RESUMO
Differentiation protocols to direct cell fate decision from pluripotent stem cells to cardiac myocytes normally achieve high purity and quality of cells. Nonetheless, the highly specialized anatomy of the heart enables the possibility that acquisition of terminal somatic differentiation from pluripotency might imply heterogeneity of non-desire cell lineages. Directed cardiac differentiation empowers differentiation of pool of cells commonly reported to contain different proportions of ventricular, atrial, and nodal-like cells. RNA sequencing (RNA-Seq) allows a precise transcriptional profiling, ensuring a quality checking of the cell identity our protocol has derived as a main outcome. Here we describe a workflow methodology on how to adapt RNA sequencing analysis for integration into the R analysis pipeline in order to characterize chamber-specific gene signatures of the major cardiac lineages of myocytes in the heart.
Assuntos
Perfilação da Expressão Gênica , Átrios do Coração/citologia , Ventrículos do Coração/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/química , RNA-Seq/métodos , Transcriptoma , Diferenciação Celular/genética , Células Cultivadas , Análise por Conglomerados , Ontologia Genética , Átrios do Coração/química , Ventrículos do Coração/química , Humanos , Análise de Componente Principal , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Fluxo de TrabalhoRESUMO
Many cardiac pathologies involve changes in tissue structure. Conventional analysis of structural features is extremely time-consuming and subject to observer bias. The possibility to determine spatial interrelations between these features is often not fully exploited. We developed a staining protocol and an ImageJ-based tool (JavaCyte) for automated histological analysis of cardiac structure, including quantification of cardiomyocyte size, overall and endomysial fibrosis, spatial patterns of endomysial fibrosis, fibroblast density, capillary density and capillary size. This automated analysis was compared to manual quantification in several well-characterized goat models of atrial fibrillation (AF). In addition, we tested inter-observer variability in atrial biopsies from the CATCH-ME consortium atrial tissue bank, with patients stratified by their cardiovascular risk profile for structural remodeling. We were able to reproduce previous manually derived histological findings in goat models for AF and AV block (AVB) using JavaCyte. Furthermore, strong correlation was found between manual and automated observations for myocyte count (r = 0.94, p < 0.001), myocyte diameter (r = 0.97, p < 0.001), endomysial fibrosis (r = 0.98, p < 0.001) and capillary count (r = 0.95, p < 0.001) in human biopsies. No significant variation between observers was observed (ICC = 0.89, p < 0.001). We developed and validated an open-source tool for high-throughput, automated histological analysis of cardiac tissue properties. JavaCyte was as accurate as manual measurements, with less inter-observer variability and faster throughput.
Assuntos
Algoritmos , Fibrilação Atrial/fisiopatologia , Automação , Átrios do Coração/química , Átrios do Coração/fisiopatologia , Idoso , Animais , Feminino , Cabras , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: This study aimed to evaluate atrium extracellular matrix remodeling in atrial fibrillation (AF) patients with severe aortic stenosis, through histological fibrosis quantification and extracellular matrix gene expression analysis, as well as serum quantification of selected protein targets. METHODS: A posthoc analysis of a prospective study was performed in a cohort of aortic stenosis patients. Between 2014 and 2019, 56 patients with severe aortic stenosis submitted to aortic valve replacement surgery in a tertiary hospital were selected. RESULTS: Fibrosis was significantly increased in the AF group when compared to sinus rhythm (SR) patients (p = 0.024). Moreover, cardiomyocyte area was significantly higher in AF patients versus SR patients (p = 0.008). Conversely, collagen III gene expression was increased in AF patients (p = 0.038). TIMP1 was less expressed in the atria of AF patients. MMP16/TIMP4 ratio was significantly decreased in AF patients (p = 0.006). TIMP1 (p = 0.004) and TIMP2 (p = 0.012) were significantly increased in the serum of AF patients. Aortic valve maximum (p = 0.0159) and mean (p = 0.031) gradients demonstrated a negative association with serum TIMP1. CONCLUSIONS: Atrial fibrillation patients with severe aortic stenosis present increased atrial fibrosis and collagen type III synthesis, with extracellular matrix remodelling demonstrated by a decrease in the MMP16/TIMP4 ratio, along with an increased serum TIMP1 and TIMP2 proteins.
