RESUMO
Allosteric modulation is a central mechanism for metabolic regulation but has yet to be described for a gut microbiota-host interaction. Phenylacetylglutamine (PAGln), a gut microbiota-derived metabolite, has previously been clinically associated with and mechanistically linked to cardiovascular disease (CVD) and heart failure (HF). Here, using cells expressing ß1- versus ß2-adrenergic receptors (ß1AR and ß2AR), PAGln is shown to act as a negative allosteric modulator (NAM) of ß2AR, but not ß1AR. In functional studies, PAGln is further shown to promote NAM effects in both isolated male mouse cardiomyocytes and failing human heart left ventricle muscle (contracting trabeculae). Finally, using in silico docking studies coupled with site-directed mutagenesis and functional analyses, we identified sites on ß2AR (residues E122 and V206) that when mutated still confer responsiveness to canonical ß2AR agonists but no longer show PAGln-elicited NAM activity. The present studies reveal the gut microbiota-obligate metabolite PAGln as an endogenous NAM of a host GPCR.
Assuntos
Microbioma Gastrointestinal , Glutamina , Miócitos Cardíacos , Receptores Adrenérgicos beta 2 , Animais , Humanos , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/genética , Regulação Alostérica , Camundongos , Masculino , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Glutamina/metabolismo , Células HEK293 , Simulação de Acoplamento Molecular , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/microbiologia , Mutagênese Sítio-Dirigida , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 1/genética , Camundongos Endogâmicos C57BLRESUMO
The combinatorial effect of genetic variants is often assumed to be additive. Although genetic variation can clearly interact non-additively, methods to uncover epistatic relationships remain in their infancy. We develop low-signal signed iterative random forests to elucidate the complex genetic architecture of cardiac hypertrophy. We derive deep learning-based estimates of left ventricular mass from the cardiac MRI scans of 29,661 individuals enrolled in the UK Biobank. We report epistatic genetic variation including variants close to CCDC141, IGF1R, TTN, and TNKS. Several loci not prioritized by univariate genome-wide association analysis are identified. Functional genomic and integrative enrichment analyses reveal a complex gene regulatory network in which genes mapped from these loci share biological processes and myogenic regulatory factors. Through a network analysis of transcriptomic data from 313 explanted human hearts, we show that these interactions are preserved at the level of the cardiac transcriptome. We assess causality of epistatic effects via RNA silencing of gene-gene interactions in human induced pluripotent stem cell-derived cardiomyocytes. Finally, single-cell morphology analysis using a novel high-throughput microfluidic system shows that cardiomyocyte hypertrophy is non-additively modifiable by specific pairwise interactions between CCDC141 and both TTN and IGF1R. Our results expand the scope of genetic regulation of cardiac structure to epistasis.
RESUMO
A specific genetic variant associated with atrial fibrillation risk, rs17171731, was identified as a regulatory variant responsible for controlling FAM13B expression. The atrial fibrillation risk allele decreases FAM13B expression, whose knockdown alters the expression of many genes in stem cell-derived cardiomyocytes, including SCN2B, and led to pro-arrhythmogenic changes in the late sodium current and Ca2+ cycling. Fam13b knockout mice had increased P-wave and QT interval duration and were more susceptible to pacing-induced arrhythmias vs control mice. FAM13B expression, its regulation, and downstream effects are potential targets for investigation of patient-specific therapeutics.
RESUMO
The combinatorial effect of genetic variants is often assumed to be additive. Although genetic variation can clearly interact non-additively, methods to uncover epistatic relationships remain in their infancy. We develop low-signal signed iterative random forests to elucidate the complex genetic architecture of cardiac hypertrophy. We derive deep learning-based estimates of left ventricular mass from the cardiac MRI scans of 29,661 individuals enrolled in the UK Biobank. We report epistatic genetic variation including variants close to CCDC141, IGF1R, TTN, and TNKS. Several loci not prioritized by univariate genome-wide association analysis are identified. Functional genomic and integrative enrichment analyses reveal a complex gene regulatory network in which genes mapped from these loci share biological processes and myogenic regulatory factors. Through a network analysis of transcriptomic data from 313 explanted human hearts, we show that these interactions are preserved at the level of the cardiac transcriptome. We assess causality of epistatic effects via RNA silencing of gene-gene interactions in human induced pluripotent stem cell-derived cardiomyocytes. Finally, single-cell morphology analysis using a novel high-throughput microfluidic system shows that cardiomyocyte hypertrophy is non-additively modifiable by specific pairwise interactions between CCDC141 and both TTN and IGF1R. Our results expand the scope of genetic regulation of cardiac structure to epistasis.
