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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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Chronic 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 this study was to identify angiogenesis-related gene networks and corresponding regulatory hubs in endothelial cells from failing human hearts. We 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 comorbidities on gene expression. In a weighted gene coexpression network analysis, we found 26 gene clusters, of which 9 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. Knockdown 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. In 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.NEW & NOTEWORTHY Gene coexpression network analysis defined 26 gene clusters expressed in endothelial cells from failing human hearts. Transcription factors CASZ1, ZNF523, and NFE2L1 were identified as hub genes of a cluster related to angiogenesis. Knockdown of CASZ1, ZNF523, or NFE2L1 in human umbilical vein endothelial cells led to a downregulation of genes from the respective cluster, confirming their regulatory function. This provides insights into the transcriptional regulation of angiogenesis in heart failure beyond classical signaling pathways.
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Redes Reguladoras de Genes , Insuficiência Cardíaca , Células Endoteliais da Veia Umbilical Humana , Humanos , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/patologia , Masculino , Pessoa de Meia-Idade , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Neovascularização Fisiológica , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Adulto , Idoso , Regulação da Expressão Gênica , Estudos de Casos e ControlesRESUMO
BACKGROUND: Catecholamines are commonly used as therapeutic drugs in intensive care medicine to maintain sufficient organ perfusion during shock. However, excessive or sustained adrenergic activation drives detrimental cardiac remodeling and may lead to heart failure. Whether catecholamine treatment in absence of heart failure causes persistent cardiac injury, is uncertain. In this experimental study, we assessed the course of cardiac remodeling and recovery during and after prolonged catecholamine treatment and investigated the molecular mechanisms involved. RESULTS: C57BL/6N wild-type mice were assigned to 14 days catecholamine treatment with isoprenaline and phenylephrine (IsoPE), treatment with IsoPE and subsequent recovery, or healthy control groups. IsoPE improved left ventricular contractility but caused substantial cardiac fibrosis and hypertrophy. However, after discontinuation of catecholamine treatment, these alterations were largely reversible. To uncover the molecular mechanisms involved, we performed RNA sequencing from isolated cardiomyocyte nuclei. IsoPE treatment resulted in a transient upregulation of genes related to extracellular matrix formation and transforming growth factor signaling. While components of adrenergic receptor signaling were downregulated during catecholamine treatment, we observed an upregulation of endothelin-1 and its receptors in cardiomyocytes, indicating crosstalk between both signaling pathways. To follow this finding, we treated mice with endothelin-1. Compared to IsoPE, treatment with endothelin-1 induced minor but longer lasting changes in cardiomyocyte gene expression. DNA methylation-guided analysis of enhancer regions identified immediate early transcription factors such as AP-1 family members Jun and Fos as key drivers of pathological gene expression following catecholamine treatment. CONCLUSIONS: The results from this study show that prolonged catecholamine exposure induces adverse cardiac remodeling and gene expression before the onset of left ventricular dysfunction which has implications for clinical practice. The observed changes depend on the type of stimulus and are largely reversible after discontinuation of catecholamine treatment. Crosstalk with endothelin signaling and the downstream transcription factors identified in this study provide new opportunities for more targeted therapeutic approaches that may help to separate desired from undesired effects of catecholamine treatment.
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Farmacologia , História do Século XX , História do Século XXI , Humanos , Farmacologia/históriaRESUMO
Endothelial cells play crucial roles in physiology and are increasingly recognized as therapeutic targets in cardiovascular disease. Here, we analyzed the regulatory landscape of cardiac endothelial cells by assessing chromatin accessibility, histone modifications, and 3D chromatin organization and confirmed the functional relevance of enhancer-promoter interactions by CRISPRi-mediated enhancer silencing. We used this dataset to explore mechanisms of transcriptional regulation in cardiovascular disease and compared six different experimental models of heart failure, hypertension, or diabetes. Enhancers that regulate gene expression in diseased endothelial cells were enriched with binding sites for a distinct set of transcription factors, including the mineralocorticoid receptor (MR), a known drug target in heart failure and hypertension. For proof of concept, we applied endothelial cell-specific MR deletion in mice to confirm MR-dependent gene expression and predicted direct MR target genes. Overall, we have compiled here a comprehensive atlas of cardiac endothelial cell enhancer elements that provides insight into the role of transcription factors in cardiovascular disease.
