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1.
Am J Hum Genet ; 108(11): 2112-2129, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34626534

RESUMO

Upregulated signal flow through RAS and the mitogen-associated protein kinase (MAPK) cascade is the unifying mechanistic theme of the RASopathies, a family of disorders affecting development and growth. Pathogenic variants in more than 20 genes have been causally linked to RASopathies, the majority having a dominant role in promoting enhanced signaling. Here, we report that SPRED2 loss of function is causally linked to a recessive phenotype evocative of Noonan syndrome. Homozygosity for three different variants-c.187C>T (p.Arg63∗), c.299T>C (p.Leu100Pro), and c.1142_1143delTT (p.Leu381Hisfs∗95)-were identified in four subjects from three families. All variants severely affected protein stability, causing accelerated degradation, and variably perturbed SPRED2 functional behavior. When overexpressed in cells, all variants were unable to negatively modulate EGF-promoted RAF1, MEK, and ERK phosphorylation, and time-course experiments in primary fibroblasts (p.Leu100Pro and p.Leu381Hisfs∗95) documented an increased and prolonged activation of the MAPK cascade in response to EGF stimulation. Morpholino-mediated knockdown of spred2a and spred2b in zebrafish induced defects in convergence and extension cell movements indicating upregulated RAS-MAPK signaling, which were rescued by expressing wild-type SPRED2 but not the SPRED2Leu381Hisfs∗95 protein. The clinical phenotype of the four affected individuals included developmental delay, intellectual disability, cardiac defects, short stature, skeletal anomalies, and a typical facial gestalt as major features, without the occurrence of the distinctive skin signs characterizing Legius syndrome. These features, in part, characterize the phenotype of Spred2-/- mice. Our findings identify the second recessive form of Noonan syndrome and document pleiotropic consequences of SPRED2 loss of function in development.


Assuntos
Mutação com Perda de Função , Síndrome de Noonan/genética , Fenótipo , Proteínas Repressoras/genética , Alelos , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Peixe-Zebra
2.
Circulation ; 144(21): 1694-1713, 2021 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-34648376

RESUMO

BACKGROUND: Barth syndrome (BTHS) is caused by mutations of the gene encoding tafazzin, which catalyzes maturation of mitochondrial cardiolipin and often manifests with systolic dysfunction during early infancy. Beyond the first months of life, BTHS cardiomyopathy typically transitions to a phenotype of diastolic dysfunction with preserved ejection fraction, blunted contractile reserve during exercise, and arrhythmic vulnerability. Previous studies traced BTHS cardiomyopathy to mitochondrial formation of reactive oxygen species (ROS). Because mitochondrial function and ROS formation are regulated by excitation-contraction coupling, integrated analysis of mechano-energetic coupling is required to delineate the pathomechanisms of BTHS cardiomyopathy. METHODS: We analyzed cardiac function and structure in a mouse model with global knockdown of tafazzin (Taz-KD) compared with wild-type littermates. Respiratory chain assembly and function, ROS emission, and Ca2+ uptake were determined in isolated mitochondria. Excitation-contraction coupling was integrated with mitochondrial redox state, ROS, and Ca2+ uptake in isolated, unloaded or preloaded cardiac myocytes, and cardiac hemodynamics analyzed in vivo. RESULTS: Taz-KD mice develop heart failure with preserved ejection fraction (>50%) and age-dependent progression of diastolic dysfunction in the absence of fibrosis. Increased myofilament Ca2+ affinity and slowed cross-bridge cycling caused diastolic dysfunction, in part, compensated by accelerated diastolic Ca2+ decay through preactivated sarcoplasmic reticulum Ca2+-ATPase. Taz deficiency provoked heart-specific loss of mitochondrial Ca2+ uniporter protein that prevented Ca2+-induced activation of the Krebs cycle during ß-adrenergic stimulation, oxidizing pyridine nucleotides and triggering arrhythmias in cardiac myocytes. In vivo, Taz-KD mice displayed prolonged QRS duration as a substrate for arrhythmias, and a lack of inotropic response to ß-adrenergic stimulation. Cellular arrhythmias and QRS prolongation, but not the defective inotropic reserve, were restored by inhibiting Ca2+ export through the mitochondrial Na+/Ca2+ exchanger. All alterations occurred in the absence of excess mitochondrial ROS in vitro or in vivo. CONCLUSIONS: Downregulation of mitochondrial Ca2+ uniporter, increased myofilament Ca2+ affinity, and preactivated sarcoplasmic reticulum Ca2+-ATPase provoke mechano-energetic uncoupling that explains diastolic dysfunction and the lack of inotropic reserve in BTHS cardiomyopathy. Furthermore, defective mitochondrial Ca2+ uptake provides a trigger and a substrate for ventricular arrhythmias. These insights can guide the ongoing search for a cure of this orphaned disease.