Assuntos
Estenose da Valva Aórtica/patologia , Remodelamento Atrial , Matriz Extracelular/patologia , Átrios do Coração/patologia , Idoso , Idoso de 80 Anos ou mais , Estenose da Valva Aórtica/sangue , Estenose da Valva Aórtica/fisiopatologia , Biomarcadores/análise , Biomarcadores/sangue , Matriz Extracelular/química , Feminino , Fibrose , Átrios do Coração/química , Átrios do Coração/fisiopatologia , Humanos , Masculino , Metaloproteinase 16 da Matriz/análise , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Inibidor Tecidual de Metaloproteinase-1/análise , Inibidor Tecidual de Metaloproteinase-1/sangue , Inibidor Tecidual de Metaloproteinase-2/sangue , Inibidores Teciduais de Metaloproteinases/análise , Inibidor Tecidual 4 de MetaloproteinaseRESUMO
BACKGROUND: Scientific research on atrial fibrosis in atrial fibrillation (AF) has mainly focused on quantitative or molecular features. The purpose of this study was to perform a clinicoarchitectural/structural investigation of fibrosis to provide one key to understanding the electrophysiological/clinical aspects of AF. METHODS: We characterized the fibrosis (amount, architecture, cellular components, and ultrastructure) in left atrial biopsies from 121 patients with persistent/long-lasting persistent AF (group 1; 59 males; 60±11 years; 91 mitral disease-related AF, 30 nonmitral disease-related AF) and from 39 patients in sinus rhythm with mitral valve regurgitation (group 2; 32 males; 59±12 years). Ten autopsy hearts served as controls. RESULTS: Qualitatively, the fibrosis exhibited the same characteristics in all cases and displayed particular architectural scenarios (which we arbitrarily subdivided into 4 stages) ranging from isolated foci to confluent sclerotic areas. The percentage of fibrosis was larger and at a more advanced stage in group 1 versus group 2 and, within group 1, in patients with rheumatic disease versus nonrheumatic cases. In patients with AF with mitral disease and no rheumatic disease, the percentage of fibrosis and the fibrosis stages correlated with both left atrial volume index and AF duration. The fibrotic areas mainly consisted of type I collagen with only a minor cellular component (especially fibroblasts/myofibroblasts; average value range 69-150 cells/mm2, depending on the areas in AF biopsies). A few fibrocytes-circulating and bone marrow-derived mesenchymal cells-were also detectable. The fibrosis-entrapped cardiomyocytes showed sarcolemmal damage and connexin 43 redistribution/internalization. CONCLUSIONS: Atrial fibrosis is an evolving and inhomogeneous histological/architectural change that progresses through different stages ranging from isolated foci to confluent sclerotic zones which-seemingly-constrain impulse conduction across restricted regions of electrotonically coupled cardiomyocytes. The fibrotic areas mainly consist of type I collagen extracellular matrix and, only to a lesser extent, mesenchymal cells.
Assuntos
Fibrilação Atrial/patologia , Átrios do Coração/patologia , Doenças das Valvas Cardíacas/patologia , Miocárdio/patologia , Cardiopatia Reumática/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Fibrilação Atrial/metabolismo , Fibrilação Atrial/fisiopatologia , Fibrilação Atrial/terapia , Função do Átrio Esquerdo , Remodelamento Atrial , Biópsia , Colágeno Tipo I/análise , Conexina 43/análise , Feminino , Fibrose , Átrios do Coração/química , Átrios do Coração/fisiopatologia , Doenças das Valvas Cardíacas/metabolismo , Doenças das Valvas Cardíacas/fisiopatologia , Doenças das Valvas Cardíacas/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Miocárdio/química , Estudos Retrospectivos , Cardiopatia Reumática/metabolismo , Cardiopatia Reumática/fisiopatologia , Cardiopatia Reumática/terapiaRESUMO
Background Atrial tissue fibrosis is linked to inflammatory cells, yet is incompletely understood. A growing body of literature associates peripheral blood levels of the antifibrotic hormone BNP (B-type natriuretic peptide) with atrial fibrillation (AF). We investigated the relationship between pro-fibrotic tissue M2 macrophage marker Cluster of Differentiation (CD)163+, atrial procollagen expression, and BNP gene expression in patients with and without AF. Methods and Results In a cross-sectional study design, right atrial tissue was procured from 37 consecutive, consenting, stable patients without heart failure or left ventricular systolic dysfunction, of whom 10 had AF and 27 were non-AF controls. Samples were analyzed for BNP and fibro-inflammatory gene expression, as well as fibrosis and CD163+. Primary analyses showed strong correlations (all P<0.008) between M2 macrophage CD163+ staining, procollagen gene expression, and myocardial BNP gene expression across the entire cohort. In secondary analyses without multiplicity adjustments, AF patients had greater left atrial volume index, more valve disease, higher serum BNP, and altered collagen turnover markers versus controls (all P<0.05). AF patients also showed higher atrial tissue M2 macrophage CD163+, collagen volume fraction, gene expression of procollagen 1 and 3, as well as reduced expression of the BNP clearance receptor NPRC (all P<0.05). Atrial procollagen 3 gene expression was correlated with fibrosis and BNP gene expression was correlated with serum BNP. Conclusions Elevated atrial tissue pro-fibrotic M2 macrophage CD163+ is associated with increased myocardial gene expression of procollagen and anti-fibrotic BNP and is higher in patients with AF. More work on modulation of BNP signaling for treatment and prevention of AF may be warranted.
