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1.
Circulation ; 141(9): 751-767, 2020 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-31948273

RESUMEN

BACKGROUND: Myocardial fibrosis is a hallmark of cardiac remodeling and functionally involved in heart failure development, a leading cause of deaths worldwide. Clinically, no therapeutic strategy is available that specifically attenuates maladaptive responses of cardiac fibroblasts, the effector cells of fibrosis in the heart. Therefore, our aim was to develop novel antifibrotic therapeutics based on naturally derived substance library screens for the treatment of cardiac fibrosis. METHODS: Antifibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts, subsequent validation, and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of human cardiac fibroblasts, modulation of apoptosis, and extracellular matrix expression. In vitro findings were confirmed in vivo with an angiotensin II-mediated murine model of cardiac fibrosis in both preventive and therapeutic settings, as well as in the Dahl salt-sensitive rat model. To investigate the mechanism underlying the antifibrotic potential of the lead compounds, treatment-dependent changes in the noncoding RNAome in primary human cardiac fibroblasts were analyzed by RNA deep sequencing. RESULTS: High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds. CONCLUSIONS: We identified the molecules bufalin and lycorine as drug candidates for therapeutic applications in cardiac fibrosis and diastolic dysfunction.


Asunto(s)
Alcaloides de Amaryllidaceae/farmacología , Bufanólidos/farmacología , Cardiomiopatías/prevención & control , Fármacos Cardiovasculares/farmacología , Fibroblastos/efectos de los fármacos , Fenantridinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Cardiomiopatías/etiología , Cardiomiopatías/metabolismo , Cardiomiopatías/fisiopatología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Diástole , Modelos Animales de Enfermedad , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis , Ensayos Analíticos de Alto Rendimiento , Humanos , Hipertensión/complicaciones , Hipertensión/fisiopatología , Masculino , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Miocardio/metabolismo , Miocardio/patología , Ratas Endogámicas Dahl , Selenoproteína P/genética , Selenoproteína P/metabolismo , Función Ventricular Izquierda/efectos de los fármacos
2.
J Mol Cell Cardiol ; 146: 43-59, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32649928

RESUMEN

Myocardial ischemia induces a multifaceted remodeling process in the heart. Novel therapeutic entry points to counteract maladaptive signalling include the modulation of non-coding RNA molecules such as long non-coding RNA (lncRNA). We here questioned if the lncRNA candidate H19 exhibits regulatory potential in the setting of myocardial infarction. Initial profiling of H19 expression revealed a dynamic expression profile of H19 with upregulation in the acute phase after murine cardiac ischemia. In vitro, we found that oxygen deficiency leads to H19 upregulation in several cardiac cell types. Repression of endogenous H19 caused multiple phenotypes in cultivated murine cardiomyocytes including enhanced cardiomyocyte apoptosis, at least partly through attenuated vitamin D signalling. Unbiased proteome analysis revealed further involvement of H19 in mRNA splicing and translation as well as inflammatory signalling pathways. To study H19 function more precisely, we investigated the phenotype of systemic H19 loss in a genetic mouse model of H19 deletion (H19 KO). Infarcted heart tissue of H19 KO mice showed a massive increase of pro-inflammatory cytokines after ischemia-reperfusion injury (I/R) without significant effects on scar formation or cardiac function but exaggerated cardiac hypertrophy indicating pathological cardiac remodeling. H19-dependent changes in cardiomyocyte-derived extracellular vesicle release and alterations in NF-κB signalling were evident. Cardiac cell fractionation experiments revealed that enhanced H19 expression in the proliferative phase after MI derived mainly from cardiac fibroblasts. Here further research is needed to elucidate its role in fibroblast activation and function. In conclusion, the lncRNA H19 is dynamically regulated after MI and involved in multiple pathways of different cardiac cell types including cardiomyocyte apoptosis and cardiac inflammation.


Asunto(s)
Pleiotropía Genética , Corazón/fisiopatología , Isquemia Miocárdica/genética , Isquemia Miocárdica/fisiopatología , ARN Largo no Codificante/metabolismo , Animales , Línea Celular , Supervivencia Celular/genética , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Inflamación/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Isquemia Miocárdica/patología , Miocitos Cardíacos/metabolismo , Oxígeno , Proteoma/metabolismo , ARN Largo no Codificante/genética , Receptores de Calcitriol/metabolismo , Remodelación Vascular/genética
3.
Circ Res ; 121(5): 575-583, 2017 Aug 18.
Artículo en Inglés | MEDLINE | ID: mdl-28630135

