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1.
Int J Mol Sci ; 22(17)2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34502235

RESUMO

Skeletal muscle is essential to maintain vital functions such as movement, breathing, and thermogenesis, and it is now recognized as an endocrine organ. Muscles release factors named myokines, which can regulate several physiological processes. Moreover, skeletal muscle is particularly important in maintaining body homeostasis, since it is responsible for more than 75% of all insulin-mediated glucose disposal. Alterations of skeletal muscle differentiation and function, with subsequent dysfunctional expression and secretion of myokines, play a key role in the pathogenesis of obesity, type 2 diabetes, and other metabolic diseases, finally leading to cardiometabolic complications. Hence, a deeper understanding of the molecular mechanisms regulating skeletal muscle function related to energy metabolism is critical for novel strategies to treat and prevent insulin resistance and its cardiometabolic complications. This review will be focused on both cellular and animal models currently available for exploring skeletal muscle metabolism and endocrine function.


Assuntos
Diabetes Mellitus Tipo 2/fisiopatologia , Modelos Animais de Doenças , Resistência à Insulina , Desenvolvimento Muscular , Músculo Esquelético/fisiopatologia , Animais , Humanos
2.
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
3.
Nat Commun ; 12(1): 5520, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535684

RESUMO

PTEN promoter hypermethylation is nearly universal and PTEN copy number loss occurs in ~25% of fusion-negative rhabdomyosarcoma (FN-RMS). Here we show Pten deletion in a mouse model of FN-RMS results in less differentiated tumors more closely resembling human embryonal RMS. PTEN loss activated the PI3K pathway but did not increase mTOR activity. In wild-type tumors, PTEN was expressed in the nucleus suggesting loss of nuclear PTEN functions could account for these phenotypes. Pten deleted tumors had increased expression of transcription factors important in neural and skeletal muscle development including Dbx1 and Pax7. Pax7 deletion completely rescued the effects of Pten loss. Strikingly, these Pten;Pax7 deleted tumors were no longer FN-RMS but displayed smooth muscle differentiation similar to leiomyosarcoma. These data highlight how Pten loss in FN-RMS is connected to a PAX7 lineage-specific transcriptional output that creates a dependency or synthetic essentiality on the transcription factor PAX7 to maintain tumor identity.


Assuntos
Fator de Transcrição PAX7/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Animais , Cruzamento , Diferenciação Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Integrases/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Knockout , Desenvolvimento Muscular , PTEN Fosfo-Hidrolase/deficiência , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rabdomiossarcoma/genética
4.
Cells ; 10(9)2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34572017

RESUMO

The present study sought to identify gene networks that are hallmarks of the developing inguinal subcutaneous adipose tissue (iWAT) and the interscapular brown adipose tissue (BAT) in the mouse. RNA profiling revealed that the iWAT of postnatal (P) day 6 mice expressed thermogenic and lipid catabolism transcripts, along with the abundance of transcripts associated with the beige adipogenesis program. This was an unexpected finding, as thermogenic BAT was believed to be the only site of nonshivering thermogenesis in the young mouse. However, the transcriptional landscape of BAT in P6 mice suggests that it is still undergoing differentiation and maturation, and that the iWAT temporally adopts thermogenic and lipolytic potential. Moreover, P6 iWAT and adult (P56) BAT were similar in their expression of immune gene networks, but P6 iWAT was unique in the abundant expression of antimicrobial proteins and virus entry factors, including a possible receptor for SARS-CoV-2. In summary, postnatal iWAT development is associated with a metabolic shift from thermogenesis and lipolysis towards fat storage. However, transcripts of beige-inducing signal pathways including ß-adrenergic receptors and interleukin-4 signaling were underrepresented in young iWAT, suggesting that the signals for thermogenic fat differentiation may be different in early postnatal life and in adulthood.