RESUMO
BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is a growing health problem without effective therapies. Epidemiological studies indicate that diabetes is a strong risk factor for HFpEF, and about 45% of patients with HFpEF are suffering from diabetes, yet the underlying mechanisms remain elusive. METHODS: Using a combination of echocardiography, hemodynamics, RNA-sequencing, molecular biology, in vitro and in vivo approaches, we investigated the roles of SIRT6 (sirtuin 6) in regulation of endothelial fatty acid (FA) transport and HFpEF in diabetes. RESULTS: We first observed that endothelial SIRT6 expression was markedly diminished in cardiac tissues from heart failure patients with diabetes. We then established an experimental mouse model of HFpEF in diabetes induced by a combination of the long-term high-fat diet feeding and a low-dose streptozocin challenge. We also generated a unique humanized SIRT6 transgenic mouse model, in which a single copy of human SIRT6 transgene was engineered at mouse Rosa26 locus and conditionally induced with the Cre-loxP technology. We found that genetically restoring endothelial SIRT6 expression in the diabetic mice ameliorated diastolic dysfunction concurrently with decreased cardiac lipid accumulation. SIRT6 gain- or loss-of-function studies showed that SIRT6 downregulated endothelial FA uptake. Mechanistically, SIRT6 suppressed endothelial expression of PPARγ through SIRT6-dependent deacetylation of histone H3 lysine 9 around PPARγ promoter region; and PPARγ reduction mediated SIRT6-dependent inhibition of endothelial FA uptake. Importantly, oral administration of small molecule SIRT6 activator MDL-800 to diabetic mice mitigated cardiac lipid accumulation and diastolic dysfunction. CONCLUSIONS: The impairment of endothelial SIRT6 expression links diabetes to HFpEF through the alteration of FA transport across the endothelial barrier. Genetic and pharmacological strategies that restored endothelial SIRT6 function in mice with diabetes alleviated experimental HFpEF by limiting FA uptake and improving cardiac metabolism, thus warranting further clinical evaluation.
Assuntos
Diabetes Mellitus Experimental , Insuficiência Cardíaca , Sirtuínas , Humanos , Camundongos , Animais , Volume Sistólico/fisiologia , Insuficiência Cardíaca/metabolismo , PPAR gama , Modelos Animais de Doenças , Sirtuínas/genética , LipídeosRESUMO
BACKGROUND: Altered kinase localization is gaining appreciation as a mechanism of cardiovascular disease. Previous work suggests GSK-3ß (glycogen synthase kinase 3ß) localizes to and regulates contractile function of the myofilament. We aimed to discover GSK-3ß's in vivo role in regulating myofilament function, the mechanisms involved, and the translational relevance. METHODS: Inducible cardiomyocyte-specific GSK-3ß knockout mice and left ventricular myocardium from nonfailing and failing human hearts were studied. RESULTS: Skinned cardiomyocytes from knockout mice failed to exhibit calcium sensitization with stretch indicating a loss of length-dependent activation (LDA), the mechanism underlying the Frank-Starling Law. Titin acts as a length sensor for LDA, and knockout mice had decreased titin stiffness compared with control mice, explaining the lack of LDA. Knockout mice exhibited no changes in titin isoforms, titin phosphorylation, or other thin filament phosphorylation sites known to affect passive tension or LDA. Mass spectrometry identified several z-disc proteins as myofilament phospho-substrates of GSK-3ß. Agreeing with the localization of its targets, GSK-3ß that is phosphorylated at Y216 binds to the z-disc. We showed pY216 was necessary and sufficient for z-disc binding using adenoviruses for wild-type, Y216F, and Y216E GSK-3ß in neonatal rat ventricular cardiomyocytes. One of GSK-3ß's z-disc targets, abLIM-1 (actin-binding LIM protein 1), binds to the z-disc domains of titin that are important for maintaining passive tension. Genetic knockdown of abLIM-1 via siRNA in human engineered heart tissues resulted in enhancement of LDA, indicating abLIM-1 may act as a negative regulator that is modulated by GSK-3ß. Last, GSK-3ß myofilament localization was reduced in left ventricular myocardium from failing human hearts, which correlated with depressed LDA. CONCLUSIONS: We identified a novel mechanism by which GSK-3ß localizes to the myofilament to modulate LDA. Importantly, z-disc GSK-3ß levels were reduced in patients with heart failure, indicating z-disc localized GSK-3ß is a possible therapeutic target to restore the Frank-Starling mechanism in patients with heart failure.
