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1.
J Transl Med ; 18(1): 174, 2020 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-32306971

RESUMO

BACKGROUND: In sepsis, the endothelial barrier becomes incompetent, with the leaking of plasma into interstitial tissues. VE-cadherin, an adherens junction protein, is the gatekeeper of endothelial cohesion. Kinins, released during sepsis, induce vascular leakage and vasodilation. They act via two G-protein coupled receptors: B1 (B1R) and B2 (B2R). B1R is inducible in the presence of pro-inflammatory cytokines, endotoxins or after tissue injury. It acts at a later stage of sepsis and elicits a sustained inflammatory response. The aim of our study was to investigate the relationships between B1R and VE-cadherin destabilization in vivo in a later phase of sepsis. METHODS: Experimental, prospective study in a university research laboratory. We used a polymicrobial model of septic shock by cecal ligation and puncture in C57BL6 male mice or C57BL6 male mice that received a specific B1R antagonist (R-954). We studied the influence of B1R on sepsis-induced vascular permeability 30 h after surgery for several organs, and VE-cadherin expression in the lung and kidneys by injecting R-954 just before surgery. The 96-h survival was determined in mice without treatment or in animals receiving R-954 as a "prophylactic" regimen (a subcutaneous injection of 200 µg/kg, prior to CLP and 24 h after CLP), or as a "curative" regimen (injection of 100 µg/kg at H6, H24 and H48 post-surgery). RESULTS: B1R inactivation helps to maintain MAP above 65 mmHg but induces different permeability profiles depending on whether or not organ perfusion is autoregulated. In our model, VE-cadherin was destabilized in vivo during septic shock. At a late stage of sepsis, the B1R blockade reduced the VE-cadherin disruption by limiting eNOS activation. The survival rate for mice that received R-954 after sepsis induction was higher than in animals that received an antagonist as a prophylactic treatment. CONCLUSIONS: B1R antagonizing reduced mortality in our model of murine septic shock by limiting the vascular permeability induced by VE-cadherin destabilization through maintenance of the macrohemodynamics, consequently limiting organ dysfunctions.


Assuntos
Cininas , Sepse , Animais , Masculino , Camundongos , Estudos Prospectivos , Receptor B1 da Bradicinina , Receptor B2 da Bradicinina , Sepse/complicações , Sepse/tratamento farmacológico
2.
Nat Chem Biol ; 11(4): 271-9, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25706338

RESUMO

Hypersecretion of norepinephrine (NE) and angiotensin II (AngII) is a hallmark of major prevalent cardiovascular diseases that contribute to cardiac pathophysiology and morbidity. Herein, we explore whether heterodimerization of presynaptic AngII AT1 receptor (AT1-R) and NE α2C-adrenergic receptor (α2C-AR) could underlie their functional cross-talk to control NE secretion. Multiple bioluminescence resonance energy transfer and protein complementation assays allowed us to accurately probe the structures and functions of the α2C-AR-AT1-R dimer promoted by ligand binding to individual protomers. We found that dual agonist occupancy resulted in a conformation of the heterodimer different from that induced by active individual protomers and triggered atypical Gs-cAMP-PKA signaling. This specific pharmacological signaling unit was identified in vivo to promote not only NE hypersecretion in sympathetic neurons but also sympathetic hyperactivity in mice. Thus, we uncovered a new process by which GPCR heterodimerization creates an original functional pharmacological entity and that could constitute a promising new target in cardiovascular therapeutics.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptor Tipo 1 de Angiotensina/agonistas , Transdução de Sinais , Agonistas alfa-Adrenérgicos/química , Animais , Biofísica , Doenças Cardiovasculares/metabolismo , AMP Cíclico/metabolismo , Dimerização , Desenho de Fármacos , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Norepinefrina/química , Células PC12 , Fosforilação , Conformação Proteica , Ratos , Receptores Adrenérgicos alfa 2/química , Sistema Nervoso Simpático/efeitos dos fármacos
3.
Nat Chem Biol ; 8(7): 622-30, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22634635

RESUMO

Functional selectivity of G protein-coupled receptor (GPCR) ligands toward different downstream signals has recently emerged as a general hallmark of this receptor class. However, pleiotropic and crosstalk signaling of GPCRs makes functional selectivity difficult to decode. To look from the initial active receptor point of view, we developed new, highly sensitive and direct bioluminescence resonance energy transfer-based G protein activation probes specific for all G protein isoforms, and we used them to evaluate the G protein-coupling activity of [(1)Sar(4)Ile(8)Ile]-angiotensin II (SII), previously described as an angiotensin II type 1 (AT(1)) receptor-biased agonist that is G protein independent but ß-arrestin selective. By multiplexing assays sensing sequential signaling events, from receptor conformations to downstream signaling, we decoded SII as an agonist stabilizing a G protein-dependent AT(1A) receptor signaling module different from that of the physiological agonist angiotensin II, both in recombinant and primary cells. Thus, a biased agonist does not necessarily select effects from the physiological agonist but may instead stabilize and create a new distinct active pharmacological receptor entity.