Assuntos
Adipócitos Bege/metabolismo , Transcrição Genética , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Ciclo Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Redes Reguladoras de Genes , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Desenvolvimento Muscular/genética , Neuropeptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais
5.
FASEB J ; 35(9): e21862, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34416035

RESUMO

Loss of muscle mass and strength after disuse followed by impaired muscle recovery commonly occurs with aging. Metformin (MET) and leucine (LEU) individually have shown positive effects in skeletal muscle during atrophy conditions but have not been evaluated in combination nor tested as a remedy to enhance muscle recovery following disuse atrophy in aging. The purpose of this study was to determine if a dual treatment of metformin and leucine (MET + LEU) would prevent disuse-induced atrophy and/or promote muscle recovery in aged mice and if these muscle responses correspond to changes in satellite cells and collagen remodeling. Aged mice (22-24 months) underwent 14 days of hindlimb unloading (HU) followed by 7 or 14 days of reloading (7 or 14 days RL). MET, LEU, or MET + LEU was administered via drinking water and were compared to Vehicle (standard drinking water) and ambulatory baseline. We observed that during HU, MET + LEU resolved whole body grip strength and soleus muscle specific force decrements caused by HU. Gastrocnemius satellite cell abundance was increased with MET + LEU treatment but did not alter muscle size during disuse or recovery conditions. Moreover, MET + LEU treatment alleviated gastrocnemius collagen accumulation caused by HU and increased collagen turnover during 7 and 14 days RL driven by a decrease in collagen IV content. Transcriptional pathway analysis revealed that MET + LEU altered muscle hallmark pathways related to inflammation and myogenesis during HU. Together, the dual treatment of MET and LEU was able to increase muscle function, satellite cell content, and reduce collagen accumulation, thus improving muscle quality during disuse and recovery in aging.


Assuntos
Envelhecimento , Colágeno/metabolismo , Leucina/uso terapêutico , Metformina/uso terapêutico , Músculo Esquelético/efeitos dos fármacos , Atrofia Muscular/prevenção & controle , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Fibrose/tratamento farmacológico , Elevação dos Membros Posteriores , Imunoglobulina G/análise , Leucina/farmacologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Força Muscular/efeitos dos fármacos , Músculo Esquelético/citologia , Músculo Esquelético/patologia , Atrofia Muscular/patologia , Tamanho do Órgão/efeitos dos fármacos , RNA-Seq , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/patologia , Transdução de Sinais/efeitos dos fármacos
6.
Gene ; 802: 145869, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34352298

RESUMO

Skeletal myoblasts are activated satellite cells capable of proliferation and differentiation. Studies on mammalian myoblast differentiation and myogenesis could be carried out in vitro thanks to the availability of mouse myoblast cell line C2C12. Lacking of muscle cell line hinders the studies of teleost fish myogenesis. Here, we established a continuous skeletal muscle cell line from juvenile rockfish (Sebastes schlegelii) muscle using explant method and subcultured more than 50 passages for over 150 days. Stable expression of myoblast-specific marker, MyoD (myoblast determination protein) and the potential of differentiation into multi-nucleated skeletal myotubes upon induction suggested the cell line were predominately composed of myoblasts. Transcriptome analysis revealed a total of 4375 genes differentially expressed at four time points after the switch to differentiation medium, which were mainly involved in proliferation and differentiation of myoblasts. KIF22 (kinesin family member 22) and POLA1 (DNA polymerase alpha 1) were identified as the key genes involved in fish myoblast proliferation whereas MYL3 (myosin light chain 3) and TNNT2 (troponin T2) were determined as the crucial genes responsible for differentiation. In all, the continuous myoblasts cultured in this study provided a cell platform for future studies on marine fish myoblast differentiation and myogenesis. The molecular process of myoblast differentiation revealed in this study will open a window into the understanding of indeterminate muscle growth of large teleost.


Assuntos
Técnicas de Cultura de Células , Linhagem Celular , Desenvolvimento Muscular/genética , Mioblastos Esqueléticos/fisiologia , Perciformes/anatomia & histologia , Animais , Criopreservação , Transcriptoma
7.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445494

RESUMO

Despite significant advances in treatment of acute coronary syndromes (ACS) many subjects still develop heart failure due to significantly reduced ejection fraction. Currently, there are no commonly available treatment strategies that replace the infarcted/dysfunctional myocardium. Therefore, understanding the mechanisms that control the regeneration of the heart muscle is important. The development of new coronary vessels plays a pivotal role in cardiac regeneration. Employing microarray expression assays and RT-qPCR validation expression pattern of genes in long-term primary cultured cells isolated form the right atrial appendage (RAA) and right atrium (RA) was evaluated. After using DAVID software, it indicated the analysis expression profiles of genes involved in ontological groups such as: "angiogenesis", "blood vessel morphogenesis", "circulatory system development", "regulation of vasculature development", and "vasculature development" associated with the process of creation new blood vessels. The performed transcriptomic comparative analysis between two different compartments of the heart muscle allowed us to indicate the presence of differences in the expression of key transcripts depending on the cell source. Increases in culture intervals significantly increased expression of SFRP2, PRRX1 genes and some other genes involved in inflammatory process, such as: CCL2, IL6, and ROBO1. Moreover, the right atrial appendage gene encoding lysyl oxidase (LOX) showed much higher expression compared to the pre-cultivation state.