Assuntos
Insuficiência Cardíaca , Miócitos Cardíacos , Animais , Conectina/genética , Conectina/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Humanos , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Fosforilação , RatosRESUMO
The Sry-related high-mobility-group box (SOX) gene family, with 20 known transcription factors in humans, plays an essential role during development and disease processes. Several SOX proteins (SOX4, 11, and 9) are required for normal heart morphogenesis. SOX9 was shown to contribute to cardiac fibrosis. However, differential expression of other SOXs and their roles in the failing human myocardium have not been explored. Here, we used the whole-transcriptome sequencing (RNA-seq), gene co-expression, and meta-analysis to examine whether any SOX factors might play a role in the failing human myocardium. RNA-seq analysis was performed for cardiac tissue samples from heart failure (HF) patients due to dilated cardiomyopathy (DCM), or hypertrophic cardiomyopathy (HCM) and healthy donors (NF). The RNA levels of 20 SOX genes from RNA-seq data were extracted and compared to the 3 groups. Four SOX genes whose RNA levels were significantly upregulated in DCM or HCM compared to NF. However, only SOX4 and SOX8 proteins were markedly increased in the HF groups. A moderate to strong correlation was observed between the RNA level of SOX4/8 and fibrotic genes among each individual. Gene co-expression network analysis identified genes associated and respond similarly to perturbations with SOX4 in cardiac tissues. Using a meta-analysis combining epigenetics and genome-wide association data, we reported several genomic variants associated with HF phenotype linked to SOX4 or SOX8. In summary, our results implicate that SOX4 and SOX8 have a role in cardiomyopathy, leading to HF in humans. The molecular mechanism associated with them in HF warrants further investigation.
Assuntos
Cardiomiopatia Dilatada , Fatores de Transcrição , Cardiomiopatia Dilatada/genética , Estudo de Associação Genômica Ampla , Humanos , RNA , Fatores de Transcrição SOX/genética , Fatores de Transcrição SOX/metabolismo , Fatores de Transcrição SOXC/genética , Fatores de Transcrição SOXE/genética , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Long noncoding RNAs (lncRNAs) have been implicated in the pathogenesis of cardiovascular diseases. We aimed to identify novel lncRNAs associated with the early response to ischemia in the heart. METHODS AND RESULTS: RNA sequencing data gathered from 81 paired left ventricle samples from patients undergoing cardiopulmonary bypass was collected before and after a period of ischemia. Novel lncRNAs were validated with Oxford Nanopore Technologies long-read sequencing. Gene modules associated with an early ischemic response were identified and the subcellular location of selected lncRNAs was determined with RNAscope. A total of 2446 mRNAs, 270 annotated lncRNAs and one novel lncRNA differed in response to ischemia (adjusted p < 0.001, absolute fold change >1.2). The novel lncRNA belonged to a gene module of highly correlated genes that also included 39 annotated lncRNAs. This module associated with ischemia (Pearson correlation coefficient = -0.69, p = 1 × 10-23) and activation of cell death pathways (p < 6 × 10-9). A further nine novel cardiac lncRNAs were identified, of which, one overlapped five cis-eQTL eSNPs for the gene RWD Domain-Containing Sumoylation Enhancer (RWDD3) and was itself correlated with RWDD3 expression (Pearson correlation coefficient -0.2, p = 0.002). CONCLUSION: We have identified 10 novel lncRNAs, one of which was associated with myocardial ischemia and may have potential as a novel therapeutic target or early marker for myocardial dysfunction.