Assuntos
Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Fibrilação Atrial/metabolismo , Remodelamento Atrial , Colágeno Tipo I/análise , Átrios do Coração/química , Macrófagos/química , Peptídeo Natriurético Encefálico/análise , Pró-Colágeno/análise , Receptores de Superfície Celular/análise , Idoso , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/genética , Fibrilação Atrial/fisiopatologia , Biomarcadores/análise , Estudos de Casos e Controles , Colágeno Tipo I/genética , Estudos Transversais , Feminino , Fibrose , Regulação da Expressão Gênica , Átrios do Coração/patologia , Átrios do Coração/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeo Natriurético Encefálico/genética , Fenótipo , Pró-Colágeno/genéticaRESUMO
BACKGROUND: Cardiac disease modelling using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) requires thorough insight into cardiac cell type differentiation processes. However, current methods to discriminate different cardiac cell types are mostly time-consuming, are costly and often provide imprecise phenotypic evaluation. DNA methylation plays a critical role during early heart development and cardiac cellular specification. We therefore investigated the DNA methylation pattern in different cardiac tissues to identify CpG loci for further cardiac cell type characterization. RESULTS: An array-based genome-wide DNA methylation analysis using Illumina Infinium HumanMethylation450 BeadChips led to the identification of 168 differentially methylated CpG loci in atrial and ventricular human heart tissue samples (n = 49) from different patients with congenital heart defects (CHD). Systematic evaluation of atrial-ventricular DNA methylation pattern in cardiac tissues in an independent sample cohort of non-failing donor hearts and cardiac patients using bisulfite pyrosequencing helped us to define a subset of 16 differentially methylated CpG loci enabling precise characterization of human atrial and ventricular cardiac tissue samples. This defined set of reproducible cardiac tissue-specific DNA methylation sites allowed us to consistently detect the cellular identity of hiPSC-CM subtypes. CONCLUSION: Testing DNA methylation of only a small set of defined CpG sites thus makes it possible to distinguish atrial and ventricular cardiac tissues and cardiac atrial and ventricular subtypes of hiPSC-CMs. This method represents a rapid and reliable system for phenotypic characterization of in vitro-generated cardiomyocytes and opens new opportunities for cardiovascular research and patient-specific therapy.
Assuntos
Metilação de DNA , Átrios do Coração/citologia , Cardiopatias Congênitas/patologia , Ventrículos do Coração/citologia , Miócitos Cardíacos/citologia , Células Cultivadas , Ilhas de CpG , Feminino , Átrios do Coração/química , Cardiopatias Congênitas/genética , Ventrículos do Coração/química , Humanos , Células-Tronco Pluripotentes Induzidas/química , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Modelos Biológicos , Miócitos Cardíacos/química , Especificidade de Órgãos , Análise de Sequência de DNA , Engenharia TecidualRESUMO
BACKGROUND: Atrial fibrillation (AF) is the most common chronic arrhythmia in elderly people and is accompanied by remodeling processes. While much is known about changes in ionic channels and in extracellular matrix, less is known about possible changes of intracellular structures. OBJECTIVE: We wanted to investigate, whether AF may also affect the structure of the Golgi apparatus and the microtubular network. METHODS: One-hundred fifty-three cardiac surgery patients were investigated [n = 24 in sinus rhythm (SR) and n = 129 with chronic AF of >1 year duration]. Tissue samples of the left atrial free wall were examined immunohistochemically. Golgi apparatus was detected by GM130 and its phosphorylated isoform. Furthermore, we investigated the length of the microtubules by α-tubulin staining. We also measured stathmin (phospho-S37), which is known to induce microtubule depolymerization. In addition, we investigated the cyclin-dependent kinase cdk5-activation, a typical stimulus for Golgi fragmentation, by measuring membrane-associated cdk5. RESULTS: We found significant fragmentation of the Golgi apparatus in AF together with a reduced fragment size. Significant more fragments of the Golgi were found lateral to the nucleus in AF, while the Golgi in SR was located more to the polar side of the nucleus, that is, in the longitudinal axis of the cell. This was accompanied by a significant reduction of the number of tubulin strands longer than 10 µm. These changes did not go along with an activation of stathmin, but with an increase in membrane association of cdk5. CONCLUSIONS: The present data may show that AF associated remodeling also involves intracellular remodeling of the Golgi-microtubular apparatus.
