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1.
Immunity ; 49(4): 615-626.e6, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30332629

RESUMO

Macrophages polarize into distinct phenotypes in response to complex environmental cues. We found that the nuclear receptor PPARγ drove robust phenotypic changes in macrophages upon repeated stimulation with interleukin (IL)-4. The functions of PPARγ on macrophage polarization in this setting were independent of ligand binding. Ligand-insensitive PPARγ bound DNA and recruited the coactivator P300 and the architectural protein RAD21. This established a permissive chromatin environment that conferred transcriptional memory by facilitating the binding of the transcriptional regulator STAT6 and RNA polymerase II, leading to robust production of enhancer and mRNAs upon IL-4 re-stimulation. Ligand-insensitive PPARγ binding controlled the expression of an extracellular matrix remodeling-related gene network in macrophages. Expression of these genes increased during muscle regeneration in a mouse model of injury, and this increase coincided with the detection of IL-4 and PPARγ in the affected tissue. Thus, a predominantly ligand-insensitive PPARγ:RXR cistrome regulates progressive and/or reinforcing macrophage polarization.


Assuntos
Epigênese Genética/imunologia , Epigenômica/métodos , Regulação da Expressão Gênica/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , PPAR gama/imunologia , Animais , Linhagem Celular , Células Cultivadas , Interleucina-4/imunologia , Interleucina-4/farmacologia , Ligantes , Ativação de Macrófagos/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , PPAR gama/genética , PPAR gama/metabolismo
2.
Proc Natl Acad Sci U S A ; 121(19): e2321438121, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38687782

RESUMO

Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader," a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development.


Assuntos
Sistemas CRISPR-Cas , Núcleo Celular , Edição de Genes , Terapia Genética , Músculo Esquelético , Distrofia Muscular de Duchenne , Edição de Genes/métodos , Animais , Camundongos , Músculo Esquelético/metabolismo , Núcleo Celular/metabolismo , Terapia Genética/métodos , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/genética , Humanos , Sinais de Localização Nuclear/genética , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Modelos Animais de Doenças , Mioblastos/metabolismo
3.
J Biol Chem ; 300(1): 105523, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38043799

RESUMO

Filopodia are slender cellular protrusions containing parallel actin bundles involved in environmental sensing and signaling, cell adhesion and migration, and growth cone guidance and extension. Myosin 10 (Myo10), an unconventional actin-based motor protein, was reported to induce filopodial initiation with its motor domain. However, the roles of the multifunctional tail domain of Myo10 in filopodial formation and elongation remain elusive. Herein, we generated several constructs of Myo10-full-length Myo10, Myo10 with a truncated tail (Myo10 HMM), and Myo10 containing four mutations to disrupt its coiled-coil domain (Myo10 CC mutant). We found that the truncation of the tail domain decreased filopodial formation and filopodial length, while four mutations in the coiled-coil domain disrupted the motion of Myo10 toward filopodial tips and the elongation of filopodia. Furthermore, we found that filopodia elongated through multiple elongation cycles, which was supported by the Myo10 tail. These findings suggest that Myo10 tail is crucial for promoting long filopodia.


Assuntos
Miosinas , Pseudópodes , Actinas/metabolismo , Adesão Celular , Miosinas/química , Miosinas/genética , Miosinas/metabolismo , Domínios Proteicos , Pseudópodes/genética , Pseudópodes/metabolismo , Células COS , Animais , Chlorocebus aethiops , Humanos
4.
Cell ; 141(4): 573-82, 2010 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-20478251

RESUMO

Myosin VI is the only type of myosin motor known to move toward the minus ends of actin filaments. This reversal in the direction of its movement is in part a consequence of the repositioning of its lever arm. In addition, myosin VI has a number of other specialized structural and functional adaptations that optimize performance of its unique cellular roles. Given that other classes of myosins may share some of these features, understanding the design principles of myosin VI will help guide the study of the functions of myosins that adopt similar strategies.


