myoblast proliferation, confirming that genomic action of TH attenuates proliferative rates. Contrarily, genomic and non-genomic signals promote muscle differentiation and the regulation of the redox state. Taken together, our data reveal that integration of genomic and non-genomic signal pathways finely regulates skeletal muscle physiology. These findings not only contribute to the understanding of the mechanisms involved in TH modulation of muscle physiology but also add insight into the interplay between different mechanisms of action of TH in muscle cells.


Assuntos
Células Musculares/fisiologia , Músculo Esquelético/fisiologia , Hormônios Tireóideos/fisiologia , Animais , Diferenciação Celular , Integrina beta3/fisiologia , Iodeto Peroxidase/fisiologia , Camundongos , Músculo Esquelético/citologia
11.
Biomed Pharmacother ; 139: 111615, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34243598

RESUMO

BACKGROUND: Severe acidosis deteriorates cardiac injury. Rat coronary arteries (RCAs) are unusually hypercontractive to extracellular (o) acidosis (EA). TMEM16A-encoded anoctamin 1 (ANO1), a Ca2+-activated chloride channel (CaCC), plays an important role in regulating coronary arterial tension. PURPOSE: We tested the possibility that the activation of CaCCs in the arterial smooth muscle cell (ASMC) contributes to EA-induced RCA constriction. METHODS: ANO1 expression was detected with immunofluorescence staining and Western blot. TMEM16A mRNA was assessed with quantitative Real-Time PCR. Cl- currents and membrane potentials were quantified with a patch clamp. The vascular tension was recorded with a myograph. Intracellular (i) level of Cl- and Ca2+ was measured with fluorescent molecular probes. RESULTS: ANO1 was expressed in all tested arterial myocytes, but was much more abundant in RCA ASMCs as compared with ASMCs isolated from rat cerebral basilar, intrarenal and mesenteric arteries. EA reduced [Cl-]i levels, augmented CaCC currents exclusively in RCA ASMCs and depolarized RCA ASMCs to a greater extent. Cl- deprivation, which depleted [Cl-]i by incubating the arteries or their ASMCs in Cl--free bath solution, decreased EA-induced [Cl-]i reduction, diminished EA-induced CaCC augmentation and time-dependently depressed EA-induced RCA constriction. Inhibitor studies showed that these EA-induced effects including RCA constriction, CaCC current augmentation, [Cl-]i reduction and/or [Ca2+]i elevation were depressed by various Cl- channel blockers, [Ca2+]i release inhibitors and L-type voltage-gated Ca2+ channel inhibitor nifedipine. ANO1 antibody attenuated all observed changes induced by EA in RCA ASMCs. CONCLUSION: The greater activity of RCA ASMC CaCCs complicated with an enhanced Ca2+ mobilization from both [Ca2+]i release and [Ca2+]o influx plays a pivotal role in the distinctive hypercontractility of RCAs to acidosis. Translation of these findings to human beings may lead to a new conception in our understanding and treating cardiac complications in severe acidosis.


Assuntos
Acidose/metabolismo , Anoctamina-1/metabolismo , Vasos Coronários/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Vasoconstrição/fisiologia , Acidose/tratamento farmacológico , Animais , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Vasos Coronários/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Nifedipino/farmacologia , Técnicas de Patch-Clamp/métodos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Vasoconstrição/efeitos dos fármacos
12.
Zhen Ci Yan Jiu ; 46(5): 368-74, 2021 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-34085458