Assuntos
Vasos Coronários/crescimento & desenvolvimento , Perfilação da Expressão Gênica/métodos , Desenvolvimento Muscular , Miocárdio/citologia , Animais , Células Cultivadas , Vasos Coronários/química , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Miocárdio/química , Análise de Sequência com Séries de Oligonucleotídeos , Cultura Primária de Células , Suínos , Sequenciamento Completo do Exoma
8.
Anim Genet ; 52(5): 598-607, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34350996

RESUMO

Fat deposition is an important economic trait in farm animals. However, it is difficult to genetically improve intramuscular fat deposition via trait-based cattle breeding. The main objectives of this study were to analyze the factors about beef flavor, and to detect functional microRNA (miRNA, miR) associated with intramuscular fat deposition in Yanbian cattle. Longissimus dorsi samples from six steers were separated into high- and low-fat groups (n = 3 each) based on the marbling score, and transcriptomic analysis was performed using miRNA sequencing. A total of 33 miRNAs and 38 genes were found to be differentially expressed in the high- and low-fat groups. Quantitative real-time polymerase chain reaction was performed to validate the sequencing results. Integrated miRNA-mRNA analysis revealed that miRNA-associated target genes were primarily associated with skeletal muscle development. However, some of the miRNAs (miR-424 etc.) and genes (ATF3 etc.) were also associated with fat metabolism. A targeted relationship between miR-22-3p and the WFIKKN2 gene and its involvement in adipocyte differentiation were confirmed experimentally. The study findings may provide potential candidate molecular targets for the selection of cattle with improved meat quality.


Assuntos
Bovinos/genética , Metabolismo dos Lipídeos/genética , MicroRNAs/genética , Desenvolvimento Muscular/genética , RNA Mensageiro/genética , Adipócitos , Animais , Células Cultivadas , Masculino , Transcriptoma
9.
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
10.
BMC Genomics ; 22(1): 593, 2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34348644

RESUMO

BACKGROUND: The mutation of insulin-like growth factor 2 (IGF2 mutation) that a single-nucleotide substitution (G→A) in the third intron of IGF2 abrogates the interaction with zinc finger BED-type containing 6 (ZBED6) and leads to increased muscle mass in pigs. IGF2 mutation knock-in (IGF2 KI) and ZBED6 knockout (ZBED6 KO) lead to changes in IGF2 expression and increase muscle mass in mice and pigs. Long noncoding RNAs (lncRNAs) may participate in numerous biological processes, including skeletal muscle development. However, the role of the ZBED6-lncRNA axis in skeletal muscle development is poorly characterized. RESULTS: In this study, we assembled transcriptomes using RNA-seq data published in previous studies by our group and identified 11,408 known lncRNAs and 2269 potential lncRNAs in seven tissues, heart, longissimus dorsi, gastrocnemius muscle, liver, spleen, lung and kidney, of ZBED6 KO (lean mass model) and WT Bama pigs. ZBED6 affected the expression of 1570 lncRNAs (differentially expressed lncRNAs [DE-lncRNAs]; log2-fold change ≥ 1, nominal p-value ≤ 0.05) in the seven examined tissues. The expressed lncRNAs (FPKM > 0.1) exhibited tissue-specific patterns in WT pigs. Specifically, 3410 lncRNAs were expressed exclusively in only one tissue. Potential functions of lncRNAs were indirectly predicted by searching their target cis- and trans-regulated protein-coding genes. LncRNAs with tissue-specific expression influence numerous genes related to tissue functions. Weighted gene coexpression network analysis (WGCNA) of 1570 DE-lncRNAs between WT and ZBED6 KO pigs was used to define the following six lncRNA modules specific to different tissues: skeletal muscle, heart, lung, spleen, kidney and liver modules. Furthermore, by conjoint analysis of longissimus dorsi data (tissue-specific expression, muscle module and DE-lncRNAs) and ChIP-PCR revealed NONSUSG002145.1 (adjusted p-values = 0.044), which is coexpressed with the IGF2 gene and binding with ZBED6, may play important roles in ZBED6 KO pig skeletal muscle development. CONCLUSIONS: These findings indicate that the identified lncRNAs may play essential roles in tissue function and regulate the mechanism of ZBED6 action in skeletal muscle development in pigs. To our knowledge, this is the first study describing lncRNAs in ZBED6 KO pigs. These results may open new research directions leading to a better understanding of the global functions of ZBED6 and of lncRNA functions in skeletal muscle development in pigs.