Assuntos
Cadeias Pesadas de Miosina/metabolismo , Actinas/metabolismo , Animais , Humanos , Modelos Moleculares , Cadeias Pesadas de Miosina/química
5.
Biochem Biophys Res Commun ; 691: 149329, 2024 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-38042035

RESUMO

The actomyosin cytoskeletal network is responsible for a variety of fundamental cellular processes. Assembly and maintenance of actin networks involve an array of associated regulatory proteins for polymerization, branching, crosslinking and contractility-driven self-organization. In this study, we make the unexpected discovery in vitro that myosin VI and myosin X, motor proteins specialized in vesicle transport and filopodia formation, are capable of crosslinking and self-organizing actin into higher-order contractile structures in the absence of other actin-associated proteins. Moreover, myosin VI alone can initiate actin elongation and branching, and assemble branched force-generating networks from crosslinked actin polymers. Additional architectural control is provided by the actin crosslinking proteins α-actinin and fascin. Our data identify critical stages of tension-mediated connectivity in network development and provide a model system for further exploration of the nonequilibrium mechanics of actomyosin self-organization.


Assuntos
Actinas , Actomiosina , Actinas/metabolismo , Actomiosina/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Citoesqueleto de Actina/metabolismo
6.
Chem Rev ; 120(1): 5-35, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31689091

RESUMO

Generating force and movement is essential for the functions of cells and organisms. A variety of molecular motors that can move on tracks within cells have evolved to serve this role. How these motors interact with their tracks and how that, in turn, leads to the generation of force and movement is key to understanding the cellular roles that these motor-track systems serve. This review is focused on the best understood of these systems, which is the molecular motor myosin that moves on tracks of filamentous (F-) actin. The review highlights both the progress and the limits of our current understanding of how force generation can be controlled by F-actin-myosin interactions. What has emerged are insights they may serve as a framework for understanding the design principles of a number of types of molecular motors and their interactions with their tracks.


Assuntos
Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Miosinas/química , Miosinas/metabolismo , Actinas/química , Actinas/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Humanos , Fenômenos Mecânicos , Modelos Moleculares , Domínios Proteicos
7.
Proc Natl Acad Sci U S A ; 115(24): 6213-6218, 2018 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-29844196

RESUMO

Myosins form a class of actin-based, ATPase motor proteins that mediate important cellular functions such as cargo transport and cell motility. Their functional cycle involves two large-scale swings of the lever arm: the force-generating powerstroke, which takes place on actin, and the recovery stroke during which the lever arm is reprimed into an armed configuration. Previous analyses of the prerecovery (postrigor) and postrecovery (prepowerstroke) states predicted that closure of switch II in the ATP binding site precedes the movement of the converter and the lever arm. Here, we report on a crystal structure of myosin VI, called pretransition state (PTS), which was solved at 2.2 Å resolution. Structural analysis and all-atom molecular dynamics simulations are consistent with PTS being an intermediate along the recovery stroke, where the Relay/SH1 elements adopt a postrecovery conformation, and switch II remains open. In this state, the converter appears to be largely uncoupled from the motor domain and explores an ensemble of partially reprimed configurations through extensive, reversible fluctuations. Moreover, we found that the free energy cost of hydrogen-bonding switch II to ATP is lowered by more than 10 kcal/mol compared with the prerecovery state. These results support the conclusion that closing of switch II does not initiate the recovery stroke transition in myosin VI. Rather, they suggest a mechanism in which lever arm repriming would be mostly driven by thermal fluctuations and eventually stabilized by the switch II interaction with the nucleotide in a ratchet-like fashion.


Assuntos
Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/metabolismo , Animais , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Suínos , Termodinâmica
8.
Trends Biochem Sci ; 41(12): 989-997, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27717739

RESUMO

How myosin interacts with actin to generate force is a subject of considerable controversy. The major debate centers on understanding at what point in force generation the inorganic phosphate is released with respect to the lever arm swing, or powerstroke. Resolving the controversy is essential for understanding how force is produced as well as the mechanisms underlying disease-causing mutations in myosin. Recent structural insights into the powerstroke have come from a high-resolution structure of myosin in a previously unseen state and from an electron cryomicroscopy (cryo-EM) 3D reconstruction of the actin-myosin-MgADP complex. Here, we argue that seemingly contradictory data from time-resolved fluorescence resonance energy transfer (FRET) studies can be reconciled, and we put forward a model for myosin force generation on actin.


Assuntos
Citoesqueleto de Actina/ultraestrutura , Actinas/química , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Miosinas/química , Fosfatos/química , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Fenômenos Biomecânicos , Domínio Catalítico , Transferência Ressonante de Energia de Fluorescência , Humanos , Mecanotransdução Celular , Miosinas/metabolismo , Fosfatos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas
9.
Am J Physiol Heart Circ Physiol ; 318(2): H378-H390, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31886717

RESUMO

Myostatin (MSTN) is a transforming growth factor (TGF)-ß superfamily member that acts as a negative regulator of muscle growth and may play a role in cardiac remodeling. We hypothesized that inhibition of activin type II receptors (ACTRII) to reduce MSTN signaling would reduce pathological cardiac remodeling in experimental heart failure (HF). C57BL/6J mice underwent left anterior descending coronary artery ligation under anesthesia to induce myocardial infarction (MI) or no ligation (sham). MI and sham animals were each randomly divided into groups (n ≥ 10 mice/group) receiving an ACTRII or ACTRII/TGFß receptor-signaling inhibiting strategy: 1) myo-Fc group (weekly 10 mg/kg Myo-Fc) or 2) Fol + TGFi group (daily 12 µg/kg follistatin plus 2 mg/kg TGFß receptor inhibitor), versus controls. ACTRII/TGFBR signaling inhibition preserved cardiac function by echocardiography and prevented an increase in brain natriuretic peptide (BNP). ACTRII/TGFBR inhibition resulted in increased phosphorylation (P) of Akt and decreased P-p38 mitogen-activated protein kinase (MAPK) in MI mice. In vitro, Akt contributed to P-SMAD2,3, P-p38, and BNP regulation in cardiomyocytes. ACTRII/TGFBR inhibition increased sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) levels and decreased unfolded protein response (UPR) markers in MI mice. ACTRII/TGFBR inhibition was associated with a decrease in cardiac fibrosis and fibrosis markers, connective tissue growth factor (CTGF), type I collagen, fibronectin, α-smooth muscle actin, and matrix metalloproteinase (MMP)-12 in MI mice. MSTN exerted a direct regulation on the UPR marker eukaryotic translation initiation factor-2α (eIf2α) in cardiomyocytes. Our study suggests that ACTRII ligand inhibition has beneficial effects on cardiac signaling and fibrosis after ischemic HF.NEW & NOTEWORTHY Activin type II receptor ligand inhibition resulted in preserved cardiac function, a decrease in cardiac fibrosis, improved SERCA2a levels, and a prevention of the unfolded protein response in mice with myocardial infarction.


Assuntos
Receptores de Activinas Tipo II/efeitos dos fármacos , Isquemia Miocárdica/tratamento farmacológico , Isquemia Miocárdica/fisiopatologia , Remodelação Ventricular/efeitos dos fármacos , Animais , Ecocardiografia , Fibrose , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Miostatina/antagonistas & inibidores , Miostatina/metabolismo , Peptídeo Natriurético Encefálico/metabolismo , Fosforilação , Resistência Física , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de Sinais/efeitos dos fármacos
10.
Artigo em Inglês | MEDLINE | ID: mdl-32081426

RESUMO

Myosin X (Myo10) has several unique design features including dimerization via an anti-parallel coiled coil and a long lever arm, which allow it to preferentially move on actin bundles. To understand the stepping behavior of single Myo10 on actin bundles, we labeled two heads of Myo10 dimers with different fluorophores. Unlike previously described for myosin V (Myo5) and VI (Myo6), which display alternating hand-over-hand stepping, Myo10 frequently took near simultaneous steps of both heads, and less frequently, 2-3 steps of one head before the other head stepped. We suggest that this behavior results from the unusual kinetic features of Myo10, in conjunction with the structural properties of the motor domain/lever arm, which will favor movement on actin bundles rather than on single filaments.