Assuntos
Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , RNA Longo não Codificante/metabolismo , Bases de Dados Genéticas , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Ventrículos do Coração/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Miocárdio/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de RNARESUMO
Left ventricular (LV) heart failure (HF) is a significant and increasing cause of death worldwide. HF is characterized by myocardial remodeling and excessive fibrosis. Transcriptional co-activator Yes-associated protein (Yap), the downstream effector of HIPPO signaling pathway, is an essential factor in cardiomyocyte survival; however, its status in human LV HF is not entirely elucidated. Here, we report that Yap is elevated in LV tissue of patients with HF, and is associated with down-regulation of its upstream inhibitor HIPPO component large tumor suppressor 1 (LATS1) activation as well as upregulation of the fibrosis marker connective tissue growth factor (CTGF). Applying the established profibrotic combined stress of TGFß and hypoxia to human ventricular cardiac fibroblasts in vitro increased Yap protein levels, down-regulated LATS1 activation, increased cell proliferation and collagen I production, and decreased ribosomal protein S6 and S6 kinase phosphorylation, a hallmark of mTOR activation, without any significant effect on mTOR and raptor protein expression or phosphorylation of mTOR or 4E-binding protein 1 (4EBP1), a downstream effector of mTOR pathway. As previously reported in various cell types, TGFß/hypoxia also enhanced cardiac fibroblast Akt and ERK1/2 phosphorylation, which was similar to our observation in LV tissues from HF patients. Further, depletion of Yap reduced TGFß/hypoxia-induced cardiac fibroblast proliferation and Akt phosphorylation at Ser 473 and Thr308, without any significant effect on TGFß/hypoxia-induced ERK1/2 activation or reduction in S6 and S6 kinase activities. Taken together, these data demonstrate that Yap is a mediator that promotes human cardiac fibroblast proliferation and suggest its possible contribution to remodeling of the LV, opening the door to further studies to decipher the cell-specific roles of Yap signaling in human HF.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proliferação de Células , Insuficiência Cardíaca/patologia , Miofibroblastos/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Estudos de Casos e Controles , Células Cultivadas , Feminino , Insuficiência Cardíaca/metabolismo , Humanos , Masculino , Miofibroblastos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição/genética , Ativação Transcricional , Proteínas de Sinalização YAPRESUMO
The association between reduced myofilament force-generating capacity (Fmax) and heart failure (HF) is clear, however the underlying molecular mechanisms are poorly understood. Here, we show impaired Fmax arises from reduced BAG3-mediated sarcomere turnover. Myofilament BAG3 expression decreases in human HF and positively correlates with Fmax. We confirm this relationship using BAG3 haploinsufficient mice, which display reduced Fmax and increased myofilament ubiquitination, suggesting impaired protein turnover. We show cardiac BAG3 operates via chaperone-assisted selective autophagy (CASA), conserved from skeletal muscle, and confirm sarcomeric CASA complex localization is BAG3/proteotoxic stress-dependent. Using mass spectrometry, we characterize the myofilament CASA interactome in the human heart and identify eight clients of BAG3-mediated turnover. To determine if increasing BAG3 expression in HF can restore sarcomere proteostasis/Fmax, HF mice were treated with rAAV9-BAG3. Gene therapy fully rescued Fmax and CASA protein turnover after four weeks. Our findings indicate BAG3-mediated sarcomere turnover is fundamental for myofilament functional maintenance.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Insuficiência Cardíaca/fisiopatologia , Miócitos Cardíacos/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Animais , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Modelos Animais de Doenças , Feminino , Terapia Genética , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/terapia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas Musculares/metabolismo , Contração Miocárdica/genética , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sarcômeros/metabolismoRESUMO