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Ascomicetos , Doenças Cardiovasculares , Insuficiência Cardíaca , Hipertensão , Animais , Camundongos , Células Endoteliais , Receptores de Mineralocorticoides/genética , Fatores de Transcrição , Elementos Facilitadores Genéticos , Expressão GênicaRESUMO
Background Epigenetic modulators have been proposed as promising new drug targets to treat adverse remodeling in heart failure. Here, we evaluated the potential of 4 epigenetic drugs, including the recently developed histone deacetylase 6 (HDAC6) inhibitor JS28, to prevent endothelin-1 induced pathological gene expression in cardiac myocytes and analyzed the chromatin binding profile of the respective inhibitor targets. Methods and Results Cardiac myocytes were differentiated and puromycin-selected from mouse embryonic stem cells and treated with endothelin-1 to induce pathological gene expression (938 differentially expressed genes, q<0.05). Dysregulation of gene expression was at least in part prevented by epigenetic inhibitors, including the pan-BRD (bromodomain-containing protein) inhibitor bromosporine (290/938 genes), the BET (bromodomain and extraterminal) inhibitor JQ1 (288/938), the broad-spectrum HDAC inhibitor suberoylanilide hydroxamic acid (227/938), and the HDAC6 inhibitor JS28 (210/938). Although the 4 compounds were similarly effective toward pathological gene expression, JS28 demonstrated the least adverse effects on physiological gene expression. Genome-wide chromatin binding profiles revealed that HDAC6 binding sites were preferentially associated with promoters of genes involved in RNA processing. In contrast, BRD4 binding was associated with genes involved in core cardiac myocyte functions, for example, myocyte contractility, and showed enrichment at enhancers and intronic regions. These distinct chromatin binding profiles of HDAC6 and BRD4 might explain the different effects of their inhibitors on pathological versus physiological gene expression. Conclusions In summary, we demonstrated, that the HDAC6 inhibitor JS28 effectively prevented the adverse effects of endothelin-1 on gene expression with minor impact on physiological gene expression in cardiac myocytes. Selective HDAC6 inhibition by JS28 appears to be a promising strategy for future evaluation in vivo and potential translation into clinical application.
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Inibidores de Histona Desacetilases , Miócitos Cardíacos , Animais , Cromatina , Endotelina-1/genética , Endotelina-1/farmacologia , Expressão Gênica , Desacetilase 6 de Histona , Inibidores de Histona Desacetilases/farmacologia , Camundongos , Proteínas Nucleares/farmacologia , Fatores de TranscriçãoRESUMO
BACKGROUND: Sepsis is associated with high platelet turnover and elevated levels of immature platelets. Changes in the platelet transcriptome and the specific impact of immature platelets on the platelet transcriptome remain unclear. Thus, this study sought to address whether and how elevated levels of immature platelets affect the platelet transcriptome in patients with sepsis. METHODS: Blood samples were obtained from patients with sepsis requiring vasopressor therapy (n = 8) and from a control group of patients with stable coronary artery disease and otherwise similar demographic characteristics (n = 8). Immature platelet fraction (IPF) was determined on a Sysmex XE 2100 analyser and platelet function was tested by impedance aggregometry. RNA from leukocyte-depleted platelets was used for transcriptome analysis by Next Generation Sequencing integrating the use of unique molecular identifiers. RESULTS: IPF (median [interquartile range]) was significantly elevated in sepsis patients (6.4 [5.3-8.7] % vs. 3.6 [2.6-4.6] %, p = 0.005). Platelet function testing revealed no differences in adenosine diphosphate- or thrombin receptor activating peptide-induced platelet aggregation between control and sepsis patients. Putative circular RNA transcripts were decreased in platelets from septic patients. Leukocyte contamination defined by CD45 abundance levels in RNA-sequencing was absent in both groups. Principal component analysis of transcripts showed only partial overlap of clustering with IPF levels. RNA sequencing showed up-regulation of 524 and down-regulation of 118 genes in platelets from sepsis patients compared to controls. Upregulated genes were mostly related to catabolic processes and protein translation. Comparison to published platelet transcriptomes showed a large overlap of changes observed in sepsis and COVID-19 but not with reticulated platelets from healthy donors. CONCLUSIONS: Patients with sepsis appear to have a less degraded platelet transcriptome as indicated by increased levels of immature platelets and decreased levels of putative circular RNA transcripts. The present data suggests that increased protein translation is a characteristic mechanism of systemic inflammation.