Assuntos
Fibrilação Atrial/patologia , Remodelamento Atrial , Complexo de Golgi/patologia , Átrios do Coração/patologia , Idoso , Fibrilação Atrial/metabolismo , Fibrilação Atrial/fisiopatologia , Autoantígenos/análise , Biomarcadores/análise , Estudos de Casos e Controles , Doença Crônica , Quinase 5 Dependente de Ciclina/análise , Feminino , Átrios do Coração/química , Átrios do Coração/fisiopatologia , Humanos , Masculino , Proteínas de Membrana/análise , Microtúbulos/química , Microtúbulos/patologia , Pessoa de Meia-Idade , Fosforilação , Estatmina/análise , Tubulina (Proteína)/análiseRESUMO
Right ventricle (RV) remodelling occurs in neonatal patients born with ventricular septal defect (VSD). The presence of a defect between the two ventricles allows for shunting of blood from the left to right side. The resulting RV hypertrophy leads to molecular remodelling which has thus far been largely investigated using right atrial (RA) tissue. In this study we used proteomic and phosphoproteomic analysis in order to determine any difference between the proteomes for RA and RV. Samples were therefore taken from the RA and RV of five infants (0.34⯱â¯0.05â¯years, mean⯱â¯SEM) with VSD who were undergoing cardiac surgery to repair the defect. Significant differences in protein expression between RV and RA were seen. 150 protein accession numbers were identified which were significantly lower in the atria, whereas none were significantly higher in the atria compared to the ventricle. 19 phosphorylation sites (representing 19 phosphoproteins) were also lower in RA. This work has identified differences in the proteome between RA and RV which reflect differences in contractile activity and metabolism. As such, caution should be used when drawing conclusions based on analysis of the RA and extrapolating to the hypertrophied RV. SIGNIFICANCE: RV hypertrophy occurs in neonatal patients born with VSD. Very little is known about how the atria responds to RV hypertrophy, especially at the protein level. Access to tissue from age-matched groups of patients is very rare, and we are in the unique position of being able to get tissue from both the atria and ventricle during reparative surgery of these infants. Our findings will be beneficial to future research into heart chamber malformations in congenital heart defects.
Assuntos
Comunicação Interventricular/metabolismo , Miocárdio/química , Proteoma/análise , Átrios do Coração/química , Comunicação Interventricular/patologia , Ventrículos do Coração/química , Ventrículos do Coração/patologia , Humanos , Hipertrofia , Lactente , Fosfoproteínas/análise , Proteômica/métodosRESUMO
BACKGROUND: Among the potential factors which may contribute to the development and perpetuation of atrial fibrillation, dysregulation of miRNAs has been suggested. Thus in this study, we have quantified the basal expressions of 662 mature human miRNAs in left atrium (LA) from patients undergoing cardiac surgery for valve repair, suffering or not from atrial fibrillation (AF) by using TaqMan® Low Density arrays (v2.0). RESULTS: Among the 299 miRNAs expressed in all patients, 42 miRNAs had altered basal expressions in patients with AF. Binding-site predictions with Targetscan (conserved sites among species) indicated that the up- and down-regulated miRNAs controlled respectively 3,310 and 5,868 genes. To identify the most relevant cellular functions under the control of the altered miRNAs, we focused on the 100 most targeted genes of each list and identified 5 functional protein-protein networks among these genes. Up-regulated networks were involved in synchronisation of circadian rythmicity and in the control of the AKT/PKC signaling pathway (i.e., proliferation/adhesion). Down-regulated networks were the IGF-1 pathway and TGF-beta signaling pathway and a network involved in RNA-mediated gene silencing, suggesting for the first time that alteration of miRNAs in AF would also perturbate the whole miRNA machinery. Then we crossed the list of miRNA predicted genes, and the list of mRNAs altered in similar patients suffering from AF and we found that respectively 44.5% and 55% of the up- and down-regulated mRNA are predicted to be conserved targets of the altered miRNAs (at least one binding site in 3'-UTR). As they were involved in the same biological processes mentioned above, these data demonstrated that a great part of the transcriptional defects previously published in LA from AF patients are likely due to defects at the post-transcriptional level and involved the miRNAs. CONCLUSIONS: Our stringent analysis permitted us to identify highly targeted protein-protein networks under the control of miRNAs in LA and, among them, to highlight those specifically affected in AF patients with altered miRNA signature. Further studies are now required to determine whether alterations of miRNA levels in AF pathology are causal or represent an adaptation to prevent cardiac electrical and structural remodeling.
Assuntos
Fibrilação Atrial/etiologia , Átrios do Coração/química , Doenças das Valvas Cardíacas/genética , MicroRNAs/análise , Transcriptoma , Regiões 3' não Traduzidas , Idade de Início , Idoso , Fibrilação Atrial/genética , Ritmo Circadiano/genética , Simulação por Computador , Suscetibilidade a Doenças , Feminino , Redes Reguladoras de Genes , Inativação Gênica , Células HEK293 , Átrios do Coração/patologia , Doenças das Valvas Cardíacas/complicações , Doenças das Valvas Cardíacas/metabolismo , Doenças das Valvas Cardíacas/cirurgia , Histona-Lisina N-Metiltransferase/genética , Humanos , Masculino , MicroRNAs/biossíntese , MicroRNAs/genética , Pessoa de Meia-Idade , Modelos Biológicos , Tamanho do Órgão , Transdução de Sinais/genética , TransfecçãoRESUMO
Atrial and B-type natriuretic peptides (ANP and BNP) are cardiac hormones important for cardiovascular and body fluid regulation. In some teleost species, an additional member of the natriuretic peptide family, ventricular NP (VNP), has been identified. In this study, we examine tissue distribution of these three NPs in the eel heart. Quantitative real-time PCR showed that anp is almost exclusively expressed in atria, bnp equally in atria and ventricles and vnp three-fold more in ventricles than in atria. The amount of bnp transcript overall in the heart was 1/10 those of anp and vnp. There was no difference in transcript levels between freshwater and seawater-acclimated fishes. Immunohistochemistry using specific antisera and in situ hybridization using gene-specific probes showed that NP signals were detected in most atrial and ventricular myocytes with some regional differences in density. Because of high sequence similarity of the three NPs, each of the three NP antisera individually was pre-incubated with 10-8 M of the other two non-targeted cardiac NPs to increase the specificity. A few atrial myocytes contained all three NPs in the same cell. Immuno-electron microscopy identified many dense-core vesicles containing ANP in atria and VNP in ventricles and some vesicles contained both ANP and VNP as demonstrated using pre-absorbed antisera. Based on these data and those of previous studies, we suggest that in eels ANP is secreted from atria in a regulatory pathway and VNP from ventricles in a constitutive pathway. In addition, VNP, not BNP, is the principal ventricular hormone in eels.
