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A functional crosstalk between epigenetic regulators and metabolic control could provide a mechanism to adapt cellular responses to environmental cues. We report that the well-known nuclear MYST family acetyl transferase MOF and a subset of its non-specific lethal complex partners reside in mitochondria. MOF regulates oxidative phosphorylation by controlling expression of respiratory genes from both nuclear and mtDNA in aerobically respiring cells. MOF binds mtDNA, and this binding is dependent on KANSL3. The mitochondrial pool of MOF, but not a catalytically deficient mutant, rescues respiratory and mtDNA transcriptional defects triggered by the absence of MOF. Mof conditional knockout has catastrophic consequences for tissues with high-energy consumption, triggering hypertrophic cardiomyopathy and cardiac failure in murine hearts; cardiomyocytes show severe mitochondrial degeneration and deregulation of mitochondrial nutrient metabolism and oxidative phosphorylation pathways. Thus, MOF is a dual-transcriptional regulator of nuclear and mitochondrial genomes connecting epigenetics and metabolism.
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Metabolismo Energético/genética , Epigênese Genética , Histona Acetiltransferases/metabolismo , Mitocôndrias Musculares/enzimologia , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Cardiomiopatia Hipertrófica/genética , Respiração Celular/genética , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Células HeLa , Insuficiência Cardíaca/genética , Histona Acetiltransferases/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/genética , Mitocôndrias Musculares/genética , Miócitos Cardíacos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação Oxidativa , Fatores de Transcrição/genéticaRESUMO
The mitochondrial proteome is built mainly by import of nuclear-encoded precursors, which are targeted mostly by cleavable presequences. Presequence processing upon import is essential for proteostasis and survival, but the consequences of dysfunctional protein maturation are unknown. We find that impaired presequence processing causes accumulation of precursors inside mitochondria that form aggregates, which escape degradation and unexpectedly do not cause cell death. Instead, cells survive via activation of a mitochondrial unfolded protein response (mtUPR)-like pathway that is triggered very early after precursor accumulation. In contrast to classical stress pathways, this immediate response maintains mitochondrial protein import, membrane potential, and translation through translocation of the nuclear HMG-box transcription factor Rox1 to mitochondria. Rox1 binds mtDNA and performs a TFAM-like function pivotal for transcription and translation. Induction of early mtUPR provides a reversible stress model to mechanistically dissect the initial steps in mtUPR pathways with the stressTFAM Rox1 as the first line of defense.
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Mitocôndrias/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Morte Celular/fisiologia , Núcleo Celular/metabolismo , DNA Mitocondrial/metabolismo , Potenciais da Membrana/fisiologia , Biossíntese de Proteínas/fisiologia , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica/fisiologiaRESUMO
IntroductionChronic heart failure is associated with adverse remodeling of the heart that is typically characterized by cardiomyocyte hypertrophy. This requires the formation of new capillaries to maintain oxygen supply. Insufficient angiogenesis promotes the transition from compensated hypertrophy into heart failure. The aim of the present study was to identify angiogenesis-related gene networks and corresponding regulatory hubs in endothelial cells from failing human hearts.Methods and resultsWe isolated left ventricular endothelial cells from patients with advanced heart failure undergoing left ventricular assist device surgery (n = 15) and healthy organ donors (n = 2) and performed RNA sequencing. Subgroup analysis revealed no impact of co-morbidities on gene expression. In a weighted coexpression network analysis, we found 26 gene clusters, of which nine clusters showed a significant positive or negative correlation with the presence of heart failure. We identified the transcription factors CASZ1 (castor zinc finger 1), ZNF523 (zinc finger protein 523), and NFE2L1 (nuclear factor erythroid 2-related factor 1) as hub genes of a cluster related to angiogenesis. Knock-down of CASZ1, ZNF523, or NFE2L1 in human umbilical vein endothelial cells led to a downregulation of genes from the respective cluster, including CD34 and platelet derived growth factor ß, confirming their regulatory function.ConclusionIn conclusion, we assessed gene networks in endothelial cells and identified transcription factors CASZ1, ZNF532, and NFE2L1 as potential regulators of angiogenesis in failing human hearts. Our study provides insights into the transcriptional regulation of angiogenesis beyond the classical vascular endothelial growth factor signaling pathway.
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Spatial genome organization is tightly controlled by several regulatory mechanisms and is essential for gene expression control. Nuclear receptors are ligand-activated transcription factors that modulate physiological and pathophysiological processes and are primary pharmacological targets. DNA binding of the important loop-forming insulator protein CCCTC-binding factor (CTCF) was modulated by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). We performed CTCF HiChIP assays to produce the first genome-wide dataset of CTCF long-range interactions in 1,25(OH)2D3-treated cells, and to determine whether dynamic changes of spatial chromatin interactions are essential for fine-tuning of nuclear receptor signaling. We detected changes in 3D chromatin organization upon vitamin D receptor (VDR) activation at 3.1% of all observed CTCF interactions. VDR binding was enriched at both differential loop anchors and within differential loops. Differential loops were observed in several putative functional roles including TAD border formation, promoter-enhancer looping, and establishment of VDR-responsive insulated neighborhoods. Vitamin D target genes were enriched in differential loops and at their anchors. Secondary vitamin D effects related to dynamic chromatin domain changes were linked to location of downstream transcription factors in differential loops. CRISPR interference and loop anchor deletion experiments confirmed the functional relevance of nuclear receptor ligand-induced adjustments of the chromatin 3D structure for gene expression regulation.
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Cromatina , Receptores de Calcitriol , Cromatina/genética , Expressão Gênica , Ligantes , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/metabolismo , Vitamina D/metabolismo , Vitamina D/farmacologiaRESUMO
BACKGROUND: Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role. METHODS: To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent-offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls. RESULTS: Gene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues. CONCLUSIONS: Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.
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Síndrome do Coração Esquerdo Hipoplásico/genética , Organogênese/genética , Heterogeneidade Genética , HumanosRESUMO
The Galaxy HiCExplorer provides a web service at https://hicexplorer.usegalaxy.eu. It enables the integrative analysis of chromosome conformation by providing tools and computational resources to pre-process, analyse and visualize Hi-C, Capture Hi-C (cHi-C) and single-cell Hi-C (scHi-C) data. Since the last publication, Galaxy HiCExplorer has been expanded considerably with new tools to facilitate the analysis of cHi-C and to provide an in-depth analysis of Hi-C data. Moreover, it supports the analysis of scHi-C data by offering a broad range of tools. With the help of the standard graphical user interface of Galaxy, presented workflows, extensive documentation and tutorials, novices as well as Hi-C experts are supported in their Hi-C data analysis with Galaxy HiCExplorer.
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Cromatina/química , Software , Gráficos por Computador , Técnicas Genéticas/normas , Internet , Conformação Molecular , Reprodutibilidade dos Testes , Análise de Célula Única/normasRESUMO
BACKGROUND: Diabetes mellitus is a worldwide epidemic that causes high mortality due to cardiovascular complications, in particular heart failure. Diabetes is associated with profound pathophysiological changes in the heart. The aim of this study was to investigate the impact of diabetes on gene expression and DNA methylation in cardiac cells. METHODS AND RESULTS: Transcriptome analysis of heart tissue from mice with streptozotocin-induced diabetes revealed only 39 genes regulated, whereas cell type-specific analysis of the diabetic heart was more sensitive and more specific than heart tissue analysis and revealed a total of 3205 differentially regulated genes in five cell types. Whole genome DNA methylation analysis with basepair resolution of distinct cardiac cell types identified highly specific DNA methylation signatures of genic and regulatory regions. Interestingly, despite marked changes in gene expression, DNA methylation remained stable in streptozotocin-induced diabetes. Integrated analysis of cell type-specific gene expression enabled us to assign the particular contribution of single cell types to the pathophysiology of the diabetic heart. Finally, analysis of gene regulation revealed ligand-receptor pairs as potential mediators of heterocellular interaction in the diabetic heart, with fibroblasts and monocytes showing the highest degree of interaction. CONCLUSION: In summary, cell type-specific analysis reveals differentially regulated gene programs that are associated with distinct biological processes in diabetes. Interestingly, despite these changes in gene expression, cell type-specific DNA methylation signatures of genic and regulatory regions remain stable in diabetes. Analysis of heterocellular interactions in the diabetic heart suggest that the interplay between fibroblasts and monocytes is of pivotal importance.
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Metilação de DNA/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Regulação da Expressão Gênica , Miocárdio/metabolismo , Miocárdio/patologia , Animais , Diabetes Mellitus Experimental/fisiopatologia , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/fisiopatologia , Perfilação da Expressão Gênica , Ligantes , Masculino , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Receptores de Superfície Celular/metabolismoRESUMO
The adult mammalian heart consists of mononuclear and binuclear cardiomyocytes (CMs) with various ploidies. However, it remains unclear whether a variation in ploidy or number of nuclei is associated with distinct functions and injury responses in CMs, including regeneration. Therefore, we investigated transcriptomes and cellular as well as nuclear features of mononucleated and binucleated CMs in adult mouse hearts with and without injury. To be able to identify the role of ploidy we analyzed control and failing human ventricular CMs because human CMs show a larger and disease-sensitive degree of polyploidization. Using transgenic Myh6-H2BmCh to identify mononucleated and binucleated mouse CMs, we found that cellular volume and RNA content were similar in both. On average nuclei of mononuclear CMs showed a 2-fold higher ploidy, as compared to binuclear CMs indicating that most mononuclear CMs are tetraploid. After myocardial infarction mononucleated and binucleated CMs in the border zone of the lesion responded with hypertrophy and corresponding changes in gene expression, as well as a low level of induction of cell cycle gene expression. Human CMs allowed us to study a wide range of polyploidy spanning from 2n to 16n. Notably, basal as well as pathological gene expression signatures and programs in failing CMs proved to be independent of ploidy. In summary, gene expression profiles were induced in proximity to injury, but independent of number of nuclei or ploidy levels in CMs.
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Adaptação Fisiológica , Núcleo Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Infarto do Miocárdio/terapia , Miócitos Cardíacos/citologia , Ploidias , Regeneração , Animais , Humanos , Masculino , Camundongos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , RNA-SeqRESUMO
RATIONALE: Structural and electrophysiological remodeling of the atria are recognized consequences of sustained atrial arrhythmias, such as atrial fibrillation. The identification of underlying key molecules and signaling pathways has been challenging because of the changing cell type composition during structural remodeling of the atria. OBJECTIVE: Thus, the aims of our study were (1) to search for transcription factors and downstream target genes, which are involved in atrial structural remodeling, (2) to characterize the significance of the transcription factor ETV1 (E twenty-six variant 1) in atrial remodeling and arrhythmia, and (3) to identify ETV1-dependent gene regulatory networks in atrial cardiac myocytes. METHODS AND RESULTS: The transcription factor ETV1 was significantly upregulated in atrial tissue from patients with permanent atrial fibrillation. Mice with cardiac myocyte-specific overexpression of ETV1 under control of the myosin heavy chain promoter developed atrial dilatation, fibrosis, thrombosis, and arrhythmia. Cardiac myocyte-specific ablation of ETV1 in mice did not alter cardiac structure and function at baseline. Treatment with Ang II (angiotensin II) for 2 weeks elicited atrial remodeling and fibrosis in control, but not in ETV1-deficient mice. To identify ETV1-regulated genes, cardiac myocytes were isolated and purified from mouse atrial tissue. Active cis-regulatory elements in mouse atrial cardiac myocytes were identified by chromatin accessibility (assay for transposase-accessible chromatin sequencing) and the active chromatin modification H3K27ac (chromatin immunoprecipitation sequencing). One hundred seventy-eight genes regulated by Ang II in an ETV1-dependent manner were associated with active cis-regulatory elements containing ETV1-binding sites. Various genes involved in Ca2+ handling or gap junction formation ( Ryr2, Jph2, Gja5), potassium channels ( Kcnh2, Kcnk3), and genes implicated in atrial fibrillation ( Tbx5) were part of this ETV1-driven gene regulatory network. The atrial ETV1-dependent transcriptome in mice showed a significant overlap with the human atrial proteome of patients with permanent atrial fibrillation. CONCLUSIONS: This study identifies ETV1 as an important component in the pathophysiology of atrial remodeling associated with atrial arrhythmias.
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Arritmias Cardíacas/genética , Remodelamento Atrial , Proteínas de Ligação a DNA/genética , Redes Reguladoras de Genes , Fatores de Transcrição/genética , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patologia , Células Cultivadas , Montagem e Desmontagem da Cromatina/genética , Conexinas/genética , Conexinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Miócitos Cardíacos/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
Galaxy HiCExplorer is a web server that facilitates the study of the 3D conformation of chromatin by allowing Hi-C data processing, analysis and visualization. With the Galaxy HiCExplorer web server, users with little bioinformatic background can perform every step of the analysis in one workflow: mapping of the raw sequence data, creation of Hi-C contact matrices, quality assessment, correction of contact matrices and identification of topological associated domains (TADs) and A/B compartments. Users can create publication ready plots of the contact matrix, A/B compartments, and TADs on a selected genomic locus, along with additional information like gene tracks or ChIP-seq signals. Galaxy HiCExplorer is freely usable at: https://hicexplorer.usegalaxy.eu and is available as a Docker container: https://github.com/deeptools/docker-galaxy-hicexplorer.
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Biologia Computacional , Genômica , Internet , Software , Cromatina/genética , Análise de Dados , Genoma/genética , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
MicroRNAs (miRs) appear to be major, yet poorly understood players in regulatory networks guiding cardiogenesis. We sought to identify miRs with unknown functions during cardiogenesis analyzing the miR-profile of multipotent Nkx2.5 enhancer cardiac progenitor cells (NkxCE-CPCs). Besides well-known candidates such as miR-1, we found about 40 miRs that were highly enriched in NkxCE-CPCs, four of which were chosen for further analysis. Knockdown in zebrafish revealed that only miR-128a affected cardiac development and function robustly. For a detailed analysis, loss-of-function and gain-of-function experiments were performed during in vitro differentiations of transgenic murine pluripotent stem cells. MiR-128a knockdown (1) increased Isl1, Sfrp5, and Hcn4 (cardiac transcription factors) but reduced Irx4 at the onset of cardiogenesis, (2) upregulated Isl1-positive CPCs, whereas NkxCE-positive CPCs were downregulated, and (3) increased the expression of the ventricular cardiomyocyte marker Myl2 accompanied by a reduced beating frequency of early cardiomyocytes. Overexpression of miR-128a (4) diminished the expression of Isl1, Sfrp5, Nkx2.5, and Mef2c, but increased Irx4, (5) enhanced NkxCE-positive CPCs, and (6) favored nodal-like cardiomyocytes (Tnnt2+, Myh6+, Shox2+) accompanied by increased beating frequencies. In summary, we demonstrated that miR-128a plays a so-far unknown role in early heart development by affecting the timing of CPC differentiation into various cardiomyocyte subtypes.
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Diferenciação Celular , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Células Cultivadas , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Humanos , Camundongos , MicroRNAs/genética , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Peixe-ZebraRESUMO
In addition to treating depression, antidepressant drugs are also a first-line treatment for neuropathic pain, which is pain secondary to lesion or pathology of the nervous system. Despite the widespread use of these drugs, the mechanism underlying their therapeutic action in this pain context remains partly elusive. The present study combined data collected in male and female mice from a model of neuropathic pain and data from the clinical setting to understand how antidepressant drugs act. We show two distinct mechanisms by which the selective inhibitor of serotonin and noradrenaline reuptake duloxetine and the tricyclic antidepressant amitriptyline relieve neuropathic allodynia. One of these mechanisms is acute, central, and requires descending noradrenergic inhibitory controls and α2A adrenoceptors, as well as the mu and delta opioid receptors. The second mechanism is delayed, peripheral, and requires noradrenaline from peripheral sympathetic endings and ß2 adrenoceptors, as well as the delta opioid receptors. We then conducted a transcriptomic analysis in dorsal root ganglia, which suggested that the peripheral component of duloxetine action involves the inhibition of neuroimmune mechanisms accompanying nerve injury, including the downregulation of the TNF-α-NF-κB signaling pathway. Accordingly, immunotherapies against either TNF-α or Toll-like receptor 2 (TLR2) provided allodynia relief. We also compared duloxetine plasma levels in the animal model and in patients and we observed that patients' drug concentrations were compatible with those measured in animals under chronic treatment involving the peripheral mechanism. Our study highlights a peripheral neuroimmune component of antidepressant drugs that is relevant to their delayed therapeutic action against neuropathic pain.SIGNIFICANCE STATEMENT In addition to treating depression, antidepressant drugs are also a first-line treatment for neuropathic pain, which is pain secondary to lesion or pathology of the nervous system. However, the mechanism by which antidepressant drugs can relieve neuropathic pain remained in part elusive. Indeed, preclinical studies led to contradictions concerning the anatomical and molecular substrates of this action. In the present work, we overcame these apparent contradictions by highlighting the existence of two independent mechanisms. One is rapid and centrally mediated by descending controls from the brain to the spinal cord and the other is delayed, peripheral, and relies on the anti-neuroimmune action of chronic antidepressant treatment.
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Amitriptilina/administração & dosagem , Antidepressivos/administração & dosagem , Cloridrato de Duloxetina/administração & dosagem , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Norepinefrina/metabolismo , Adulto , Idoso , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Manejo da Dor/métodos , Receptor A2A de Adenosina/metabolismoRESUMO
Stress is a precipitating agent in neuropsychiatric disease and initiates relapse to drug-seeking behavior in addicted patients. Targeting the stress system in protracted abstinence from drugs of abuse with anxiolytics may be an effective treatment modality for substance use disorders. α2A-adrenergic receptors (α2A-ARs) in extended amygdala structures play key roles in dampening stress responses. Contrary to early thinking, α2A-ARs are expressed at non-noradrenergic sites in the brain. These non-noradrenergic α2A-ARs play important roles in stress responses, but their cellular mechanisms of action are unclear. In humans, the α2A-AR agonist guanfacine reduces overall craving and uncouples craving from stress, yet minimally affects relapse, potentially due to competing actions in the brain. Here, we show that heteroceptor α2A-ARs postsynaptically enhance dorsal bed nucleus of the stria terminalis (dBNST) neuronal activity in mice of both sexes. This effect is mediated by hyperpolarization-activated cyclic nucleotide-gated cation channels because inhibition of these channels is necessary and sufficient for excitatory actions. Finally, this excitatory action is mimicked by clozapine-N-oxide activation of the Gi-coupled DREADD hM4Di in dBNST neurons and its activation elicits anxiety-like behavior in the elevated plus maze. Together, these data provide a framework for elucidating cell-specific actions of GPCR signaling and provide a potential mechanism whereby competing anxiogenic and anxiolytic actions of guanfacine may affect its clinical utility in the treatment of addiction.SIGNIFICANCE STATEMENT Stress affects the development of neuropsychiatric disorders including anxiety and addiction. Guanfacine is an α2A-adrenergic receptor (α2A-AR) agonist with actions in the bed nucleus of the stria terminalis (BNST) that produces antidepressant actions and uncouples stress from reward-related behaviors. Here, we show that guanfacine increases dorsal BNST neuronal activity through actions at postsynaptic α2A-ARs via a mechanism that involves hyperpolarization-activated cyclic nucleotide gated cation channels. This action is mimicked by activation of the designer receptor hM4Di expressed in the BNST, which also induces anxiety-like behaviors. Together, these data suggest that postsynaptic α2A-ARs in BNST have excitatory actions on BNST neurons and that these actions can be phenocopied by the so-called "inhibitory" DREADDs, suggesting that care must be taken regarding interpretation of data obtained with these tools.
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Ansiedade/fisiopatologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/fisiologia , Neurônios/fisiologia , Receptores Adrenérgicos alfa 2/fisiologia , Núcleos Septais/fisiologia , Estresse Psicológico/fisiopatologia , Agonistas de Receptores Adrenérgicos alfa 2/administração & dosagem , Animais , Catecolaminas/metabolismo , Feminino , Guanfacina/administração & dosagem , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Núcleos Septais/diagnóstico por imagem , Núcleos Septais/metabolismoRESUMO
RATIONALE: Epigenetic mechanisms are crucial for cell identity and transcriptional control. The heart consists of different cell types, including cardiac myocytes, endothelial cells, fibroblasts, and others. Therefore, cell type-specific analysis is needed to gain mechanistic insight into the regulation of gene expression in cardiac myocytes. Although cytosolic mRNA represents steady-state levels, nuclear mRNA more closely reflects transcriptional activity. To unravel epigenetic mechanisms of transcriptional control, cell type-specific analysis of nuclear mRNA and epigenetic modifications is crucial. OBJECTIVE: The aim was to purify cardiac myocyte nuclei from hearts of different species by magnetic- or fluorescent-assisted sorting and to determine the nuclear and cellular RNA expression profiles and epigenetic marks in a cardiac myocyte-specific manner. METHODS AND RESULTS: Frozen cardiac tissue samples were used to isolate cardiac myocyte nuclei. High sorting purity was confirmed for cardiac myocyte nuclei isolated from mice, rats, and humans. Deep sequencing of nuclear RNA revealed a major fraction of nascent, unspliced RNA in contrast to results obtained from purified cardiac myocytes. Cardiac myocyte nuclear and cellular RNA expression profiles showed differences, especially for metabolic genes. Genome-wide maps of the transcriptional elongation mark H3K36me3 were generated by chromatin-immunoprecipitation. Transcriptome and epigenetic data confirmed the high degree of cardiac myocyte-specificity of our protocol. An integrative analysis of nuclear mRNA and histone mark occurrence indicated a major impact of the chromatin state on transcriptional activity in cardiac myocytes. CONCLUSIONS: This study establishes cardiac myocyte-specific sorting of nuclei as a universal method to investigate epigenetic and transcriptional processes in cardiac myocytes of different origins. These data sets provide novel insight into cardiac myocyte transcription.
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Epigênese Genética/fisiologia , Miócitos Cardíacos/fisiologia , Transcrição Gênica/fisiologia , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ratos , Ratos Wistar , Especificidade da EspécieRESUMO
RATIONALE: In chronic heart failure, increased adrenergic activation contributes to structural remodeling and altered gene expression. Although adrenergic signaling alters histone modifications, it is unknown, whether it also affects other epigenetic processes, including DNA methylation and its recognition. OBJECTIVE: The aim of this study was to identify the mechanism of regulation of the methyl-CpG-binding protein 2 (MeCP2) and its functional significance during cardiac pressure overload and unloading. METHODS AND RESULTS: MeCP2 was identified as a reversibly repressed gene in mouse hearts after transverse aortic constriction and was normalized after removal of the constriction. Similarly, MeCP2 repression in human failing hearts resolved after unloading by a left ventricular assist device. The cluster miR-212/132 was upregulated after transverse aortic constriction or on activation of α1- and ß1-adrenoceptors and miR-212/132 led to repression of MeCP2. Prevention of MeCP2 repression by a cardiomyocyte-specific, doxycycline-regulatable transgenic mouse model aggravated cardiac hypertrophy, fibrosis, and contractile dysfunction after transverse aortic constriction. Ablation of MeCP2 in cardiomyocytes facilitated recovery of failing hearts after reversible transverse aortic constriction. Genome-wide expression analysis, chromatin immunoprecipitation experiments, and DNA methylation analysis identified mitochondrial genes and their transcriptional regulators as MeCP2 target genes. Coincident with its repression, MeCP2 was removed from its target genes, whereas DNA methylation of MeCP2 target genes remained stable during pressure overload. CONCLUSIONS: These data connect adrenergic activation with a microRNA-MeCP2 epigenetic pathway that is important for cardiac adaptation during the development and recovery from heart failure.
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Adaptação Fisiológica/fisiologia , Epigênese Genética/fisiologia , Insuficiência Cardíaca/metabolismo , Proteína 2 de Ligação a Metil-CpG/biossíntese , Receptores Adrenérgicos/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Doença Crônica , Insuficiência Cardíaca/genética , Humanos , Proteína 2 de Ligação a Metil-CpG/antagonistas & inibidores , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Ratos , Receptores Adrenérgicos/genéticaRESUMO
AIMS: Biglycan, a small leucine-rich proteoglycan, has been shown to play an important role in stabilizing fibrotic scars after experimental myocardial infarction. However, the role of biglycan in the development and regression of cardiomyocyte hypertrophy and fibrosis during cardiac pressure overload and unloading remains elusive. Thus, the aim of the present study was to assess the effect of biglycan on cardiac remodeling in a mouse model of left ventricular pressure overload and unloading. METHODS AND RESULTS: Left ventricular pressure overload induced by transverse aortic constriction (TAC) in mice resulted in left ventricular dysfunction, fibrosis and increased biglycan expression. Fluorescence- and magnetic-assisted sorting of cardiac cell types revealed upregulation of biglycan in the fibroblast population, but not in cardiomyocytes, endothelial cells or leukocytes after TAC. Removal of the aortic constriction (rTAC) after short-term pressure overload (3weeks) improved cardiac contractility and reversed ventricular hypertrophy but not fibrosis in wild-type (WT) mice. Biglycan ablation (KO) enhanced functional recovery but did not resolve cardiac fibrosis. After long-term TAC for 9weeks, ablation of biglycan attenuated the development of cardiac hypertrophy and fibrosis. In vitro, biglycan induced hypertrophy of neonatal rat cardiomyocytes and led to activation of a hypertrophic gene program. Putative downstream mediators of biglycan signaling include Rcan1, Abra and Tnfrsf12a. These genes were concordantly induced by TAC in WT but not in biglycan KO mice. CONCLUSIONS: Left ventricular pressure overload induces biglycan expression in cardiac fibroblasts. Ablation of biglycan improves cardiac function and attenuates left ventricular hypertrophy and fibrosis after long-term pressure overload. In vitro biglycan induces hypertrophy of cardiomyocytes, suggesting that biglycan may act as a signaling molecule between cell types to modulate cardiac remodeling.
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Biglicano/deficiência , Biglicano/metabolismo , Cardiomegalia/etiologia , Cardiomegalia/metabolismo , Disfunção Ventricular Esquerda/fisiopatologia , Animais , Cardiomegalia/diagnóstico , Modelos Animais de Doenças , Ecocardiografia , Feminino , Fibrose , Hipertrofia Ventricular Esquerda/diagnóstico , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/metabolismo , Masculino , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Proteoma , Proteômica , Ratos , Remodelação VentricularRESUMO
Macrophages in the arterial intima sustain chronic inflammation during atherogenesis. Under hypercholesterolemic conditions murine Ly6C(high) monocytes surge in the blood and spleen, infiltrate nascent atherosclerotic plaques, and differentiate into macrophages that proliferate locally as disease progresses. Spleen tyrosine kinase (SYK) may participate in downstream signaling of various receptors that mediate these processes. We tested the effect of the SYK inhibitor fostamatinib on hypercholesterolemia-associated myelopoiesis and plaque formation in Apoe(-/-) mice during early and established atherosclerosis. Mice consuming a high cholesterol diet supplemented with fostamatinib for 8 weeks developed less atherosclerosis. Histologic and flow cytometric analysis of aortic tissue showed that fostamatinib reduced the content of Ly6C(high) monocytes and macrophages. SYK inhibition limited Ly6C(high) monocytosis through interference with GM-CSF/IL-3 stimulated myelopoiesis, attenuated cell adhesion to the intimal surface, and blocked M-CSF stimulated monocyte to macrophage differentiation. In Apoe(-/-) mice with established atherosclerosis, however, fostamatinib treatment did not limit macrophage accumulation or lesion progression despite a significant reduction in blood monocyte counts, as lesional macrophages continued to proliferate. Thus, inhibition of hypercholesterolemia-associated monocytosis, monocyte infiltration, and differentiation by SYK antagonism attenuates early atherogenesis but not established disease when local macrophage proliferation dominates lesion progression.
Assuntos
Aterosclerose/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Monócitos/efeitos dos fármacos , Mielopoese/efeitos dos fármacos , Oxazinas/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridinas/uso terapêutico , Aminopiridinas , Animais , Aterosclerose/imunologia , Aterosclerose/prevenção & controle , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos , Feminino , Macrófagos/efeitos dos fármacos , Camundongos , Morfolinas , Oxazinas/farmacologia , Piridinas/farmacologia , Pirimidinas , Distribuição Aleatória , Quinase SykRESUMO
Growth/differentiation factor-15 (Gdf-15) is a member of the transforming growth factor-ß (Tgf-ß) superfamily and has been shown to be a potent neurotrophic factor for midbrain dopaminergic (DAergic) neurons both in vitro and in vivo. Gdf-15 has also been shown to be involved in inflammatory processes. The aim of this study was to identify the role of endogenous Gdf-15 in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) mouse model of Parkinson's disease (PD) by comparing Gdf-15 (+/+) and Gdf-15 (-/-) mice. At 4 days and 14 days post-MPTP administration, both Gdf-15 (+/+) and Gdf-15 (-/-) mice showed a similar decline in DAergic neuron numbers and in striatal dopamine (DA) levels. This was followed by a comparable restorative phase at 90 days and 120 days, indicating that the absence of Gdf-15 does not affect the susceptibility or the recovery capacity of the nigrostriatal system after MPTP administration. The MPTP-induced microglial and astrocytic response was not significantly altered between the two genotypes. However, pro-inflammatory and anti-inflammatory cytokine profiling revealed the differential expression of markers in Gdf-15 (+/+) and Gdf-15 (-/-) mice after MPTP administration. Thus, the MPTP mouse model fails to uncover a major role of endogenous Gdf-15 in the protection of MPTP-lesioned nigrostriatal DAergic neurons, in contrast to its capacity to protect the 6-hydroxydopamine-intoxicated nigrostriatal system.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Fator 15 de Diferenciação de Crescimento/deficiência , Neostriado/metabolismo , Neostriado/patologia , Substância Negra/metabolismo , Substância Negra/patologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/administração & dosagem , Animais , Biomarcadores/metabolismo , Proliferação de Células , Citocinas/metabolismo , Fator 15 de Diferenciação de Crescimento/metabolismo , Mediadores da Inflamação/metabolismo , Camundongos , Neuroglia/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Pasteurella multocida toxin (PMT) persistently activates heterotrimeric G proteins of the Gαq/11 , Gα12/13 and Gαi family without interaction with G protein-coupled receptors (GPCRs). We show that PMT acts on heart tissue in vivo and on cardiomyocytes and cardiac fibroblasts in vitro by deamidation of heterotrimeric G proteins. Increased normalized ventricle weights and fibrosis were detected after intraperitoneal administration of PMT in combination with the GPCR agonist phenylephrine. In neonatal rat cardiomyocytes, PMT stimulated the mitogen-activated protein kinase pathway, which is crucial for the development of cellular hypertrophy. The toxin induced phosphorylation of the canonical phosphorylation sites of the extracellular-regulated kinase 1/2 and, additionally, caused phosphorylation of the recently recognized autophosphorylation site, which appears to be important for the development of cellular hypertrophy. Moreover, PMT stimulated the small GTPases Rac1 and RhoA. Both switch proteins are involved in cardiomyocyte hypertrophy. In addition, PMT stimulated RhoA and Rac1 in neonatal rat cardiac fibroblasts. RhoA and Rac1 have been implicated in the regulation of connective tissue growth factor (CTGF) secretion and expression. Accordingly, we show that PMT treatment increased secretion and expression of CTGF in cardiac fibroblasts. Altogether, the data indicate that PMT is an inducer of pathological remodelling of cardiac cells and identifies the toxin as a promising tool for studying heterotrimeric G protein-dependent signalling in cardiac cells.