RESUMO
BACKGROUND: Organ fibrosis due to excessive production of extracellular matrix by resident fibroblasts is estimated to contribute to >45% of deaths in the Western world, including those due to cardiovascular diseases such as heart failure. Here, we screened for small molecule inhibitors with a common ability to suppress activation of fibroblasts across organ systems. METHODS: High-content imaging of cultured cardiac, pulmonary, and renal fibroblasts was used to identify nontoxic compounds that blocked induction of markers of activation in response to the profibrotic stimulus, transforming growth factor-ß1. SW033291, which inhibits the eicosanoid-degrading enzyme, 15-hydroxyprostaglandin dehydrogenase, was chosen for follow-up studies with cultured adult rat ventricular fibroblasts and human cardiac fibroblasts (CF), and for evaluation in mouse models of cardiac fibrosis and diastolic dysfunction. Additional mechanistic studies were performed with CFs treated with exogenous eicosanoids. RESULTS: Nine compounds, including SW033291, shared a common ability to suppress transforming growth factor-ß1-mediated activation of cardiac, pulmonary, and renal fibroblasts. SW033291 dose-dependently inhibited transforming growth factor-ß1-induced expression of activation markers (eg, α-smooth muscle actin and periostin) in adult rat ventricular fibroblasts and normal human CFs, and reduced contractile capacity of the cells. Remarkably, the 15-hydroxyprostaglandin dehydrogenase inhibitor also reversed constitutive activation of fibroblasts obtained from explanted hearts from patients with heart failure. SW033291 blocked cardiac fibrosis induced by angiotensin II infusion and ameliorated diastolic dysfunction in an alternative model of systemic hypertension driven by combined uninephrectomy and deoxycorticosterone acetate administration. Mechanistically, SW033291-mediated stimulation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase signaling was required for the compound to block CF activation. Of the 12 exogenous eicosanoids that were tested, only 12(S)-hydroxyeicosatetraenoic acid, which signals through the G protein-coupled receptor, GPR31, recapitulated the suppressive effects of SW033291 on CF activation. CONCLUSIONS: Inhibition of degradation of eicosanoids, arachidonic acid-derived fatty acids that signal through G protein-coupled receptors, is a potential therapeutic strategy for suppression of pathological organ fibrosis. In the heart, we propose that 15-hydroxyprostaglandin dehydrogenase inhibition triggers CF-derived autocrine/paracrine signaling by eicosanoids, including 12(S)-hydroxyeicosatetraenoic acid, to stimulate extracellular signal-regulated kinase 1/2 and block conversion of fibroblasts into activated cells that secrete excessive amounts of extracellular matrix and contribute to heart failure pathogenesis.
Assuntos
Insuficiência Cardíaca , Camundongos , Ratos , Humanos , Animais , Fator de Crescimento Transformador beta1/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miocárdio/metabolismo , Insuficiência Cardíaca/metabolismo , Fibroblastos/metabolismo , Fibrose , Células CultivadasRESUMO
BACKGROUND: Diastolic dysfunction (DD) is associated with the development of heart failure and contributes to the pathogenesis of other cardiac maladies, including atrial fibrillation. Inhibition of histone deacetylases (HDACs) has been shown to prevent DD by enhancing myofibril relaxation. We addressed the therapeutic potential of HDAC inhibition in a model of established DD with preserved ejection fraction. METHODS: Four weeks after uninephrectomy and implantation with deoxycorticosterone acetate pellets, when DD was clearly evident, 1 cohort of mice was administered the clinical-stage HDAC inhibitor ITF2357/Givinostat. Echocardiography, blood pressure measurements, and end point invasive hemodynamic analyses were performed. Myofibril mechanics and intact cardiomyocyte relaxation were assessed ex vivo. Cardiac fibrosis was evaluated by picrosirius red staining and second harmonic generation microscopy of left ventricle (LV) sections, RNA sequencing of LV mRNA, mass spectrometry-based evaluation of decellularized LV biopsies, and atomic force microscopy determination of LV stiffness. Mechanistic studies were performed with primary rat and human cardiac fibroblasts. RESULTS: HDAC inhibition normalized DD without lowering blood pressure in this model of systemic hypertension. In contrast to previous models, myofibril relaxation was unimpaired in uninephrectomy/deoxycorticosterone acetate mice. Furthermore, cardiac fibrosis was not evident in any mouse cohort on the basis of picrosirius red staining or second harmonic generation microscopy. However, mass spectrometry revealed induction in the expression of >100 extracellular matrix proteins in LVs of uninephrectomy/deoxycorticosterone acetate mice, which correlated with profound tissue stiffening based on atomic force microscopy. ITF2357/Givinostat treatment blocked extracellular matrix expansion and LV stiffening. The HDAC inhibitor was subsequently shown to suppress cardiac fibroblast activation, at least in part, by blunting recruitment of the profibrotic chromatin reader protein BRD4 (bromodomain-containing protein 4) to key gene regulatory elements. CONCLUSIONS: These findings demonstrate the potential of HDAC inhibition as a therapeutic intervention to reverse existing DD and establish blockade of extracellular matrix remodeling as a second mechanism by which HDAC inhibitors improve ventricular filling. Our data reveal the existence of pathophysiologically relevant covert or hidden cardiac fibrosis that is below the limit of detection of histochemical stains such as picrosirius red, highlighting the need to evaluate fibrosis of the heart using diverse methodologies.