Assuntos
RNA Longo não Codificante , Animais , Íntrons , Camundongos , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , RNA Longo não Codificante/genética , Proteínas Repressoras/genética , Suínos/genética , Transcriptoma
11.
Int J Mol Sci ; 22(16)2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34445082

RESUMO

Cultured meat is an emerging alternative food technology which aims to deliver a more ethical, sustainable, and healthy muscle-tissue-derived food item compared to conventional meat. As start-up companies are rapidly forming and accelerating this technology, many aspects of this multi-faceted science have still not been investigated in academia. In this study, we investigated if bovine satellite cells with the ability to proliferate and undergo myogenic differentiation could be isolated after extended tissue storage, for the purpose of increasing the practicality for cultured meat production. Proliferation of bovine satellite cells isolated on the day of arrival or after 2 and 5 days of tissue storage were analyzed by metabolic and DNA-based assays, while their myogenic characteristics were investigated using RT-qPCR and immunofluorescence. Extended tissue storage up to 5 days did not negatively affect proliferation nor the ability to undergo fusion and create myosin heavy chain-positive myotubes. The expression patterns of myogenic and muscle-specific genes were also not affected after tissue storage. In fact, the data indicated a positive trend in terms of myogenic potential after tissue storage, although it was non-significant. These results suggest that the timeframe of which viable myogenic satellite cells can be isolated and used for cultured meat production can be greatly extended by proper tissue storage.


Assuntos
Bovinos , Desenvolvimento Muscular , Carne Vermelha , Células Satélites de Músculo Esquelético/citologia , Animais , Bovinos/metabolismo , Células Cultivadas , Indústria Alimentícia/métodos , Carne Vermelha/provisão & distribuição , Células Satélites de Músculo Esquelético/metabolismo , Técnicas de Cultura de Tecidos/métodos
12.
Int J Pharm ; 606: 120841, 2021 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-34216768

RESUMO

Recapitulation of in vivo environments that drive muscle cells to organize into a physiologically relevant 3D architecture remains a major challenge for muscle tissue engineering. To recreate electrophysiology of muscle tissues, electroactive biomaterials have been used to stimulate muscle cells with exogenous electrical fields. In particular, the use of electroactive biomaterials with an anisotropic micro-/nanostructure that closely mimic the native skeletal-muscle extracellular matrix (ECM) is desirable for skeletal muscle tissue engineering. Herein, we present a hierarchically organized, anisotropic, and conductive Polycaprolactone/gold (PCL/Au) scaffold for guiding myoblasts alignment and promoting the elongation and maturation of myotubes under electrical stimulation. Culturing with H9c2 myoblasts cells indicated that the nanotopographic cues was crucial for nuclei alignment, while the presence of microscale grooves effectively enhanced both the formation and elongation of myotubes. The anisotropic structure also leads to anisotropic conductivity. Under electrical stimulation, the elongation and maturation of myotubes were significantly enhanced along the anisotropic scaffold. Specifically, compared to the unstimulated group (0 V), the myotube area percentage increased by 1.4, 1.9 and 2.4 times in the 1 V, 2 V, 3 V groups, respectively. In addition, the myotube average length in the 1 V group increased by 1.3 times compared to that of the unstimulated group, and significantly increased by 1.8 and 2.0 times in the 2 V, 3 V groups, respectively. Impressively, the longest myotubes reached more than 4 mm in both 2 V and 3 V groups. Overall, our conductive, anisotropic 3D nano/microfibrous scaffolds with the application of electrical stimulation provides a desirable platform for skeletal muscle tissue engineering.