Mutations to the sarcomere-localized cochaperone protein Bcl2-associated athanogene 3 (BAG3) are associated with dilated cardiomyopathy (DCM) and display greater penetrance in male patients. Decreased protein expression of BAG3 is also associated with nongenetic heart failure; however, the factors regulating cardiac BAG3 expression are unknown. Using left ventricular (LV) tissue from nonfailing and DCM human samples, we found that whole LV BAG3 expression was not significantly impacted by DCM or sex; however, myofilament localized BAG3 was significantly decreased in males with DCM. Females with DCM displayed no changes in BAG3 compared with nonfailing. This sex difference appears to be estrogen independent, as estrogen treatment in ovariectomized female rats had no impact on BAG3 expression. BAG3 gene expression in noncardiac cells is primarily regulated by the heat shock transcription factor-1 (HSF-1). We show whole LV HSF-1 expression and nuclear localized/active HSF-1 each displayed a striking positive correlation with whole LV BAG3 expression. We further found that HSF-1 localizes to the sarcomere Z-disc in cardiomyocytes and that this myofilament-associated HSF-1 pool decreases in heart failure. The decrease of HSF-1 was more pronounced in male patients and tightly correlated with myofilament BAG3 expression. Together our findings indicate that cardiac BAG3 expression and myofilament localization are differentially impacted by sex and disease and are linked to HSF-1.NEW & NOTEWORTHY Myofilament BAG3 expression decreases in male patients with nonischemic DCM but is preserved in female patients with DCM. BAG3 expression in the human heart is tightly linked to HSF-1 expression and nuclear translocation. HSF-1 localizes to the sarcomere Z-disc in the human heart. HSF-1 expression in the myofilament fraction decreases in male patients with DCM and positively correlates with myofilament BAG3.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Cardiomiopatia Dilatada/metabolismo , Ventrículos do Coração/metabolismo , Fatores de Transcrição de Choque Térmico/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Sarcômeros/metabolismo , Adulto , Idoso , Animais , Feminino , Expressão Gênica , Humanos , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Miocárdio/patologia , Miócitos Cardíacos/patologia , Miofibrilas/metabolismo , Ovariectomia , Ratos , Sarcômeros/patologia , Fatores SexuaisAssuntos
Cardiomiopatias/patologia , Redes Reguladoras de Genes/genética , Insuficiência Cardíaca/patologia , Ventrículos do Coração/metabolismo , Transcriptoma , Biomarcadores/metabolismo , Cardiomiopatias/complicações , Cardiomiopatias/genética , Regulação para Baixo , Perfilação da Expressão Gênica/métodos , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/genética , Humanos , Miocárdio/metabolismo , Fenótipo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , Transdução de Sinais/genética , Regulação para CimaRESUMO
BACKGROUND: Ventricular tachyarrhythmias and sudden cardiac death show a circadian pattern of occurrence in patients with heart failure. In the rodent ventricle, a significant portion of genes, including some ion channels, shows a circadian pattern of expression. However, genes that define electrophysiological properties in failing human heart ventricles have not been examined for a circadian expression pattern. METHODS: Ventricular tissue samples were collected from patients at the time of cardiac transplantation. Two sets of samples (n=37 and 46, one set with a greater arrhythmic history) were selected to generate pseudo-time series according to their collection time. A third set (n=27) of samples was acquired from the nonfailing ventricles of brain-dead donors. The expression of 5 known circadian clock genes and 19 additional ion channel genes plausibly important to electrophysiological properties were analyzed by real-time polymerase chain reaction and then analyzed for the percentage of expression variation attributed to a 24-hour circadian pattern. RESULTS: The 5 known circadian clock gene transcripts showed a strong circadian expression pattern. Compared with rodent hearts, the human circadian clock gene transcripts showed a similar temporal order of acrophases but with a ≈7.6 hours phase shift. Five of the ion channel genes also showed strong circadian expression. Comparable studies of circadian clock gene expression in samples recovered from nonheart failure brain-dead donors showed acrophase shifts, or weak or complete loss of circadian rhythmicity, suggesting alterations in circadian gene expression. CONCLUSIONS: Ventricular tissue from failing human hearts display a circadian pattern of circadian clock gene expression but phase-shifted relative to rodent hearts. At least 5 ion channels show a circadian expression pattern in the ventricles of failing human hearts, which may underlie a circadian pattern of ventricular tachyarrhythmia/sudden cardiac death. Nonfailing hearts from brain-dead donors show marked differences in circadian clock gene expression patterns, suggesting fundamental deviations from circadian expression.
