RESUMO
Previous studies have established that transmural gradients of the fast transient outward K+ current (Ito,f) correlate with regional differences in action potential (AP) profile and excitation-contraction coupling (ECC) with high Ito,f expression in the epimyocardium (EPI) being associated with short APs and low contractility and vice versa. Herein, we investigated the effects of altering the Ito,f gradients on transmural contractile properties using mice lacking Irx5 (Irx5-KO) or lacking Kcnd2 (KV4.2-KO) or both. Irx5-KO mice exhibited decreased global LV contractility in association with elevated Ito,f, as well as reduced cell shortening and Ca2+ transient amplitudes in cardiomyocytes isolated from the endomyocardium (ENDO) but not in cardiomyocytes from the EPI. Transcriptional profiling revealed that the primary effect of Irx5 ablation on ECC-related genes was to increase Ito,f gene expression (i.e., Kcnd2 and Kcnip2) in the ENDO, but not the EPI. By contrast, KV4.2-KO mice showed selective increases in cell shortening and Ca2+ transients in isolated EPI cardiomyocytes, leading to enhanced ventricular contractility and mice lacking both Irx5 and Kcnd2 displayed elevated ventricular contractility, comparable to KV4.2-KO mice, demonstrating a dominant role of Irx5-dependent modulation of Ito,f in the regulation of contractility. Our findings show that the transmural electromechanical heterogeneities in the healthy ventricles depend on the Irx5-dependent Ito,f gradients. These observations provide a useful framework for assessing the molecular mechanisms underlying the alterations in contractile heterogeneity seen in the diseased heart.NEW & NOTEWORTHY Irx5 is a vital transcription factor that establishes the transmural heterogeneity of ventricular myocyte contractility, thereby ensuring proper contractile function in the healthy heart. Regional differences in excitation-contraction coupling in the ventricular myocardium are primarily mediated through the inverse relationship between Irx5 and the fast transient outward K+ current (Ito,f) across the ventricular wall.
Assuntos
Ventrículos do Coração , Miocárdio , Potenciais de Ação/fisiologia , Animais , Ventrículos do Coração/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Potássio Shal/genética , Canais de Potássio Shal/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Ischemic heart disease is the leading cause of death in the United States, Canada, and worldwide. Severe disease is characterized by coronary artery occlusion, loss of blood flow to the myocardium, and necrosis of tissue, with subsequent remodeling of the heart wall, including fibrotic scarring. The current study aims to demonstrate the efficacy of quantitating infarct size via two-dimensional (2-D) echocardiographic akinetic length and four-dimensional (4-D) echocardiographic infarct volume and surface area as in vivo analysis techniques. We further describe and evaluate a new surface area strain analysis technique for estimating myocardial infarction (MI) size after ischemic injury. Experimental MI was induced in mice via left coronary artery ligation. Ejection fraction and infarct size were measured through 2-D and 4-D echocardiography. Infarct size established via histology was compared with ultrasound-based metrics via linear regression analysis. Two-dimensional echocardiographic akinetic length (r = 0.76, P = 0.03), 4-D echocardiographic infarct volume (r = 0.85, P = 0.008), and surface area (r = 0.90, P = 0.002) correlate well with histology. Although both 2-D and 4-D echocardiography were reliable measurement techniques to assess infarct, 4-D analysis is superior in assessing asymmetry of the left ventricle and the infarct. Strain analysis performed on 4-D data also provides additional infarct sizing techniques, which correlate with histology (surface strain: r = 0.94, P < 0.001, transmural thickness: r = 0.76, P = 0.001). Two-dimensional echocardiographic akinetic length, 4-D echocardiography ultrasound, and strain provide effective in vivo methods for measuring fibrotic scarring after MI.NEW & NOTEWORTHY Our study supports that both 2-D and 4-D echocardiographic analysis techniques are reliable in quantifying infarct size though 4-D ultrasound provides a more holistic image of LV function and structure, especially after myocardial infarction. Furthermore, 4-D strain analysis correctly identifies infarct size and regional LV dysfunction after MI. Therefore, these techniques can improve functional insight into the impact of pharmacological interventions on the pathophysiology of cardiac disease.