Assuntos
Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/etiologia , Síndrome de Barth/complicações , Síndrome de Barth/genética , Canais de Cálcio/deficiência , Contração Miocárdica/genética , Trifosfato de Adenosina/biossíntese , Animais , Síndrome de Barth/metabolismo , Biomarcadores , Encéfalo/metabolismo , Cálcio/metabolismo , Diástole , Modelos Animais de Doenças , Suscetibilidade a Doenças , Acoplamento Excitação-Contração/genética , Testes de Função Cardíaca , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Músculo Esquelético/metabolismo , Miócitos Cardíacos/metabolismo , NADP/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Volume Sistólico , Sístole
3.
Proc Natl Acad Sci U S A ; 114(12): E2420-E2429, 2017 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-28255084

RESUMO

In recent years, the myocardium has been rediscovered under the lenses of immunology, and lymphocytes have been implicated in the pathogenesis of cardiomyopathies with different etiologies. Aging is an important risk factor for heart diseases, and it also has impact on the immune system. Thus, we sought to determine whether immunological activity would influence myocardial structure and function in elderly mice. Morphological, functional, and molecular analyses revealed that the age-related myocardial impairment occurs in parallel with shifts in the composition of tissue-resident leukocytes and with an accumulation of activated CD4+ Foxp3- (forkhead box P3) IFN-γ+ T cells in the heart-draining lymph nodes. A comprehensive characterization of different aged immune-deficient mouse strains revealed that T cells significantly contribute to age-related myocardial inflammation and functional decline. Upon adoptive cell transfer, the T cells isolated from the mediastinal lymph node (med-LN) of aged animals exhibited increased cardiotropism, compared with cells purified from young donors or from other irrelevant sites. Nevertheless, these cells caused rather mild effects on cardiac functionality, indicating that myocardial aging might stem from a combination of intrinsic and extrinsic (immunological) factors. Taken together, the data herein presented indicate that heart-directed immune responses may spontaneously arise in the elderly, even in the absence of a clear tissue damage or concomitant infection. These observations might shed new light on the emerging role of T cells in myocardial diseases, which primarily affect the elderly population.


Assuntos
Envelhecimento/imunologia , Linfócitos T CD4-Positivos/imunologia , Miocárdio/imunologia , Transferência Adotiva , Animais , Coração/crescimento & desenvolvimento , Humanos , Linfonodos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
4.
J Mol Cell Cardiol ; 129: 13-26, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30771306

RESUMO

Cardiac functionality is dependent on a balanced protein turnover. Accordingly, regulated protein decay is critical to maintain cardiac function. Here we demonstrate that deficiency of SPRED2, an intracellular repressor of ERK-MAPK signaling markedly expressed in human heart, resulted in impaired autophagy, heart failure, and shortened lifespan. SPRED2-/- mice showed cardiomyocyte hypertrophy, cardiac fibrosis, impaired electrical excitability, and severe arrhythmias. Mechanistically, cardiomyocyte dysfunction resulted from ERK hyperactivation and dysregulated autophagy, observed as accumulation of vesicles, vacuolar structures, and degenerated mitochondria. The diminished autophagic flux in SPRED2-/- hearts was reflected by a reduced LC3-II/LC3-I ratio and by decreased Atg7, Atg4B and Atg16L expression. Furthermore, the autophagosomal adaptors p62/SQSTM1 and NBR1 and lysosomal Cathepsin D accumulated in SPRED2-/- hearts. In wild-type hearts, SPRED2 interacted physically with p62/SQSTM1, NBR1, and Cathepsin D, indicating that SPRED2 is required for autophagolysosome formation in regular autophagy. Restored inhibition of MAPK signaling by selumetinib led to an increase in autophagic flux in vivo. Therefore, our study identifies SPRED2 as a novel, indispensable regulator of cardiac autophagy. Vice versa, SPRED2 deficiency impairs autophagy, leading to cardiac dysfunction and life-threatening arrhythmias.


Assuntos
Arritmias Cardíacas/metabolismo , Autofagia , Mortalidade Prematura , Proteínas Repressoras/deficiência , Adulto , Aldosterona/farmacologia , Animais , Arritmias Cardíacas/complicações , Arritmias Cardíacas/patologia , Arritmias Cardíacas/fisiopatologia , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Biomarcadores/metabolismo , Pressão Sanguínea , Cardiomegalia/complicações , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Catepsina D/metabolismo , Colágeno/metabolismo , Fenômenos Eletrofisiológicos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Condução Cardíaco/fisiopatologia , Hemodinâmica , Humanos , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Miocárdio/ultraestrutura , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Proteínas Repressoras/metabolismo , Vacúolos/metabolismo , Vacúolos/ultraestrutura
5.
Circulation ; 138(5): 494-508, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-29626067

RESUMO

BACKGROUND: Peripheral vascular resistance has a major impact on arterial blood pressure levels. Endothelial C-type natriuretic peptide (CNP) participates in the local regulation of vascular tone, but the target cells remain controversial. The cGMP-producing guanylyl cyclase-B (GC-B) receptor for CNP is expressed in vascular smooth muscle cells (SMCs). However, whereas endothelial cell-specific CNP knockout mice are hypertensive, mice with deletion of GC-B in vascular SMCs have unaltered blood pressure. METHODS: We analyzed whether the vasodilating response to CNP changes along the vascular tree, ie, whether the GC-B receptor is expressed in microvascular types of cells. Mice with a floxed GC-B ( Npr2) gene were interbred with Tie2-Cre or PDGF-Rß-Cre ERT2 lines to develop mice lacking GC-B in endothelial cells or in precapillary arteriolar SMCs and capillary pericytes. Intravital microscopy, invasive and noninvasive hemodynamics, fluorescence energy transfer studies of pericyte cAMP levels in situ, and renal physiology were combined to dissect whether and how CNP/GC-B/cGMP signaling modulates microcirculatory tone and blood pressure. RESULTS: Intravital microscopy studies revealed that the vasodilatatory effect of CNP increases toward small-diameter arterioles and capillaries. CNP consistently did not prevent endothelin-1-induced acute constrictions of proximal arterioles, but fully reversed endothelin effects in precapillary arterioles and capillaries. Here, the GC-B receptor is expressed both in endothelial and mural cells, ie, in pericytes. It is notable that the vasodilatatory effects of CNP were preserved in mice with endothelial GC-B deletion, but abolished in mice lacking GC-B in microcirculatory SMCs and pericytes. CNP, via GC-B/cGMP signaling, modulates 2 signaling cascades in pericytes: it activates cGMP-dependent protein kinase I to phosphorylate downstream targets such as the cytoskeleton-associated vasodilator-activated phosphoprotein, and it inhibits phosphodiesterase 3A, thereby enhancing pericyte cAMP levels. These pathways ultimately prevent endothelin-induced increases of pericyte calcium levels and pericyte contraction. Mice with deletion of GC-B in microcirculatory SMCs and pericytes have elevated peripheral resistance and chronic arterial hypertension without a change in renal function. CONCLUSIONS: Our studies indicate that endothelial CNP regulates distal arteriolar and capillary blood flow. CNP-induced GC-B/cGMP signaling in microvascular SMCs and pericytes is essential for the maintenance of normal microvascular resistance and blood pressure.


Assuntos
Pressão Arterial/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Hipertensão/metabolismo , Microcirculação/efeitos dos fármacos , Microvasos/efeitos dos fármacos , Peptídeo Natriurético Tipo C/farmacologia , Pericitos/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Técnicas Biossensoriais , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , GMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Transferência Ressonante de Energia de Fluorescência , Predisposição Genética para Doença , Hipertensão/genética , Hipertensão/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/metabolismo , Microvasos/fisiopatologia , Peptídeo Natriurético Tipo C/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Fenótipo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/deficiência , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptores do Fator Natriurético Atrial/deficiência , Receptores do Fator Natriurético Atrial/genética
6.
Ann Neurol ; 82(5): 729-743, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29023958

RESUMO

OBJECTIVE: Cardiac diseases are established risk factors for ischemic stroke incidence and severity. Conversely, there is increasing evidence that brain ischemia can cause cardiac dysfunction. The mechanisms underlying this neurogenic heart disease are incompletely understood. Although it is established that ischemic stroke is associated with cardiac arrhythmias, myocardial damage, elevated cardiac enzymes, and plasma catecholamines in the acute phase, nothing is known about the delayed consequences of ischemic stroke on cardiovascular function. METHODS: To determine the long-term cardiac consequences of a focal cerebral ischemia, we subjected young and aged mice to a 30-minute transient middle cerebral artery occlusion and analyzed cardiac function by serial transthoracic echocardiography and hemodynamic measurements up to week 8 after surgery. Finally, animals were treated with metoprolol to evaluate a pharmacologic treatment option to prevent the development of heart failure. RESULTS: Focal cerebral ischemia induced a long-term cardiac dysfunction with a reduction in left ventricular ejection fraction and an increase in left ventricular volumes; this development was associated with higher peripheral sympathetic activity. Metoprolol treatment prevented the development of chronic cardiac dysfunction by decelerating extracellular cardiac remodeling and inhibiting sympathetic signaling relevant to chronic autonomic dysfunction. INTERPRETATION: Focal cerebral ischemia in mice leads to the development of chronic systolic dysfunction driven by increased sympathetic activity. If these results can be confirmed in a clinical setting, treating physicians should be attentive to clinical signs of heart failure in every patient after ischemic stroke. Therapeutically, the successful ß-blockade with metoprolol in mice could also have future clinical implications. Ann Neurol 2017;82:729-743.


Assuntos
Isquemia Encefálica/fisiopatologia , Hemodinâmica/fisiologia , Acidente Vascular Cerebral/fisiopatologia , Sistema Nervoso Simpático/fisiopatologia , Antagonistas de Receptores Adrenérgicos beta 1/uso terapêutico , Animais , Isquemia Encefálica/sangue , Isquemia Encefálica/complicações , Ecocardiografia , Epinefrina/sangue , Insuficiência Cardíaca/prevenção & controle , Hemodinâmica/efeitos dos fármacos , Hidrocortisona/sangue , Infarto da Artéria Cerebral Média , Masculino , Metoprolol/uso terapêutico , Camundongos , Peptídeo Natriurético Encefálico/sangue , Norepinefrina/sangue , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/complicações , Sistema Nervoso Simpático/efeitos dos fármacos
8.
Circ Res ; 116(8): 1304-11, 2015 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-25688144

RESUMO

RATIONALE: Cyclic nucleotides are second messengers that regulate cardiomyocyte function through compartmentalized signaling in discrete subcellular microdomains. However, the role of different microdomains and their changes in cardiac disease are not well understood. OBJECTIVE: To directly visualize alterations in ß-adrenergic receptor-associated cAMP and cGMP microdomain signaling in early cardiac disease. METHODS AND RESULTS: Unexpectedly, measurements of cell shortening revealed augmented ß-adrenergic receptor-stimulated cardiomyocyte contractility by atrial natriuretic peptide/cGMP signaling in early cardiac hypertrophy after transverse aortic constriction, which was in sharp contrast to well-documented ß-adrenergic and natriuretic peptide signaling desensitization during chronic disease. Real-time cAMP analysis in ß1- and ß2-adrenergic receptor-associated membrane microdomains using a novel membrane-targeted Förster resonance energy transfer-based biosensor transgenically expressed in mice revealed that this unexpected atrial natriuretic peptide effect is brought about by spatial redistribution of cGMP-sensitive phosphodiesterases 2 and 3 between both receptor compartments. Functionally, this led to a significant shift in cGMP/cAMP cross-talk and, in particular, to cGMP-driven augmentation of contractility in vitro and in vivo. CONCLUSIONS: Redistribution of cGMP-regulated phosphodiesterases and functional reorganization of receptor-associated microdomains occurs in early cardiac hypertrophy, affects cGMP-mediated contractility, and might represent a previously not recognized therapeutically relevant compensatory mechanism to sustain normal heart function.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Fator Natriurético Atrial/farmacologia , Cardiomegalia/enzimologia , GMP Cíclico/metabolismo , Isoproterenol/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Receptores Adrenérgicos beta/efeitos dos fármacos , Animais , Técnicas Biossensoriais , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Feminino , Transferência Ressonante de Energia de Fluorescência , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Microdomínios da Membrana/enzimologia , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Transporte Proteico , Receptor Cross-Talk/efeitos dos fármacos , Receptores Adrenérgicos beta/metabolismo , Receptores Adrenérgicos beta 1/efeitos dos fármacos , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Receptores Adrenérgicos beta 2/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Fatores de Tempo
9.
Basic Res Cardiol ; 111(2): 22, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26909880

RESUMO

The cardiac hormone atrial natriuretic peptide (ANP) regulates systemic and pulmonary arterial blood pressure by activation of its cyclic GMP-producing guanylyl cyclase-A (GC-A) receptor. In the lung, these hypotensive effects were mainly attributed to smooth muscle-mediated vasodilatation. It is unknown whether pulmonary endothelial cells participate in the homeostatic actions of ANP. Therefore, we analyzed GC-A/cGMP signalling in lung endothelial cells and the cause and functional impact of lung endothelial GC-A dysfunction. Western blot and cGMP determinations showed that cultured human and murine pulmonary endothelial cells exhibit prominent GC-A expression and activity which were markedly blunted by hypoxia, a condition known to trigger pulmonary hypertension (PH). To elucidate the consequences of impaired endothelial ANP signalling, we studied mice with genetic endothelial cell-restricted ablation of the GC-A receptor (EC GC-A KO). Notably, EC GC-A KO mice exhibit PH already under resting, normoxic conditions, with enhanced muscularization of small arteries and perivascular infiltration of inflammatory cells. These alterations were aggravated on exposure of mice to chronic hypoxia. Lung endothelial GC-A dysfunction was associated with enhanced expression of angiotensin converting enzyme (ACE) and increased pulmonary levels of Angiotensin II. Angiotensin II/AT1-blockade with losartan reversed pulmonary vascular remodelling and perivascular inflammation of EC GC-A KO mice, and prevented their increment by chronic hypoxia. This experimental study indicates that endothelial effects of ANP are critical to prevent pulmonary vascular remodelling and PH. Chronic endothelial ANP/GC-A dysfunction, e.g. provoked by hypoxia, is associated with activation of the ACE-angiotensin pathway in the lung and PH.


Assuntos
Fator Natriurético Atrial/metabolismo , Células Endoteliais/metabolismo , Hipertensão Pulmonar/enzimologia , Hipóxia/metabolismo , Receptores do Fator Natriurético Atrial/metabolismo , Angiotensina II/metabolismo , Animais , Endotelina-1/metabolismo , Hipertensão Pulmonar/etiologia , Pulmão/metabolismo , Camundongos , Camundongos Knockout , Peptidil Dipeptidase A/metabolismo , Receptores do Fator Natriurético Atrial/genética
10.
Dev Dyn ; 244(4): 591-606, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25690936

RESUMO

BACKGROUND: Pluripotency, self-renewal, and differentiation are special features of embryonic stem (ES) cells, thereby providing valuable perspectives in regenerative medicine. Developmental processes require a fine-tuned organization, mainly regulated by the well-known JAK/STAT, PI3K/AKT, and ERK/MAPK pathways. SPREDs (Sprouty related proteins with EVH1 domain) were discovered as inhibitors of the ERK/MAPK signaling pathway, whereas nothing was known about their functions in ES cells and during early differentiation, so far. RESULTS: We generated SPRED1 and SPRED2 overexpressing and SPRED2 knockout murine ES cells to analyze the functions of SPRED proteins in ES cells and during early differentiation. Overexpression of SPREDs increases significantly the self-renewal and clonogenicity of murine ES cells, whereas lack of SPRED2 reduces proliferation and increases apoptosis. During early differentiation in embryoid bodies, SPREDs promote the pluripotent state and inhibit differentiation whereby mesodermal differentiation into cardiomyocytes is considerably delayed and inhibited. LIF- and growth factor-stimulation revealed that SPREDs inhibit ERK/MAPK activation in murine ES cells. However, no effects were detectable on LIF-induced activation of the JAK/STAT3, or PI3K/AKT signaling pathway by SPRED proteins. CONCLUSIONS: We show that SPREDs promote self-renewal and inhibit mesodermal differentiation of murine ES cells by selective suppression of the ERK/MAPK signaling pathway in pluripotent cells.


Assuntos
Células-Tronco Embrionárias/metabolismo , Fator Inibidor de Leucemia/metabolismo , Mesoderma/metabolismo , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose , Diferenciação Celular , Proliferação de Células , Células-Tronco Embrionárias/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais
11.
Dev Dyn ; 243(5): 699-711, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24288149

RESUMO

BACKGROUND: ATIPs (Angiotensin receptor type 2 [AT2] interacting proteins) are described as AT2 interacting protein variants, whereas their expression and functions during development are not known yet. RESULTS: Here, we provide a detailed expression pattern of ATIP variants during mouse development by visualizing Mtus1 promotor activity, Mtus1 RNA, and subsequent ATIP protein expression. ATIPs are strongly expressed in the developing cardiovascular system, including the vascular plexus of the yolk sac and the fetal vascular part of the placenta. Moreover, ATIP is expressed spatially distinct during eye and limb bud development, and in later stages in lung and nervous system. The three murine ATIP isoforms are expressed in a distinct manner, whereupon isoform 1 and 4 are correlated to cardiovascular, lung, and limb bud development and isoform 3 is restricted to brain and eye development. Interestingly, Mtus1 expression is not necessarily correlated to Agtr2 expression, suggesting novel but yet unknown functions for ATIP, independent of AT2 signaling. CONCLUSIONS: ATIPs seem to be mainly involved in the developmental regulation of the cardiovascular system and may act in different AT2-dependent and -independent manners. Hence, these results deliver valuable information to further elucidate the different functions of ATIPs in the processes of mammalian development.


Assuntos
Sistema Cardiovascular/embriologia , Proteínas de Transporte/biossíntese , Embrião de Mamíferos/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas Supressoras de Tumor/biossíntese , Angiotensina II/genética , Angiotensina II/metabolismo , Animais , Sistema Cardiovascular/citologia , Proteínas de Transporte/genética , Embrião de Mamíferos/citologia , Camundongos , Camundongos Transgênicos , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Proteínas Supressoras de Tumor/genética
12.
Am J Pathol ; 182(4): 1205-18, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23462508

RESUMO

Despite recent advances in understanding the relevance of cell adhesion-related signaling in the pathogenesis of ischemic cardiomyopathy (ICM) in animal models, substantial questions remain unanswered in the human setting. We have previously shown that the neural cell adhesion molecule CD56 [neural cell adhesion molecule (NCAM1)] is specifically overexpressed in ICM; it was the aim of the current study to further elucidate the role of CD56 in the pathogenesis of human ICM. We used quantitative real-time PCR and IHC in human ICM and a rat model of coronary obstruction to demonstrate that CD56(140kD), the only extraneuronally expressed NCAM1 isoform with a cytoplasmic protein domain capable of inducing intracellular signaling, is the only up-regulated CD56 isoform in failing cardiomyocytes in human ICM in vivo. In subsequent analyses of the cellular effects of CD56(140kD) overexpression in the development of ICM using differential whole transcriptome expression analyses and functional in vitro cardiomyocyte cell culture assays, we further show that the up-regulation of CD56(140kD) is associated with profound gene expression changes, increased apoptosis, and reduced Ca(2+) signaling in failing human cardiomyocytes. Because apoptosis and Ca(2+)-related sarcomeric dysfunction are molecular hallmarks of ICM in humans, our results provide strong evidence that CD56(140kD) up-regulation plays a pivotal role in the pathogenesis of ICM and may be a target for future immunotherapeutic strategies in the treatment of this common and often fatal disease.


Assuntos
Antígeno CD56/metabolismo , Cardiomiopatias/patologia , Isquemia Miocárdica/patologia , Animais , Apoptose , Antígeno CD56/genética , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomiopatias/complicações , Cardiomiopatias/genética , Proliferação de Células , Modelos Animais de Doenças , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Humanos , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Peso Molecular , Proteínas Mutantes/metabolismo , Isquemia Miocárdica/complicações , Isquemia Miocárdica/genética , Miocárdio/metabolismo , Miocárdio/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Isoformas de Proteínas/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real
13.
Eur Heart J ; 34(16): 1233-44, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22199120

RESUMO

AIMS: Cardiac hypertrophy is a common and often lethal complication of arterial hypertension. Elevation of myocyte cyclic GMP levels by local actions of endogenous atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) or by pharmacological inhibition of phosphodiesterase-5 was shown to counter-regulate pathological hypertrophy. It was suggested that cGMP-dependent protein kinase I (cGKI) mediates this protective effect, although the role in vivo is under debate. Here, we investigated whether cGKI modulates myocyte growth and/or function in the intact organism. METHODS AND RESULTS: To circumvent the systemic phenotype associated with germline ablation of cGKI, we inactivated the murine cGKI gene selectively in cardiomyocytes by Cre/loxP-mediated recombination. Mice with cardiomyocyte-restricted cGKI deletion exhibited unaltered cardiac morphology and function under resting conditions. Also, cardiac hypertrophic and contractile responses to ß-adrenoreceptor stimulation by isoprenaline (at 40 mg/kg/day during 1 week) were unaltered. However, angiotensin II (Ang II, at 1000 ng/kg/min for 2 weeks) or transverse aortic constriction (for 3 weeks) provoked dilated cardiomyopathy with marked deterioration of cardiac function. This was accompanied by diminished expression of the [Ca(2+)]i-regulating proteins SERCA2a and phospholamban (PLB) and a reduction in PLB phosphorylation at Ser16, the specific target site for cGKI, resulting in altered myocyte Ca(2+)i homeostasis. In isolated adult myocytes, CNP, but not ANP, stimulated PLB phosphorylation, Ca(2+)i-handling, and contractility via cGKI. CONCLUSION: These results indicate that the loss of cGKI in cardiac myocytes compromises the hypertrophic program to pathological stimulation, rendering the heart more susceptible to dysfunction. In particular, cGKI mediates stimulatory effects of CNP on myocyte Ca(2+)i handling and contractility.


Assuntos
Cardiomiopatia Dilatada/enzimologia , Proteína Quinase Dependente de GMP Cíclico Tipo I/fisiologia , Estresse Fisiológico/fisiologia , Agonistas Adrenérgicos beta/farmacologia , Análise de Variância , Angiotensina II/farmacologia , Animais , Aorta , Pressão Sanguínea/efeitos dos fármacos , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cardiomiopatia Dilatada/genética , Cardiotônicos/farmacologia , Constrição , Proteína Quinase Dependente de GMP Cíclico Tipo I/deficiência , Proteína Quinase Dependente de GMP Cíclico Tipo I/genética , Ecocardiografia , Deleção de Genes , Hemodinâmica/efeitos dos fármacos , Isoproterenol/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Peptídeo Natriurético Tipo C/fisiologia , Fosforilação/fisiologia , Transdução de Sinais/fisiologia , Vasoconstritores/farmacologia
14.
Cell Commun Signal ; 11: 56, 2013 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-23937664

RESUMO

BACKGROUND: In the heart, cytoplasmic actin networks are thought to have important roles in mechanical support, myofibrillogenesis, and ion channel function. However, subcellular localization of cytoplasmic actin isoforms and proteins involved in the modulation of the cytoplasmic actin networks are elusive. Mena and VASP are important regulators of actin dynamics. Due to the lethal phenotype of mice with combined deficiency in Mena and VASP, however, distinct cardiac roles of the proteins remain speculative. In the present study, we analyzed the physiological functions of Mena and VASP in the heart and also investigated the role of the proteins in the organization of cytoplasmic actin networks. RESULTS: We generated a mouse model, which simultaneously lacks Mena and VASP in the heart. Mena/VASP double-deficiency induced dilated cardiomyopathy and conduction abnormalities. In wild-type mice, Mena and VASP specifically interacted with a distinct αII-Spectrin splice variant (SH3i), which is in cardiomyocytes exclusively localized at Z- and intercalated discs. At Z- and intercalated discs, Mena and ß-actin localized to the edges of the sarcomeres, where the thin filaments are anchored. In Mena/VASP double-deficient mice, ß-actin networks were disrupted and the integrity of Z- and intercalated discs was markedly impaired. CONCLUSIONS: Together, our data suggest that Mena, VASP, and αII-Spectrin assemble cardiac multi-protein complexes, which regulate cytoplasmic actin networks. Conversely, Mena/VASP deficiency results in disrupted ß-actin assembly, Z- and intercalated disc malformation, and induces dilated cardiomyopathy and conduction abnormalities.


Assuntos
Actinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas dos Microfilamentos/metabolismo , Miócitos Cardíacos/metabolismo , Fosfoproteínas/metabolismo , Espectrina/metabolismo , Animais , Cardiomiopatia Dilatada/metabolismo , Moléculas de Adesão Celular/genética , Citoplasma/metabolismo , Proteínas do Citoesqueleto/genética , Sistema de Condução Cardíaco/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Fosfoproteínas/genética , Espectrina/química , Domínios de Homologia de src
15.
FEBS Open Bio ; 13(1): 118-132, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36352324

RESUMO

Recent evidence demonstrated that alterations in the QT interval duration on the ECG are not only determined by mutations in genes for ion channels, but also by modulators of ion channels. Changes in the QT interval duration beyond certain thresholds are pathological and can lead to sudden cardiac death. We here focus on the ion channel modulator nitric oxide synthase 1 adaptor protein (Nos1ap). Whole-cell patch-clamp measurements of a conditional transgenic mouse model exhibiting cardiac-specific Nos1ap over-expression revealed a Nos1ap-dependent increase of L-type calcium channel nitrosylation, which led to increased susceptibility to ventricular tachycardias associated with a decrease in QT duration and shortening of APD90 duration. Survival was significantly reduced (60% after 12 weeks vs. 100% in controls). Examination of the structural features of the hearts of transgenic mice revealed constant heart dimensions and wall thickness without abnormal fibrosis content or BNP production after 3 months of Nos1ap over-expression compared to controls. Nos1ap over-expression did not alter cGMP production or ROS concentration. Our study showed that myocardial over-expression of Nos1ap leads to the shortening of the QT interval and reduces the survival rate of transgenic animals, perhaps via the development of ventricular arrhythmias. We conclude that Nos1ap overexpression causes targeted subcellular localization of Nos1 to the CaV1.2 with a subsequent decrease of ADP90 and the QT interval. This causes detrimental cardiac arrhythmias in transgenic mice.


Assuntos
Síndrome do QT Longo , Camundongos , Animais , Síndrome do QT Longo/genética , Camundongos Transgênicos , Genótipo , Eletrocardiografia , Arritmias Cardíacas , Proteínas Adaptadoras de Transdução de Sinal/genética
16.
JCI Insight ; 8(13)2023 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-37227779

RESUMO

Excessive activation of cardiac fibroblasts (CFs) in response to injury provokes cardiac fibrosis, stiffness, and failure. The local mediators counterregulating this response remain unclear. Exogenous C-type natriuretic peptide (CNP) exerts antifibrotic effects in preclinical models. To unravel the role of the endogenous hormone, we generated mice with fibroblast-restricted deletion (KO) of guanylyl cyclase-B (GC-B), the cGMP-synthesizing CNP receptor. CNP activated GC-B/cGMP signaling in human and murine CFs, preventing proliferative and promigratory effects of angiotensin II (Ang II) and TGF-ß. Fibroblast-specific GC-B-KO mice showed enhanced fibrosis in response to Ang II infusions. Moreover, after 2 weeks of mild pressure overload induced by transverse aortic constriction (TAC), such KO mice had augmented cardiac fibrosis and hypertrophy, together with systolic and diastolic contractile dysfunction. This was associated with increased expression of the profibrotic genes encoding collagen I, III, and periostin. Notably, such responses to Ang II and TAC were greater in female as compared with male KO mice. Enhanced Ang II-induced CNP expression in female hearts and augmented GC-B expression and activity in female CFs may contribute to this sex disparity. The results show that paracrine CNP signaling in CFs has antifibrotic and antihypertrophic effects. The CNP/GC-B/cGMP pathway might be a target for therapies combating pathological cardiac remodeling.


Assuntos
Peptídeo Natriurético Tipo C , Remodelação Ventricular , Camundongos , Animais , Masculino , Feminino , Humanos , Peptídeo Natriurético Tipo C/genética , Peptídeo Natriurético Tipo C/farmacologia , Vasodilatadores/farmacologia , Fibrose , Angiotensina II/farmacologia , Fibroblastos/metabolismo
17.
J Biol Chem ; 286(11): 9477-88, 2011 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-21199868

RESUMO

Sprouty-related proteins with EVH1 (enabled/vasodilator-stimulated phosphoprotein homology 1) domain (SPREDs) are inhibitors of MAPK signaling. To elucidate SPRED2 in vivo function, we characterized body homeostasis in SPRED2(-/-) mice. They showed a doubled daily water uptake, induced by elevated serum osmolality, originating from increased blood salt load. Accordingly, serum aldosterone was doubled, accompanied by augmented adrenal aldosterone synthase (AS) expression. Surprisingly, serum vasopressin (AVP) was unaltered, and, as evidenced by halved angiotensin II (Ang II) levels, the renin angiotensin system (RAS) was down-regulated. Adrenocorticotropic hormone (ACTH) was significantly elevated in SPRED2(-/-) mice, together with its secretagogue corticotropin-releasing hormone (CRH) and its downstream target corticosterone. ERK phosphorylation in brains was augmented, and hypothalamic CRH mRNA levels were elevated, both contributing to the increased CRH release. Our data were supported by CRH promoter reporter assays in hypothalamic mHypoE-44 cells, revealing a SPRED-dependent inhibition of Ets (ERK/E-twenty-six)-dependent transcription. Furthermore, SPRED suppressed CRH production in these cells. In conclusion, our study suggests that SPRED2 deficiency leads to an increased MAPK signaling, which results in an augmented CRH promoter activity. The subsequent CRH overproduction causes an up-regulation of downstream hypothalamic-pituitary-adrenal (HPA) hormone secretion. This constitutes a possible trigger for the observed compulsive grooming in SPRED2(-/-) mice and may, together with hyperplasia of aldosterone-producing cells, contribute to the hyperaldosteronism and homeostatic imbalances.


Assuntos
Sistema Hipotálamo-Hipofisário/metabolismo , Hipotálamo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sistema Hipófise-Suprarrenal/metabolismo , Proteínas Repressoras/metabolismo , Animais , Hormônio Liberador da Corticotropina/genética , Hormônio Liberador da Corticotropina/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Fosforilação/fisiologia , Proteínas Repressoras/genética
18.
J Exp Med ; 203(3): 513-8, 2006 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-16533887

RESUMO

Formation of fibrin is critical for limiting blood loss at a site of blood vessel injury (hemostasis), but may also contribute to vascular thrombosis. Hereditary deficiency of factor XII (FXII), the protease that triggers the intrinsic pathway of coagulation in vitro, is not associated with spontaneous or excessive injury-related bleeding, indicating FXII is not required for hemostasis. We demonstrate that deficiency or inhibition of FXII protects mice from ischemic brain injury. After transient middle cerebral artery occlusion, the volume of infarcted brain in FXII-deficient and FXII inhibitor-treated mice was substantially less than in wild-type controls, without an increase in infarct-associated hemorrhage. Targeting FXII reduced fibrin formation in ischemic vessels, and reconstitution of FXII-deficient mice with human FXII restored fibrin deposition. Mice deficient in the FXII substrate factor XI were similarly protected from vessel-occluding fibrin formation, suggesting that FXII contributes to pathologic clotting through the intrinsic pathway. These data demonstrate that some processes involved in pathologic thrombus formation are distinct from those required for normal hemostasis. As FXII appears to be instrumental in pathologic fibrin formation but dispensable for hemostasis, FXII inhibition may offer a selective and safe strategy for preventing stroke and other thromboembolic diseases.