Assuntos
Ritmo Circadiano , Expressão Gênica , Insuficiência Cardíaca/genética , Miocárdio/metabolismo , Adulto , Feminino , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Humanos , Canais Iônicos/biossíntese , Canais Iônicos/genética , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: The mesenchymal stem cell (MSC), known to remodel in disease and have an extensive secretome, has recently been isolated from the human heart. However, the effects of normal and diseased cardiac MSCs on myocyte electrophysiology remain unclear. We hypothesize that in disease the inflammatory secretome of cardiac human MSCs (hMSCs) remodels and can regulate arrhythmia substrates. METHODS: hMSCs were isolated from patients with or without heart failure from tissue attached to extracted device leads and from samples taken from explanted/donor hearts. Failing hMSCs or nonfailing hMSCs were cocultured with normal human cardiac myocytes derived from induced pluripotent stem cells. Using fluorescent indicators, action potential duration, Ca2+ alternans, and spontaneous calcium release (SCR) incidence were determined. RESULTS: Failing and nonfailing hMSCs from both sources exhibited similar trilineage differentiation potential and cell surface marker expression as bone marrow hMSCs. Compared with nonfailing hMSCs, failing hMSCs prolonged action potential duration by 24% (P<0.001, n=15), increased Ca2+ alternans by 300% (P<0.001, n=18), and promoted spontaneous calcium release activity (n=14, P<0.013) in human cardiac myocytes derived from induced pluripotent stem cells. Failing hMSCs exhibited increased secretion of inflammatory cytokines IL (interleukin)-1ß (98%, P<0.0001) and IL-6 (460%, P<0.02) compared with nonfailing hMSCs. IL-1ß or IL-6 in the absence of hMSCs prolonged action potential duration but only IL-6 increased Ca2+ alternans and promoted spontaneous calcium release activity in human cardiac myocytes derived from induced pluripotent stem cells, replicating the effects of failing hMSCs. In contrast, nonfailing hMSCs prevented Ca2+ alternans in human cardiac myocytes derived from induced pluripotent stem cells during oxidative stress. Finally, nonfailing hMSCs exhibited >25× higher secretion of IGF (insulin-like growth factor)-1 compared with failing hMSCs. Importantly, IGF-1 supplementation or anti-IL-6 treatment rescued the arrhythmia substrates induced by failing hMSCs. CONCLUSIONS: We identified device leads as a novel source of cardiac hMSCs. Our findings show that cardiac hMSCs can regulate arrhythmia substrates by remodeling their secretome in disease. Importantly, therapy inhibiting (anti-IL-6) or mimicking (IGF-1) the cardiac hMSC secretome can rescue arrhythmia substrates.
Assuntos
Potenciais de Ação , Arritmias Cardíacas/metabolismo , Sinalização do Cálcio , Insuficiência Cardíaca/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mediadores da Inflamação/metabolismo , Células-Tronco Mesenquimais/metabolismo , Miócitos Cardíacos/metabolismo , Comunicação Parácrina , Adulto , Idoso , Arritmias Cardíacas/patologia , Arritmias Cardíacas/fisiopatologia , Estudos de Casos e Controles , Linhagem da Célula , Células Cultivadas , Técnicas de Cocultura , Feminino , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Cinética , Masculino , Células-Tronco Mesenquimais/patologia , Pessoa de Meia-Idade , Miócitos Cardíacos/patologia , FenótipoRESUMO
BACKGROUND: Acetylation and methylation of histones alter the chromatin structure and accessibility that affect transcriptional regulators binding to enhancers and promoters. The binding of transcriptional regulators enables the interaction between enhancers and promoters, thus affecting gene expression. However, our knowledge of these epigenetic alternations in patients with heart failure remains limited. METHODS AND RESULTS: From the comprehensive analysis of major histone modifications, 3-dimensional chromatin interactions, and transcriptome in left ventricular (LV) tissues from dilated cardiomyopathy (DCM) patients and non-heart failure (NF) donors, differential active enhancer and promoter regions were identified between NF and DCM. Moreover, the genome-wide average promoter signal is significantly lower in DCM than in NF. Super-enhancer (SE) analysis revealed that fewer SEs were found in DCM LVs than in NF ones, and three unique SE-associated genes between NF and DCM were identified. Moreover, SEs are enriched within the genomic region associated with long-range chromatin interactions. The differential enhancer-promoter interactions were observed in the known heart failure gene loci and are correlated with the gene expression levels. Motif analysis identified known cardiac factors and possible novel players for DCM. CONCLUSIONS: We have established the cistrome of four histone modifications and chromatin interactome for enhancers and promoters in NF and DCM tissues. Differential histone modifications and enhancer-promoter interactions were found in DCM, which were associated with gene expression levels of a subset of disease-associated genes in human heart failure.