RESUMO

OBJECTIVE: To observe the effect of electroacupuncture (EA) preconditioning on left-cardiac function, contents of serum TNF-α and IL-6 and expression of myocardial farnesoid X receptor(FXR), small heterodimer partner (SHP), apoptosis inducing factor (AIF) and heat shock proteins 70 (HSP70) genes in myocardial ischemia-reperfusion injury (MIRI) rats, so as to explore its mechanisms underlying improvement of ischemic myocardial injury. METHODS: Forty male Wistar rats were randomly divided into normal control, sham operation, MIRI model and EA pretreatment groups, with 10 rats in each group. Rats of the sham operation group received exposure of the thorax and heart. The MIRI model was established by occlusion of the anterior descending branch of the left coronary artery (LAD). EA (2 Hz/100 Hz and 1 mA) was applied to bilateral "Neiguan" (PC6), "Zusanli" (ST36) and "Guanyuan" (CV4) for 20 min, once a day for 7 days. The left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP)and maximal rates of rise and fall of left ventricular pressure (±dp/dtmax) were detected, the contents of serum TNF-α and IL-6 were detected by using enzyme-linked immunosorbent assay (ELISA), and the expression of FXR, SHP, AIF and HSP70 apoptotic genes in the myocardial tissue were measured by fluorescent quantitative RT-PCR. RESULTS: Compared with the normal control group, the LVEDP, contents of serum TNF-α and IL-6, and the expression levels of myocardial FXR, SHP, AIF and HSP70 mRNAs were significantly increased (P<0.05), while LVSP and ±dp/dtmax levels were obviously decreased in the model group (P<0.05). In comparison with the model group, MIRI-induced increases of LVEDP, TNF-α and IL-6 contents, and FXR, SHP and AIF mRNA expression and decreases of ±dp/dtmax and LVSP levels were reversed(P<0.05), except HSP70 mRNA expression with significantly increased (P<0.05) in the EA pretreatment group. CONCLUSION: EA pretreatment can protect the left ventricular function of the ischemic heart in MIRI rats, which may be related to its effects in reliving peripheral inflammation and regulating the expression levels of apoptosis-related factors FXR, SHP, AIF and HSP70 in the myocardium.


Assuntos
Eletroacupuntura , Traumatismo por Reperfusão Miocárdica , Pontos de Acupuntura , Animais , Apoptose/genética , Masculino , Células Musculares , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/terapia , Ratos , Ratos Wistar
13.
Molecules ; 26(9)2021 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-34063700

RESUMO

Momordica charantia is a popular vegetable associated with effective complementary and alternative diabetes management in some parts of the world. However, the molecular mechanism is less commonly investigated. In this study, we investigated the association between a major cucurbitane triterpenoid isolated from M. charantia, 3ß,7ß,25-trihydroxycucurbita-5,23(E)-dien-19-al (THCB) and peroxisome proliferator activated receptor gamma (PPARγ) activation and its related activities using cell culture and molecular biology techniques. In this study, we report on both M. charantia fruit crude extract and THCB in driving the luciferase activity of Peroxisome Proliferator Response Element, associated with PPARγ activation. Other than that, THCB also induced adipocyte differentiation at far less intensity as compared to the full agonist rosiglitazone. In conjunction, THCB treatment on adipocytes also resulted in upregulation of PPAR gamma target genes expression; AP2, adiponectin, LPL and CD34 at a lower magnitude compared to rosiglitazone's induction. THCB also induced glucose uptake into muscle cells and the mechanism is via Glut4 translocation to the cell membrane. In conclusion, THCB acts as one of the many components in M. charantia to induce hypoglycaemic effect by acting as PPARγ ligand and inducing glucose uptake activity in the muscles by means of Glut4 translocation.


Assuntos
Momordica/química , PPAR gama/metabolismo , Triterpenos/química , Células 3T3-L1 , Adipócitos/citologia , Animais , Diferenciação Celular , Membrana Celular/metabolismo , Glucose/metabolismo , Hepatócitos/citologia , Hipoglicemia/tratamento farmacológico , Insulina/química , Ligantes , Camundongos , Células Musculares/citologia , Domínios Proteicos , Rosiglitazona/farmacologia , Triterpenos/farmacologia
14.
Aging (Albany NY) ; 13(11): 14557-14570, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078750

RESUMO

GRSF1 is a mitochondrial RNA-binding protein important for maintaining mitochondrial function. We found that GRSF1 is highly expressed in cultured skeletal myoblasts differentiating into myotubes. To understand the physiological function of GRSF1 in vivo, we generated mice in which GRSF1 was specifically ablated in skeletal muscle. The conditional knockout mice (Grsf1cKO) appeared normal until 7-9 months of age. Importantly, however, a reduction of muscle endurance compared to wild-type controls was observed in 16- to 18-month old Grsf1cKO mice. Transcriptomic analysis revealed more than 200 mRNAs differentially expressed in Grsf1cKO muscle at this age. Notably, mRNAs encoding proteins involved in mitochondrial function, inflammation, and ion transport, including Mgarp, Cxcl10, Nfkb2, and Sln mRNAs, were significantly elevated in aged Grsf1cKO muscle. Our findings suggest that GRSF1 deficiency exacerbates the functional decline of aged skeletal muscle, likely through multiple downstream effector proteins.