Assuntos
Fator Natriurético Atrial/metabolismo , Enguias/metabolismo , Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Peptídeo Natriurético Encefálico/metabolismo , Sequência de Aminoácidos , Animais , Fator Natriurético Atrial/química , Fator Natriurético Atrial/genética , Enguias/genética , Átrios do Coração/química , Ventrículos do Coração/química , Miócitos Cardíacos/química , Miócitos Cardíacos/metabolismo , Peptídeo Natriurético Encefálico/química , Peptídeo Natriurético Encefálico/genética , RNA Mensageiro/genética , Vesículas Secretórias/química , Vesículas Secretórias/metabolismo , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: The aim of this pilot study was to detect correlations of microbiological DNA, inflammatory proteins, and infection parameters in patients with periodontal disease (PD) and valvular heart disease (VHD). METHODS: A perioperative comprehensive dental examination for the investigation of periodontal status, including sampling of specific subgingival bacteria, was performed in 10 patients with indication for surgery of aortic valve stenosis with or without concomitant myocardial revascularization. Standard protocol biopsies were taken from right atrium (A), left septal myocardium (M), and aortic valve (V). Eleven periodontal pathogens DNA in oral and cardiac tissue samples (A/M/V) were analyzed using polymerase chain reaction. For cardiac tissue samples, Western blot analysis of LPS-binding protein (LBP), immunohistochemical (IHC) detection of LBP-big42, LPS-binding protein receptor (CD14), and macrophages (CD68), as well as inflammation scoring measurement were performed. RESULTS: Periodontitis was present in all patients with severe intensity in 7, moderate in 2 and mild in one patient. Same bacterial DNA was detected in A, M, and V in different distribution, and detection was more often in atrium than in myocardium or valve tissue. Morphological investigation revealed increased extracellular inflammatory cell migration. In IHC markers of LBP, CD68 and CD14 showed positive findings for all patients in atrium and myocardium. CONCLUSION: Our results demonstrate the presence of oral bacterial DNA in human cardiac tissue, as well as inflammatory markers potentially indicating connection of PD and VHD. Further investigation is necessary to confirm these preliminary data.
Assuntos
Estenose da Valva Aórtica/microbiologia , Valva Aórtica/microbiologia , DNA Bacteriano/genética , Átrios do Coração/microbiologia , Periodontite/microbiologia , Proteínas de Fase Aguda/análise , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Valva Aórtica/química , Valva Aórtica/cirurgia , Estenose da Valva Aórtica/complicações , Estenose da Valva Aórtica/diagnóstico , Estenose da Valva Aórtica/metabolismo , Proteínas de Transporte/análise , Feminino , Átrios do Coração/química , Septos Cardíacos/química , Septos Cardíacos/microbiologia , Implante de Prótese de Valva Cardíaca , Humanos , Mediadores da Inflamação/análise , Receptores de Lipopolissacarídeos/análise , Masculino , Glicoproteínas de Membrana/análise , Pessoa de Meia-Idade , Periodontite/complicações , Periodontite/diagnóstico , Projetos Piloto , Dados Preliminares , Fatores de Risco , Índice de Gravidade de DoençaRESUMO
Aims: Quantitative real-time RT-PCR (RT-qPCR) has become the method of choice for mRNA quantification, but requires an accurate normalization based on the use of reference genes showing invariant expression across various pathological conditions. Only few data exist on appropriate reference genes for the human heart. The objective of this study was to determine a set of suitable reference genes in human atrial and ventricular tissues, from right and left cavities in control and in cardiac diseases. Methods and results: We assessed the expression of 16 reference genes (ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, IPO8, PGK1, POLR2A, PPIA, RPLP0, TBP, TFRC, UBC, YWHAZ, 18S) in tissues from: right and left ventricles from healthy controls and heart failure (HF) patients; right-atrial tissue from patients in sinus rhythm with (SRd) or without (SRnd) atrial dilatation, patients with paroxysmal (pAF) or chronic (cAF) atrial fibrillation or with HF; and left-atrial tissue from patients in SR or cAF. Consensual analysis (by geNorm and Normfinder algorithms, BestKeeper software tool and comparative delta-Ct method) of the variability scores obtained for each reference gene expression shows that the most stably expressed genes are: GAPDH, GUSB, IPO8, POLR2A, and YWHAZ when comparing either right and left ventricle or ventricle from healthy controls and HF patients; GAPDH, IPO8, POLR2A, PPIA, and RPLP0 when comparing either right and left atrium or right atria from all pathological groups. ACTB, TBP, TFRC, and 18S genes were identified as the least stable. Conclusions: The overall most stable reference genes across different heart cavities and disease conditions were GAPDH, IPO8, POLR2A and PPIA. YWHAZ or GUSB could be added to this set for some specific experiments. This study should provide useful guidelines for reference gene selection in RT-qPCR studies in human heart.