RESUMEN

RATIONALE: Cardiac fibroblasts (CFs) drive extracellular matrix remodeling after pressure overload, leading to fibrosis and diastolic dysfunction. Recent studies described the role of long noncoding RNAs (lncRNAs) in cardiac pathologies. Nevertheless, detailed reports on lncRNAs regulating CF biology and describing their implication in cardiac remodeling are still missing. OBJECTIVE: Here, we aimed at characterizing lncRNA expression in murine CFs after chronic pressure overload to identify CF-enriched lncRNAs and investigate their function and contribution to cardiac fibrosis and diastolic dysfunction. METHODS AND RESULTS: Global lncRNA profiling identified several dysregulated transcripts. Among them, the lncRNA maternally expressed gene 3 (Meg3) was found to be mostly expressed by CFs and to undergo transcriptional downregulation during late cardiac remodeling. In vitro, Meg3 regulated the production of matrix metalloproteinase-2 (MMP-2). GapmeR-mediated silencing of Meg3 in CFs resulted in the downregulation of Mmp-2 transcription, which, in turn, was dependent on P53 activity both in the absence and in the presence of transforming growth factor-ß I. Chromatin immunoprecipitation showed that further induction of Mmp-2 expression by transforming growth factor-ß I was blocked by Meg3 silencing through the inhibition of P53 binding on the Mmp-2 promoter. Consistently, inhibition of Meg3 in vivo after transverse aortic constriction prevented cardiac MMP-2 induction, leading to decreased cardiac fibrosis and improved diastolic performance. CONCLUSIONS: Collectively, our findings uncover a critical role for Meg3 in the regulation of MMP-2 production by CFs in vitro and in vivo, identifying a new player in the development of cardiac fibrosis and potential new target for the prevention of cardiac remodeling.


Asunto(s)
Fibroblastos/metabolismo , Insuficiencia Cardíaca Diastólica/metabolismo , Insuficiencia Cardíaca Diastólica/prevención & control , Miocitos Cardíacos/metabolismo , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/metabolismo , Animales , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Cardiomiopatías/prevención & control , Células Cultivadas , Fibroblastos/patología , Fibrosis/metabolismo , Fibrosis/patología , Fibrosis/prevención & control , Insuficiencia Cardíaca Diastólica/patología , Masculino , Metaloproteinasa 2 de la Matriz/biosíntesis , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/patología , Ratas , Remodelación Ventricular/fisiología
4.
Circulation ; 134(24): 1973-1990, 2016 Dec 13.
Artículo en Inglés | MEDLINE | ID: mdl-27780851

RESUMEN

BACKGROUND: The transcription factor GATA2 orchestrates the expression of many endothelial-specific genes, illustrating its crucial importance for endothelial cell function. The capacity of this transcription factor in orchestrating endothelial-important microRNAs (miRNAs/miR) is unknown. METHODS: Endothelial GATA2 was functionally analyzed in human endothelial cells in vitro. Endogenous short interfering RNA-mediated knockdown and lentiviral-based overexpression were applied to decipher the capacity of GATA2 in regulating cell viability and capillary formation. Next, the GATA2-dependent miR transcriptome was identified by using a profiling approach on the basis of quantitative real-time polymerase chain reaction. Transcriptional control of miR promoters was assessed via chromatin immunoprecipitation, luciferase promoter assays, and bisulfite sequencing analysis of sites in proximity. Selected miRs were modulated in combination with GATA2 to identify signaling pathways at the angiogenic cytokine level via proteome profiler and enzyme-linked immunosorbent assays. Downstream miR targets were identified via bioinformatic target prediction and luciferase reporter gene assays. In vitro findings were translated to a mouse model of carotid injury in an endothelial GATA2 knockout background. Nanoparticle-mediated delivery of proangiogenic miR-126 was tested in the reendothelialization model. RESULTS: GATA2 gain- and loss-of-function experiments in human umbilical vein endothelial cells identified a key role of GATA2 as master regulator of multiple endothelial functions via miRNA-dependent mechanisms. Global miRNAnome-screening identified several GATA2-regulated miRNAs including miR-126 and miR-221. Specifically, proangiogenic miR-126 was regulated by GATA2 transcriptionally and targeted antiangiogenic SPRED1 and FOXO3a contributing to GATA2-mediated formation of normal vascular structures, whereas GATA2 deficiency led to vascular abnormalities. In contrast to GATA2 deficiency, supplementation with miR-126 normalized vascular function and expression profiles of cytokines contributing to proangiogenic paracrine effects. GATA2 silencing resulted in endothelial DNA hypomethylation leading to induced expression of antiangiogenic miR-221 by GATA2-dependent demethylation of a putative CpG island in the miR-221 promoter. Mechanistically, a reverted GATA2 phenotype by endogenous suppression of miR-221 was mediated through direct proangiogenic miR-221 target genes ICAM1 and ETS1. In a mouse model of carotid injury, GATA2 was reduced, and systemic supplementation of miR-126-coupled nanoparticles enhanced miR-126 availability in the carotid artery and improved reendothelialization of injured carotid arteries in vivo. CONCLUSIONS: GATA2-mediated regulation of miR-126 and miR-221 has an important impact on endothelial biology. Hence, modulation of GATA2 and its targets miR-126 and miR-221 is a promising therapeutic strategy for treatment of many vascular diseases.