Assuntos
Envelhecimento/metabolismo , Músculo Esquelético/metabolismo , Resistência Física , Proteínas de Ligação a Poli(A)/deficiência , Animais , Diferenciação Celular/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Musculares/metabolismo , Desenvolvimento Muscular/genética , Proteínas de Ligação a Poli(A)/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo
15.
PLoS One ; 16(5): e0250741, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33961664

RESUMO

The interactions between skeletal muscle and bone have been recently noted, and muscle-derived humoral factors related to bone metabolism play crucial roles in the muscle/bone relationships. We previously reported that extracellular vesicles from mouse muscle C2C12 cells (Myo-EVs) suppress osteoclast formation in mice. Although mechanical stress is included in extrinsic factors which are important for both muscle and bone, the detailed roles of mechanical stress in the muscle/bone interactions have still remained unknown. In present study, we examined the effects of fluid flow shear stress (FFSS) to C2C12 cells on the physiological actions of muscle cell-derived EV. Applying FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in mouse bone marrow cells in the presence of receptor activator nuclear factor κB ligand (RANKL). Moreover, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed mitochondria biogenesis genes during osteoclast formation with RANKL treatment. In addition, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in Raw264.7 cells in the presence of RANKL. Small RNA-seq-analysis showed that FFSS elevated the expression of miR196a-5p and miR155-5p with the suppressive actions of osteoclast formation and low expression in mouse bone cells. On the other hand, muscle cell-derived EVs with or without FFSS to C2C12 cells did not affect the expression of osteogenic genes, alkaline phosphatase activity and mineralization in mouse osteoblasts. In conclusion, we first showed that FFSS to C2C12 cells enhances the suppressive effects of muscle cell-derived EVs on osteoclast formation in mouse cells. Muscle cell-derived EVs might be partly involved in the effects of mechanical stress on the muscle/bone relationships.


Assuntos
Osso e Ossos/fisiologia , Vesículas Extracelulares/metabolismo , Hidrodinâmica , Células Musculares/citologia , Resistência ao Cisalhamento , Estresse Mecânico , Animais , Fenômenos Biomecânicos , Linhagem Celular , Camundongos
16.
Nat Commun ; 12(1): 2577, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972551

RESUMO

Inter-tissue interaction is fundamental to multicellularity. Although the basement membrane (BM) is located at tissue interfaces, its mode of action in inter-tissue interactions remains poorly understood, mainly because the molecular and structural details of the BM at distinct inter-tissue interfaces remain unclear. By combining quantitative transcriptomics and immunohistochemistry, we systematically identify the cellular origin, molecular identity and tissue distribution of extracellular matrix molecules in mouse hair follicles, and reveal that BM composition and architecture are exquisitely specialized for distinct inter-tissue interactions, including epithelial-fibroblast, epithelial-muscle and epithelial-nerve interactions. The epithelial-fibroblast interface, namely, hair germ-dermal papilla interface, makes asymmetrically organized side-specific heterogeneity in the BM, defined by the newly characterized interface, hook and mesh BMs. One component of these BMs, laminin α5, is required for hair cycle regulation and hair germ-dermal papilla anchoring. Our study highlights the significance of BM heterogeneity in distinct inter-tissue interactions.


Assuntos
Membrana Basal/citologia , Matriz Extracelular/metabolismo , Folículo Piloso/metabolismo , Laminina/metabolismo , Transcriptoma/genética , Animais , Membrana Basal/metabolismo , Membrana Basal/ultraestrutura , Células Epiteliais/metabolismo , Matriz Extracelular/genética , Feminino , Fibroblastos/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Família Multigênica , Células Musculares/metabolismo , Neurônios/metabolismo , Análise de Célula Única
17.
J Vet Med Sci ; 83(7): 1022-1030, 2021 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-33967186

RESUMO

This study aimed to investigate the function of estrogen receptors (ERs) in myoregeneration and intermuscular adipogenesis. Ovariectomized (OVX) ERα knockout (KO) mice and ERß KO mice were used to assess the effect of estrogen on the myoregenerative process. Tibialis anterior muscle was collected on days 7, 10, and 14 after cardiotoxin injection to assess myotube morphology and adipogenesis area. Regenerated myotubes from OVX-ERß KO mice were consistently smaller in diameter than those from OVX-ERα KO and OVX-wild-type mice, whereas the adipogenesis area of OVX-ERß KO mice was consistently greater than that of the other types. Therefore, ERß may be an influential factor in promoting myoregeneration and adipogenesis inhibition compared to ERα.