Assuntos
Fibrilação Atrial/genética , Perfilação da Expressão Gênica/métodos , Genes Essenciais , Átrios do Coração/química , Cardiopatias/genética , Ventrículos do Coração/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma , Proteínas 14-3-3/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , RNA Polimerases Dirigidas por DNA/genética , Europa (Continente) , Feminino , Perfilação da Expressão Gênica/normas , Marcadores Genéticos , Glucuronidase/genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Masculino , Pessoa de Meia-Idade , Peptidilprolil Isomerase/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Reprodutibilidade dos Testes , beta Carioferinas/genéticaRESUMO
The purpose of this study was to evaluate structural and optical properties of atrial tissue from common animal models and to compare it with human atria. We aimed to do this in a format that will be useful for development of better ablation tools and/or new means for visualizing atrial lesions. Human atrial tissue from clinically relevant age group was compared and contrasted with atrial tissue of large animal models commonly available for research purposes. These included pigs, sheep, dogs and cows. The presented data include area measurements of smooth atrial surface available for ablation and estimates of thickness of collagen and muscle for five different species. We also described methods to quantify presence of collagen and overall thickness of atrial wall. Provided information enables placement of atrial lesions to locations with clinically relevant atrial wall thickness and macroscopic structure ultimately helping investigators to develop better ablation and imaging tools. It also highlights the impact of collagen thickness on optical measurements and lesion visualization.
Assuntos
Átrios do Coração/anatomia & histologia , Átrios do Coração/química , Técnicas de Ablação/métodos , Fatores Etários , Idoso , Animais , Bovinos , Colágeno/análise , Cães , Feminino , Átrios do Coração/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Ovinos , Especificidade da Espécie , SuínosRESUMO
BACKGROUND: The epigenetic changes underlying the development of atrial fibrillation (AF) remain incompletely understood. Limited evidence suggests that abnormal DNA methylation might be involved in the pathogenesis of AF. In the present study, we evaluated the methylation status of genomic DNA from myocardial tissue in AF patients and sinus rhythm (SR) patients systematically. HYPOTHESIS: DNA methylation dysregulations will be associated with valvular AF. METHODS: Right atrial myocardial tissue was obtained from rheumatic valvular patients who had undergone valve replacement surgery (SR group, n = 10; AF group, n = 10). The global DNA methylation level, the promoter methylation level of the natriuretic peptide receptor-A gene (NPRA), and its correlation with the mRNA expression level of DNA methyltransferase genes were detected. RESULTS: The global DNA methylation level was significantly higher in the AF group than in the SR group (P < 0.05). The NPRA mRNA expression was decreased and the NPRA gene was hypermethylated in the AF group (P < 0.05). Meanwhile, the NPRA mRNA expression level has a negative correlation with the mean methylation level in the promoter region of the NPRA gene. CONCLUSIONS: DNA methylation dysregulations may be relevant in the pathogenesis of AF. DNA methyltransferase 3B likely plays an essential role in the DNA methylation dysregulations in AF.
Assuntos
Fibrilação Atrial/genética , Metilação de DNA , Epigênese Genética , Átrios do Coração/química , Receptores do Fator Natriurético Atrial/genética , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/enzimologia , Estudos de Casos e Controles , DNA (Citosina-5-)-Metiltransferases/genética , Átrios do Coração/enzimologia , Humanos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , DNA Metiltransferase 3BRESUMO
A 66-year-old female patient was referred to our hospital for resection of a right atrial mass. Four months earlier, she had suffered an acute cerebrovascular accident due to occlusion of the sylvian segment of the right middle cerebral artery from atheromatous tight stenosis in the right internal carotid artery. Later, investigations with transthoracic and transesophageal echocardiography revealed a 3.4-cm right atrial mass that was resected surgically. Microscopic evaluation revealed a well-circumscribed nodular tumor, located within the interatrial septum, and corresponding to an angioleiomyoma (ALM). This tumor differs histologically from atrial myxoma. ALM is a ubiquitous benign tumor but has never been reported to occur in the atrium. ALM can mimic cardiac myxoma and should be considered in the differential diagnosis of atrial tumors.