Asunto(s)
Enfermedades de las Arterias Carótidas/terapia , Factor de Transcripción GATA2/metabolismo , MicroARNs/uso terapéutico , Regiones no Traducidas 3' , Proteínas Adaptadoras Transductoras de Señales , Animales , Antagomirs/metabolismo , Secuencia de Bases , Enfermedades de las Arterias Carótidas/patología , Modelos Animales de Enfermedad , Proteína Forkhead Box O3/antagonistas & inhibidores , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Factor de Transcripción GATA2/antagonistas & inhibidores , Factor de Transcripción GATA2/genética , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Molécula 1 de Adhesión Intercelular/química , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lentivirus/genética , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/antagonistas & inhibidores , MicroARNs/metabolismo , Nanopartículas/química , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Alineación de Secuencia
5.
J Am Coll Cardiol ; 68(14): 1557-71, 2016 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-27687198

RESUMEN

BACKGROUND: Aging populations show higher incidences of myocardial infarction (MI) and heart failure (HF). Cardiac remodeling post-MI leads to progressive impaired cardiac function caused by a disarray of several processes including derailed autophagy. Microribonucleic acids (miRNAs) are known to be key players in cardiovascular disease but their involvement in cardiac autophagy and aging is not well understood. OBJECTIVES: This study sought to identify new miRNA candidates that regulate cardiac autophagy and aging. METHODS: We exploited a high-throughput, fluorescence-activated cell sorting-based green fluorescent protein-LC3 detection method to measure the autophagic flux in cardiomyocytes after transfection of a precursor miRNA library consisting of 380 miRNAs. This was followed by a series of molecular and in vivo studies. RESULTS: Together with additional expression screenings, we identified miR-22 as an abundant and strong inhibitor of the cardiac autophagy process. Cardiac miR-22 expression levels increased during aging of mice as well as in aging neonatal cardiomyocytes in vitro by a P53-dependent mechanism. Inhibition of miR-22 in aging cardiomyocytes in vitro activated autophagy and inhibited cellular hypertrophy. Pharmacological inhibition of miR-22 post-MI in older mice activated cardiac autophagy, prevented post-infarction remodeling, and improved cardiac function compared with control subjects. Interestingly, similar effects were less pronounced in younger mice with significantly lower cardiac miR-22 expression levels. In addition, circulating levels of miR-22 in 154 patients with systolic HF were highly associated with early mortality. CONCLUSIONS: We concluded that miR-22 is an important regulator of cardiac autophagy and a potential therapeutic target, especially in the older myocardium. Finally, circulating miR-22 provides prognostic information for HF patients, highlighting miR-22 as a promising therapeutic and biomarker candidate for cardiovascular disorders.


Asunto(s)
Autofagia/genética , MicroARNs/fisiología , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/genética , Anciano , Animales , Biomarcadores/sangre , Células Cultivadas , Modelos Animales de Enfermedad , Insuficiencia Cardíaca/sangre , Insuficiencia Cardíaca/genética , Humanos , Ratones , MicroARNs/antagonistas & inhibidores , MicroARNs/sangre , Infarto del Miocardio/patología , Miocitos Cardíacos
6.
Sci Transl Med ; 8(326): 326ra22, 2016 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-26888430

RESUMEN

Recent studies highlighted long noncoding RNAs (lncRNAs) to play an important role in cardiac development. However, understanding of lncRNAs in cardiac diseases is still limited. Global lncRNA expression profiling indicated that several lncRNA transcripts are deregulated during pressure overload-induced cardiac hypertrophy in mice. Using stringent selection criteria, we identified Chast (cardiac hypertrophy-associated transcript) as a potential lncRNA candidate that influences cardiomyocyte hypertrophy. Cell fractionation experiments indicated that Chast is specifically up-regulated in cardiomyocytes in vivo in transverse aortic constriction (TAC)-operated mice. In accordance, CHAST homolog in humans was significantly up-regulated in hypertrophic heart tissue from aortic stenosis patients and in human embryonic stem cell-derived cardiomyocytes upon hypertrophic stimuli. Viral-based overexpression of Chast was sufficient to induce cardiomyocyte hypertrophy in vitro and in vivo. GapmeR-mediated silencing of Chast both prevented and attenuated TAC-induced pathological cardiac remodeling with no early signs on toxicological side effects. Mechanistically, Chast negatively regulated Pleckstrin homology domain-containing protein family M member 1 (opposite strand of Chast), impeding cardiomyocyte autophagy and driving hypertrophy. These results indicate that Chast can be a potential target to prevent cardiac remodeling and highlight a general role of lncRNAs in heart diseases.