Assuntos
Receptor Tipo 1 de Angiotensina/metabolismo , Técnicas Biossensoriais , Linhagem Celular , Proteínas de Ligação ao GTP/metabolismo , Humanos , Conformação Proteica , Receptor Tipo 1 de Angiotensina/agonistas , Receptor Tipo 1 de Angiotensina/química
4.
Circ Res ; 110(5): 688-700, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22302788

RESUMO

RATIONALE: Cardiac tissue cohesion relying on highly ordered cardiomyocytes (CM) interactions is critical because most cardiomyopathies are associated with tissue remodeling and architecture alterations. OBJECTIVE: Eph/ephrin system constitutes a ubiquitous system coordinating cellular communications which recently emerged as a major regulator in adult organs. We examined if eph/ephrin could participate in cardiac tissue cyto-organization. METHODS AND RESULTS: We reported the expression of cardiac ephrin-B1 in both endothelial cells and for the first time in CMs where ephrin-B1 localized specifically at the lateral membrane. Ephrin-B1 knock-out (KO) mice progressively developed cardiac tissue disorganization with loss of adult CM rod-shape and sarcomeric and intercalated disk structural disorganization confirmed in CM-specific ephrin-B1 KO mice. CMs lateral membrane exhibited abnormal structure by electron microscopy and notably increased stiffness by atomic force microscopy. In wild-type CMs, ephrin-B1 interacted with claudin-5/ZO-1 complex at the lateral membrane, whereas the complex disappeared in KO/CM-specific ephrin-B1 KO mice. Ephrin-B1 deficiency resulted in decreased mRNA expression of CM basement membrane components and disorganized fibrillar collagen matrix, independently of classical integrin/dystroglycan system. KO/CM-specific ephrin-B1 KO mice exhibited increased left ventricle diameter and delayed atrioventricular conduction. Under pressure overload stress, KO mice were prone to death and exhibited striking tissue disorganization. Finally, failing CMs displayed downregulated ephrin-B1/claudin-5 gene expression linearly related to the ejection fraction. CONCLUSIONS: Ephrin-B1 is necessary for cardiac tissue architecture cohesion by stabilizing the adult CM morphology through regulation of its lateral membrane. Because decreased ephrin-B1 is associated with molecular/functional cardiac defects, it could represent a new actor in the transition toward heart failure.


Assuntos
Comunicação Celular/fisiologia , Efrina-B1/fisiologia , Proteínas de Membrana/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Células Cultivadas , Colágeno/fisiologia , Colágeno/ultraestrutura , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Endotélio Vascular/ultraestrutura , Efrina-B1/deficiência , Efrina-B1/genética , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Miócitos Cardíacos/citologia , Miócitos Cardíacos/ultraestrutura , Sarcômeros/diagnóstico por imagem , Sarcômeros/fisiologia , Ultrassonografia
5.
Eur Heart J ; 33(18): 2360-9, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22028387

RESUMO

AIMS: Activation of cardiac fibroblasts and their differentiation into myofibroblasts is a key event in the progression of cardiac fibrosis that leads to end-stage heart failure. Apelin, an adipocyte-derived factor, exhibits a number of cardioprotective properties; however, whether apelin is involved in cardiac fibroblast activation and myofibroblast formation remains unknown. The aim of this study was to determine the effects of apelin in activated cardiac fibroblasts, the potential related mechanisms and impact on cardiac fibrotic remodelling process. METHODS AND RESULTS: In vitro experiments were performed in mouse cardiac fibroblasts obtained from normal and pressure-overload hearts. Pretreatment of naive cardiac fibroblasts with apelin (1-100 nM) inhibited Transforming growth factor-ß (TGF-ß)-mediated expression of the myofibroblast marker α-smooth muscle actin (α-SMA) and collagen production. Furthermore, apelin decreased the spontaneous collagen production in cardiac fibroblasts isolated from hearts after aortic banding. Knockdown strategy and pharmacological inhibition revealed that prevention of collagen accumulation by apelin was mediated by a reduction in sphingosine kinase 1 (SphK1) activity. In vivo studies using the aortic banding model indicated that pretreatment with apelin attenuated the development of myocardial fibrotic remodelling and inhibited cardiac SphK1 activity and α-SMA expression. Moreover, administration of apelin 2 weeks after aortic banding prevented cardiac remodelling by inhibiting myocyte hypertrophy, cardiac fibrosis, and ventricular dysfunction. CONCLUSION: Our data provide the first evidence that apelin inhibits TGF-ß-stimulated activation of cardiac fibroblasts through a SphK1-dependent mechanism. We also demonstrated that the administration of apelin during the phase of reactive fibrosis prevents structural remodelling of the myocardium and ventricular dysfunction. These findings may have important implications for designing future therapies for myocardial performance during fibrotic remodelling, affecting the clinical management of patients with progressive heart failure.


Assuntos
Colágeno/biossíntese , Fibroblastos/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Miócitos Cardíacos/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/farmacologia , Adipocinas , Animais , Apelina , Inibidores Enzimáticos/farmacologia , Hemodinâmica/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Fator de Crescimento Transformador beta/farmacologia , Remodelação Ventricular/fisiologia
6.
Elife ; 122023 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-36649053

RESUMO

The rod-shaped adult cardiomyocyte (CM) harbors a unique architecture of its lateral surface with periodic crests, relying on the presence of subsarcolemmal mitochondria (SSM) with unknown role. Here, we investigated the development and functional role of CM crests during the postnatal period. We found in rodents that CM crest maturation occurs late between postnatal day 20 (P20) and P60 through both SSM biogenesis, swelling and crest-crest lateral interactions between adjacent CM, promoting tissue compaction. At the functional level, we showed that the P20-P60 period is dedicated to the improvement of relaxation. Interestingly, crest maturation specifically contributes to an atypical CM hypertrophy of its short axis, without myofibril addition, but relying on CM lateral stretching. Mechanistically, using constitutive and conditional CM-specific knock-out mice, we identified ephrin-B1, a lateral membrane stabilizer, as a molecular determinant of P20-P60 crest maturation, governing both the CM lateral stretch and the diastolic function, thus highly suggesting a link between crest maturity and diastole. Remarkably, while young adult CM-specific Efnb1 KO mice essentially exhibit an impairment of the ventricular diastole with preserved ejection fraction and exercise intolerance, they progressively switch toward systolic heart failure with 100% KO mice dying after 13 months, indicative of a critical role of CM-ephrin-B1 in the adult heart function. This study highlights the molecular determinants and the biological implication of a new late P20-P60 postnatal developmental stage of the heart in rodents during which, in part, ephrin-B1 specifically regulates the maturation of the CM surface crests and of the diastolic function.


Assuntos
Efrina-B1 , Miócitos Cardíacos , Animais , Camundongos , Diástole , Miofibrilas
7.
Stem Cells ; 27(11): 2734-43, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19591227

RESUMO

Recent studies showed that mesenchymal stem cells (MSCs) transplantation significantly decreased cardiac fibrosis; however, the mechanisms involved in these effects are still poorly understood. In this work, we investigated whether the antifibrotic properties of MSCs involve the regulation of matrix metalloproteinases (MMPs) and matrix metalloproteinase endogenous inhibitor (TIMP) production by cardiac fibroblasts. In vitro experiments showed that conditioned medium from MSCs decreased viability, alpha-smooth muscle actin expression, and collagen secretion of cardiac fibroblasts. These effects were concomitant with the stimulation of MMP-2/MMP-9 activities and membrane type 1 MMP expression. Experiments performed with fibroblasts from MMP2-knockout mice demonstrated that MMP-2 plays a preponderant role in preventing collagen accumulation upon incubation with conditioned medium from MSCs. We found that MSC-conditioned medium also decreased the expression of TIMP2 in cardiac fibroblasts. In vivo studies showed that intracardiac injection of MSCs in a rat model of postischemic heart failure induced a significant decrease in ventricular fibrosis. This effect was associated with the improvement of morphological and functional cardiac parameters. In conclusion, we showed that MSCs modulate the phenotype of cardiac fibroblasts and their ability to degrade extracellular matrix. These properties of MSCs open new perspectives for understanding the mechanisms of action of MSCs and anticipate their potential therapeutic or side effects.


Assuntos
Colagenases/metabolismo , Fibroblastos/metabolismo , Fibrose/prevenção & controle , Células-Tronco Mesenquimais/fisiologia , Infarto do Miocárdio/patologia , Actinas/metabolismo , Animais , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , Meios de Cultivo Condicionados/farmacologia , Ecocardiografia , Fibroblastos/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Imuno-Histoquímica , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Knockout , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/terapia , Reação em Cadeia da Polimerase , Pontos Quânticos , Ratos , Inibidor Tecidual de Metaloproteinase-2/metabolismo
8.
J Mol Cell Cardiol ; 46(4): 518-25, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19167402

RESUMO

Platelet activation occurs in different acute and chronic heart diseases including myocardial infarction, obstructive hypertrophic cardiomyopathy and valve stenosis. Recent studies suggested that some factors secreted by activated platelets may participate in cardiac remodeling. In the present study, we investigated whether platelets and platelet-released serotonin (5-HT) are directly involved in the functional regulation of cardiac fibroblasts. Treatment of neonatal rat cardiac fibroblasts with platelet lysate, 5-HT and the 5-HT2A receptor agonist DOI increased the expression of alpha-SMA protein, a marker of fibroblast differentiation into myofibroblasts. Platelet lysate, 5-HT and DOI also induced a time-dependent stimulation of cardiac fibroblast migration that was inhibited by the 5-HT2A receptor antagonist ketanserin. Finally, incubation of cardiac fibroblasts with platelet lysate or 5-HT enhanced secretion of TGF-beta1 and expression of MMP-3 and MMP-13. As observed for fibroblast migration, these effects were prevented by ketanserin. These results demonstrated for the first time that factors released from platelet directly regulate cardiac fibroblasts by enhancing secretion of TGF-beta1 and MMPs and promoting their migration and differentiation. 5-HT released by platelets appears to be a major contributor of platelet effects which are mediated through 5-HT2A receptors.


Assuntos
Plaquetas/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Miocárdio/citologia , Receptor 5-HT2A de Serotonina/metabolismo , Serotonina/farmacologia , Actinas/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Miocárdio/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Fator de Crescimento Transformador beta1/metabolismo
9.
Stem Cells ; 26(7): 1749-57, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18467662

RESUMO

Bone marrow mesenchymal stem cells (MSCs) have shown great potential in cell therapy of solid organs. Approaches to improving the ability of grafted MSCs to survive and secrete paracrine factors represent one of the challenges for the further development of this novel therapy. In the present study, we designed a strategy of ex vivo pretreatment with the pineal hormone melatonin to improve survival, paracrine activity, and efficiency of MSCs. Using a rat model of acute renal failure, we showed that melatonin pretreatment strongly increased survival of MSCs after intraparenchymal injection. This effect was concomitant with overstimulation of angiogenesis, proliferation of renal cells, and accelerated recovery of renal function. To gain insight into the mechanisms involved in the effects observed in vivo, melatonin was tested in vitro on cultured MSCs. Our results show that through stimulation of specific melatonin receptors, melatonin induced an overexpression of the antioxidant enzyme catalase and superoxide dismutase-1 and increased the resistance of MSCs to hydrogen peroxide-dependent apoptosis. Compared with untreated cells, MSCs incubated with melatonin displayed a higher expression of basic fibroblast growth factor and hepatocyte growth factor. In addition, conditioned culture media from melatonin-treated MSCs stimulated tube formation by endothelial progenitor cells and proliferation of proximal tubule cells in culture. In conclusion, our results show that melatonin behaves as a preconditioning agent increasing survival, paracrine activity, and efficiency of MSCs. The use of this molecule for pretreatment of stem cells may represent a novel and safe approach to improving the beneficial effects of cell therapy of solid organs.


Assuntos
Células da Medula Óssea/citologia , Sobrevivência Celular/efeitos dos fármacos , Isquemia/patologia , Rim/efeitos dos fármacos , Rim/metabolismo , Melatonina/metabolismo , Células-Tronco Mesenquimais/citologia , Animais , Proliferação de Células , Fator 2 de Crescimento de Fibroblastos/biossíntese , Fator de Crescimento de Hepatócito/biossíntese , Humanos , Neovascularização Patológica , Ratos , Ratos Endogâmicos Lew , Traumatismo por Reperfusão/metabolismo
10.
Circ Res ; 100(1): 41-9, 2007 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-17158340

RESUMO

The mitochondrial enzyme monoamine oxidase (MAO), its isoform MAO-A, plays a major role in reactive oxygen species-dependent cardiomyocyte apoptosis and postischemic cardiac damage. In the current study, we investigated whether sphingolipid metabolism can account for mediating MAO-A- and reactive oxygen species-dependent cardiomyocyte apoptosis. In H9c2 cardiomyoblasts, MAO-A-dependent reactive oxygen species generation led to mitochondria-mediated apoptosis, along with sphingosine kinase-1 (SphK1) inhibition. These phenomena were associated with generation of proapoptotic ceramide and decrease in prosurvival sphingosine 1-phosphate. These events were mimicked by inhibition of SphK1 with either pharmacological inhibitor or small interfering RNA, as well as by extracellular addition of C(2)-ceramide or H(2)O(2). In contrast, enforced expression of SphK1 protected H9c2 cells from serotonin- or H(2)O(2)-induced apoptosis. Analysis of cardiac tissues from wild-type mice subjected to ischemia/reperfusion revealed significant upregulation of ceramide and inhibition of SphK1. It is noteworthy that SphK1 inhibition, ceramide accumulation, and concomitantly infarct size and cardiomyocyte apoptosis were significantly decreased in MAO-A-deficient animals. In conclusion, we show for the first time that the upregulation of ceramide/sphingosine 1-phosphate ratio is a critical event in MAO-A-mediated cardiac cell apoptosis. In addition, we provide the first evidence linking generation of reactive oxygen species with SphK1 inhibition. Finally, we propose sphingolipid metabolites as key mediators of postischemic/reperfusion cardiac injury.


Assuntos
Apoptose/fisiologia , Monoaminoxidase/metabolismo , Miócitos Cardíacos/fisiologia , Estresse Oxidativo/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Ceramidas/metabolismo , Ceramidas/farmacologia , Regulação para Baixo , Resistência a Medicamentos/fisiologia , Peróxido de Hidrogênio/farmacologia , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/fisiologia , Monoaminoxidase/deficiência , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Oxidantes/farmacologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Serotonina/farmacologia , Esfingolipídeos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Regulação para Cima
11.
Cardiovasc Res ; 115(6): 1078-1091, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30329023

RESUMO

AIMS: This study explored the lateral crest structures of adult cardiomyocytes (CMs) within healthy and diseased cardiac tissue. METHODS AND RESULTS: Using high-resolution electron and atomic force microscopy, we performed an exhaustive quantitative analysis of the three-dimensional (3D) structure of the CM lateral surface in different cardiac compartments from various mammalian species (mouse, rat, cow, and human) and determined the technical pitfalls that limit its observation. Although crests were observed in nearly all CMs from all heart compartments in all species, we showed that their heights, dictated by the subsarcolemmal mitochondria number, substantially differ between compartments from one species to another and tightly correlate with the sarcomere length. Differences in crest heights also exist between species; for example, the similar cardiac compartments in cows and humans exhibit higher crests than rodents. Unexpectedly, we found that lateral surface crests establish tight junctional contacts with crests from neighbouring CMs. Consistently, super-resolution SIM or STED-based immunofluorescence imaging of the cardiac tissue revealed intermittent claudin-5-claudin-5 interactions in trans via their extracellular part and crossing the basement membrane. Finally, we found a loss of crest structures and crest-crest contacts in diseased human CMs and in an experimental mouse model of left ventricle barometric overload. CONCLUSION: Overall, these results provide the first evidence for the existence of differential CM surface crests in the cardiac tissue as well as the existence of CM-CM direct physical contacts at their lateral face through crest-crest interactions. We propose a model in which this specific 3D organization of the CM lateral membrane ensures the myofibril/myofiber alignment and the overall cardiac tissue cohesion. A potential role in the control of sarcomere relaxation and of diastolic ventricular dysfunction is also discussed. Whether the loss of CM surface crests constitutes an initial and common event leading to the CM degeneration and the setting of heart failure will need further investigation.


Assuntos
Membrana Celular/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Idoso , Idoso de 80 Anos ou mais , Animais , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Bovinos , Membrana Celular/metabolismo , Claudina-5/metabolismo , Microscopia Crioeletrônica , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Mitocôndrias Cardíacas/ultraestrutura , Miócitos Cardíacos/metabolismo , Ratos Wistar , Sarcômeros/ultraestrutura , Especificidade da Espécie , Junções Íntimas/metabolismo , Junções Íntimas/ultraestrutura
12.
Sci Rep ; 7(1): 7885, 2017 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-28801617

RESUMO

Biased agonism at G protein coupled receptors emerges as an opportunity for development of drugs with enhanced benefit/risk balance making biased ligand identification a priority. However, ligand biased signature, classically inferred from ligand activity across multiple pathways, displays high variability in recombinant systems. Functional assays usually necessity receptor/effector overexpression that should be controlled among assays to allow comparison but this calibration currently fails. Herein, we demonstrate that Gα expression level dictates the biased profiling of agonists and, to a lesser extent of ß-blockers, in a Gα isoform- and receptor-specific way, depending on specific G protein activity in different membrane territories. These results have major therapeutic implications since they suggest that the ligand bias phenotype is not necessarily maintained in pathological cell background characterized by fluctuations in G protein expression. Thus, we recommend implementation of G protein stoichiometry as a new parameter in biased ligand screening programs.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Células HEK293 , Humanos , Ligantes , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores Acoplados a Proteínas G/genética
13.
Circulation ; 112(21): 3297-305, 2005 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-16286591

RESUMO

BACKGROUND: Serotonin (5-hydroxytryptamine [5-HT]), released by activated platelets during cardiac ischemia, is metabolized by the mitochondrial enzyme monoamine oxidase A (MAO-A). Because hydrogen peroxide is one of the byproducts of 5-HT degradation by MAO-A, we investigated the potential role of reactive oxygen species generated by MAOs in 5-HT-dependent cardiomyocyte death and post-ischemia-reperfusion cardiac damage. METHODS AND RESULTS: Treatment of isolated adult rat cardiomyocytes with 5-HT induced intracellular oxidative stress and cell apoptosis. The apoptotic cascade triggered by 5-HT involves release of cytochrome c, upregulation of proapoptotic Bax protein, and downregulation of antiapoptotic Bcl-2 protein. These effects were prevented by inhibition of amine transporter or MAO, antioxidants, or iron chelation. In contrast, cardiomyocyte apoptosis was only slightly affected by the 5-HT(2B) receptor antagonist SB 206553. In vivo, inhibition of MAO-A largely reduced myocardial ultrastructural damage induced by 30 minutes of ischemia followed by 60 minutes of reperfusion in the rat heart. Cardioprotective effects of MAO inhibitors were associated with the prevention of postischemic oxidative stress, neutrophil accumulation, and mitochondrial-dependent cell death and were not reverted by SB 206553. Administration of MAO-A inhibitors during ischemia was still effective in preventing cardiac damage. CONCLUSIONS: Our results supply the first direct evidence that oxidative stress induced by MAO is responsible for receptor-independent apoptotic effects of 5-HT in cardiomyocytes and postischemic myocardial injury. These findings provide new insight into the mechanisms of 5-HT action in the heart and may constitute the basis for novel therapies.


Assuntos
Apoptose/fisiologia , Monoaminoxidase/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Serotonina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3 , Caspases/metabolismo , Células Cultivadas , Ventrículos do Coração/citologia , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/fisiologia , Masculino , Inibidores da Monoaminoxidase/farmacologia , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Fagócitos/efeitos dos fármacos , Fagócitos/fisiologia , Ratos , Ratos Sprague-Dawley , Serotonina/metabolismo , Proteína X Associada a bcl-2/metabolismo
14.
Hypertension ; 68(6): 1365-1374, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27698068

RESUMO

Hyperactivity of the renin-angiotensin-aldosterone system through the angiotensin II (Ang II)/Ang II type 1 receptor (AT1-R) axis constitutes a hallmark of hypertension. Recent findings indicate that only a subset of AT1-R signaling pathways is cardiodeleterious, and their selective inhibition by biased ligands promotes therapeutic benefit. To date, only synthetic biased ligands have been described, and whether natural renin-angiotensin-aldosterone system peptides exhibit functional selectivity at AT1-R remains unknown. In this study, we systematically determined efficacy and potency of Ang II, Ang III, Ang IV, and Ang-(1-7) in AT1-R-expressing HEK293T cells on the activation of cardiodeleterious G-proteins and cardioprotective ß-arrestin2. Ang III and Ang IV fully activate similar G-proteins than Ang II, the prototypical AT1-R agonist, despite weaker potency of Ang IV. Interestingly, Ang-(1-7) that binds AT1-R fails to promote G-protein activation but behaves as a competitive antagonist for Ang II/Gi and Ang II/Gq pathways. Conversely, all renin-angiotensin-aldosterone system peptides act as agonists on the AT1-R/ß-arrestin2 axis but display biased activities relative to Ang II as indicated by their differences in potency and AT1-R/ß-arrestin2 intracellular routing. Importantly, we reveal Ang-(1-7) a known Mas receptor-specific ligand, as an AT1-R-biased agonist, selectively promoting ß-arrestin activation while blocking the detrimental Ang II/AT1-R/Gq axis. This original pharmacological profile of Ang-(1-7) at AT1-R, similar to that of synthetic AT1-R-biased agonists, could, in part, contribute to its cardiovascular benefits. Accordingly, in vivo, Ang-(1-7) counteracts the phenylephrine-induced aorta contraction, which was blunted in AT1-R knockout mice. Collectively, these data suggest that Ang-(1-7) natural-biased agonism at AT1-R could fine-tune the physiology of the renin-angiotensin-aldosterone system.


Assuntos
Angiotensina II/farmacologia , Angiotensina I/metabolismo , Cardiotônicos/metabolismo , Células HEK293/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Animais , Aorta Abdominal/efeitos dos fármacos , Aorta Abdominal/fisiologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Células HEK293/efeitos dos fármacos , Humanos , Músculos , Fenilefrina/farmacologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Sistema Renina-Angiotensina/fisiologia , Sensibilidade e Especificidade , Transdução de Sinais , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia , beta-Arrestinas/metabolismo
15.
FASEB J ; 16(9): 1129-31, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12039844

RESUMO

Reactive oxygen species (ROS) contribute to the ischemia-reperfusion injury. In kidney, the intracellular sources of ROS during ischemia-reperfusion are still unclear. In the present study, we investigated the role of the catecholamine-degrading enzyme monoamine oxidases (MAOs) in hydrogen peroxide (H2O2) generation after reperfusion and their involvement in cell events leading to tissue injury and recovery. In a rat model of renal ischemia-reperfusion, we show concomitant MAO-dependent H2O2 production and lipid peroxidation in the early reperfusion period. Rat pretreatment with the irreversible MAO inhibitor pargyline resulted in the following: i) prevented H2O2 production and lipid peroxidation; ii) decreased tubular cell apoptosis and necrosis, measured by TUNEL staining and histomorphological criteria; and iii) increased tubular cell proliferation as determined by proliferating cell nuclear antigen expression. MAO inhibition also prevented Jun N-terminal kinase phosphorylation and promoted extracellular signal-regulated kinase activation, two mitogen-activated protein kinases described as a part of a "death" and "survival" pathway after ischemia-reperfusion. This work demonstrates the crucial role of MAOs in mediating the production of injurious ROS, which contribute to acute apoptotic and necrotic cell death induced by renal ischemia-reperfusion in vivo. Targeted inhibition of these oxidases could provide a new avenue for therapy to prevent renal damage and promote renal recovery after ischemia-reperfusion.


Assuntos
Apoptose , Rim/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monoaminoxidase/metabolismo , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/patologia , Animais , Divisão Celular/efeitos dos fármacos , Ativação Enzimática , Peróxido de Hidrogênio/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Rim/enzimologia , Rim/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Modelos Biológicos , Inibidores da Monoaminoxidase/farmacologia , Necrose , Pargilina/farmacologia , Ratos , Regeneração , Traumatismo por Reperfusão/metabolismo
16.
FASEB J ; 17(8): 872-4, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12670883

RESUMO

The pineal hormone melatonin has been reported to protect tissue from oxidative damage. This study was designed to determine whether melatonin could prevent cell events leading to tissue injury and renal dysfunction after ischemia/reperfusion (I/R). Using an in vivo rat model of I/R, we show a significant increase in kidney malondialdehyde concentrations, reflecting lipid peroxidation, and cell apoptosis measured by TUNEL staining. This apoptotic cell death was associated with an increase in the activity of the proapoptotic factor caspase-3, determined by fluorometric protease activity assay. Histomorphological analysis of ischemic kidneys revealed that the most extensive tubular necrosis occurred at 24 and 48 h after reperfusion, which correlated with peak elevations in blood urea nitrogen and creatinine. Rat pretreatment with melatonin prevented lipid peroxidation, cell apoptosis, and necrosis and blocked caspase-3 activity. The prevention of tissue injury was associated with the improvement of renal function as shown by the decrease in blood urea nitrogen and creatinine concentrations. The demonstration that melatonin prevents postreperfusion apoptotic and necrotic cell death and improves renal function suggests that melatonin may represent a novel therapeutic approach for prevention of I/R injury.


Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Rim/efeitos dos fármacos , Melatonina/farmacologia , Traumatismo por Reperfusão/fisiopatologia , Animais , Nitrogênio da Ureia Sanguínea , Caspase 3 , Creatinina/sangue , Ativação Enzimática/efeitos dos fármacos , Marcação In Situ das Extremidades Cortadas , Rim/irrigação sanguínea , Rim/fisiopatologia , Túbulos Renais/irrigação sanguínea , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Necrose , Estresse Oxidativo/efeitos dos fármacos , Ratos
17.
PLoS One ; 8(11): e79068, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24244423

RESUMO

Sympathetic nervous system (SNS) plays a key role in cardiac homeostasis and its deregulations always associate with bad clinical outcomes. To date, little is known about molecular mechanisms regulating cardiac sympathetic innervation. The aim of the study was to determine the role of fibroblasts in heart sympathetic innervation. RT-qPCR and western-blots analysis performed in cardiomyocytes and fibroblasts isolated from healthy adult rat hearts revealed that Pro-Nerve growth factor (NGF) and pro-differentiating mature NGF were the most abundant neurotrophins expressed in cardiac fibroblasts while barely detectable in cardiomyocytes. When cultured with cardiac fibroblasts or fibroblast-conditioned medium, PC12 cells differentiated into/sympathetic-like neurons expressing axonal marker Tau-1 at neurites in contact with cardiomyocytes. This was prevented by anti-NGF blocking antibodies suggesting a paracrine action of NGF secreted by fibroblasts. When co-cultured with cardiomyocytes to mimic neurocardiac synapse, differentiated PC12 cells exhibited enhanced norepinephrine secretion as quantified by HPLC compared to PC12 cultured alone while co-culture with fibroblasts had no effect. However, when supplemented to PC12-cardiomyocytes co-culture, fibroblasts allowed long-term survival of the neurocardiac synapse. Activated fibroblasts (myofibroblasts) isolated from myocardial infarction rat hearts exhibited significantly higher mature NGF expression than normal fibroblasts and also promoted PC12 cells differentiation. Within the ischemic area lacking cardiomyocytes and neurocardiac synapses, tyrosine hydroxylase immunoreactivity was increased and associated with local anarchical and immature sympathetic hyperinnervation but tissue norepinephrine content was similar to that of normal cardiac tissue, suggesting depressed sympathetic function. Collectively, these findings demonstrate for the first time that fibroblasts are essential for the setting of cardiac sympathetic innervation and neurocardiac synapse stability. They also suggest that neurocardiac synapse functionality relies on a triptych with tight interaction between sympathetic nerve endings, cardiomyocytes and fibroblasts. Deregulations of this triptych may be involved in pathophysiology of cardiac diseases.


Assuntos
Axônios/metabolismo , Fibroblastos/metabolismo , Miocárdio/metabolismo , Fator de Crescimento Neural/metabolismo , Sistema Nervoso Simpático/metabolismo , Sinapses/metabolismo , Animais , Técnicas de Cocultura , Fibroblastos/citologia , Miocárdio/citologia , Células PC12 , Ratos , Ratos Endogâmicos Lew , Sistema Nervoso Simpático/citologia
18.
PLoS One ; 7(7): e34893, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22829864

RESUMO

Serotonin (5-HT) regulates different cardiac functions by acting directly on cardiomyocytes, fibroblasts and endothelial cells. Today, it is widely accepted that activated platelets represent a major source of 5-HT. In contrast, a supposed production of 5-HT in the heart is still controversial. To address this issue, we investigated the expression and localization of 5-HT synthesizing enzyme tryptophan hydroxylase (TPH) and L-aromatic amino acid decarboxylase (AADC) in the heart. We also evaluated their involvement in cardiac production of 5-HT. TPH1 was weakly expressed in mouse and rat heart and appeared restricted to mast cells. Degranulation of mast cells by compound 48/80 did not modify 5-HT cardiac content in mice. Western blots and immunolabelling experiments showed an abundant expression of AADC in the mouse and rat heart and its co-localization with endothelial cells. Incubation of cardiac homogenate with the AADC substrate (5-hydroxy-L-tryptophan) 5-HTP or intraperitoneal injection of 5-HTP in mice significantly increased cardiac 5-HT. These effects were prevented by the AADC inhibitor benserazide. Finally, 5-HTP administration in mice increased phosphorylation of aortic nitric oxide synthase 3 at Ser (1177) as well as accumulation of nitrates in cardiac tissue. This suggests that the increase in 5-HT production by AADC leads to activation of endothelial and cardiac nitric oxide pathway. These data show that endothelial AADC plays an important role in cardiac synthesis of 5-HT and possibly in 5-HT-dependent regulation of nitric oxide generation.