Assuntos
Desenvolvimento Muscular , Tecidos Suporte , Diferenciação Celular , Fibras Musculares Esqueléticas , Músculo Esquelético , Engenharia Tecidual
13.
Biomaterials ; 275: 120973, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34224984

RESUMO

Skeletal muscle stem cells (MuSCs) are essential for efficacious muscle repair, making MuSCs promising therapeutic targets for tissue engineering and regenerative medicine. MuSCs are presented with a diverse and temporally defined set of cues from their microenvironment during regeneration that direct stem cell expansion, differentiation, and return to quiescence. Understanding the complex interplay among these biophysical and biochemical cues is necessary to develop therapies targeting or employing MuSCs. To probe the role of mechanical cues presented by the extracellular matrix, we leverage chemically defined hydrogel substrates with controllable stiffness and adhesive ligand composition to characterize the MuSC response to matrix cues presented during early and late phases of regeneration. We demonstrate that relatively soft hydrogels recapitulating healthy muscle stiffness promote MuSC activation and expansion, while relatively stiff hydrogels impair MuSC proliferation and arrest myogenic progression. These effects are seen on soft and stiff hydrogels presenting laminin-111 and exacerbated on hydrogels presenting RGD adhesive peptides. Soluble factors present in the MuSC niche during different phases of regeneration, prostaglandin E2 and oncostatin M, synergize with matrix-presented cues to enhance stem cell expansion on soft substrates and block myogenic progression on stiff substrates. To determine if temporally varied matrix stiffness reminiscent of the regenerating microenvironment alters MuSC fate, we developed a photoresponsive hydrogel system with accelerated reaction kinetics that can be rapidly softened on demand. MuSCs cultured on these materials revealed that the cellular response to a stiff microenvironment is fixed within the first three days of culture, as subsequent softening back to a healthy stiffness did not rescue MuSC proliferation or myogenic progression. These results highlight the importance of temporally controlled biophysical and biochemical cues in regulating MuSC fate that can be harnessed to improve regenerative medicine approaches to restore skeletal muscle tissue.


Assuntos
Sinais (Psicologia) , Mioblastos , Diferenciação Celular , Hidrogéis , Desenvolvimento Muscular , Músculo Esquelético , Regeneração , Nicho de Células-Tronco
14.
J Vet Med Sci ; 83(9): 1369-1377, 2021 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-34248106

RESUMO

Maternal obesity and diabetes are known to be involved in fetal myogenesis, but the later stages of myogenesis are not well understood. In this study, we investigated the influence of a hyperglycemic environment on L6 skeletal myoblast differentiation and the function of omega-7 palmitoleic acids. Exposure to a high concentration of glucose (25 mM) in high-glucose culture medium (HG) increased the expression of myogenic genes (MyoD, Myogenin, MRF4, Myhc2x, and Myhc2a) and the synthesis of myosin. HG also activated the PI3K/AKT pathway revealed muscle cell differentiation. Furthermore, the levels of reactive oxygen species (ROS) and an inflammatory cytokine (Tnfaip3; tumor necrosis factor alpha-induced protein 3), which are crucial for the growth and differentiation of skeletal muscle, were increased by HG. Palmitoleic acids suppressed the expression levels of myogenic regulatory genes and increased the expression level of a cell proliferation-related gene (Pax3). Trans-palmitoleic acid and eicosapentaenoic acid (TPA and EPA) increased the phosphorylation level of MAPK/ERK1/2 and downregulated ROS generation and Tnfaip3 expression. In contrast, cis-palmitoleic acid inactivated MAPK/ERK1/2, leading to increased ROS generation. In conclusion, a hyperglycemic environment mediated by HG induced excessive muscle differentiation. Palmitoleic acids inhibited myoblast differentiation by downregulating muscle-specific genes. Moreover, trans-palmitoleic acids may have beneficial antioxidant and/or anti-inflammatory effects in cells.


