RESUMEN
Heart failure is a leading cause of death that develops subsequent to deleterious hypertrophic cardiac remodelling. MAPK pathways play a key role in coordinating the induction of gene expression during hypertrophy. Induction of the immediate early gene (IEG) response including activator protein 1 (AP-1) complex factors is a necessary and early event in this process. How MAPK and IEG expression are coupled during cardiac hypertrophy is not resolved. Here, in vitro, in rodent models and in human samples, we demonstrate that MAPK-stimulated IEG induction depends on the mitogen and stress-activated protein kinase (MSK) and its phosphorylation of histone H3 at serine 28 (pH3S28). pH3S28 in IEG promoters in turn recruits Brg1, a BAF60 ATP-dependent chromatin remodelling complex component, initiating gene expression. Without MSK activity and IEG induction, the hypertrophic response is suppressed. These studies provide new mechanistic insights into the role of MAPK pathways in signalling to the epigenome and regulation of gene expression during cardiac hypertrophy.
Asunto(s)
Ensamble y Desensamble de Cromatina , Histonas , Cardiomegalia/genética , Expresión Génica , Histonas/metabolismo , Humanos , Fosforilación , Serina/metabolismoRESUMEN
Pharmaceutical agents despite their efficacy to treat disease can cause additional unwanted cardiovascular side effects. Cardiotoxicity is characterized by changes in either the function and/or structure of the myocardium. Over recent years, functional cardiotoxicity has received much attention, however morphological damage to the myocardium and/or loss of viability still requires improved detection and mechanistic insights. A human 3D cardiac microtissue containing human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), cardiac endothelial cells and cardiac fibroblasts was used to assess their suitability to detect drug induced changes in cardiac structure. Histology and clinical pathology confirmed these cardiac microtissues were morphologically intact, lacked a necrotic/apoptotic core and contained all relevant cell constituents. High-throughput methods to assess mitochondrial membrane potential, endoplasmic reticulum integrity and cellular viability were developed and 15 FDA approved structural cardiotoxins and 14 FDA approved non-structural cardiotoxins were evaluated. We report that cardiac microtissues provide a high-throughput experimental model that is both able to detect changes in cardiac structure at clinically relevant concentrations and provide insights into the phenotypic mechanisms of this liability.
Asunto(s)
Imagenología Tridimensional , Miocardio/patología , Antineoplásicos/farmacología , Biomarcadores/metabolismo , Cardiotoxinas/química , Supervivencia Celular/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Supervivencia Tisular/efectos de los fármacosRESUMEN
G-protein coupled receptor (GPCR) mediated activation of the MAPK signalling cascade is a key pathway in the induction of hypertrophic remodelling of the heart - a response to pathological cues including hypertension and myocardial infarction. While levels of pro-hypertrophic hormone agonists of GPCRs increase during periods of greater workload to enhance cardiac output, hypertrophy does not necessarily result. Here we investigated the relationship between the duration of exposure to the pro-hypertrophic GPCR agonist endothelin-1 (ET-1) and the induction of hypertrophic remodelling in neonatal rat ventricular myocytes (NRVM) and in the adult rat heart in vivo. Notably, a 15min pulse of ET-1 was sufficient to induce markers of hypertrophy that were present when measured at 24h in vivo and 48h in vitro. The persistence of ET-1 action was insensitive to ET type A receptor (ETA receptor) antagonism with BQ123. The extended effects of ET-1 were dependent upon sustained MAPK signalling and involved persistent transcription. Inhibitors of endocytosis however conferred sensitivity upon the hypertrophic response to BQ123, suggesting that endocytosis of ETA receptors following ligand binding preserves their active state by protection against antagonist. Contrastingly, α1 adrenergic-induced hypertrophic responses required the continued presence of agonist and were sensitive to antagonist. These studies shed new light on strategies to pharmacologically intervene in the action of different pro-hypertrophic mediators.
Asunto(s)
Cardiomegalia/genética , Cardiomegalia/patología , Endotelina-1/farmacología , Miocitos Cardíacos/metabolismo , Receptor de Endotelina A/metabolismo , Transducción de Señal , Transcripción Genética/efectos de los fármacos , Animales , Animales Recién Nacidos , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Masculino , Miocitos Cardíacos/efectos de los fármacos , Fenilefrina/farmacología , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Components of the type II CRISPR-Cas complex in bacteria have been used successfully in eukaryotic cells to facilitate rapid and accurate cell line engineering, animal model generation and functional genomic screens. Such developments are providing new opportunities for drug target identification and validation, particularly with the application of pooled genetic screening. As CRISPR-Cas is a relatively new genetic screening tool, it is important to assess its functionality in a number of different cell lines and to analyse potential improvements that might increase the sensitivity of a given screen. To examine critical aspects of screening quality, we constructed ultra-complex libraries containing sgRNA sequences targeting a collection of essential genes. We examined the performance of screening in both haploid and hypotriploid cell lines, using two alternative guide design algorithms and two tracrRNA variants in a time-resolved analysis. Our data indicate that a simple adaptation of the tracrRNA substantially improves the robustness of guide loss during a screen. This modification minimises the requirement for high numbers of sgRNAs targeting each gene, increasing hit scoring and creating a powerful new platform for successful screening.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Marcación de Gen/métodos , Ingeniería Genética/métodos , Pruebas Genéticas/métodos , Secuencia de Bases , Línea Celular Tumoral , Células HEK293 , Células HL-60 , Humanos , ARN Guía de Kinetoplastida/genética , Reproducibilidad de los ResultadosRESUMEN
UNLABELLED: Endothelin-1 (ET-1) is a critical autocrine and paracrine regulator of cardiac physiology and pathology. Produced locally within the myocardium in response to diverse mechanical and neurohormonal stimuli, ET-1 acutely modulates cardiac contractility. During pathological cardiovascular conditions such as ischaemia, left ventricular hypertrophy and heart failure, myocyte expression and activity of the entire ET-1 system is enhanced, allowing the peptide to both initiate and maintain maladaptive cellular responses. Both the acute and chronic effects of ET-1 are dependent on the activation of intracellular signalling pathways, regulated by the inositol-trisphosphate and diacylglycerol produced upon activation of the ET(A) receptor. Subsequent stimulation of protein kinases C and D, calmodulin-dependent kinase II, calcineurin and MAPKs modifies the systolic calcium transient, myofibril function and the activity of transcription factors that coordinate cellular remodelling. The precise nature of the cellular response to ET-1 is governed by the timing, localization and context of such signals, allowing the peptide to regulate both cardiomyocyte physiology and instigate disease. LINKED ARTICLES: This article is part of a themed section on Endothelin. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.168.issue-1.