Asunto(s)
ARN Largo no Codificante/metabolismo , Remodelación Ventricular/genética , Animales , Secuencia de Bases , Cardiomegalia/genética , Cardiomegalia/patología , Cardiomegalia/fisiopatología , Regulación de la Expresión Génica , Humanos , Masculino , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Factores de Transcripción NFATC/metabolismo , Presión , ARN Largo no Codificante/genética , Transducción de Señal , Investigación Biomédica Traslacional
7.
J Clin Invest ; 124(5): 2136-46, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24743145

RESUMEN

In response to stress, the heart undergoes extensive cardiac remodeling that results in cardiac fibrosis and pathological growth of cardiomyocytes (hypertrophy), which contribute to heart failure. Alterations in microRNA (miRNA) levels are associated with dysfunctional gene expression profiles associated with many cardiovascular disease conditions; however, miRNAs have emerged recently as paracrine signaling mediators. Thus, we investigated a potential paracrine miRNA crosstalk between cardiac fibroblasts and cardiomyocytes and found that cardiac fibroblasts secrete miRNA-enriched exosomes. Surprisingly, evaluation of the miRNA content of cardiac fibroblast-derived exosomes revealed a relatively high abundance of many miRNA passenger strands ("star" miRNAs), which normally undergo intracellular degradation. Using confocal imaging and coculture assays, we identified fibroblast exosomal-derived miR-21_3p (miR-21*) as a potent paracrine-acting RNA molecule that induces cardiomyocyte hypertrophy. Proteome profiling identified sorbin and SH3 domain-containing protein 2 (SORBS2) and PDZ and LIM domain 5 (PDLIM5) as miR-21* targets, and silencing SORBS2 or PDLIM5 in cardiomyocytes induced hypertrophy. Pharmacological inhibition of miR-21* in a mouse model of Ang II-induced cardiac hypertrophy attenuated pathology. These findings demonstrate that cardiac fibroblasts secrete star miRNA-enriched exosomes and identify fibroblast-derived miR-21* as a paracrine signaling mediator of cardiomyocyte hypertrophy that has potential as a therapeutic target.


Asunto(s)
Cardiomegalia/metabolismo , Exosomas/metabolismo , Fibroblastos/metabolismo , MicroARNs/metabolismo , Miocitos Cardíacos/metabolismo , Comunicación Paracrina , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Animales , Cardiomegalia/patología , Exosomas/patología , Fibroblastos/patología , Silenciador del Gen , Ratones , Proteínas de Microfilamentos/biosíntesis , Miocitos Cardíacos/patología , Ratas
8.
Nat Commun ; 3: 1078, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23011132

RESUMEN

Pathological growth of cardiomyocytes (hypertrophy) is a major determinant for the development of heart failure, one of the leading medical causes of mortality worldwide. Here we show that the microRNA (miRNA)-212/132 family regulates cardiac hypertrophy and autophagy in cardiomyocytes. Hypertrophic stimuli upregulate cardiomyocyte expression of miR-212 and miR-132, which are both necessary and sufficient to drive the hypertrophic growth of cardiomyocytes. MiR-212/132 null mice are protected from pressure-overload-induced heart failure, whereas cardiomyocyte-specific overexpression of the miR-212/132 family leads to pathological cardiac hypertrophy, heart failure and death in mice. Both miR-212 and miR-132 directly target the anti-hypertrophic and pro-autophagic FoxO3 transcription factor and overexpression of these miRNAs leads to hyperactivation of pro-hypertrophic calcineurin/NFAT signalling and an impaired autophagic response upon starvation. Pharmacological inhibition of miR-132 by antagomir injection rescues cardiac hypertrophy and heart failure in mice, offering a possible therapeutic approach for cardiac failure.


Asunto(s)
Autofagia/genética , Cardiomegalia/genética , MicroARNs/genética , Miocitos Cardíacos/metabolismo , Oligonucleótidos/genética , Animales , Antagomirs , Calcineurina/genética , Células Cultivadas , Masculino , Ratones , Ratones Transgénicos , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
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