Assuntos
Angiomioma/patologia , Átrios do Coração/patologia , Neoplasias Cardíacas/patologia , Idoso , Angiomioma/química , Angiomioma/diagnóstico por imagem , Angiomioma/cirurgia , Biomarcadores Tumorais/análise , Biópsia , Ecocardiografia Transesofagiana , Eletrocardiografia , Feminino , Átrios do Coração/química , Átrios do Coração/diagnóstico por imagem , Átrios do Coração/cirurgia , Neoplasias Cardíacas/química , Neoplasias Cardíacas/diagnóstico por imagem , Neoplasias Cardíacas/cirurgia , Humanos , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Resultado do Tratamento , Carga TumoralRESUMO
Surgeries to correct congenital heart diseases are increasing in Brazil and worldwide. However, even with the advances in surgical techniques and perfusion, some cases, especially the more complex ones, can develop heart failure and death. A retrospective study of patients who underwent surgery for correction of congenital heart diseases with cardiopulmonary bypass (CPB) in a university tertiary-care hospital that died, showed infarction in different stages of evolution and scattered microcalcifications in the myocardium, even without coronary obstruction. CPB is a process routinely used during cardiac surgery for congenital heart disease. However, CPB has been related to increased endogenous catecholamines that can lead to major injuries in cardiomyocytes. The mechanisms involved are not completely understood. The aim of this study was to evaluate the alterations induced in the ß-adrenergic receptors and GRK-2 present in atrial cardiomyocytes of infants with congenital heart disease undergoing surgical repair with CPB and correlate the alterations with functional and biochemical markers of ischemia/myocardial injury. The study consisted of right atrial biopsies of infants undergoing surgical correction in HC-FMRPUSP. Thirty-three cases were selected. Atrial biopsies were obtained at the beginning of CPB (group G1) and at the end of CPB (group G2). Real-time PCR, Western blotting, and immunofluorescence analysis were conducted to evaluate the expression of ß1, ß2-adrenergic receptors, and GRK-2 in atrial myocardium. Cardiac function was evaluated by echocardiography and biochemical analysis (N-terminal pro-brain natriuretic peptide (NT-ProBNP), lactate, and cardiac troponin I). We observed an increase in serum lactate, NT-proBNP, and troponin I at the end of CPB indicating tissue hypoxia/ischemia. Even without major clinical consequences in cardiac function, these alterations were followed by a significant increase in gene expression of ß1 and ß2 receptors and GRK-2, suggesting that this is one of the mechanisms responsible for the exacerbated response of cardiomyocytes to circulating catecholamines. These alterations could explain the irreversible myocardial damage and lipid peroxidation of membranes classically attributed to catecholamine excess, observed in some infants who develop heart failure and postoperative death. Although other factors may be involved, this study confirms that CPB acts as a potent inducer of increased gene expression of ß- adrenergic receptors and GRK-2, making the myocardium of these infants more susceptible to the effects of circulating endogenous catecholamines, which may contribute to the development of irreversible myocardial damage and death.
Assuntos
Ponte Cardiopulmonar/efeitos adversos , Quinase 2 de Receptor Acoplado a Proteína G/genética , Átrios do Coração/metabolismo , Cardiopatias Congênitas/cirurgia , Insuficiência Cardíaca/genética , Receptores Adrenérgicos beta/genética , Biomarcadores/análise , Biópsia , Catecolaminas/metabolismo , Feminino , Quinase 2 de Receptor Acoplado a Proteína G/análise , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Expressão Gênica , Átrios do Coração/química , Átrios do Coração/patologia , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Humanos , Lactente , Recém-Nascido , Masculino , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos beta/análise , Receptores Adrenérgicos beta/metabolismo , Estudos RetrospectivosRESUMO
AIMS: Chronic inflammation in the atrial myocardium was shown to play an important role in the development of atrial fibrosis in patients with atrial fibrillation (AF). However, it is not clear to what extent atrial inflammatory reaction associated with AF extends on the ventricular myocardium. Our aim was to assess the extent of fibrosis and lymphomononuclear infiltration in human ventricular myocardium and explore its association with AF. METHODS AND RESULTS: Medical records from consecutive autopsies were checked for presence of AF. Heart specimens from 30 patients died from cardiovascular causes (64 ± 12 years, 17 men) were collected in three equal groups: no AF, paroxysmal AF, and permanent AF. Tissue samples were taken from the Bachmann's bundle, crista terminalis, posterior left atrium, left ventricle and right ventricle free walls and stained with Masson's trichrome for analysis of fibrosis extent. Immunohistochemistry was performed using antibodies against CD3- and CD45-antigens and quantified as number of antigen-positive cells per 1 mm2. Fibrosis extent, CD3+ and CD45+ cell counts were elevated in AF patients at all sites (P < 0.001 for all). Fibrosis extent demonstrated correlation with both CD3+ and CD45+ cell counts in the right (r = 0.781, P < 0.001 for CD45+ and r = 0.720, P < 0.001 for CD3+) and the left (r = 0.515, P = 0.004 for CD45+ and r = 0.573, P = 0.001 for CD3+) ventricles. Neither fibrosis nor inflammatory cell count showed association with either age or comorbidities. CONCLUSION: Histological signs of chronic inflammation affecting ventricular myocardium are strongly associated with AF and demonstrate significant correlation with fibrosis extent that cannot be explained by cardiovascular comorbidities otherwise.