Assuntos
Isquemia Encefálica/metabolismo , Deficiência do Fator XII/metabolismo , Fator XII/metabolismo , Hemostasia , Trombose/metabolismo , Animais , Inibidores dos Fatores de Coagulação Sanguínea/administração & dosagem , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Isquemia Encefálica/patologia , Isquemia Encefálica/prevenção & controle , Deficiência do Fator XI/tratamento farmacológico , Deficiência do Fator XI/metabolismo , Deficiência do Fator XI/patologia , Fator XII/antagonistas & inibidores , Deficiência do Fator XII/patologia , Feminino , Fibrina/metabolismo , Hemostasia/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Trombose/tratamento farmacológico , Trombose/patologia
19.
J Immunol ; 184(12): 6575-84, 2010 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-20483741

RESUMO

The monomeric GTPase Rap1 controls functional activation of beta2 integrins in leukocytes. In this article, we describe a novel mechanism by which the chemoattractant fMLP activates Rap1 and inside-out signaling of beta2 integrins. We found that fMLP-induced activation of Rap1 in human polymorphonuclear leukocytes or neutrophils and differentiated PLB-985 cells was blocked by inhibitors of the NO/guanosine-3',5'-cyclic monophosphate-dependent protein kinase (cGKI) pathway [N-(3-(aminomethyl)benzyl)acetamidine, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, DT-3 peptide, 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphothioate, Rp-isomer triethylammonium salt-guanosine-3',5'-cyclic monophosphate], indicating that the downstream signaling events in Rap1 activation involve the production of NO and guanosine-3',5'-cyclic monophosphate, as well as the activation of cGKI. Silencing the expression of vasodilator-stimulated phosphoprotein (VASP), a substrate of cGKI, in resting PLB-985 cells or mice neutrophils led to constitutive activation of Rap1. In parallel, silencing VASP in differentiated PLB-985 cells led to recruitment of C3G, a guanine nucleotide exchange factor for Rap1, to the plasma membrane. Expression of murine GFP-tagged phosphodeficient VASP Ser235Ala mutant (murine serine 235 of VASP corresponds to human serine 239) in PLB-985 cells blunted fMLP-induced translocation of C3G to the membrane and activation of Rap1. Thus, bacterial fMLP triggers cGKI-dependent phosphorylation of human VASP on serine 239 and, thereby, controls membrane recruitment of C3G, which is required for activation of Rap1 and beta2 integrin-dependent antibacterial functions of neutrophils.


Assuntos
Antígenos CD18/imunologia , Moléculas de Adesão Celular/imunologia , Proteínas dos Microfilamentos/imunologia , Neutrófilos/imunologia , Fosfoproteínas/imunologia , Transdução de Sinais/imunologia , Animais , Proteínas de Bactérias/imunologia , Western Blotting , Antígenos CD18/metabolismo , Moléculas de Adesão Celular/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/imunologia , Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Neutrófilos/metabolismo , Fosfoproteínas/metabolismo , Transporte Proteico/fisiologia , Proteínas rap1 de Ligação ao GTP/imunologia , Proteínas rap1 de Ligação ao GTP/metabolismo
20.
Circulation ; 122(16): 1588-603, 2010 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-20921441

RESUMO

BACKGROUND: We previously demonstrated that conditional overexpression of neuronal nitric oxide synthase (nNOS) inhibited L-type Ca2+ channels and decreased myocardial contractility. However, nNOS has multiple targets within the cardiac myocyte. We now hypothesize that nNOS overexpression is cardioprotective after ischemia/reperfusion because of inhibition of mitochondrial function and a reduction in reactive oxygen species generation. METHODS AND RESULTS: Ischemia/reperfusion injury in wild-type mice resulted in nNOS accumulation in the mitochondria. Similarly, transgenic nNOS overexpression caused nNOS abundance in mitochondria. nNOS translocation into the mitochondria was dependent on heat shock protein 90. Ischemia/reperfusion experiments in isolated hearts showed a cardioprotective effect of nNOS overexpression. Infarct size in vivo was also significantly reduced. nNOS overexpression also caused a significant increase in mitochondrial nitrite levels accompanied by a decrease of cytochrome c oxidase activity. Accordingly, O(2) consumption in isolated heart muscle strips was decreased in nNOS-overexpressing nNOS(+)/αMHC-tTA(+) mice already under resting conditions. Additionally, we found that the reactive oxygen species concentration was significantly decreased in hearts of nNOS-overexpressing nNOS(+)/αMHC-tTA(+) mice compared with noninduced nNOS(+)/αMHC-tTA(+) animals. CONCLUSION: We demonstrated that conditional transgenic overexpression of nNOS resulted in myocardial protection after ischemia/reperfusion injury. Besides a reduction in reactive oxygen species generation, this might be caused by nitrite-mediated inhibition of mitochondrial function, which reduced myocardial oxygen consumption already under baseline conditions.


Assuntos
Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/complicações , Traumatismo por Reperfusão Miocárdica/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Animais , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias Cardíacas/metabolismo , Modelos Animais , Infarto do Miocárdio/patologia , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Consumo de Oxigênio/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Disfunção Ventricular Esquerda/fisiopatologia
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