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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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Epigenetic modulators such as lysine-specific demethylase 1 (LSD1) and histone deacetylases (HDACs) are drug targets for cancer, neuropsychiatric disease, or inflammation, but inhibitors of these enzymes exhibit considerable side effects. For a potential local treatment with reduced systemic toxicity, we present here soft drug candidates as new LSD1 and HDAC inhibitors. A soft drug is a compound that is degraded in vivo to less active metabolites after having achieved its therapeutic function. This has been successfully applied for corticosteroids in the clinic, but soft drugs targeting epigenetic enzymes are scarce, with the HDAC inhibitor remetinostat being the only example. We have developed new methyl ester-containing inhibitors targeting LSD1 or HDACs and compared the biological activities of these to their respective carboxylic acid cleavage products. In vitro activity assays, cellular experiments, and a stability assay identified potent HDAC and LSD1 soft drug candidates that are superior to their corresponding carboxylic acids in cellular models.
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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
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
PURPOSE OF REVIEW: Development, physiological growth and the response of the heart to injury are accompanied by changes of the transcriptome and epigenome of cardiac myocytes. Recently, cell sorting and next generation sequencing techniques have been applied to determine cardiac myocyte-specific transcriptional and epigenetic mechanisms. This review provides a comprehensive overview of studies analysing the transcriptome and epigenome of cardiac myocytes in mouse and human hearts during development, physiological growth and disease. RECENT FINDINGS: Adult cardiac myocytes express > 12,600 genes, and their expression levels correlate positively with active histone marks and inversely with gene body DNA methylation. DNA methylation accompanied the perinatal switch in sarcomere or metabolic isoform gene expression in cardiac myocytes, but remained rather stable in heart disease. DNA methylation and histone marks identified > 100,000 cis-regulatory regions in the cardiac myocyte epigenome with a dynamic spectrum of transcription factor binding sites. The ETS-related transcription factor ETV1 was identified as an atrial-specific element involved in the pathogenesis of atrial fibrillation. Thus, dynamic development of the atrial vs. ventricular cardiac myocyte epigenome provides a basis to identify location and time-dependent mechanisms of epigenetic control to shape pathological gene expression during heart disease. Identifying the four dimensions of the cardiac myocyte epigenome, atrial vs. ventricular location, time during development and growth, and disease-specific signals, may ultimately lead to new treatment strategies for heart disease.
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Epigênese Genética/genética , Epigênese Genética/fisiologia , Epigenoma , Coração Fetal/fisiologia , Coração/crescimento & desenvolvimento , Miócitos Cardíacos/metabolismo , Adulto , Animais , Regulação da Expressão Gênica , Ventrículos do Coração , Humanos , CamundongosRESUMO
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
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
OBJECTIVE: Endothelial cells (ECs) are a highly specialized cell type with marked diversity between different organs or vascular beds. Cardiac ECs are an important player in cardiac physiology and pathophysiology but are not sufficiently characterized yet. Thus, the aim of the present study was to analyze the cardiac EC transcriptome. APPROACH AND RESULTS: We applied fluorescence-assisted cell sorting to isolate pure ECs from adult mouse hearts. RNAseq revealed 1288 genes predominantly expressed in cardiac ECs versus heart tissue including several transcription factors. We found an overrepresentation of corresponding transcription factor binding motifs within the promotor region of EC-enriched genes, suggesting that they control the EC transcriptome. Cardiac ECs exhibit a distinct gene expression profile when compared with renal, cerebral, or pulmonary ECs. For example, we found the Meox2/Tcf15, Fabp4, and Cd36 signaling cascade higher expressed in cardiac ECs which is a key regulator of fatty acid uptake and involved in the development of atherosclerosis. CONCLUSIONS: The results from this study provide a comprehensive resource of gene expression and transcriptional control in cardiac ECs. The cardiac EC transcriptome exhibits distinct differences in gene expression compared with other cardiac cell types and ECs from other organs. We identified new candidate genes that have not been investigated in ECs yet as promising targets for future evaluation.
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Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica/métodos , Transcriptoma , Animais , Antígenos CD/genética , Caderinas/genética , Separação Celular/métodos , Biologia Computacional , Vasos Coronários/citologia , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Camundongos Transgênicos , Regiões Promotoras Genéticas , Transdução de Sinais/genética , Transcrição GênicaRESUMO
The mineralocorticoid aldosterone is a key regulator of water and electrolyte homeostasis. Numerous recent developments have advanced the field of mineralocorticoid pharmacologynamely, clinical trials have shown the beneficial effects of aldosterone antagonists in chronic heart failure and post-myocardial infarction treatment. Experimental studies using cell type-specific gene targeting of the mineralocorticoid receptor (MR) gene in mice have revealed the importance of extrarenal aldosterone signaling in cardiac myocytes, endothelial cells, vascular smooth cells, and macrophages. In addition, several molecular pathways involving signal transduction via the classical MR as well as the G protein-coupled receptor GPER mediate the diverse spectrum of effects of aldosterone on cells. This knowledge has initiated the development of new pharmacological ligands to specifically interfere with targets on different levels of aldosterone signaling. For example, aldosterone synthase inhibitors such as LCI699 and the novel nonsteroidal MR antagonist BAY 94-8862 have been tested in clinical trials. Interference with the interaction between MR and its coregulators seems to be a promising strategy toward the development of selective MR modulators.
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Aldosterona/metabolismo , Vasos Sanguíneos/metabolismo , Doenças Cardiovasculares/metabolismo , Miocárdio/metabolismo , Receptores de Mineralocorticoides/metabolismo , Animais , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/fisiopatologia , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/fisiopatologia , Desenho de Fármacos , Humanos , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Terapia de Alvo Molecular , Receptores de Mineralocorticoides/efeitos dos fármacos , Transdução de SinaisRESUMO
Genetic factors are known to modulate cardiac susceptibility to ventricular hypertrophy and failure. To determine how strain influences the transcriptional response to pressure overload-induced heart failure (HF) and which of these changes accurately reflect the human disease, we analyzed the myocardial transcriptional profile of mouse strains with high (C57BL/6J) and low (129S1/SvImJ) susceptibility for HF development, which we compared to that of human failing hearts. Following transverse aortic constriction (TAC), C57BL/6J mice developed overt HF while 129S1/SvImJ did not. Despite a milder aortic constriction, impairment of ejection fraction and ventricular remodeling (dilation, fibrosis) was more pronounced in C57BL/6J mice. Similarly, changes in myocardial gene expression were more robust in C57BL/6J (461 genes) compared to 129S1/SvImJ mice (71 genes). When comparing these patterns to human dilated cardiomyopathy (1344 genes), C57BL/6J mice tightly grouped to human hearts. Overlay and bioinformatic analysis of the transcriptional profiles of C57BL/6J mice and human failing hearts identified six co-regulated genes (POSTN, CTGF, FN1, LOX, NOX4, TGFB2) with established link to HF development. Pathway enrichment analysis identified angiotensin and IGF-1 signaling as most enriched putative upstream regulator and pathway, respectively, shared between TAC-induced HF in C57BL/6J mice and in human failing hearts. TAC-induced heart failure in C57BL/6J mice more closely reflects the gene expression pattern of human dilated cardiomyopathy compared to 129S1/SvImJ mice. Unbiased as well as targeted gene expression and pathway analyses identified periostin, angiotensin signaling, and IGF-1 signaling as potential causes of increased HF susceptibility in C57BL/6J mice and as potentially useful drug targets for HF treatment.
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Cardiomiopatia Dilatada/genética , Regulação da Expressão Gênica , Insuficiência Cardíaca/genética , Hipertrofia Ventricular Esquerda/genética , Função Ventricular Esquerda/genética , Animais , Cardiomiopatia Dilatada/complicações , Cardiomiopatia Dilatada/fisiopatologia , Estudos de Casos e Controles , Modelos Animais de Doenças , Progressão da Doença , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Predisposição Genética para Doença , Insuficiência Cardíaca/fisiopatologia , Hipertrofia Ventricular Esquerda/complicações , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Fenótipo , Especificidade da Espécie , Transcriptoma , Remodelação Ventricular/genéticaAssuntos
Diabetes Mellitus Tipo 2 , Insuficiência Renal Crônica , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Humanos , Naftiridinas , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/epidemiologiaRESUMO
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
Arterial hypertension causes left ventricular (LV) myocyte hypertrophy. Alterations in nuclear Ca2+ may be involved in regulation of histone acetylation, transcription and hypertrophy. Regulation of nuclear Ca2+ in hypertension, however, is unknown. Therefore, we elucidated cellular mechanisms underlying nuclear Ca2+ regulation in LV myocytes from hypertensive versus normotensive rats and evaluated possible consequences for Ca2+-dependent regulation of histone acetylation. LV myocytes and myocyte nuclei were isolated from young spontaneously hypertensive rats (SHR) shortly after development of hypertension. Normotensive Wistar-Kyoto rats (WKY) served as controls. Cytoplasmic and nucleoplasmic Ca2+ transients (CaTs) were imaged simultaneously using linescan confocal microscopy and Fluo-4. LV myocytes and nuclei from SHR exhibited hypertrophy. Cytoplasmic and nucleoplasmic CaTs were increased in SHR. The increase in nucleoplasmic Ca2+, however, exceeded the increase in cytoplasmic Ca2+, indicating enhanced nuclear Ca2+ signaling in SHR. Ca2+ load of sarcoplasmic reticulum and perinuclear Ca2+ stores was also increased in SHR, while fractional release from both stores remained unchanged. Intranuclear Ca2+ propagation was accelerated in SHR, associated with preserved density of nuclear envelope invaginations and elevated nuclear expression of nucleoporins and SR-Ca2+-ATPase, SERCA2a. Nuclear Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) expression was elevated and histone deacetylases exhibited redistribution from nucleus to cytosol associated with increased histone acetylation in SHR. Thus, in early hypertension, there is remodeling of nuclear Ca2+ handling resulting in enhanced nuclear Ca2+ signaling. Enhanced nuclear Ca2+ signaling, in turn, increases nuclear localization and activity of CaMKIIδ driving nuclear export of histone deacetylases and increased histone acetylation.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Hipertensão/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Hipertensão/etiologia , Masculino , Membrana Nuclear/metabolismo , Ratos , Ratos Endogâmicos SHR , Retículo Sarcoplasmático/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
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.
Assuntos
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
It has been hypothesized that α2-adrenoceptors (α2-ARs) may be involved in the pathomechanism of colitis; however, the results are conflicting because both aggravation and amelioration of colonic inflammation have been described in response to α2-AR agonists. Therefore, we aimed to analyze the role of α2-ARs in acute murine colitis. The experiments were carried out in wild-type, α2A-, α2B-, and α2C-AR knockout (KO) C57BL/6 mice. Colitis was induced by dextran sulfate sodium (DSS, 2%); alpha2-AR ligands were injected i.p. The severity of colitis was determined both macroscopically and histologically. Colonic myeloperoxidase (MPO) and cytokine levels were measured by enzyme-linked immunosorbent assay and proteome profiler array, respectively. The nonselective α2-AR agonist clonidine induced a modest aggravation of DSS-induced colitis. It accelerated the disease development and markedly enhanced the weight loss of animals, but did not influence the colon shortening, tissue MPO levels, or histologic score. Clonidine induced similar changes in α2B- and α2C-AR KO mice, whereas it failed to affect the disease activity index scores and caused only minor weight loss in α2A-AR KO animals. In contrast, selective inhibition of α2A-ARs by BRL 44408 significantly delayed the development of colitis; reduced the colonic levels of MPO and chemokine (C-C motif) ligand 3, chemokine (C-X-C motif) ligand 2 (CXCL2), CXCL13, and granulocyte-colony stimulating factor; and elevated that of tissue inhibitor of metalloproteinases-1. In this work, we report that activation of α2-ARs aggravates murine colitis, an effect mediated by the α2A-AR subtype, and selective inhibition of these receptors reduces the severity of gut inflammation.