Assuntos
Cardiomiopatia Dilatada/genética , Cromatina/metabolismo , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/metabolismo , Sequência de Bases , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Epigênese Genética , Ontologia Genética , Genoma Humano , Insuficiência Cardíaca/genética , Ventrículos do Coração/patologia , Humanos , Lisina/metabolismo , Masculino , Metilação , Proteínas Musculares/metabolismo , Motivos de Nucleotídeos/genética , Regiões Promotoras GenéticasRESUMO
Heart failure is a leading cause of mortality, yet our understanding of the genetic interactions underlying this disease remains incomplete. Here, we harvest 1352 healthy and failing human hearts directly from transplant center operating rooms, and obtain genome-wide genotyping and gene expression measurements for a subset of 313. We build failing and non-failing cardiac regulatory gene networks, revealing important regulators and cardiac expression quantitative trait loci (eQTLs). PPP1R3A emerges as a regulator whose network connectivity changes significantly between health and disease. RNA sequencing after PPP1R3A knockdown validates network-based predictions, and highlights metabolic pathway regulation associated with increased cardiomyocyte size and perturbed respiratory metabolism. Mice lacking PPP1R3A are protected against pressure-overload heart failure. We present a global gene interaction map of the human heart failure transition, identify previously unreported cardiac eQTLs, and demonstrate the discovery potential of disease-specific networks through the description of PPP1R3A as a central regulator in heart failure.
Assuntos
Redes Reguladoras de Genes/genética , Insuficiência Cardíaca/genética , Miócitos Cardíacos/patologia , Fosfoproteínas Fosfatases/metabolismo , Animais , Benzenoacetamidas , Células Cultivadas , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Estudo de Associação Genômica Ampla , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Humanos , Masculino , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Fosfoproteínas Fosfatases/genética , Cultura Primária de Células , Piridinas , Locos de Características Quantitativas/genética , Ratos , Ratos Sprague-Dawley , Análise de Sequência de RNA/métodosRESUMO
BACKGROUND: Limited knowledge exists of the extent of epigenetic alterations, such as DNA methylation, in heart failure (HF). We conducted targeted DNA methylation sequencing to identify DNA methylation alterations in coding and noncoding RNA (ncRNA) across different etiological subtypes of HF. METHODS AND RESULTS: A targeted bisulfite sequence capture sequencing platform was applied to DNA extracted from cardiac interventricular septal tissue of 30 male HF patients encompassing causes including hypertrophic obstructive cardiomyopathy, ischemic cardiomyopathy, dilated cardiomyopathy, and 9 control patients with nonfailing hearts. We detected 62 678 differentially methylated regions in the studied HF cohort. By comparing each HF subgroup to the nonfailing control group, we identified 195 unique differentially methylated regions: 5 in hypertrophic obstructive cardiomyopathy, 151 in dilated cardiomyopathy, and 55 in ischemic cardiomyopathy. These translated to 4 genes/1 ncRNA in hypertrophic obstructive cardiomyopathy, 131 genes/17 ncRNA in dilated cardiomyopathy, and 51 genes/5 ncRNA in ischemic cardiomyopathy. Subsequent gene/ncRNA expression analysis was assessed using quantitative reverse transcription polymerase chain reaction and revealed 6 genes: 4 hypermethylated ( HEY2, MSR1, MYOM3, and COX17), 2 hypomethylated ( CTGF and MMP2); and 2 microRNA: 1 hypermethylated (miR-24-1), 1 hypomethylated (miR-155) with significantly upregulated or downregulated expression levels consistent with the direction of methylation in the particular HF subgroup. CONCLUSIONS: For the first time DNA methylation alterations and associated gene expression changes were identified in etiologically variant pathological HF tissue. The methylation-sensitive and disease-associated genes/ncRNA identified from this study represent a unique cohort of loci that demonstrate a plausible potential as a novel diagnostic and therapeutic target in HF and warrant further investigation.