Assuntos
Matriz Extracelular/fisiologia , Sopros Cardíacos/tratamento farmacológico , Inibidores de Histona Desacetilases/uso terapêutico , Remodelação Ventricular/fisiologia , Animais , Modelos Animais de Doenças , Feminino , Inibidores de Histona Desacetilases/farmacologia , Humanos , Masculino , CamundongosRESUMO
Pulmonary hypertension (PH) is associated with structural remodeling of pulmonary arteries (PAs) because of excessive proliferation of fibroblasts, endothelial cells, and smooth muscle cells (SMCs). The peptide hormone angiotensin II (ANG II) contributes to pulmonary vascular remodeling, in part, through its ability to trigger extracellular signal-regulated kinase (ERK1/2) activation. Here, we demonstrate that the ERK1/2 phosphatase, dual-specificity phosphatase 5 (DUSP5), functions as a negative regulator of ANG II-mediated SMC proliferation and PH. In contrast to wild-type controls, Dusp5 null mice infused with ANG II developed PH and right ventricular (RV) hypertrophy. PH in Dusp5 null mice was associated with thickening of the medial layer of small PAs, suggesting an in vivo role for DUSP5 as a negative regulator of ANG II-dependent SMC proliferation. Consistent with this, overexpression of DUSP5 blocked ANG II-mediated proliferation of cultured human pulmonary artery SMCs (hPASMCs) derived from patients with idiopathic PH or from failed donor controls. Collectively, the data support a role for DUSP5 as a feedback inhibitor of ANG II-mediated ERK signaling and PASMC proliferation and suggest that disruption of this circuit leads to adverse cardiopulmonary remodeling.NEW & NOTEWORTHY Dual-specificity phosphatases (DUSPs) serve critical roles in the regulation of mitogen-activated protein kinases, but their functions in the cardiovascular system remain poorly defined. Here, we provide evidence that DUSP5, which resides in the nucleus and specifically dephosphorylates extracellular signal-regulated kinase (ERK1/2), blocks pulmonary vascular smooth muscle cell proliferation. In response to angiotensin II infusion, mice lacking DUSP5 develop pulmonary hypertension and right ventricular cardiac hypertrophy. These findings illustrate DUSP5-mediated suppression of ERK signaling in the lungs as a protective mechanism.
Assuntos
Proliferação de Células/genética , Fosfatases de Especificidade Dupla/genética , Ventrículos do Coração/metabolismo , Hipertensão Pulmonar/genética , Hipertrofia Ventricular Direita/genética , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar/metabolismo , Remodelação Vascular/genética , Angiotensina II/farmacologia , Animais , Estudos de Casos e Controles , Células Cultivadas , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Humanos , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/induzido quimicamente , Hipertrofia Ventricular Direita/fisiopatologia , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , Vasoconstritores/farmacologiaRESUMO
RATIONALE: Small molecule inhibitors of the acetyl-histone binding protein BRD4 have been shown to block cardiac fibrosis in preclinical models of heart failure (HF). However, since the inhibitors target BRD4 ubiquitously, it is unclear whether this chromatin reader protein functions in cell type-specific manner to control pathological myocardial fibrosis. Furthermore, the molecular mechanisms by which BRD4 stimulates the transcriptional program for cardiac fibrosis remain unknown. OBJECTIVE: We sought to test the hypothesis that BRD4 functions in a cell-autonomous and signal-responsive manner to control activation of cardiac fibroblasts, which are the major extracellular matrix-producing cells of the heart. METHODS AND RESULTS: RNA-sequencing, mass spectrometry, and cell-based assays employing primary adult rat ventricular fibroblasts demonstrated that BRD4 functions as an effector of TGF-ß (transforming growth factor-ß) signaling to stimulate conversion of quiescent cardiac fibroblasts into Periostin (Postn)-positive cells that express high levels of extracellular matrix. These findings were confirmed in vivo through whole-transcriptome analysis of cardiac fibroblasts from mice subjected to transverse aortic constriction and treated with the small molecule BRD4 inhibitor, JQ1. Chromatin immunoprecipitation-sequencing revealed that BRD4 undergoes stimulus-dependent, genome-wide redistribution in cardiac fibroblasts, becoming enriched on a subset of enhancers and super-enhancers, and leading to RNA polymerase II activation and expression of downstream target genes. Employing the Sertad4 (SERTA domain-containing protein 4) locus as a prototype, we demonstrate that dynamic chromatin targeting of BRD4 is controlled, in part, by p38 MAPK (mitogen-activated protein kinase) and provide evidence of a critical function for Sertad4 in TGF-ß-mediated cardiac fibroblast activation. CONCLUSIONS: These findings define BRD4 as a central regulator of the pro-fibrotic cardiac fibroblast phenotype, establish a p38-dependent signaling circuit for epigenetic reprogramming in heart failure, and uncover a novel role for Sertad4. The work provides a mechanistic foundation for the development of BRD4 inhibitors as targeted anti-fibrotic therapies for the heart.