Assuntos
Descarboxilases de Aminoácido-L-Aromático/metabolismo , Miocárdio/metabolismo , Nitratos/metabolismo , Serotonina/metabolismo , 5-Hidroxitriptofano/farmacologia , Animais , Inibidores das Descarboxilases de Aminoácidos Aromáticos , Western Blotting , Cromatografia Líquida de Alta Pressão , Coração/efeitos dos fármacos , Coração/embriologia , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triptofano Hidroxilase/metabolismo
19.
Stem Cells Dev ; 19(10): 1571-8, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20073575

RESUMO

Early death of grafted bone marrow mesenchymal stem cells (MSCs) represents a major limit to their use in cell therapy of solid organs. It is well known that oxidative stress plays a major role in cell death. We have recently shown that the serotonin-degrading enzyme monoamine oxidase A (MAO-A) generates large amount of hydrogen peroxide (H2O2) responsible for cell apoptosis. Hydrogen peroxide generation requires 5-HT internalization into the cell and its degradation by MAO-A. In the present study, we investigated whether MAO-A is expressed in MSCs and we defined its role in serotonin-dependent MSCs apoptosis. RT-PCR analysis and western blots showed that the serotonin transporter (SERT) and the 2 MAO isoenzymes, A and B, are expressed in MSCs. As shown by enzyme assays using [14C]serotonin or [14C]ß-phenylethylamine as selective MAO-A or MAO-B substrates, MAO-A is largely predominant in MSCs. Incubation of MSCs with the MAO substrate tyramine led to a time-dependent generation of H2O2 that was prevented by the MAO inhibitor pargyline. Finally, exposure of the cells to serotonin promoted an increase in MSCs apoptosis prevented by pargyline and the SERT inhibitor imipramine. The pro-apoptotic effect of serotonin was associated to a decrease in the expression of the anti-apoptotic factor Bcl-2. In conclusion, these results show for the first time that the 5-HT-degrading enzyme MAO-A is an important source of H2O2 in MSCs and plays a major role in 5-HT-dependent MSCs apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Células-Tronco Mesenquimais/enzimologia , Monoaminoxidase/metabolismo , Oxidantes/metabolismo , Serotonina/farmacologia , Inibidores da Captação Adrenérgica/farmacologia , Animais , Células Cultivadas , Citocromos c/metabolismo , Imipramina/farmacologia , Isoenzimas/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Inibidores da Monoaminoxidase/farmacologia , Pargilina/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Endogâmicos Lew , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Simpatomiméticos/farmacologia , Tiramina/farmacologia , Proteína X Associada a bcl-2/metabolismo
20.
Am J Physiol Renal Physiol ; 292(5): F1592-8, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17244889

RESUMO

Renal dopamine, synthesized by proximal tubules, plays an important role in the regulation of renal sodium excretion. Although the renal dopaminergic system has been extensively investigated in both physiological and pathological situations, the mechanisms whereby dopamine is stored and secreted by proximal tubule cells remain obscure. In the present study we investigated whether vesicular monoamine transporters (VMAT)-1 and -2, which participate in amine storing and secretion, are expressed in rat renal proximal tubules, and we defined their involvement in dopamine secretion. By combining RT-PCR, Western blot, and immunocytochemistry we showed that VMAT-1 is the predominant isoform expressed in isolated proximal tubule cells. These results were confirmed by immunohistochemistry analysis of rat renal cortex showing that VMAT-1 was found in proximal tubules but not in glomeruli. Functional studies showed that, as previously reported for VMAT-dependent amine transporters, dopamine release by cultured proximal tubule cells was partially inhibited by disruption of intracellular H(+) gradient. In addition, dopamine secretion was prevented by the VMAT-1/VMAT-2 inhibitor reserpine but not by the VMAT-2 inhibitor tetrabenazine. Finally, we demonstrated that tubular VMAT-1 mRNA and protein expression were significantly upregulated during a high-sodium diet. In conclusion, our results show for the first time the expression of a VMAT in the renal proximal tubule and its involvement in regulation of dopamine secretion. These data represent the first step toward the comprehension of the role of this transporter in renal dopamine handling and its involvement in pathological situations.


Assuntos
Dopamina/metabolismo , Túbulos Renais Proximais/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/fisiologia , Animais , Western Blotting , Células Cultivadas , Dopamina/biossíntese , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Túbulos Renais Proximais/citologia , Masculino , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Cloreto de Sódio na Dieta/administração & dosagem , Cloreto de Sódio na Dieta/farmacologia , Proteínas Vesiculares de Transporte de Monoamina/metabolismo
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