Assuntos
Metilação de DNA/genética , Epigênese Genética/genética , Insuficiência Cardíaca/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Coração/fisiopatologia , Humanos , Masculino , FenótipoRESUMO
Exercise and heart disease both induce cardiac remodeling, but only disease causes fibrosis and compromises heart function. The cardioprotective benefits of exercise have been attributed to changes in cardiomyocyte physiology, but the impact of exercise on cardiac fibroblasts (CFs) is unknown. Here, RNA-sequencing reveals rapid divergence of CF transcriptional programs during exercise and disease. Among the differentially expressed programs, NRF2-dependent antioxidant genes - including metallothioneins (Mt1 and Mt2) - are induced in CFs during exercise and suppressed by TGF-ß/p38 signaling in disease. In vivo, mice lacking Mt1/2 exhibit signs of cardiac dysfunction in exercise, including cardiac fibrosis, vascular rarefaction, and functional decline. Mechanistically, exogenous MTs derived from fibroblasts are taken up by cultured cardiomyocytes, reducing oxidative damage-dependent cell death. Importantly, suppression of MT expression is conserved in human heart failure. Taken together, this study defines the acute transcriptional response of CFs to exercise and disease and reveals a cardioprotective mechanism that is lost in disease.
RESUMO
Patients with diabetes are at significantly higher risk of developing heart failure. Increases in advanced glycation end products are a proposed pathophysiological link, but their impact and mechanism remain incompletely understood. Methylglyoxal (MG) is a glycolysis byproduct, elevated in diabetes, and modifies arginine and lysine residues. We show that left ventricular myofilament from patients with diabetes and heart failure (dbHF) exhibited increased MG modifications compared with nonfailing controls (NF) or heart failure patients without diabetes. In skinned NF human and mouse cardiomyocytes, acute MG treatment depressed both calcium sensitivity and maximal calcium-activated force in a dose-dependent manner. Importantly, dbHF myocytes were resistant to myofilament functional changes from MG treatment, indicating that myofilaments from dbHF patients already had depressed function arising from MG modifications. In human dbHF and MG-treated mice, mass spectrometry identified increased MG modifications on actin and myosin. Cosedimentation and in vitro motility assays indicate that MG modifications on actin and myosin independently depress calcium sensitivity, and mechanistically, the functional consequence requires actin/myosin interaction with thin-filament regulatory proteins. MG modification of the myofilament may represent a critical mechanism by which diabetes induces heart failure, as well as a therapeutic target to avoid the development of or ameliorate heart failure in these patients.
Assuntos
Diabetes Mellitus Tipo 2/complicações , Insuficiência Cardíaca/patologia , Ventrículos do Coração/fisiopatologia , Aldeído Pirúvico/metabolismo , Sarcômeros/patologia , Actinas/metabolismo , Adulto , Animais , Arginina/metabolismo , Cardiomiopatia Dilatada/patologia , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Feminino , Glicólise , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/citologia , Ventrículos do Coração/patologia , Humanos , Lisina/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Miosinas/metabolismo , Aldeído Pirúvico/administração & dosagem , Sarcômeros/metabolismo , Sarcômeros/fisiologia , Análise de Célula ÚnicaRESUMO
Biofeedback has been shown to have some level of efficacy for the treatment of a number of chronic medical conditions; however, individualized biofeedback treatment is not always feasible. While group- based interventions are growing in practice due to numerous advantages, the dearth of research examining the efficacy of Group Biofeedback (GBF) suggests that this treatment modality may not be commonly utilized. Thus, the current paper highlights some advantages and constructively addresses potential challenges of utilizing GBF. Obstacles specific to GBF include equipment for participants, need for support staffing, and billing. However, the potential benefits are numerous, and pertain to cost-effectiveness, improved patient access, and additive benefits specific to group-based treatment. We offer a six-session GBF protocol to be used to guide future clinical work in this area. We hope that through the ideas and protocol presented in this paper, biofeedback practitioners will be more inclined to implement GBF.