Assuntos
Cromatina/metabolismo , Insuficiência Cardíaca/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Miofibroblastos/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Azepinas/farmacologia , Azepinas/uso terapêutico , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Elementos Facilitadores Genéticos , Epigênese Genética , Matriz Extracelular/metabolismo , Feminino , Fibrose , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/genética , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Ligação Proteica , RNA Polimerase II/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Transcriptoma , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Triazóis/farmacologia , Triazóis/uso terapêutico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Pulmonary arterial hypertension (PAH) is a progressive disease defined by a chronic elevation in pulmonary arterial pressure with extensive pulmonary vascular remodeling and perivascular inflammation characterized by an accumulation of macrophages, lymphocytes, dendritic cells, and mast cells. Although the exact etiology of the disease is unknown, clinical as well as preclinical data strongly implicate a role for serotonin (5-HT) in the process. Here, we investigated the chronic effects of pharmacological inhibition of tryptophan hydroxylase 1 (TPH1), the rate-limiting enzyme in peripheral 5-HT biosynthesis, in two preclinical models of pulmonary hypertension (PH), the monocrotaline (MCT) rat and the semaxanib (SUGEN, Medinoah, Suzhou, China)-hypoxia rat. In both PH models, ethyl (S)-8-(2-amino-6-((R)-1-(5-chloro-[1,1'-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate and ethyl (S)-8-(2-amino-6-((R)-1-(3',4'-dimethyl-3-(3-methyl-1 H-pyrazol-1-yl)-[1,1'-biphenyl]-4-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate, novel orally active TPH1 inhibitors with nanomolar in vitro potency, decreased serum, gut, and lung 5-HT levels in a dose-dependent manner and significantly reduced pulmonary arterial pressure, and pulmonary vessel wall thickness and occlusion in male rats. In the MCT rat model, decreases in lung 5-HT significantly correlated with reductions in histamine levels and mast cell number (P < 0.001, r2 = 0.88). In contrast, neither ambrisentan nor tadalafil, which are vasodilators approved for the treatment of PAH, reduced mast cell number or 5-HT levels, nor were they as effective in treating the vascular remodeling as were the TPH1 inhibitors. When administered in combination with ambrisentan, the TPH1 inhibitors showed an additive effect on pulmonary vascular remodeling and pressures. These data demonstrate that in addition to reducing vascular remodeling, TPH1 inhibition has the added benefit of reducing the perivascular mast cell accumulation associated with PH.
Assuntos
Inibidores Enzimáticos/farmacologia , Hipertensão Pulmonar/fisiopatologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/fisiopatologia , Triptofano Hidroxilase/antagonistas & inibidores , Remodelação Vascular/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Ratos , Ratos Sprague-Dawley , Serotonina/sangue , Serotonina/metabolismo , Triptofano Hidroxilase/metabolismoRESUMO
BackgroundHistone deacetylase (HDAC) inhibitors are promising therapeutics for various forms of cardiac diseases. The purpose of this study was to assess cardiac HDAC catalytic activity and expression in children with single ventricle (SV) heart disease of right ventricular morphology, as well as in a rodent model of right ventricular hypertrophy (RVH).MethodsHomogenates of right ventricle (RV) explants from non-failing controls and children born with a SV were assayed for HDAC catalytic activity and HDAC isoform expression. Postnatal 1-day-old rat pups were placed in hypoxic conditions, and echocardiographic analysis, gene expression, HDAC catalytic activity, and isoform expression studies of the RV were performed.ResultsClass I, IIa, and IIb HDAC catalytic activity and protein expression were elevated in the hearts of children born with a SV. Hypoxic neonatal rats demonstrated RVH, abnormal gene expression, elevated class I and class IIb HDAC catalytic activity, and protein expression in the RV compared with those in the control.ConclusionsThese data suggest that myocardial HDAC adaptations occur in the SV heart and could represent a novel therapeutic target. Although further characterization of the hypoxic neonatal rat is needed, this animal model may be suitable for preclinical investigations of pediatric RV disease and could serve as a useful model for future mechanistic studies.
Assuntos
Cardiopatias Congênitas/enzimologia , Ventrículos do Coração/enzimologia , Histona Desacetilases/metabolismo , Hipertrofia Ventricular Direita/enzimologia , Função Ventricular Direita , Remodelação Ventricular , Adaptação Fisiológica , Adolescente , Animais , Animais Recém-Nascidos , Estudos de Casos e Controles , Criança , Feminino , Regulação Enzimológica da Expressão Gênica , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/patologia , Cardiopatias Congênitas/fisiopatologia , Ventrículos do Coração/anormalidades , Ventrículos do Coração/fisiopatologia , Histona Desacetilases/genética , Humanos , Hipertrofia Ventricular Direita/genética , Hipertrofia Ventricular Direita/patologia , Hipertrofia Ventricular Direita/fisiopatologia , Lactente , Isoenzimas , Masculino , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Chronic cardiac hypertrophy is maladaptive and contributes to the pathogenesis of heart failure. The objective of this study was to identify small molecule inhibitors of pathological cardiomyocyte hypertrophy. High content screening was performed with primary neonatal rat ventricular myocytes (NRVMs) cultured on 96-well plates and treated with a library of 3241 distinct small molecules. Non-toxic hit compounds that blocked hypertrophy in response to phenylephrine (PE) and phorbol myristate acetate (PMA) were identified based on their ability to reduce cell size and inhibit expression of atrial natriuretic factor (ANF), which is a biomarker of pathological cardiac hypertrophy. Many of the hit compounds are existing drugs that have not previously been evaluated for benefit in the setting of cardiovascular disease. One such compound, the anti-malarial drug artesunate, blocked left ventricular hypertrophy (LVH) and improved cardiac function in adult mice subjected to transverse aortic constriction (TAC). These findings demonstrate that phenotypic screening with primary cardiomyocytes can be used to discover anti-hypertrophic lead compounds for heart failure drug discovery. Using annotated libraries of compounds with known selectivity profiles, this screening methodology also facilitates chemical biological dissection of signaling networks that control pathological growth of the heart.
Assuntos
Cardiomegalia/metabolismo , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Animais , Cardiomegalia/diagnóstico por imagem , Cardiomegalia/tratamento farmacológico , Células Cultivadas , Modelos Animais de Doenças , Hemodinâmica/efeitos dos fármacos , Masculino , Camundongos , Imagem Molecular/métodos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas , Remodelação Ventricular/efeitos dos fármacosRESUMO
Fibrosis, which is defined as excessive accumulation of fibrous connective tissue, contributes to the pathogenesis of numerous diseases involving diverse organ systems. Cardiac fibrosis predisposes individuals to myocardial ischemia, arrhythmias and sudden death, and is commonly associated with diastolic dysfunction. Histone deacetylase (HDAC) inhibitors block cardiac fibrosis in pre-clinical models of heart failure. However, which HDAC isoforms govern cardiac fibrosis, and the mechanisms by which they do so, remains unclear. Here, we show that selective inhibition of class I HDACs potently suppresses angiotensin II (Ang II)-mediated cardiac fibrosis by targeting two key effector cell populations, cardiac fibroblasts and bone marrow-derived fibrocytes. Class I HDAC inhibition blocks cardiac fibroblast cell cycle progression through derepression of the genes encoding the cyclin-dependent kinase (CDK) inhibitors, p15 and p57. In contrast, class I HDAC inhibitors block agonist-dependent differentiation of fibrocytes through a mechanism involving repression of ERK1/2 signaling. These findings define novel roles for class I HDACs in the control of pathological cardiac fibrosis. Furthermore, since fibrocytes have been implicated in the pathogenesis of a variety of human diseases, including heart, lung and kidney failure, our results suggest broad utility for isoform-selective HDAC inhibitors as anti-fibrotic agents that function, in part, by targeting these circulating mesenchymal cells.
Assuntos
Angiotensina II/metabolismo , Fibroblastos/efeitos dos fármacos , Fibrose/fisiopatologia , Inibidores de Histona Desacetilases/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular , Fibroblastos/metabolismo , Fibrose/tratamento farmacológico , Citometria de Fluxo , Humanos , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Isoformas de Proteínas/farmacologiaRESUMO
Little is known about the function of the cytoplasmic histone deacetylase HDAC6 in striated muscle. Here, we addressed the role of HDAC6 in cardiac and skeletal muscle remodeling induced by the peptide hormone angiotensin II (ANG II), which plays a central role in blood pressure control, heart failure, and associated skeletal muscle wasting. Comparable with wild-type (WT) mice, HDAC6 null mice developed cardiac hypertrophy and fibrosis in response to ANG II. However, whereas WT mice developed systolic dysfunction upon treatment with ANG II, cardiac function was maintained in HDAC6 null mice treated with ANG II for up to 8 wk. The cardioprotective effect of HDAC6 deletion was mimicked in WT mice treated with the small molecule HDAC6 inhibitor tubastatin A. HDAC6 null mice also exhibited improved left ventricular function in the setting of pressure overload mediated by transverse aortic constriction. HDAC6 inhibition appeared to preserve systolic function, in part, by enhancing cooperativity of myofibrillar force generation. Finally, we show that HDAC6 null mice are resistant to skeletal muscle wasting mediated by chronic ANG-II signaling. These findings define novel roles for HDAC6 in striated muscle and suggest potential for HDAC6-selective inhibitors for the treatment of cardiac dysfunction and muscle wasting in patients with heart failure.