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Plaquetas/metabolismo , Sepse/genética , Transcriptoma/genética , Idoso , Sequência de Bases/genética , Plaquetas/patologia , Fracionamento Celular/métodos , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Ativação Plaquetária/genética , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Contagem de Plaquetas , Testes de Função Plaquetária , RNA Circular/análise , RNA Circular/genética , Sepse/sangue , Análise de Sequência de RNA/métodosRESUMO
Pulmonary hypertension (PH) is a devastating condition characterized by pulmonary vascular remodelling, leading to progressive increase in pulmonary artery pressure and subsequent right ventricular failure. Aldosterone and the mineralocorticoid receptor (MR), a nuclear transcription factor, are key drivers of cardiovascular disease and MR antagonists are well-established in heart failure. Now, a growing body of evidence points at a detrimental role of MR in PH. Pharmacological MR blockade attenuated PH and prevented RV failure in experimental models. Mouse models with cell selective MR deletion suggest that this effect is mediated by MR in endothelial cells. While the evidence from experimental studies appears convincing, the available clinical data on MR antagonist use in patients with PH is more controversial. Integrated analysis of clinical data together with MR-dependent molecular alterations may provide insights why some patients respond to MRA treatment while others do not. Potential ways to identify MRA 'responders' include the analysis of underlying PH causes, stage of disease, or sex, as well as new biomarkers.
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Insuficiência Cardíaca , Hipertensão Pulmonar , Aldosterona , Animais , Células Endoteliais , Insuficiência Cardíaca/tratamento farmacológico , Humanos , Hipertensão Pulmonar/tratamento farmacológico , Camundongos , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Biologia Molecular , Receptores de MineralocorticoidesRESUMO
Ligand binding to G proteincoupled receptors (GPCRs), such as the α2a-adrenergic receptor (α2aAR), results in the activation of heterotrimeric G proteins, which consist of functionally distinct Gα subunits and Gßγ dimers. α2aAR-dependent inhibition of synaptic transmission regulates functions such as spontaneous locomotor activity, anesthetic sparing, and working memory enhancement and requires the soluble NSF attachment protein receptor (SNARE) complex, a Gßγ effector. To understand how the Gßγ-SNARE complex underlies the α2aAR-dependent inhibition of synaptic transmission, we examined the specificity of Gßγ subunits for the SNARE complex in adrenergic neurons, in which auto-α2aARs respond to epinephrine released from these neurons, and nonadrenergic neurons, in which hetero-α2aARs respond to epinephrine released from other neurons. We performed a quantitative, targeted multiple reaction monitoring proteomic analysis of Gß and Gγ subunits bound to the SNARE complex in synaptosomes from mouse brains. In the absence of stimulation of auto-α2aARs, Gß1 and Gγ3 interacted with the SNARE complex. However, Gß1, Gß2, and Gγ3 were found in the complex when auto-α2aARs were activated by epinephrine. Further understanding of the specific usage of distinct Gßγ subunits in vivo may provide insights into the homeostatic regulation of synaptic transmission and the mechanisms of dysfunction that occur in neurological diseases.