Assuntos
Infarto do Miocárdio/diagnóstico por imagem , Ultrassonografia/métodos , Algoritmos , Animais , Débito Cardíaco , Feminino , Ventrículos do Coração/diagnóstico por imagem , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Imageamento Tridimensional/métodos , Imageamento Tridimensional/normas , Masculino , Camundongos , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Sensibilidade e Especificidade , Ultrassonografia/normasRESUMO
OBJECTIVE: Obesity, a leading cause of several metabolic abnormalities, is mainly caused by imbalanced energy homeostasis. IRX3 and IRX5 have been suggested as genetic determinants of obesity in connection with the intronic variants of the FTO gene, the strongest genetic risk factor of polygenic obesity in humans. Although the causal effects of Irx3 and its cooperation with Irx5 in obesity and associated metabolic abnormalities have been demonstrated in vivo, the function of Irx5 in energy homeostasis remains unclear. Here we aim to decipher the actions of Irx5 in the regulation of obesity and metabolic abnormalities. METHODS: We employed a mouse model homozygous for an Irx5-knockout (Irx5KO) allele and determined its metabolic phenotype in the presence or absence of a high-fat diet challenge. To investigate the function of Irx5 in the regulation of energy homeostasis, adipose thermogenesis and hypothalamic leptin response were assessed, and single-cell RNA sequencing (scRNA-seq) in the hypothalamic arcuate-median eminence (ARC-ME) was conducted. RESULTS: Irx5KO mice were leaner and resistant to diet-induced obesity as well as associated metabolic abnormalities, primarily through loss of adiposity. Assessments of energy expenditure and long-term dietary intake revealed that an increase in basal metabolic rate with adipose thermogenesis and a reduction of food intake with improved hypothalamic leptin response in Irx5KO mice may contribute to the anti-obesity effects. Utilizing scRNA-seq and marker gene analyses, we demonstrated the number of ARC-ME neurons was elevated in Irx5KO mice, suggesting a direct role for Irx5 in hypothalamic feeding control. CONCLUSIONS: Our study demonstrates that Irx5 is a genetic factor determining body mass/composition and obesity and regulates both energy expenditure and intake.
Assuntos
Leptina , Obesidade , Humanos , Animais , Camundongos , Leptina/metabolismo , Obesidade/genética , Obesidade/metabolismo , Dieta Hiperlipídica , Hipotálamo/metabolismo , Metabolismo Energético/genética , Camundongos Knockout , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismoRESUMO
BACKGROUND: Cardiac hypertrophy is a key biological response to injurious stresses such as pressure overload and, when excessive, can lead to heart failure. Innate immune activation by danger signals, through intracellular pattern recognition receptors such as nucleotide-binding oligomerization domain 1 (Nod1) and its adaptor receptor-interacting protein 2 (RIP2), might play a major role in cardiac remodeling and progression to heart failure. We hypothesize that Nod1/RIP2 are major contributors to cardiac hypertrophy, but may not be sufficient to fully express the phenotype alone. METHODS: To elucidate the contribution of Nod1/RIP2 signaling to cardiac hypertrophy, we randomized Nod1-/-, RIP2-/-, or wild-type mice to transverse aortic constriction or sham operations. Cardiac hypertrophy, fibrosis, and cardiac function were examined in these mice. RESULTS: Nod1 and RIP2 proteins were upregulated in the heart after transverse aortic constriction, and this was paralleled by increased expression of mitochondrial proteins, including mitochondrial antiviral signaling protein (MAVS). Nod1-/- and RIP2-/- mice subjected to transverse aortic constriction exhibited better survival, improved cardiac function, and decreased cardiac hypertrophy. Downstream signal transduction pathways that regulate inflammation and fibrosis, including NF (nuclear factor) κB and MAPK (mitogen-activated protein kinase)-GATA4/p300, were reduced in both Nod1-/- and RIP2-/- mice after transverse aortic constriction compared with wild-type mice. Coimmunoprecipitation of extracted cardiac proteins and confocal immunofluorescence microscopy showed that Nod1/RIP2 interaction was robust and that this complex also included MAVS as an essential component. Suppression of MAVS expression attenuated the complex formation, NF κB signaling, and myocyte hypertrophy. Interrogation of mitochondrial function compared in the presence or ablation of MAVS revealed that MAVS serves to suppress mitochondrial energy output and mediate fission/fusion related dynamic changes. The latter is possibly linked to mitophagy during cardiomyocytes stress, which may provide an intriguing link between innate immune activation and mitochondrial energy balance under stress or injury conditions. CONCLUSIONS: We have identified that innate immune Nod1/RIP2 signaling is a major contributor to cardiac remodeling after stress. This process is critically joined by and regulated through the mitochondrial danger signal adapter MAVS. This novel complex coordinates remodeling, inflammatory response, and mitochondrial energy metabolism in stressed cardiomyocytes. Thus, Nod1/RIP2/MAVS signaling complex may represent an attractive new therapeutic approach toward heart failure.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Cardiomegalia/imunologia , Metabolismo Energético/fisiologia , Imunidade Inata/fisiologia , Proteína Adaptadora de Sinalização NOD1/imunologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Recém-Nascidos , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/imunologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D). Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes. However, no direct connection between the obesity-associated variants and FTO expression or function has been made. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition.
Assuntos
Proteínas de Homeodomínio/genética , Íntrons/genética , Oxigenases de Função Mista/genética , Obesidade/genética , Oxo-Ácido-Liases/genética , Proteínas/genética , Fatores de Transcrição/genética , Tecido Adiposo/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Animais , Metabolismo Basal/genética , Índice de Massa Corporal , Peso Corporal/genética , Encéfalo/metabolismo , Diabetes Mellitus Tipo 2/genética , Dieta , Genes Dominantes/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Hipotálamo/metabolismo , Masculino , Camundongos , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Magreza/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
BACKGROUND: Genomewide association studies can be used to identify disease-relevant genomic regions, but interpretation of the data is challenging. The FTO region harbors the strongest genetic association with obesity, yet the mechanistic basis of this association remains elusive. METHODS: We examined epigenomic data, allelic activity, motif conservation, regulator expression, and gene coexpression patterns, with the aim of dissecting the regulatory circuitry and mechanistic basis of the association between the FTO region and obesity. We validated our predictions with the use of directed perturbations in samples from patients and from mice and with endogenous CRISPR-Cas9 genome editing in samples from patients. RESULTS: Our data indicate that the FTO allele associated with obesity represses mitochondrial thermogenesis in adipocyte precursor cells in a tissue-autonomous manner. The rs1421085 T-to-C single-nucleotide variant disrupts a conserved motif for the ARID5B repressor, which leads to derepression of a potent preadipocyte enhancer and a doubling of IRX3 and IRX5 expression during early adipocyte differentiation. This results in a cell-autonomous developmental shift from energy-dissipating beige (brite) adipocytes to energy-storing white adipocytes, with a reduction in mitochondrial thermogenesis by a factor of 5, as well as an increase in lipid storage. Inhibition of Irx3 in adipose tissue in mice reduced body weight and increased energy dissipation without a change in physical activity or appetite. Knockdown of IRX3 or IRX5 in primary adipocytes from participants with the risk allele restored thermogenesis, increasing it by a factor of 7, and overexpression of these genes had the opposite effect in adipocytes from nonrisk-allele carriers. Repair of the ARID5B motif by CRISPR-Cas9 editing of rs1421085 in primary adipocytes from a patient with the risk allele restored IRX3 and IRX5 repression, activated browning expression programs, and restored thermogenesis, increasing it by a factor of 7. CONCLUSIONS: Our results point to a pathway for adipocyte thermogenesis regulation involving ARID5B, rs1421085, IRX3, and IRX5, which, when manipulated, had pronounced pro-obesity and anti-obesity effects. (Funded by the German Research Center for Environmental Health and others.).
Assuntos
Adipócitos/metabolismo , Obesidade/genética , Proteínas/genética , Termogênese/genética , Alelos , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Animais , Sequência de Bases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Epigenômica , Expressão Gênica , Engenharia Genética , Humanos , Camundongos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Obesidade/metabolismo , Fenótipo , Edição de RNA , Risco , Termogênese/fisiologiaRESUMO
The Iroquois homeobox (Irx) homeodomain transcription factors are important for several aspects of embryonic development. In the developing heart, individual Irx genes are important for certain postnatal cardiac functions, including cardiac repolarization (Irx5) and rapid ventricular conduction (Irx3). Irx genes are expressed in dynamic and partially overlapping patterns in the developing heart. Here we show in mice that Irx3 and Irx5 have redundant function in the endocardium to regulate atrioventricular canal morphogenesis and outflow tract formation. Our data suggest that direct transcriptional repression of Bmp10 by Irx3 and Irx5 in the endocardium is required for ventricular septation. A postnatal deletion of Irx3 and Irx5 in the myocardium leads to prolongation of atrioventricular conduction, due in part to activation of expression of the Na(+) channel protein Nav1.5. Surprisingly, combined postnatal loss of Irx3 and Irx5 results in a restoration of the repolarization gradient that is altered in Irx5 mutant hearts, suggesting that postnatal Irx3 activity can be repressed by Irx5. Our results have uncovered complex genetic interactions between Irx3 and Irx5 in embryonic cardiac development and postnatal physiology.
Assuntos
Coração/embriologia , Coração/fisiologia , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Imunoprecipitação da Cromatina , Eletrofisiologia , Feminino , Ventrículos do Coração/embriologia , Ventrículos do Coração/metabolismo , Proteínas de Homeodomínio/genética , Imunoprecipitação , Camundongos , Gravidez , Fatores de Transcrição/genéticaRESUMO
Numerous cardiac transcription factors play overlapping roles in both the specification and proliferation of the cardiac tissues and chambers during heart development. It has become increasingly apparent that cardiac transcription factors also play critical roles in the regulation of expression of many functional genes in the prenatal and postnatal hearts. Accordingly, mutations of cardiac transcription factors cannot only result in congenital heart defects but also alter heart function thereby predisposing to heart disease and cardiac arrhythmias. In this review, we summarize the roles of Iroquois homeobox (Irx) family of transcription factors in heart development and function. In all, 6 Irx genes are expressed with distinct and overlapping patterns in the mammalian heart. Studies in several animal models demonstrate that Irx genes are important for the establishment of ventricular chamber properties, the ventricular conduction system, as well as heterogeneity of the ventricular repolarization. The molecular mechanisms by which Irx proteins regulate gene expression and the clinical relevance of Irx functions in the heart are discussed.
Assuntos
Coração/crescimento & desenvolvimento , Proteínas de Homeodomínio/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Coração/fisiopatologia , Sistema de Condução Cardíaco/crescimento & desenvolvimento , Sistema de Condução Cardíaco/metabolismo , Cardiopatias/genética , Cardiopatias/metabolismo , Cardiopatias/fisiopatologia , Ventrículos do Coração/crescimento & desenvolvimento , Ventrículos do Coração/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Fatores de Transcrição/genéticaRESUMO
Rapid electrical conduction in the His-Purkinje system tightly controls spatiotemporal activation of the ventricles. Although recent work has shed much light on the regulation of early specification and morphogenesis of the His-Purkinje system, less is known about how transcriptional regulation establishes impulse conduction properties of the constituent cells. Here we show that Iroquois homeobox gene 3 (Irx3) is critical for efficient conduction in this specialized tissue by antithetically regulating two gap junction-forming connexins (Cxs). Loss of Irx3 resulted in disruption of the rapid coordinated spread of ventricular excitation, reduced levels of Cx40, and ectopic Cx43 expression in the proximal bundle branches. Irx3 directly represses Cx43 transcription and indirectly activates Cx40 transcription. Our results reveal a critical role for Irx3 in the precise regulation of intercellular gap junction coupling and impulse propagation in the heart.
Assuntos
Fascículo Atrioventricular/fisiologia , Sistema de Condução Cardíaco , Proteínas de Homeodomínio/fisiologia , Ramos Subendocárdicos/fisiologia , Fatores de Transcrição/fisiologia , Animais , Conexina 43/genética , Conexinas/genética , Junções Comunicantes , Regulação da Expressão Gênica , Genes Homeobox , Ventrículos do Coração , Camundongos , Transcrição GênicaRESUMO
The cardiac conduction system (CCS) is a network of specialized cardiomyocytes that coordinates electrical impulse generation and propagation for synchronized heart contractions. Although the components of the CCS, including the sinoatrial node, atrioventricular node, His bundle, bundle branches, and Purkinje fibers, were anatomically discovered more than 100 years ago, their molecular constituents and regulatory mechanisms remain incompletely understood. