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1.
Eur Phys J E Soft Matter ; 45(5): 44, 2022 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-35532848

RESUMO

We present an in-depth investigation of a fully automated Fourier-based analysis to determine the cell size and the width of its distribution in 3D biological tissues. The results are thoroughly tested using generated images, and we offer valuable criteria for image acquisition settings to optimize accuracy. We demonstrate that the most important parameter is the number of cells in the field of view, and we show that accurate measurements can already be made on volume only containing [Formula: see text] cells. The resolution in z is also not so important, and a reduced number of in-depth images, of order of one per cell, already provides a measure of the mean cell size with less than 5% error. The technique thus appears to be a very promising tool for very fast live local volume cell measurement in 3D tissues in vivo while strongly limiting photobleaching and phototoxicity issues.


Assuntos
Processamento de Imagem Assistida por Computador , Tamanho Celular , Análise de Fourier , Processamento de Imagem Assistida por Computador/métodos
2.
J R Soc Interface ; 19(190): 20220026, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35537474

RESUMO

Although the polygonal shape of epithelial cells has been drawing the attention of scientists for several centuries, only a decade and a half ago it was demonstrated that distributions of polygon types (DOPTs) are similar in proliferative epithelia of many different plant and animal species. In this study, we show that hyper-proliferation of cancer cells disrupts this universal paradigm and results in randomly organized epithelial structures. Examining non-synchronized and synchronized HeLa cervix cells, we suppose that the spread of cell sizes is the main parameter controlling the DOPT in the cancer cell monolayers. To test this hypothesis, we develop a theory of morphologically similar random polygonal packings. By analysing differences between tumoural and normal epithelial cell monolayers, we conclude that the latter have more ordered structures because of their lower proliferation rates and, consequently, more effective relaxation of mechanical stress associated with cell division and growth. To explain the structural features of normal proliferative epithelium, we take into account the spread of cell sizes in the monolayer. The proposed theory also rationalizes some highly ordered unconventional post-mitotic epithelia.


Assuntos
Células Epiteliais , Neoplasias , Animais , Divisão Celular , Tamanho Celular , Epitélio , Estresse Mecânico
3.
PLoS One ; 17(4): e0267207, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35442970

RESUMO

Physical parameters of the pathogenic cells, like its volume, shape, and stiffness, are important biomarkers for diseases, chemical changes within the cell, and overall cell health. The response of pathogenic bacteria and viruses to different chemical disinfectants is studied widely. Some of the routinely employed techniques to measure these changes require elaborate and expensive equipment which limits any study to a non-mobile research lab facility. Recently, we showed a micropore-based electro-fluidic technique to have great promise in measuring subtle changes in cell volumes at high throughput and resolution. This method, however, requires commercial amplifiers, which makes this technique expensive and incompatible for in-field use. In this paper, we develop a home-built amplifier to make this technique in-field compatible and apply it to measure changes in bacterial volumes upon exposure to alcohol. First, we introduce our low-cost and portable transimpedance amplifier and characterize the maximum range, absolute error percentage, and RMS noise of the amplifier in the measured current signal, along with the amplifier's bandwidth, and compared these characteristics with the commercial amplifiers. Using our home-built amplifier, we demonstrate a high throughput detection of ~1300 cells/second and resolve cell diameter changes down to 1 µm. Finally, we demonstrate measurement of cell volume changes in E. coli bacteria when exposed to ethanol (5% v/v), which is otherwise difficult to measure via imaging techniques. Our low-cost amplifier (~100-fold lower than commercial alternatives) is battery-run, completely portable for point-of-care applications, and the electro-fluidic devices are currently being tested for in-field applications.