Assuntos
Desenvolvimento Muscular , Fosfatidilinositol 3-Quinases , Animais , Diferenciação Celular , Ácidos Graxos Monoinsaturados , Feminino , Músculo Esquelético , Gravidez
15.
Methods Mol Biol ; 2319: 51-60, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331242

RESUMO

Cardiovascular disease is a worldwide health issue that affects millions of lives every year, and thus, researchers are in need of high-throughput model systems with which to investigate mechanisms of disease and to develop and test potential therapies. The use of human-derived induced pluripotent stem cells (iPSCs) differentiated into cardiomyocytes aims to address this need. While cardiac differentiation protocols have been established previously in iPSCs, optimization of cardiac differentiation remains crucial to obtaining high quality cardiomyocytes for future experimental analyses. Important factors to consider include cell density and rate of proliferation, temporal regulation of media changes throughout the differentiation process, and the concentration of the chemicals utilized. In this chapter, we present a detailed protocol to outline the process of differentiating cardiomyocytes from human iPSCs via modulation of Wnt signaling, characterization of cardiomyocytes by immunofluorescence, as well as guidelines for troubleshooting and optimizing these techniques.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Células-Tronco Pluripotentes Induzidas/citologia , Desenvolvimento Muscular , Miócitos Cardíacos/citologia , Via de Sinalização Wnt , Imunofluorescência , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo
16.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198655

RESUMO

A decline in the body's motor functions has been linked to decreased muscle mass and function in the oral cavity and throat; however, aging of the junctions of the muscles and bones has also been identified as an associated factor. Basic and clinical studies on the muscles, tendons and bones, each considered independently, have been published. In recent years, however, research has focused on muscle attachment as the muscle-tendon-bone complex from various perspectives, and there is a growing body of knowledge on SRY-box9 (Sox9) and Mohawk(Mkx), which has been identified as a common controlling factor and a key element. Myostatin, a factor that inhibits muscle growth, has been identified as a potential key element in the mechanisms of lifetime structural maintenance of the muscle-tendon-bone complex. Findings in recent studies have also uncovered aspects of the mechanisms of motor organ complex morphostasis in the superaged society of today and will lay the groundwork for treatments to prevent motor function decline in older adults.


Assuntos
Desenvolvimento Ósseo/fisiologia , Morfogênese , Desenvolvimento Muscular/fisiologia , Tendões/crescimento & desenvolvimento , Animais , Pesquisa Biomédica , Humanos , Proteínas Musculares/metabolismo
17.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204426

RESUMO

The last decade has witnessed the identification of several families affected by hereditary non-syndromic hearing loss (NSHL) caused by mutations in the SMPX gene and the loss of function has been suggested as the underlying mechanism. In the attempt to confirm this hypothesis we generated an Smpx-deficient zebrafish model, pointing out its crucial role in proper inner ear development. Indeed, a marked decrease in the number of kinocilia together with structural alterations of the stereocilia and the kinocilium itself in the hair cells of the inner ear were observed. We also report the impairment of the mechanotransduction by the hair cells, making SMPX a potential key player in the construction of the machinery necessary for sound detection. This wealth of evidence provides the first possible explanation for hearing loss in SMPX-mutated patients. Additionally, we observed a clear muscular phenotype consisting of the defective organization and functioning of muscle fibers, strongly suggesting a potential role for the protein in the development of muscle fibers. This piece of evidence highlights the need for more in-depth analyses in search for possible correlations between SMPX mutations and muscular disorders in humans, thus potentially turning this non-syndromic hearing loss-associated gene into the genetic cause of dysfunctions characterized by more than one symptom, making SMPX a novel syndromic gene.


Assuntos
Orelha Interna/embriologia , Orelha Interna/metabolismo , Proteínas Musculares/deficiência , Músculos/embriologia , Músculos/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Desenvolvimento Embrionário , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Células Ciliadas Auditivas/metabolismo , Mecanotransdução Celular/genética , Desenvolvimento Muscular/genética , Organogênese/genética , Fenótipo , Transporte Proteico
18.
DNA Cell Biol ; 40(9): 1167-1176, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34255539

RESUMO

Skeletal muscle has great plasticity. An increase in protein degradation can cause muscle atrophy. Atrogin-1 and muscle ring finger-1 (MuRF1) are dramatically upregulated in various muscle atrophy. Inhibition of Atrogin-1 and MuRF1 protects against muscle atrophy. MiR-29 plays an important regulatory role in skeletal muscle development. However, the function of miR-29 in skeletal muscle protein metabolism is not clear. To investigate the function of miR-29, we generated miR-29 knockout mice and the miR-29ab1 cluster overexpression mice. The disruption of miR-29 led to severe atrophy of skeletal muscle during puberty, and the muscle-specific overexpression of the miR-29ab1 cluster protected against denervation-induced and fasting-induced muscle atrophy. Furthermore, the overexpression of miR-29a, b mimics in myotubes resisted the muscle atrophy. MuRF1 was the direct target gene of miR-29a, b. These results demonstrate that miR-29ab1 cluster plays a critical role in the maintenance of skeletal muscle. MiR-29ab1 cluster is the excellent inhibitor of MuRF1, ultimately indicating that miR-29ab1 cluster is good therapeutic molecule candidate for adulthood.