Assuntos
Adipogenia , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Células Musculares/citologia , Regeneração , Animais , Estradiol , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Estrogênios , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovariectomia/veterinária
18.
Genes Cells ; 26(7): 495-512, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33960573

RESUMO

Mesenchymal stem cells (MSCs) are the likely precursors of multiple lines of mesenchymal cells. The existence of bona fide MSCs with self-renewal capacity and differentiation potential into all mesenchymal lineages, however, has been unclear because of the lack of MSC-specific marker(s) that are not expressed by the terminally differentiated progeny. Meflin, a glycosylphosphatidylinositol-anchored protein, is an MSC marker candidate that is specifically expressed in rare stromal cells in all tissues. Our previous report showed that Meflin expression becomes down-regulated in bone marrow-derived MSCs cultured on plastic, making it difficult to examine the self-renewal and differentiation of Meflin-positive cells at the single-cell level. Here, we traced the lineage of Meflin-positive cells in postnatal and adult mice, showing that those cells differentiated into white and brown adipocytes, osteocytes, chondrocytes and skeletal myocytes. Interestingly, cells derived from Meflin-positive cells formed clusters of differentiated cells, implying the in situ proliferation of Meflin-positive cells or their lineage-committed progenitors. These results, taken together with previous findings that Meflin expression in cultured MSCs was lost upon their multilineage differentiation, suggest that Meflin is a useful potential marker to localize MSCs and/or their immature progenitors in multiple tissues.


Assuntos
Diferenciação Celular , Linhagem da Célula , Imunoglobulinas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Condrócitos/citologia , Condrócitos/metabolismo , Imunoglobulinas/genética , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Células Musculares/citologia , Células Musculares/metabolismo , Osteócitos/citologia , Osteócitos/metabolismo
19.
Cells ; 10(4)2021 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-33801626

RESUMO

Muscle tissue is often removed during hamstring tendon graft preparation for anterior cruciate ligament (ACL) reconstruction. The purpose of the study was to test whether preservation of muscle remnants on a tendon graft is beneficial to the graft healing process following ACL reconstruction. Co-culturing of tendon-derived cells (TDCs) and muscle-derived cells (MDCs) was performed at various ratios, and their potential for cell viability and multilineage differentiation was compared to a single TDC cell group. Ligamentous and chondrogenic differentiation was most enhanced when a small population of MDCs was co-cultured with TDCs (6:2 co-culture group). Cell viability and osteogenic differentiation were proportionally enhanced with increasing MDC population size. MDCs co-cultured with TDCs possess both the ability to enhance cell viability and differentiate into other cell lineages.


Assuntos
Diferenciação Celular , Tendões dos Músculos Isquiotibiais/transplante , Células Musculares/citologia , Preservação Biológica , Adolescente , Adulto , Becaplermina/farmacologia , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Condrogênese/efeitos dos fármacos , Técnicas de Cocultura , Colágeno/biossíntese , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ligamentos/citologia , Masculino , Células Musculares/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Adulto Jovem
20.
Proc Natl Acad Sci U S A ; 118(11)2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33836595

RESUMO

Protein aggregation is associated with a wide range of degenerative human diseases with devastating consequences, as exemplified by Alzheimer's, Parkinson's, and Huntington's diseases. In vitro kinetic studies have provided a mechanistic understanding of the aggregation process at the molecular level. However, it has so far remained largely unclear to what extent the biophysical principles of amyloid formation learned in vitro translate to the complex environment of living organisms. Here, we take advantage of the unique properties of a Caenorhabditis elegans model expressing a fluorescently tagged polyglutamine (polyQ) protein, which aggregates into discrete micrometer-sized inclusions that can be directly visualized in real time. We provide a quantitative analysis of protein aggregation in this system and show that the data are described by a molecular model where stochastic nucleation occurs independently in each cell, followed by rapid aggregate growth. Global fitting of the image-based aggregation kinetics reveals a nucleation rate corresponding to 0.01 h-1 per cell at 1 mM intracellular protein concentration, and shows that the intrinsic molecular stochasticity of nucleation accounts for a significant fraction of the observed animal-to-animal variation. Our results highlight how independent, stochastic nucleation events in individual cells control the overall progression of polyQ aggregation in a living animal. The key finding that the biophysical principles associated with protein aggregation in small volumes remain the governing factors, even in the complex environment of a living organism, will be critical for the interpretation of in vivo data from a wide range of protein aggregation diseases.


Assuntos
Peptídeos/metabolismo , Agregação Patológica de Proteínas/metabolismo , Amiloide/metabolismo , Animais , Caenorhabditis elegans , Cinética , Modelos Moleculares , Células Musculares/metabolismo , Agregados Proteicos
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