Assuntos
Angiotensina II , Cardiomegalia/enzimologia , Insuficiência Cardíaca/enzimologia , Histona Desacetilases/metabolismo , Músculo Esquelético/enzimologia , Atrofia Muscular/enzimologia , Miocárdio/enzimologia , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Cardiomegalia/prevenção & controle , Modelos Animais de Doenças , Fibrose , Insuficiência Cardíaca/induzido quimicamente , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/prevenção & controle , Desacetilase 6 de Histona , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/deficiência , Histona Desacetilases/genética , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/patologia , Atrofia Muscular/prevenção & controle , Miocárdio/patologia , Transdução de Sinais , Volume Sistólico , Sístole , Fatores de Tempo , Função Ventricular Esquerda , Remodelação VentricularRESUMO
BACKGROUND: Patients with pulmonary arterial hypertension (PAH) are treated with vasodilators, including endothelin receptor antagonists (ERAs), phosphodiesterase-5 (PDE-5) inhibitors, soluble guanylyl cyclase activators, and prostacyclin. Despite recent advances in pharmacotherapy for individuals with PAH, morbidity and mortality rates in this patient population remain unacceptably high. Here, we tested the hypothesis that combination therapy with two PAH drugs that target distinct biochemical pathways will provide superior efficacy relative to monotherapy in the rat SU5416 plus hypoxia (SU-Hx) model of severe angioproliferative PAH, which closely mimics the human condition. METHODS: Male Sprague Dawley rats were injected with a single dose of SU5416, which is a VEGF receptor antagonist, and exposed to hypobaric hypoxia for three weeks. Rats were subsequently housed at Denver altitude and treated daily with the PDE-5 inhibitor, tadalafil (TAD), the type A endothelin receptor (ETA) antagonist, ambrisentan (AMB), or a combination of TAD and AMB for four additional weeks. RESULTS: Monotherapy with TAD or AMB led to modest reductions in pulmonary arterial pressure (PAP) and right ventricular (RV) hypertrophy. In contrast, echocardiography and invasive hemodynamic measurements revealed that combined TAD/AMB nearly completely reversed pulmonary hemodynamic impairment, RV hypertrophy, and RV functional deficit in SU-Hx rats. Efficacy of TAD/AMB was associated with dramatic reductions in pulmonary vascular remodeling, including suppression of endothelial cell plexiform lesions, which are common in human PAH. CONCLUSIONS: Combined therapy with two vasodilators that are approved for the treatment of human PAH provides unprecedented efficacy in the rat SU-Hx preclinical model of severe, angioproliferative PAH.
Assuntos
Hipertensão Pulmonar/terapia , Hipertrofia Ventricular Direita/terapia , Inibidores da Fosfodiesterase 5/uso terapêutico , Receptores de Endotelina/efeitos dos fármacos , Animais , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
RATIONALE: Histone deacetylase (HDAC) inhibitors are efficacious in models of hypertension-induced left ventricular heart failure. The consequences of HDAC inhibition in the context of pulmonary hypertension with associated right ventricular cardiac remodeling are poorly understood. OBJECTIVE: This study was performed to assess the utility of selective small-molecule inhibitors of class I HDACs in a preclinical model of pulmonary hypertension. METHODS AND RESULTS: Rats were exposed to hypobaric hypoxia for 3 weeks in the absence or presence of a benzamide HDAC inhibitor, MGCD0103, which selectively inhibits class I HDACs 1, 2, and 3. The compound reduced pulmonary arterial pressure more dramatically than tadalafil, a standard-of-care therapy for human pulmonary hypertension that functions as a vasodilator. MGCD0103 improved pulmonary artery acceleration time and reduced systolic notching of the pulmonary artery flow envelope, which suggests a positive impact of the HDAC inhibitor on pulmonary vascular remodeling and stiffening. Similar results were obtained with an independent class I HDAC-selective inhibitor, MS-275. Reduced pulmonary arterial pressure in MGCD0103-treated animals was associated with blunted pulmonary arterial wall thickening because of suppression of smooth muscle cell proliferation. Right ventricular function was maintained in MGCD0103-treated animals. Although the class I HDAC inhibitor only modestly reduced right ventricular hypertrophy, it had multiple beneficial effects on the right ventricle, which included suppression of pathological gene expression, inhibition of proapoptotic caspase activity, and repression of proinflammatory protein expression. CONCLUSIONS: By targeting distinct pathogenic mechanisms, isoform-selective HDAC inhibitors have potential as novel therapeutics for pulmonary hypertension that will complement vasodilator standards of care.
Assuntos
Proliferação de Células/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/efeitos dos fármacos , Hipertensão Pulmonar/prevenção & controle , Músculo Liso Vascular/citologia , Remodelação Ventricular/efeitos dos fármacos , Animais , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/fisiopatologia , Hipertensão Pulmonar/etiologia , Hipóxia/complicações , Músculo Liso Vascular/efeitos dos fármacos , Piridinas/farmacologia , Piridinas/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fluxo Sanguíneo Regional/fisiologiaRESUMO
Cardiac hypertrophy is an independent predictor of adverse outcomes in patients with heart failure, and thus represents an attractive target for novel therapeutic intervention. JQ1, a small molecule inhibitor of bromodomain and extraterminal (BET) acetyl-lysine reader proteins, was identified in a high throughput screen designed to discover novel small molecule regulators of cardiomyocyte hypertrophy. JQ1 dose-dependently blocked agonist-dependent hypertrophy of cultured neonatal rat ventricular myocytes (NRVMs) and reversed the prototypical gene program associated with pathological cardiac hypertrophy. JQ1 also blocked left ventricular hypertrophy (LVH) and improved cardiac function in adult mice subjected to transverse aortic constriction (TAC). The BET family consists of BRD2, BRD3, BRD4 and BRDT. BRD4 protein expression was increased during cardiac hypertrophy, and hypertrophic stimuli promoted recruitment of BRD4 to the transcriptional start site (TSS) of the gene encoding atrial natriuretic factor (ANF). Binding of BRD4 to the ANF TSS was associated with increased phosphorylation of local RNA polymerase II. These findings define a novel function for BET proteins as signal-responsive regulators of cardiac hypertrophy, and suggest that small molecule inhibitors of these epigenetic reader proteins have potential as therapeutics for heart failure.
Assuntos
Cardiomegalia/metabolismo , Proteínas de Transporte/metabolismo , Animais , Azepinas/farmacologia , Cardiomegalia/tratamento farmacológico , Cardiomegalia/patologia , Proteínas de Transporte/química , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Modelos Biológicos , Ligação Proteica/efeitos dos fármacos , Ratos , Triazóis/farmacologiaRESUMO
The transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a key determinant of cardiac metabolic function by regulating genes governing fatty acid oxidation and mitochondrial biogenesis. PGC-1α expression is reduced in many cardiac diseases, and gene deletion of PGC-1α results in impaired cardiomyocyte metabolism and function. Reduced fuel supply generally induces PGC-1α expression, but the specific role of oxygen deprivation is unclear, and the mechanisms governing PGC-1α gene expression in these situations are poorly understood. During hypoxia of primary rat cardiomyocytes up to 12 h, we found that PGC-1α expression was downregulated via a histone deacetylation-dependent mechanism. Conversely, extended hypoxia to 24 h concomitant with glucose depletion upregulated PGC-1α expression via an AMP-activated protein kinase (AMPK)-mediated mechanism. Our previous work demonstrated that estrogen-related receptor-α (ERRα) regulates PGC-1α expression, and we show here that overexpression of ERRα was sufficient to attenuate PGC-1α downregulation in hypoxia. We confirmed that chronic hypoxia downregulated cardiac PGC-1α expression in a hypoxic but nonischemic hypobaric rat model of pulmonary hypertension. Our data demonstrate that depletion of oxygen or fuel results in repression or induction, respectively, of PGC-1α expression via discrete mechanisms, which may contribute to cardiac energetic derangement during hypoxia, ischemia, and failure.
Assuntos
Adenilato Quinase/metabolismo , Regulação da Expressão Gênica/fisiologia , Histonas/metabolismo , Hipóxia/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas de Ligação a RNA/biossíntese , Fatores de Transcrição/biossíntese , Animais , Imunoprecipitação da Cromatina , Modelos Animais de Doenças , Histona Desacetilases/metabolismo , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Hipóxia/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Proteínas de Ligação a RNA/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/genéticaRESUMO
Acute kidney injury (AKI) is common in patients, causes systemic sequelae, and predisposes patients to long-term cardiovascular disease. To date, studies of the effects of AKI on cardiovascular outcomes have only been performed in male mice. We recently demonstrated that male mice developed diastolic dysfunction, hypertension and reduced cardiac ATP levels versus sham 1 year after AKI. The effects of female sex on long-term cardiac outcomes after AKI are unknown. Therefore, we examined the 1-year cardiorenal outcomes following a single episode of bilateral renal ischemia-reperfusion injury in female C57BL/6 mice using a model with similar severity of AKI and performed concomitantly to recently published male cohorts. To match the severity of AKI between male and female mice, females received 34 min of ischemia time compared to 25 min in males. Serial renal function, echocardiograms and blood pressure assessments were performed throughout the 1-year study. Renal histology, and cardiac and plasma metabolomics and mitochondrial function in the heart and kidney were evaluated at 1 year. Measured glomerular filtration rates (GFR) were similar between male and female mice throughout the 1-year study period. One year after AKI, female mice had preserved diastolic function, normal blood pressure, and preserved levels of cardiac ATP. Compared to males, females demonstrated pathway enrichment in arginine metabolism and amino acid related energy production in both the heart and plasma, and glutathione in the plasma. Cardiac mitochondrial respiration in Complex I of the electron transport chain demonstrated improved mitochondrial function in females compared to males, regardless of AKI or sham. This is the first study to examine the long-term cardiac effects of AKI on female mice and indicate that there are important sex-related cardiorenal differences. The role of female sex in cardiovascular outcomes after AKI merits further investigation.