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Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Receptores Adrenérgicos alfa 2/metabolismo , Proteínas SNARE , Animais , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Masculino , Camundongos Transgênicos , Proteínas SNARE/metabolismoRESUMO
Successful treatment of acute myeloid leukemia (AML) with chimeric antigen receptor (CAR) T cells is hampered by toxicity on normal hematopoietic progenitor cells and low CAR T cell persistence. Here, we develop third-generation anti-CD123 CAR T cells with a humanized CSL362-based ScFv and a CD28-OX40-CD3ζ intracellular signaling domain. This CAR demonstrates anti-AML activity without affecting the healthy hematopoietic system, or causing epithelial tissue damage in a xenograft model. CD123 expression on leukemia cells increases upon 5'-Azacitidine (AZA) treatment. AZA treatment of leukemia-bearing mice causes an increase in CTLA-4negative anti-CD123 CAR T cell numbers following infusion. Functionally, the CTLA-4negative anti-CD123 CAR T cells exhibit superior cytotoxicity against AML cells, accompanied by higher TNFα production and enhanced downstream phosphorylation of key T cell activation molecules. Our findings indicate that AZA increases the immunogenicity of AML cells, enhancing recognition and elimination of malignant cells by highly efficient CTLA-4negative anti-CD123 CAR T cells.
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Azacitidina/administração & dosagem , Imunoterapia Adotiva/métodos , Subunidade alfa de Receptor de Interleucina-3/imunologia , Leucemia Mieloide/terapia , Anticorpos de Cadeia Única/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Doença Aguda , Animais , Linhagem Celular Tumoral , Células Cultivadas , Citotoxicidade Imunológica , Metilação de DNA/efeitos dos fármacos , Inibidores Enzimáticos/administração & dosagem , Células HEK293 , Células HL-60 , Humanos , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Estimativa de Kaplan-Meier , Leucemia Mieloide/imunologia , Leucemia Mieloide/patologia , Camundongos Knockout , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismoRESUMO
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 , HumanosAssuntos
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
An amino acid exchange (P209L) in the HSPB8 binding site of the human co-chaperone BAG3 gives rise to severe childhood cardiomyopathy. To phenocopy the disease in mice and gain insight into its mechanisms, we generated humanized transgenic mouse models. Expression of human BAG3P209L-eGFP in mice caused Z-disc disintegration and formation of protein aggregates. This was accompanied by massive fibrosis resulting in early-onset restrictive cardiomyopathy with increased mortality as observed in patients. RNA-Seq and proteomics revealed changes in the protein quality control system and increased autophagy in hearts from hBAG3P209L-eGFP mice. The mutation renders hBAG3P209L less soluble in vivo and induces protein aggregation, but does not abrogate hBAG3 binding properties. In conclusion, we report a mouse model mimicking the human disease. Our data suggest that the disease mechanism is due to accumulation of hBAG3P209L and mouse Bag3, causing sequestering of components of the protein quality control system and autophagy machinery leading to sarcomere disruption.
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Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Cardiomiopatia Restritiva/genética , Cardiomiopatia Restritiva/metabolismo , Animais , Autofagia , Sítios de Ligação , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Cardiomiopatias/terapia , Cardiomiopatia Restritiva/terapia , Criança , Modelos Animais de Doenças , Regulação da Expressão Gênica , Terapia Genética , Coração , Proteínas de Choque Térmico , Humanos , Camundongos , Camundongos Transgênicos , Chaperonas Moleculares/metabolismo , Mutação , Ligação Proteica , Proteômica , Sarcômeros/metabolismoRESUMO
[Figure: see text].
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Anti-Hipertensivos/farmacologia , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Eplerenona/farmacologia , Hipertensão Pulmonar/tratamento farmacológico , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Remodelação Vascular/efeitos dos fármacos , Animais , Anti-Hipertensivos/uso terapêutico , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Eplerenona/uso terapêutico , Hipertensão Pulmonar/metabolismo , Masculino , Camundongos , Camundongos Knockout , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Resultado do TratamentoRESUMO
Two-pore channels (TPCs) are key components for regulating Ca2+ current from endosomes and lysosomes to the cytosol. This locally restricted Ca2+ current forms the basis for fusion and fission events between endolysosomal membranes and thereby for intracellular trafficking processes. Here, we study the function of TPC1 and TPC2 for uptake, recycling, and degradation of epidermal growth factor receptor (EGFR) using a set of TPC knockout cells. RNA sequencing analysis revealed multiple changes in the expression levels of EGFR pathway-related genes in TPC1-deficient cells. We propose that a prolonged presence of activated EGFRs in endolysosomal signaling platforms, caused by genetic inactivation of TPCs, does not only affect EGFR signaling pathways but also increases de novo synthesis of EGFR. Increased basal phospho-c-Jun levels contribute to the high EGFR expression in TPC-deficient cells. Our data point to a role of TPCs not only as important regulators for the EGFR transportation network but also for EGFR-signaling and expression.
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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
Genetic factors undoubtedly affect the development of congenital heart disease (CHD) but still remain ill defined. We sought to identify genetic risk factors associated with CHD and to accomplish a functional analysis of SNP-carrying genes. We performed a genome-wide association study (GWAS) of 4034 White patients with CHD and 8486 healthy controls. One SNP on chromosome 5q22.2 reached genome-wide significance across all CHD phenotypes and was also indicative for septal defects. One region on chromosome 20p12.1 pointing to the MACROD2 locus identified 4 highly significant SNPs in patients with transposition of the great arteries (TGA). Three highly significant risk variants on chromosome 17q21.32 within the GOSR2 locus were detected in patients with anomalies of thoracic arteries and veins (ATAV). Genetic variants associated with ATAV are suggested to influence the expression of WNT3, and the variant rs870142 related to septal defects is proposed to influence the expression of MSX1. We analyzed the expression of all 4 genes during cardiac differentiation of human and murine induced pluripotent stem cells in vitro and by single-cell RNA-Seq analyses of developing murine and human hearts. Our data show that MACROD2, GOSR2, WNT3, and MSX1 play an essential functional role in heart development at the embryonic and newborn stages.
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Loci Gênicos , Cardiopatias Congênitas/genética , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Animais , Feminino , Estudo de Associação Genômica Ampla , Alemanha/epidemiologia , Cardiopatias Congênitas/epidemiologia , Humanos , Masculino , Camundongos , Fatores de RiscoRESUMO
BACKGROUND: Reticulated platelets (RP) are the youngest circulating platelets in blood. An increased amount of this subpopulation is associated with higher cardiovascular risk and mortality. OBJECTIVES: It is unknown to what extent intrinsic properties of RP contribute to their hyperreactive features. This study is the first providing a multifactorial approach based on ultrastructural, transcriptional, and functional analysis of RP compared to non-RP sorted by flow cytometry. METHODS: Reticulated platelets and non-RP were sorted after platelet staining with SYTO 13. Employing transmission electron microscopy, 1089 micrographs were analyzed for platelet size, amounts of intracellular structures, and anatomical surrogates indicating activation. Long and small RNA-sequencing (RNA-seq) were performed for analyzing differential gene expression. Functional analysis of P-selectin-an upregulated mRNA in RP-was performed in healthy subjects and patients on P2Y12 -inhibitors. RESULTS: Electron micrographs uncovered distinct ultrastructural differences in RP versus non-RP. Cross sections were 1.9-fold larger in RP (P < .0001). Amounts of α-granules, dense granules, open canalicular system-openings, and mitochondria were increased in RP, which persisted after adjustment for platelet size. Long RNA-seq showed 1212 upregulated transcripts that are predominantly associated to platelet shape change, aggregation, and activation; 1264 mRNAs were downregulated in RP. Small RNA-seq did not reveal any differentially expressed transcripts. Functional analysis displayed higher P-selectin expression as compared to non-RP upon ADP- or TRAP-stimulation. CONCLUSIONS: Our results demonstrate that altered intrinsic structural and molecular properties contribute to the hyperreactivity of RP. These properties and an increased amount of RP may account for the association with cardiovascular risk.
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Plaquetas , Citometria de Fluxo , Humanos , Contagem de PlaquetasRESUMO
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.