11.
Radiology ; 295(3): 616-625, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32286193

RESUMO

Background Upper extremity MRI and proton MR spectroscopy are increasingly considered to be outcome measures in Duchenne muscular dystrophy (DMD) clinical trials. Purpose To demonstrate the feasibility of acquiring upper extremity MRI and proton (1H) MR spectroscopy measures of T2 and fat fraction in a large, multicenter cohort (ImagingDMD) of ambulatory and nonambulatory individuals with DMD; compare upper and lower extremity muscles by using MRI and 1H MR spectroscopy; and correlate upper extremity MRI and 1H MR spectroscopy measures to function. Materials and Methods In this prospective cross-sectional study, MRI and 1H MR spectroscopy and functional assessment data were acquired from participants with DMD and unaffected control participants at three centers (from January 28, 2016, to April 24, 2018). T2 maps of the shoulder, upper arm, forearm, thigh, and calf were generated from a spin-echo sequence (repetition time msec/echo time msec, 3000/20-320). Fat fraction maps were generated from chemical shift-encoded imaging (eight echo times). Fat fraction and 1H2O T2 in the deltoid and biceps brachii were measured from single-voxel 1H MR spectroscopy (9000/11-243). Groups were compared by using Mann-Whitney test, and relationships between MRI and 1H MR spectroscopy and arm function were assessed by using Spearman correlation. Results This study evaluated 119 male participants with DMD (mean age, 12 years ± 3 [standard deviation]) and 38 unaffected male control participants (mean age, 12 years ± 3). Deltoid and biceps brachii muscles were different in participants with DMD versus control participants in all age groups by using quantitative T2 MRI (P < .001) and 1H MR spectroscopy fat fraction (P < .05). The deltoid, biceps brachii, and triceps brachii were affected to the same extent (P > .05) as the soleus and medial gastrocnemius. Negative correlations were observed between arm function and MRI (T2: range among muscles, ρ = -0.53 to -0.73 [P < .01]; fat fraction, ρ = -0.49 to -0.70 [P < .01]) and 1H MR spectroscopy fat fraction (ρ = -0.64 to -0.71; P < .01). Conclusion This multicenter study demonstrated early and progressive involvement of upper extremity muscles in Duchenne muscular dystrophy (DMD) and showed the feasibility of MRI and 1H MR spectroscopy to track disease progression over a wide range of ages in participants with DMD. © RSNA, 2020 Online supplemental material is available for this article.


Assuntos
Braço/diagnóstico por imagem , Perna (Membro)/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Músculo Esquelético/diagnóstico por imagem , Distrofia Muscular de Duchenne/diagnóstico por imagem , Espectroscopia de Prótons por Ressonância Magnética/métodos , Adolescente , Estudos de Casos e Controles , Criança , Estudos de Coortes , Estudos Transversais , Progressão da Doença , Estudos de Viabilidade , Humanos , Masculino , Avaliação de Resultados em Cuidados de Saúde , Estudos Prospectivos
12.
Mol Cell ; 48(1): 75-86, 2012 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-22940248

RESUMO

Myosin VI is the only known reverse-direction myosin motor. It has an unprecedented means of amplifying movements within the motor involving rearrangements of the converter subdomain at the C terminus of the motor and an unusual lever arm projecting from the converter. While the average step size of a myosin VI dimer is 30-36 nm, the step size is highly variable, presenting a challenge to the lever arm mechanism by which all myosins are thought to move. Herein, we present structures of myosin VI that reveal regions of compliance that allow an uncoupling of the lead head when movement is modeled on actin. The location of the compliance restricts the possible actin binding sites and predicts the observed stepping behavior. The model reveals that myosin VI, unlike plus-end directed myosins, does not use a pure lever arm mechanism, but instead steps with a mechanism analogous to the kinesin neck-linker uncoupling model.