Assuntos
Injúria Renal AgudaRESUMO
Passive stiffness of the heart is determined largely by extracellular matrix and titin, which functions as a molecular spring within sarcomeres. Titin stiffening is associated with the development of diastolic dysfunction (DD), while augmented titin compliance appears to impair systolic performance in dilated cardiomyopathy. We found that myofibril stiffness was elevated in mice lacking histone deacetylase 6 (HDAC6). Cultured adult murine ventricular myocytes treated with a selective HDAC6 inhibitor also exhibited increased myofibril stiffness. Conversely, HDAC6 overexpression in cardiomyocytes led to decreased myofibril stiffness, as did ex vivo treatment of mouse, rat, and human myofibrils with recombinant HDAC6. Modulation of myofibril stiffness by HDAC6 was dependent on 282 amino acids encompassing a portion of the PEVK element of titin. HDAC6 colocalized with Z-disks, and proteomics analysis suggested that HDAC6 functions as a sarcomeric protein deacetylase. Finally, increased myofibril stiffness in HDAC6-deficient mice was associated with exacerbated DD in response to hypertension or aging. These findings define a role for a deacetylase in the control of myofibril function and myocardial passive stiffness, suggest that reversible acetylation alters titin compliance, and reveal the potential of targeting HDAC6 to manipulate the elastic properties of the heart to treat cardiac diseases.
Assuntos
Miofibrilas , Sarcômeros , Animais , Conectina/química , Conectina/genética , Conectina/metabolismo , Desacetilase 6 de Histona/genética , Desacetilase 6 de Histona/metabolismo , Humanos , Camundongos , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Ratos , Sarcômeros/metabolismoRESUMO
Small molecule histone deacetylase (HDAC) inhibitors block adverse cardiac remodeling in animal models of heart failure. The efficacious compounds target class I, class IIb and, to a lesser extent, class IIa HDACs. It is hypothesized that a selective inhibitor of a specific HDAC class (or an isoform within that class) will provide a favorable therapeutic window for the treatment of heart failure, although the optimal selectivity profile for such a compound remains unknown. Genetic studies have suggested that class I HDACs promote pathological cardiac remodeling, while class IIa HDACs are protective. In contrast, nothing is known about the function or regulation of class IIb HDACs in the heart. We developed assays to quantify catalytic activity of distinct HDAC classes in left and right ventricular cardiac tissue from animal models of hypertensive heart disease. Class I and IIa HDAC activity was elevated in some but not all diseased tissues. In contrast, catalytic activity of the class IIb HDAC, HDAC6, was consistently increased in stressed myocardium, but not in a model of physiologic hypertrophy. HDAC6 catalytic activity was also induced by diverse extracellular stimuli in cultured cardiac myocytes and fibroblasts. These findings suggest an unforeseen role for HDAC6 in the heart, and highlight the need for pre-clinical evaluation of HDAC6-selective inhibitors to determine whether this HDAC isoform is pathological or protective in the setting of cardiovascular disease.
Assuntos
Histona Desacetilases/metabolismo , Hipertensão/enzimologia , Miocárdio/enzimologia , Adenoviridae/genética , Animais , Doenças Cardiovasculares , Células Cultivadas , Ventrículos do Coração/enzimologia , Desacetilase 6 de Histona , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/biossíntese , Histona Desacetilases/genética , Hipertensão/patologia , Masculino , Camundongos , Miócitos Cardíacos/enzimologia , Reação em Cadeia da Polimerase , Isoformas de Proteínas , Interferência de RNA , RNA Interferente Pequeno , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Remodelação VentricularRESUMO
Growing epidemiological data demonstrate that acute kidney injury (AKI) is associated with long-term cardiovascular morbidity and mortality. Here, the authors present a 1-year study of cardiorenal outcomes following bilateral ischemia-reperfusion injury in male mice. These data suggest that AKI causes long-term dysfunction in the cardiac metabolome, which is associated with diastolic dysfunction and hypertension. Mice treated with the histone deacetylase inhibitor, ITF2357, had preservation of cardiac function and remained normotensive throughout the study. ITF2357 did not protect against the development of kidney fibrosis after AKI.