Assuntos
Fibrilação Atrial/patologia , Átrios do Coração/patologia , Ventrículos do Coração/patologia , Miocardite/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Fibrilação Atrial/fisiopatologia , Autopsia , Biomarcadores/análise , Biópsia , Complexo CD3/análise , Estudos de Casos e Controles , Feminino , Fibrose , Átrios do Coração/química , Átrios do Coração/fisiopatologia , Ventrículos do Coração/química , Ventrículos do Coração/fisiopatologia , Humanos , Imuno-Histoquímica , Antígenos Comuns de Leucócito/análise , Masculino , Pessoa de Meia-Idade , Miocardite/fisiopatologia , Prognóstico , Função Ventricular Esquerda , Função Ventricular Direita , Remodelação VentricularAssuntos
Adenocarcinoma/secundário , Neoplasias Colorretais/patologia , Átrios do Coração/patologia , Veia Cava Inferior/patologia , Adenocarcinoma/química , Adenocarcinoma/diagnóstico por imagem , Idoso , Biomarcadores Tumorais/análise , Biópsia com Agulha de Grande Calibre , Fator de Transcrição CDX2/análise , Neoplasias Colorretais/química , Neoplasias Colorretais/diagnóstico por imagem , Átrios do Coração/química , Átrios do Coração/diagnóstico por imagem , Humanos , Imuno-Histoquímica , Masculino , Tomografia Computadorizada por Raios X , Veia Cava Inferior/química , Veia Cava Inferior/diagnóstico por imagemRESUMO
BACKGROUND: Mitral stenosis (MS) has the highest incidence of atrial fibrillation (AF) in chronic rheumatic valvular disease. There are very few studies in isolated MS comparing histopathological changes in patients with sinus rhythm (SR) and AF. OBJECTIVES: To analyze the histological changes associated with isolated MS and compare between changes in AF and SR. METHODS: This was a prospective study in patients undergoing valve replacement surgery for symptomatic isolated MS who were divided into 2 groups, Group I AF (n = 13) and Group II SR (n = 10). Intra-operative biopsies performed from 5 different sites from both atria were analyzed for 10 histopathologic changes commonly associated with AF. RESULTS: On multivariate analysis, myocytolysis (odds ratio [OR]: 1.48, P = 0.05) was found to be associated with AF, whereas myocyte hypertrophy (OR: 0.21, P = 0.003), and glycogen deposition (OR: 0.43, P = 0.002) was associated with SR. Interstitial fibrosis the commonest change was uniformly distributed across both atria irrespective of the rhythm. CONCLUSION: In rheumatic MS, SR is associated with myocyte hypertrophy whereas AF is associated with myocytolysis. Endocardial inflammation is more common in left atrial appendage irrespective of rhythm. Interstitial fibrosis is seen in >90% of patients distributed in both the atria and is independent of the rhythm. Amyloid and Aschoff bodies are uncommon and the rest of the changes are uniformly distributed across both the atria.
Assuntos
Fibrilação Atrial/patologia , Átrios do Coração/patologia , Estenose da Valva Mitral/patologia , Cardiopatia Reumática/patologia , Adolescente , Adulto , Idoso , Fibrilação Atrial/etiologia , Fibrilação Atrial/metabolismo , Biópsia , Cardiomegalia/etiologia , Cardiomegalia/patologia , Feminino , Fibrose , Glicogênio/análise , Átrios do Coração/química , Implante de Prótese de Valva Cardíaca , Humanos , Masculino , Pessoa de Meia-Idade , Estenose da Valva Mitral/etiologia , Estenose da Valva Mitral/metabolismo , Estenose da Valva Mitral/cirurgia , Análise Multivariada , Razão de Chances , Estudos Prospectivos , Cardiopatia Reumática/complicações , Cardiopatia Reumática/metabolismo , Cardiopatia Reumática/cirurgia , Fatores de Risco , Adulto JovemRESUMO
Atrial fibrillation (AF) is associated with increased morbidity and mortality. Atrial-selective potassium (K(+)) channel blockers may represent a novel therapeutic target. The best validated atrial-specific ion currents are the acetylcholine-activated inward-rectifier K(+) current IK,ACh and ultrarapidly activating delayed-rectifier K(+) current IKur. Two-pore domain and small-conductance Ca(2+)-activated K(+) channels and Kv1.1 channels may also contribute to the atrial repolarization. We review the molecular and electrophysiologic characteristics of atrial-selective K(+) channels and their potential pathophysiologic role in AF. We summarize currently available K(+) channel blockers focusing on the most important compounds.