Assuntos
Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/metabolismo , Actinas/química , Actinas/metabolismo , Animais , Sítios de Ligação , Fenômenos Biofísicos , Calmodulina/química , Calmodulina/metabolismo , Complacência (Medida de Distensibilidade) , Cristalografia por Raios X , Modelos Biológicos , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Miosinas/química , Miosinas/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Suínos
13.
Adv Exp Med Biol ; 1239: 7-19, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32451853

RESUMO

Directed movements on actin filaments within the cell are powered by molecular motors of the myosin superfamily. On actin filaments, myosin motors convert the energy from ATP into force and movement. Myosin motors power such diverse cellular functions as cytokinesis, membrane trafficking, organelle movements, and cellular migration. Myosin generates force and movement via a number of structural changes associated with hydrolysis of ATP, binding to actin, and release of the ATP hydrolysis products while bound to actin. Herein we provide an overview of those structural changes and how they relate to the actin-myosin ATPase cycle. These structural changes are the basis of chemo-mechanical transduction by myosin motors.


Assuntos
Miosinas/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Hidrólise , Movimento , Miosinas/metabolismo
14.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 51(2): 200-206, 2020 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-32220188

RESUMO

OBJEVTIVE: To explore the thoracic ascending aortic (TAA) pathophysiological characteristics of heterozygous mutant Myh11 R247C/+ mice under the norepinephrine-induced hypertension mode. METHODS: Female heterozygous mutant Myh11 R247C/+ and wild type Myh11 +/+ mice were selected as experimental group (HET group) and control group (WT group),respectively. The hypertensive model was induced by intraperitoneal injection of norepinephrine (NE),and TAA diameter and invasive blood pressure (Bp) data were collected dynamically in real time using high-frequency ultrasound imaging and invasive arterial blood pressure monitoring technique,so as to indirectly analyze TAA compliance of two groups of mice. At the same time,the incidences of hemothorax and TAA rupture were further analyzed by autopsy and histology. RESULTS: After injection of NE,heterozygous mice did not show a higher Bp increase percentage in systole or diastole comparing with wildtype mice. However,heterozygous mice exhibited 17% and 32% higher TAA diameter dilation percentage than wildtype ones in systole and diastole respectively. Two heterozygous mice had TAA dissection and rupture,and the incidence of hemothorax in heterozygous mice (3/5) was higher than that in wildtype (0/5). CONCLUSION: It was very likely that the altered TAA wall compliance of mutant Myh11 R247C/+ mice had led to a higher TAA dilation degree than that in wildtype,and even could be the potential reason of TAA dissection and rupture.


Assuntos
Aneurisma Roto/genética , Aorta Torácica/patologia , Aneurisma da Aorta Torácica/genética , Hipertensão , Cadeias Pesadas de Miosina/genética , Animais , Aorta , Aneurisma da Aorta Torácica/fisiopatologia , Modelos Animais de Doenças , Feminino , Hipertensão/complicações , Camundongos , Mutação , Norepinefrina
15.
Muscle Nerve ; 60(4): 464-473, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31323135

RESUMO

INTRODUCTION: Dysferlin loss-of-function mutations cause muscular dystrophy, accompanied by impaired membrane repair and muscle weakness. Growth promoting strategies including insulin-like growth factor 1 (IGF-1) could provide benefit but may cause strength loss or be ineffective. The objective of this study was to determine whether locally increased IGF-1 promotes functional muscle hypertrophy in dysferlin-null (Dysf-/- ) mice. METHODS: Muscle-specific transgenic expression and postnatal viral delivery of Igf1 were used in Dysf-/- and control mice. Increased IGF-1 levels were confirmed by enzyme-linked immunosorbent assay. Testing for skeletal muscle mass and function was performed in male and female mice. RESULTS: Muscle hypertrophy occurred in response to increased IGF-1 in mice with and without dysferlin. Male mice showed a more robust response compared with females. Increased IGF-1 did not cause loss of force per cross-sectional area in Dysf-/- muscles. DISCUSSION: We conclude that increased local IGF-1 promotes functional hypertrophy when dysferlin is absent and reestablishes IGF-1 as a potential therapeutic for dysferlinopathies.


Assuntos
Disferlina/genética , Fator de Crescimento Insulin-Like I/genética , Músculo Esquelético/metabolismo , Animais , Diafragma/metabolismo , Diafragma/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Músculo Esquelético/patologia , Distrofias Musculares/genética , Tamanho do Órgão
16.
Exp Cell Res ; 370(1): 168-173, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29928867

RESUMO

This paper describes a method to construct three-dimensional (3D) contractile human skeletal muscle tissues from a cell line. The 3D tissue was fabricated as a fiber-based structure and cultured for two weeks under tension by anchoring its both ends. While myotubes from the immortalized human skeletal myocytes used in this study never contracted in the conventional two-dimensional (2D) monolayer culture, myotubes in the 3D tissue showed spontaneous contraction at a high frequency and also reacted to the electrical stimulation. Immunofluorescence revealed that the myotubes in the 3D tissues had sarcomeres and expressed ryanodine receptor (RyR) and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). In addition, intracellular calcium oscillations in the myotubes in the 3D tissue were observed. These results indicated that the 3D culture enabled the myocyte cell line to reach a more highly matured state compared to 2D culture. Since contraction is the most significant feature of skeletal muscle, we believe that our 3D human muscle tissue with the contractile ability would be a useful tool for both basic biology research and drug discovery as one of the muscle-on-chips.


Assuntos
Fibras Musculares Esqueléticas/citologia , Músculo Esquelético/citologia , Animais , Sinalização do Cálcio/fisiologia , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Células Cultivadas , Humanos , Camundongos , Desenvolvimento Muscular/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sarcômeros/metabolismo , Sarcômeros/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Engenharia Tecidual/métodos
17.
Proc Natl Acad Sci U S A ; 113(27): E3824-33, 2016 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-27325775

RESUMO

The well-known, muscle-specific smooth muscle myosin light chain kinase (MLCK) (smMLCK) and skeletal muscle MLCK (skMLCK) are dedicated protein kinases regulated by an autoregulatory segment C terminus of the catalytic core that blocks myosin regulatory light chain (RLC) binding and phosphorylation in the absence of Ca(2+)/calmodulin (CaM). Although it is known that a more recently discovered cardiac MLCK (cMLCK) is necessary for normal RLC phosphorylation in vivo and physiological cardiac performance, information on cMLCK biochemical properties are limited. We find that a fourth uncharacterized MLCK, MLCK4, is also expressed in cardiac muscle with high catalytic domain sequence similarity with other MLCKs but lacking an autoinhibitory segment. Its crystal structure shows the catalytic domain in its active conformation with a short C-terminal "pseudoregulatory helix" that cannot inhibit catalysis as a result of missing linker regions. MLCK4 has only Ca(2+)/CaM-independent activity with comparable Vmax and Km values for different RLCs. In contrast, the Vmax value of cMLCK is orders of magnitude lower than those of the other three MLCK family members, whereas its Km (RLC and ATP) and KCaM values are similar. In contrast to smMLCK and skMLCK, which lack activity in the absence of Ca(2+)/CaM, cMLCK has constitutive activity that is stimulated by Ca(2+)/CaM. Potential contributions of autoregulatory segment to cMLCK activity were analyzed with chimeras of skMLCK and cMLCK. The constitutive, low activity of cMLCK appears to be intrinsic to its catalytic core structure rather than an autoinhibitory segment. Thus, RLC phosphorylation in cardiac muscle may be regulated by two different protein kinases with distinct biochemical regulatory properties.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Sequência de Aminoácidos , Animais , Camundongos , Conformação Molecular
18.
Proc Natl Acad Sci U S A ; 113(13): E1844-52, 2016 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-26976594

RESUMO

Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly bound, whereas myosin is strongly bound to actin. We have generated an 8-Å cryoEM reconstruction of this state for myosin V and used molecular dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The ADP-bound structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This state is accomplished by a previously unseen conformation of the ß-sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5° rotation of the myosin lever arm, coupled to a ß-sheet rearrangement. Thus, the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release that is essential for processive myosins, such as myosin V.


Assuntos
Actinas/metabolismo , Difosfato de Adenosina/metabolismo , Miosina Tipo V/química , Miosina Tipo V/metabolismo , Actinas/química , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica
19.
Lancet ; 390(10101): 1489-1498, 2017 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-28728956

RESUMO

BACKGROUND: Duchenne muscular dystrophy (DMD) is a severe, progressive, and rare neuromuscular, X-linked recessive disease. Dystrophin deficiency is the underlying cause of disease; therefore, mutation-specific therapies aimed at restoring dystrophin protein production are being explored. We aimed to assess the efficacy and safety of ataluren in ambulatory boys with nonsense mutation DMD. METHODS: We did this multicentre, randomised, double-blind, placebo-controlled, phase 3 trial at 54 sites in 18 countries located in North America, Europe, the Asia-Pacific region, and Latin America. Boys aged 7-16 years with nonsense mutation DMD and a baseline 6-minute walk distance (6MWD) of 150 m or more and 80% or less of the predicted normal value for age and height were randomly assigned (1:1), via permuted block randomisation (block size of four) using an interactive voice-response or web-response system, to receive ataluren orally three times daily (40 mg/kg per day) or matching placebo. Randomisation was stratified by age (<9 years vs ≥9 years), duration of previous corticosteroid use (6 months to <12 months vs ≥12 months), and baseline 6MWD (<350 m vs ≥350 m). Patients, parents and caregivers, investigational site personnel, PTC Therapeutics employees, and all other study personnel were masked to group allocation until after database lock. The primary endpoint was change in 6MWD from baseline to week 48. We additionally did a prespecified subgroup analysis of the primary endpoint, based on baseline 6MWD, which is reflective of anticipated rates of disease progression over 1 year. The primary analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number NCT01826487. FINDINGS: Between March 26, 2013, and Aug 26, 2014, we randomly assigned 230 patients to receive ataluren (n=115) or placebo (n=115); 228 patients comprised the intention-to-treat population. The least-squares mean change in 6MWD from baseline to week 48 was -47·7 m (SE 9·3) for ataluren-treated patients and -60·7 m (9·3) for placebo-treated patients (difference 13·0 m [SE 10·4], 95% CI -7·4 to 33·4; p=0·213). The least-squares mean change for ataluren versus placebo in the prespecified subgroups was -7·7 m (SE 24·1, 95% CI -54·9 to 39·5; p=0·749) in the group with a 6MWD of less than 300 m, 42·9 m (15·9, 11·8-74·0; p=0·007) in the group with a 6MWD of 300 m or more to less than 400 m, and -9·5 m (17·2, -43·2 to 24·2; p=0·580) in the group with a 6MWD of 400 m or more. Ataluren was generally well tolerated and most treatment-emergent adverse events were mild to moderate in severity. Eight (3%) patients (n=4 per group) reported serious adverse events; all except one event in the placebo group (abnormal hepatic function deemed possibly related to treatment) were deemed unrelated to treatment. INTERPRETATION: Change in 6MWD did not differ significantly between patients in the ataluren group and those in the placebo group, neither in the intention-to-treat population nor in the prespecified subgroups with a baseline 6MWD of less than 300 m or 400 m or more. However, we recorded a significant effect of ataluren in the prespecified subgroup of patients with a baseline 6MWD of 300 m or more to less than 400 m. Baseline 6MWD values within this range were associated with a more predictable rate of decline over 1 year; this finding has implications for the design of future DMD trials with the 6-minute walk test as the endpoint. FUNDING: PTC Therapeutics.


Assuntos
Códon sem Sentido/genética , Distrofia Muscular de Duchenne/tratamento farmacológico , Oxidiazóis/administração & dosagem , Adolescente , Criança , Método Duplo-Cego , Distrofina/deficiência , Distrofina/genética , Saúde Global , Humanos , Masculino , Distrofia Muscular de Duchenne/genética , Resultado do Tratamento , Caminhada
20.
Am J Physiol Heart Circ Physiol ; 315(5): H1463-H1476, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30141986

RESUMO

Clinical and experimental studies have suggested that the duration of left ventricular assist device (LVAD) support may affect remodeling of the failing heart. We aimed to 1) characterize the changes in Ca2+/calmodulin-dependent protein kinase type-IIδ (CaMKIIδ), growth signaling, structural proteins, fibrosis, apoptosis, and gene expression before and after LVAD support and 2) assess whether the duration of support correlated with improvement or worsening of reverse remodeling. Left ventricular apex tissue and serum pairs were collected in patients with dilated cardiomyopathy ( n = 25, 23 men and 2 women) at LVAD implantation and after LVAD support at cardiac transplantation/LVAD explantation. Normal cardiac tissue was obtained from healthy hearts ( n = 4) and normal serum from age-matched control hearts ( n = 4). The duration of LVAD support ranged from 48 to 1,170 days (median duration: 270 days). LVAD support was associated with CaMKIIδ activation, increased nuclear myocyte enhancer factor 2, sustained histone deacetylase-4 phosphorylation, increased circulating and cardiac myostatin (MSTN) and MSTN signaling mediated by SMAD2, ongoing structural protein dysregulation and sustained fibrosis and apoptosis (all P < 0.05). Increased CaMKIIδ phosphorylation, nuclear myocyte enhancer factor 2, and cardiac MSTN significantly correlated with the duration of support. Phosphorylation of SMAD2 and apoptosis decreased with a shorter duration of LVAD support but increased with a longer duration of LVAD support. Further study is needed to define the optimal duration of LVAD support in patients with dilated cardiomyopathy. NEW & NOTEWORTHY A long duration of left ventricular assist device support may be detrimental for myocardial recovery, based on myocardial tissue experiments in patients with prolonged support showing significantly worsened activation of Ca2+/calmodulin-dependent protein kinase-IIδ, increased nuclear myocyte enhancer factor 2, increased myostatin and its signaling by SMAD2, and apoptosis as well as sustained histone deacetylase-4 phosphorylation, structural protein dysregulation, and fibrosis.


Assuntos
Cardiomiopatia Dilatada/terapia , Insuficiência Cardíaca/terapia , Ventrículos do Coração/metabolismo , Coração Auxiliar , Miocárdio/metabolismo , Função Ventricular Esquerda , Apoptose , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomiopatia Dilatada/complicações , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/fisiopatologia , Estudos de Casos e Controles , Feminino , Fibrose , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/fisiopatologia , Histona Desacetilases/metabolismo , Humanos , Fatores de Transcrição MEF2/metabolismo , Masculino , Pessoa de Meia-Idade , Miostatina/metabolismo , Fosforilação , Desenho de Prótese , Recuperação de Função Fisiológica , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Fatores de Tempo , Resultado do Tratamento , Remodelação Ventricular
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