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Hepatic and circulating endothelin-1 (ET-1) are increased in patients with cirrhosis and in cirrhotic animals. However, the distinct roles of ET receptor subtypes ETA and ETB in cirrhosis and portal hypertension (PHT) have not been clearly elucidated. Thus, we studied the effects of selective ET-1 antagonists (ETA-ant or ETB-ant) and nonselective ET-1 antagonist (ETA/B-ant) on hepatic hemodynamics in cirrhotic rats. Liver fibrosis and PHT were induced by complete bile duct ligation (BDL) in rats. Two weeks after BDL or sham surgery, hemodynamic responses were measured during intraportal infusion of incremental doses of the following ET-ants: (i) BQ-123, (ii) BQ-788, and (iii) bosentan. After equilibration with vehicle, doses of ET-ants were infused for 30 min periods, and steady-state systemic and hepatic hemodynamics, portal venous pressure (PVP), and hepatic blood flow (HBF) were measured. BDL induced significant PHT and elevated concentrations of plasma ET-1 compared with sham. ETA-ant decreased PVP of cirrhotic rats but had no effect on sham, whereas ETB-ant increased PVP in sham but had no effect in BDL. Nonselective ETA/B-ant decreased PVP of BDL similarly to ETA-ant. Both ETA-ant and ETB-ant decreased local HBF, whereas a nonselective antagonist did not change HBF in sham; however no significant changes were observed in HBF of BDL rats with any of the antagonists. These findings suggest ETA activation contributes to PHT in cirrhotic rats, whereas ETB-mediated portal depressor effects are attenuated in cirrhotic rats compared with noncirrhotic rats.
Assuntos
Antagonistas dos Receptores de Endotelina , Hemodinâmica/efeitos dos fármacos , Cirrose Hepática/fisiopatologia , Fígado/irrigação sanguínea , Animais , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Bosentana , Modelos Animais de Doenças , Endotelina-1/sangue , Endotelina-1/genética , Expressão Gênica/genética , Hemodinâmica/fisiologia , Células Estreladas do Fígado/metabolismo , Hipertensão Portal/etiologia , Hipertensão Portal/fisiopatologia , Fígado/efeitos dos fármacos , Fígado/fisiopatologia , Cirrose Hepática/sangue , Cirrose Hepática/complicações , Cirrose Hepática/metabolismo , Masculino , Oligopeptídeos/administração & dosagem , Oligopeptídeos/farmacologia , Peptídeos Cíclicos/administração & dosagem , Peptídeos Cíclicos/farmacologia , Piperidinas/administração & dosagem , Piperidinas/farmacologia , Pressão na Veia Porta/efeitos dos fármacos , Pressão na Veia Porta/fisiologia , Ratos , Ratos Sprague-Dawley , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fluxo Sanguíneo Regional/fisiologia , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacologiaRESUMO
Tubastatin A, a tetrahydro-γ-carboline-capped selective HDAC6 inhibitor (HDAC6i), was rationally designed 10 years ago, and has become the best investigated HDAC6i to date. It shows efficacy in various neurological disease animal models, as HDAC6 plays a crucial regulatory role in axonal transport deficits, protein aggregation, as well as oxidative stress. In this work, we provide new insights into this HDAC6i by investigating the molecular basis of its interactions with HDAC6 through X-ray crystallography, determining its functional capability to elevate the levels of acetylated α-tubulin in vitro and in vivo, correlating PK/PD profiles to determine effective doses in plasma and brain, and finally assessing its therapeutic potential toward psychiatric diseases through use of the SmartCube screening platform.
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BACKGROUND: Cardiovascular disease and myocardial infarction are associated with high mortality and morbidity and a more effective treatment remains a major clinical need. The intramyocardial injection of biomaterials has been investigated as a potential treatment for heart failure by providing mechanical support to the myocardium and reducing stress on cardiomyocytes. Another treatment approach that has been explored is therapeutic angiogenesis that requires careful spatiotemporal control of angiogenic drug delivery. An injectable sulfonated reversible thermal gel composed of a polyurea conjugated with poly(N-isopropylacrylamide) and sulfonate groups has been developed for intramyocardial injection with angiogenic factors for the protection of cardiac function after a myocardial infarction. RESULTS: The thermal gel allowed for the sustained, localized release of VEGF in vivo with intramyocardial injection after two weeks. A myocardial infarction reperfusion injury model was used to evaluate therapeutic benefits to cardiac function and vascularization. Echocardiography presented improved cardiac function, infarct size and ventricular wall thinning were reduced, and immunohistochemistry showed improved vascularization with thermal gel injections. The thermal gel alone showed cardioprotective and vascularization properties, and slightly improved further with the additional delivery of VEGF. An inflammatory response evaluation demonstrated the infiltration of macrophages due to the myocardial infarction was more significant compared to the foreign body inflammatory response to the thermal gel. Detecting DNA fragments of apoptotic cells also demonstrated potential anti-apoptotic effects of the thermal gel. CONCLUSION: The intramyocardial injection of the sulfonated reversible thermal gel has cardioprotective and vascularization properties for the treatment of myocardial infarction.