Assuntos
MicroRNAs/fisiologia , Desenvolvimento Muscular , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Mioblastos
19.
Nucleic Acids Res ; 49(14): 8060-8077, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34289068

RESUMO

Skeletal muscle regeneration is mediated by myoblasts that undergo epigenomic changes to establish the gene expression program of differentiated myofibers. mSWI/SNF chromatin remodeling enzymes coordinate with lineage-determining transcription factors to establish the epigenome of differentiated myofibers. Bromodomains bind to acetylated lysines on histone N-terminal tails and other proteins. The mutually exclusive ATPases of mSWI/SNF complexes, BRG1 and BRM, contain bromodomains with undefined functional importance in skeletal muscle differentiation. Pharmacological inhibition of mSWI/SNF bromodomain function using the small molecule PFI-3 reduced differentiation in cell culture and in vivo through decreased myogenic gene expression, while increasing cell cycle-related gene expression and the number of cells remaining in the cell cycle. Comparative gene expression analysis with data from myoblasts depleted of BRG1 or BRM showed that bromodomain function was required for a subset of BRG1- and BRM-dependent gene expression. Reduced binding of BRG1 and BRM after PFI-3 treatment showed that the bromodomain is required for stable chromatin binding at target gene promoters to alter gene expression. Our findings demonstrate that mSWI/SNF ATPase bromodomains permit stable binding of the mSWI/SNF ATPases to promoters required for cell cycle exit and establishment of muscle-specific gene expression.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Cromatina/genética , DNA Helicases/genética , Desenvolvimento Muscular/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Adenosina Trifosfatases/genética , Animais , Compostos Azabicíclicos/farmacologia , Diferenciação Celular/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Histonas/genética , Humanos , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Piridinas/farmacologia , Fatores de Transcrição/antagonistas & inibidores
20.
Am J Physiol Regul Integr Comp Physiol ; 321(3): R352-R363, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34287074

RESUMO

Fetal skeletal muscle growth requires myoblast proliferation, differentiation, and fusion into myofibers in addition to protein accretion for fiber hypertrophy. Oxygen is an important regulator of this process. Therefore, we hypothesized that fetal anemic hypoxemia would inhibit skeletal muscle growth. Studies were performed in late-gestation fetal sheep that were bled to anemic and therefore hypoxemic conditions beginning at ∼125 days of gestation (term = 148 days) for 9 ± 0 days (n = 19) and compared with control fetuses (n = 16). A metabolic study was performed on gestational day ∼134 to measure fetal protein kinetic rates. Myoblast proliferation and myofiber area were determined in biceps femoris (BF), tibialis anterior (TA), and flexor digitorum superficialis (FDS) muscles. mRNA expression of muscle regulatory factors was determined in BF. Fetal arterial hematocrit and oxygen content were 28% and 52% lower, respectively, in anemic fetuses. Fetal weight and whole body protein synthesis, breakdown, and accretion rates were not different between groups. Hindlimb length, however, was 7% shorter in anemic fetuses. TA and FDS muscles weighed less, and FDS myofiber area was smaller in anemic fetuses compared with controls. The percentage of Pax7+ myoblasts that expressed Ki67 was lower in BF and tended to be lower in FDS from anemic fetuses indicating reduced myoblast proliferation. There was less MYOD and MYF6 mRNA expression in anemic versus control BF consistent with reduced myoblast differentiation. These results indicate that fetal anemic hypoxemia reduced muscle growth. We speculate that fetal muscle growth may be improved by strategies that increase oxygen availability.


Assuntos
Proliferação de Células/fisiologia , Desenvolvimento Fetal/fisiologia , Hipóxia/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Animais , Feminino , Retardo do Crescimento Fetal/metabolismo , Feto/metabolismo , Membro Posterior/metabolismo , Desenvolvimento Muscular/fisiologia , Ovinos
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