Assuntos
Escherichia coli , Sistemas Automatizados de Assistência Junto ao Leito , Amplificadores Eletrônicos , Tamanho Celular , Fontes de Energia Elétrica
4.
Biophys J ; 121(9): 1643-1659, 2022 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-35378081

RESUMO

Caveola membrane structures harbor mechanosensitive chloride channels (MCCs; including chloride channel 2, chloride channel 3, and SWELL1, also known as LRRC8A) that form a swelling-activated chloride current (ICl,swell) and play an important role in cell volume regulation and mechanoelectrical signal transduction. However, the role of the muscle-specific caveolar scaffolding protein caveolin-3 (Cav3) in regulation of MCC expression, activity, and contribution to membrane integrity in response to mechanical stress remains unclear. Here we showed that Cav3-transfected (Cav3-positive) HEK293 cells were significantly resistant to extreme (<20 milliosmole) hypotonic swelling compared with native (Cav3-negative) HEK293 cells; the percentage of cells with membrane damage decreased from 45% in Cav3-negative cells to 17% in Cav3-positive cells (p < 0.05). This mechanoprotection was significantly reduced (p < 0.05) when cells were exposed to the ICl,swell-selective inhibitor 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (10 µM). These results were recapitulated in isolated mouse ventricular myocytes, where the percentage of cardiomyocytes with membrane damage increased from 47% in control cells to 78% in 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid-treated cells (p < 0.05). A higher resistance to hypotonic swelling in Cav3-positive HEK293 cells was accompanied by a significant twofold increase of ICl,swell current density and SWELL1 protein expression, whereas ClC-2/3 protein levels remained unchanged. Förster resonance energy transfer analysis showed a less than 10-nm membrane and intracellular association between Cav3 and SWELL1. Cav3/SWELL1 membrane Förster resonance energy transfer efficiency was halved in mild (220 milliosmole) hypotonic solution as well as after disruption of caveola structures via cholesterol depletion by 1-h treatment with 10 mM methyl-ß-cyclodextrin. A close association between Cav3 and SWELL1 was confirmed by co-immunoprecipitation analysis. Our findings indicate that, in the MCCs tested, SWELL1 abundance and activity are regulated by Cav3 and that their association relies on membrane tension and caveola integrity. This study highlights the mechanoprotective role of Cav3, which is facilitated by complimentary SWELL1 expression and activity.


Assuntos
Caveolina 3/metabolismo , Cloretos , Animais , Ácido Butírico , Tamanho Celular , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Camundongos
5.
Adipocyte ; 11(1): 175-189, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35436409

RESUMO

Adipose tissue (AT) dysfunctions, such as adipocyte hypertrophy, macrophage infiltration and secretory adiposopathy (low plasma adiponectin/leptin, A/L, ratio), associate with metabolic disorders. However, no study has compared the relative contribution of these markers to cardiometabolic risk in women of varying age and adiposity. Body composition, regional AT distribution, lipid-lipoprotein profile, glucose homeostasis and plasma A and L levels were determined in 67 women (age: 40-62 years; BMI: 17-41 kg/m2). Expression of macrophage infiltration marker CD68 and adipocyte size were measured from subcutaneous abdominal (SCABD) and omental (OME) fat. AT dysfunction markers correlated with most lipid-lipoprotein levels. The A/L ratio was negatively associated with fasting insulinemia and HOMA-IR, while SCABD or OME adipocyte size and SCABD CD68 expression were positively related to these variables. Combination of tertiles of largest adipocyte size and lowest A/L ratio showed the highest HOMA-IR. Multiple regression analyses including these markers and TAG levels revealed that the A/L ratio was the only predictor of fasting insulinemia and HOMA-IR. The contribution of the A/L ratio was superseded by adipose cell size in the model where the latter replaced TAGs. Finally, leptinemia was a better predictor of IR than adipocyte size and the A/L ratio in our participants sample.


Assuntos
Resistência à Insulina , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Adiposidade , Adulto , Biomarcadores/metabolismo , Tamanho Celular , Feminino , Humanos , Lipídeos , Lipoproteínas/metabolismo , Masculino , Pessoa de Meia-Idade , Obesidade/metabolismo
6.
Can Vet J ; 63(4): 365-372, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35368402

RESUMO

The objective of this prospective case-control study of 125 horses with gastrointestinal tract-induced colic was to determine whether heart rate (HR) and packed cell volume (PCV) can predict surgical versus medical treatment and its short-term survival (time of discharge of the patient). Sixty-four horses were treated medically and 61 surgically (29 small intestinal and 32 large intestinal cases). At admission, both PCV and HR were higher in horses treated surgically than in horses treated medically; however, with longer duration of colic before presentation, the PCV was higher in the small intestinal surgical group only. In addition, both PCV and HR were higher, and the duration of colic was longer in non-survivors compared to survivors. Binary logistic regression demonstrated a significant association between HR and type of treatment, and both HR and PCV were predictive of survival. Simple parameters such as HR and PCV provide useful information for management of colic cases.


L'hématocrite et la fréquence cardiaque pour prédire les cas médicaux et chirurgicaux et leur survie à court terme chez les chevaux souffrant de coliques d'origine gastro-intestinale. L'objectif de cette étude prospective cas-témoins de 125 chevaux souffrant de coliques induites par le tractus gastro-intestinal était de déterminer si la fréquence cardiaque (HR) et l'hématocrite (PCV) peuvent prédire le traitement chirurgical par rapport au traitement médical et sa survie à court terme (temps de congé du patient). Soixante-quatre chevaux ont été traités médicalement et 61 chirurgicalement (29 cas d'intestin grêle et 32 cas de gros intestin). A l'admission, le PCV et le HR étaient plus élevés chez les chevaux traités chirurgicalement que chez les chevaux traités médicalement; cependant, avec une durée plus longue des coliques avant la présentation, le PCV était plus élevé uniquement dans le groupe de chirurgie de l'intestin grêle. De plus, le PCV et le HR étaient plus élevés, et la durée des coliques était plus longue chez les non-survivants que chez les survivants. La régression logistique binaire a démontré une association significative entre le HR et le type de traitement, et le HR et le PCV étaient tous deux prédictifs de la survie. Des paramètres simples tels que HR et PCV fournissent des informations utiles pour la gestion des cas de coliques.(Traduit par Dr Serge Messier).


Assuntos
Cólica , Doenças dos Cavalos , Animais , Estudos de Casos e Controles , Tamanho Celular , Cólica/cirurgia , Cólica/veterinária , Trato Gastrointestinal , Frequência Cardíaca , Doenças dos Cavalos/cirurgia , Cavalos
7.
Elife ; 112022 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-35416768

RESUMO

Mechanics has been a central focus of physical biology in the past decade. In comparison, how cells manage their size is less understood. Here, we show that a parameter central to both the physics and the physiology of the cell, its volume, depends on a mechano-osmotic coupling. We found that cells change their volume depending on the rate at which they change shape, when they spontaneously spread or when they are externally deformed. Cells undergo slow deformation at constant volume, while fast deformation leads to volume loss. We propose a mechanosensitive pump and leak model to explain this phenomenon. Our model and experiments suggest that volume modulation depends on the state of the actin cortex and the coupling of ion fluxes to membrane tension. This mechano-osmotic coupling defines a membrane tension homeostasis module constantly at work in cells, causing volume fluctuations associated with fast cell shape changes, with potential consequences on cellular physiology.


Assuntos
Actinas , Actinas/metabolismo , Membrana Celular/metabolismo , Forma Celular , Tamanho Celular , Retroalimentação , Pressão Osmótica
8.
Proc Natl Acad Sci U S A ; 119(12): e2119381119, 2022 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-35294282

RESUMO

Cytokinesis is the final step of cell division during which a contractile ring forms a furrow that partitions the cytoplasm in two. How furrow ingression is spatiotemporally regulated and how it is adapted to complex cellular environments and developmental transitions remain poorly understood. Here, we examine furrow ingression dynamics in the context of the early mouse embryo and find that cell size is a powerful determinant of furrow ingression speed during reductive cell divisions. In addition, the emergence of cell polarity and the assembly of the apical domain in outer cells locally inhibits the recruitment of cytokinesis components and thereby negatively regulates furrow ingression specifically on one side of the furrow. We show that this biasing of cytokinesis is not dependent upon cell­cell adhesion or shape but rather is cell intrinsic and is caused by a paucity of cytokinetic machinery in the apical domain. The results thus reveal that in the mouse embryo cell polarity directly regulates the recruitment of cytokinetic machinery in a cell-autonomous manner and that subcellular organization can instigate differential force generation and constriction speed in different zones of the cytokinetic furrow.


Assuntos
Polaridade Celular , Citocinese , Animais , Divisão Celular , Tamanho Celular , Desenvolvimento Embrionário , Camundongos
9.
PLoS Biol ; 20(3): e3001551, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35349578

RESUMO

Significant increases in sedimentation rate accompany the evolution of multicellularity. These increases should lead to rapid changes in ecological distribution, thereby affecting the costs and benefits of multicellularity and its likelihood to evolve. However, how genetic and cellular traits control this process, their likelihood of emergence over evolutionary timescales, and the variation in these traits as multicellularity evolves are still poorly understood. Here, using isolates of the ichthyosporean genus Sphaeroforma-close unicellular relatives of animals with brief transient multicellular life stages-we demonstrate that sedimentation rate is a highly variable and evolvable trait affected by at least 2 distinct physical mechanisms. First, we find extensive (>300×) variation in sedimentation rates for different Sphaeroforma species, mainly driven by size and density during the unicellular-to-multicellular life cycle transition. Second, using experimental evolution with sedimentation rate as a focal trait, we readily obtained, for the first time, fast settling and multicellular Sphaeroforma arctica isolates. Quantitative microscopy showed that increased sedimentation rates most often arose by incomplete cellular separation after cell division, leading to clonal "clumping" multicellular variants with increased size and density. Strikingly, density increases also arose by an acceleration of the nuclear doubling time relative to cell size. Similar size- and density-affecting phenotypes were observed in 4 additional species from the Sphaeroforma genus, suggesting that variation in these traits might be widespread in the marine habitat. By resequencing evolved isolates to high genomic coverage, we identified mutations in regulators of cytokinesis, plasma membrane remodeling, and chromatin condensation that may contribute to both clump formation and the increase in the nuclear number-to-volume ratio. Taken together, this study illustrates how extensive cellular control of density and size drive sedimentation rate variation, likely shaping the onset and further evolution of multicellularity.


Assuntos
Citocinese , Animais , Tamanho Celular , Fenótipo
11.
Dev Cell ; 57(5): 598-609.e5, 2022 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-35245444

RESUMO

Organ morphogenesis involves dynamic changes of tissue properties while cells adapt to their mechanical environment through mechanosensitive pathways. How mechanical cues influence cell behaviors during morphogenesis remains unclear. Here, we studied the formation of the zebrafish atrioventricular canal (AVC) where cardiac valves develop. We show that the AVC forms within a zone of tissue convergence associated with the increased activation of the actomyosin meshwork and cell-orientation changes. We demonstrate that tissue convergence occurs with a reduction of cell volume triggered by mechanical forces and the mechanosensitive channel TRPP2/TRPV4. Finally, we show that the extracellular matrix component hyaluronic acid controls cell volume changes. Together, our data suggest that multiple force-sensitive signaling pathways converge to modulate cell volume. We conclude that cell volume reduction is a key cellular feature activated by mechanotransduction during cardiovascular morphogenesis. This work further identifies how mechanical forces and extracellular matrix influence tissue remodeling in developing organs.


Assuntos
Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Tamanho Celular , Valvas Cardíacas/metabolismo , Mecanotransdução Celular , Morfogênese , Canais de Cátion TRPV/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
12.
Dev Cell ; 57(5): 566-568, 2022 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-35290779

RESUMO

Dramatic shape changes occur during heart morphogenesis to build a functional organ. In this issue of Developmental Cell, Vignes et al. show that formation of the cardiac valve during zebrafish heart development is associated with a decrease in cellular volume that is regulated by heart mechanics and hyaluronic acid.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Peixe-Zebra , Animais , Tamanho Celular , Coração , Valvas Cardíacas , Morfogênese , Peixe-Zebra/genética
13.
Nat Commun ; 13(1): 1660, 2022 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-35351906

RESUMO

How cell size and number are determined during organ development remains a fundamental question in cell biology. Here, we identified a GRAS family transcription factor, called SCARECROW-LIKE28 (SCL28), with a critical role in determining cell size in Arabidopsis. SCL28 is part of a transcriptional regulatory network downstream of the central MYB3Rs that regulate G2 to M phase cell cycle transition. We show that SCL28 forms a dimer with the AP2-type transcription factor, AtSMOS1, which defines the specificity for promoter binding and directly activates transcription of a specific set of SIAMESE-RELATED (SMR) family genes, encoding plant-specific inhibitors of cyclin-dependent kinases and thus inhibiting cell cycle progression at G2 and promoting the onset of endoreplication. Through this dose-dependent regulation of SMR transcription, SCL28 quantitatively sets the balance between cell size and number without dramatically changing final organ size. We propose that this hierarchical transcriptional network constitutes a cell cycle regulatory mechanism that allows to adjust cell size and number to attain robust organ growth.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ciclo Celular/genética , Tamanho Celular , Redes Reguladoras de Genes , Fatores de Transcrição/metabolismo
15.
Cell Physiol Biochem ; 56(S2): 12-30, 2022 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-35133721

RESUMO

Astrocytes are the second most abundant cell type in the central nervous system and serve various functions, many of which maintain homeostasis of the intracellular milieu in the face of constant change. In order to accomplish these important functions, astrocytes must regulate their cell volume. In astrocytes, cell volume regulation involves multiple channels and transporters, including AQP4, TRPV4, TRPM4, VRAC, Na+/K+ ATPase, NKCC1 and Kir4.1. AQP4 is a bidirectional water channel directly involved in astrocyte cell volume regulation. AQP4 also forms heteromultimeric complexes with other channels and transporters involved in cell volume regulation. TRPV4, a mechanosensitive channel in involved in osmotic regulation in various cell types, forms a complex with AQP4 to decrease cell volume in response to cell swelling. TRPM4 also forms a complex with AQP4 and SUR1 in response to injury resulting in cell swelling. Another complex forms between Na+/K+ ATPase, AQP4, and mGluR5 to regulate the perisynaptic space. NKCC1 is a co-transporter involved in cell volume increases either independently through cotransport of water or a functional interaction with AQPs. VRAC is implicated in regulatory volume decreases and may also functionally interact with AQP4. Although Kir4.1 colocalizes with AQP4, its role in cell volume regulation is debated. In diseases where fluid/electrolyte homeostasis is disturbed such as stroke, ischemic injury, inflammation, traumatic brain injury and hydrocephalus, cell volume regulation is challenged, sometimes past the point of recovery. Thus, a greater understanding of signaling pathways which regulate transport proteins as well as the functional and physical interactions that exist between transporters will provide a basis for the development of pharmaceutical targets to treat these prevalent and often devastating diseases.


Assuntos
Aquaporina 4 , Astrócitos , Tamanho Celular , Sistema Nervoso Central , Homeostase
16.
Water Res ; 212: 118127, 2022 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-35121420

RESUMO

Cyanobacteria harmful blooms can represent a major risk for public health due to potential release of toxins and other noxious compounds in the water. A continuous and high-resolution monitoring of the cyanobacteria population is required due to their rapid dynamics, which has been increasingly done using in-situ fluorescence of phycocyanin (f-PC) and chlorophyll a (f-Chl a). Appropriate in-situ fluorometers calibration is essential because f-PC and f-Chl a are affected by biotic and abiotic factors, including species composition. Measurement of f-PC and f-Chl a in mixed species assemblages during different growth phases - representative of most field conditions - has received little attention. We hypothesized that f-PC and f-Chl a of mixed assemblages of cyanobacteria may be accurately estimated if taxa composition and fluorescence characteristics are known. We also hypothesized that species with different morphologies would have different fluorescence per unit cell and biomass. We tested these hypotheses in a controlled culture experiment in which photosynthetic pigment fluorescence, chemical pigment extraction, optical density and microscopic enumeration of four common cyanobacteria species (Aphanocapsa sp, Microcystis aeruginosa, Dolichospermum circinale and Raphidiopsis raciborskii) were quantified. Both monocultures and mixed cultures were monitored from exponential to late stationary growth phases. The sum of fluorescence of individual species calculated for mixed samples was not significantly different than measured fluorescence of mixed cultures. Estimated and measured f-PC and f-Chl a of mixed cultures had higher correlations and smaller absolute median errors when estimations were based on fluorescence per biomass instead of fluorescence per cell. Largest errors were overestimations of measured fluorescence for species with different morphologies. Fluorescence per cell was significantly different among most species, while fluorescence per unit biomass was not, indicating that conversion of fluorescence to biomass reduces species-specific bias. This study presents new information on the effect of species composition on cyanobacteria fluorescence. Best practices of deployment and operation of fluorometers, and data-driven models supporting in-situ fluorometers calibration are discussed as suitable solutions to minimize taxa-specific bias in fluorescence estimates.


Assuntos
Cianobactérias , Ficocianina , Tamanho Celular , Clorofila/análise , Clorofila A , Monitoramento Ambiental , Fluorescência
17.
Elife ; 112022 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-35188101

RESUMO

The prevalence of multicellular organisms is due in part to their ability to form complex structures. How cells pack in these structures is a fundamental biophysical issue, underlying their functional properties. However, much remains unknown about how cell packing geometries arise, and how they are affected by random noise during growth - especially absent developmental programs. Here, we quantify the statistics of cellular neighborhoods of two different multicellular eukaryotes: lab-evolved 'snowflake' yeast and the green alga Volvox carteri. We find that despite large differences in cellular organization, the free space associated with individual cells in both organisms closely fits a modified gamma distribution, consistent with maximum entropy predictions originally developed for granular materials. This 'entropic' cellular packing ensures a degree of predictability despite noise, facilitating parent-offspring fidelity even in the absence of developmental regulation. Together with simulations of diverse growth morphologies, these results suggest that gamma-distributed cell neighborhood sizes are a general feature of multicellularity, arising from conserved statistics of cellular packing.


Assuntos
Evolução Molecular Direcionada , Volvox/genética , Leveduras/genética , Tamanho Celular , Filogenia , Volvox/citologia , Volvox/fisiologia , Leveduras/citologia , Leveduras/fisiologia
18.
Phys Rev Lett ; 128(4): 048103, 2022 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-35148133

RESUMO

We explore the relationship between the nonequilibrium generation of myosin-induced active stress within the F-actin cytoskeleton and the pressure-volume relationship of cellular aggregates as models of simple tissues. We find that due to active stress, aggregate surface tension depends upon its size. As a result, both pressure and cell number density depend on size and violate equilibrium assumptions. However, the relationship between them resembles an equilibrium equation of state with an effective temperature. This suggests that bulk and surface properties of aggregates balance to yield a constant average work performed by each cell on their environment in regulating tissue size. These results describe basic physical principles that govern the size of cell aggregates.


Assuntos
Citoesqueleto de Actina , Actinas , Agregação Celular , Modelos Biológicos , Miosinas , Tamanho Celular , Tensão Superficial
19.
Biochem J ; 479(3): 401-424, 2022 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-35147166

RESUMO

The extracellular signal-regulated kinase 1/2 (ERK1/2) cascade promotes cardiomyocyte hypertrophy and is cardioprotective, with the three RAF kinases forming a node for signal integration. Our aims were to determine if BRAF is relevant for human heart failure, whether BRAF promotes cardiomyocyte hypertrophy, and if Type 1 RAF inhibitors developed for cancer (that paradoxically activate ERK1/2 at low concentrations: the 'RAF paradox') may have the same effect. BRAF was up-regulated in heart samples from patients with heart failure compared with normal controls. We assessed the effects of activated BRAF in the heart using mice with tamoxifen-activated Cre for cardiomyocyte-specific knock-in of the activating V600E mutation into the endogenous gene. We used echocardiography to measure cardiac dimensions/function. Cardiomyocyte BRAFV600E induced cardiac hypertrophy within 10 d, resulting in increased ejection fraction and fractional shortening over 6 weeks. This was associated with increased cardiomyocyte size without significant fibrosis, consistent with compensated hypertrophy. The experimental Type 1 RAF inhibitor, SB590885, and/or encorafenib (a RAF inhibitor used clinically) increased ERK1/2 phosphorylation in cardiomyocytes, and promoted hypertrophy, consistent with a 'RAF paradox' effect. Both promoted cardiac hypertrophy in mouse hearts in vivo, with increased cardiomyocyte size and no overt fibrosis. In conclusion, BRAF potentially plays an important role in human failing hearts, activation of BRAF is sufficient to induce hypertrophy, and Type 1 RAF inhibitors promote hypertrophy via the 'RAF paradox'. Cardiac hypertrophy resulting from these interventions was not associated with pathological features, suggesting that Type 1 RAF inhibitors may be useful to boost cardiomyocyte function.


Assuntos
Cardiomegalia/patologia , Sistema de Sinalização das MAP Quinases/fisiologia , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas B-raf/fisiologia , Animais , Carbamatos/farmacologia , Carbamatos/toxicidade , Cardiomegalia/metabolismo , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Dimerização , Técnicas de Introdução de Genes , Insuficiência Cardíaca/patologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Mutação Puntual , Conformação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/biossíntese , Ratos , Ratos Sprague-Dawley , Sulfonamidas/farmacologia , Sulfonamidas/toxicidade
20.
Int J Mol Sci ; 23(3)2022 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-35163456

RESUMO

Dysregulated energy metabolism is a major contributor to a multitude of pathologies, including obesity and diabetes. Understanding the regulation of metabolic homeostasis is of utmost importance for the identification of therapeutic targets for the treatment of metabolically driven diseases. We previously identified the deubiquitinase OTUB1 as substrate for the cellular oxygen sensor factor-inhibiting HIF (FIH) with regulatory effects on cellular energy metabolism, but the physiological relevance of OTUB1 is unclear. Here, we report that the induced global deletion of OTUB1 in adult mice (Otub1 iKO) elevated energy expenditure, reduced age-dependent body weight gain, facilitated blood glucose clearance and lowered basal plasma insulin levels. The respiratory exchange ratio was maintained, indicating an unaltered nutrient oxidation. In addition, Otub1 deletion in cells enhanced AKT activity, leading to a larger cell size, higher ATP levels and reduced AMPK phosphorylation. AKT is an integral part of insulin-mediated signaling and Otub1 iKO mice presented with increased AKT phosphorylation following acute insulin administration combined with insulin hypersensitivity. We conclude that OTUB1 is an important regulator of metabolic homeostasis.


Assuntos
Trifosfato de Adenosina/metabolismo , Cisteína Endopeptidases/genética , Deleção de Genes , Resistência à Insulina/genética , Insulina/administração & dosagem , Oxigenases de Função Mista/metabolismo , Adenilato Quinase/metabolismo , Animais , Glicemia , Peso Corporal , Tamanho Celular , Células Cultivadas , Cisteína Endopeptidases/metabolismo , Metabolismo Energético , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Insulina/efeitos adversos , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo
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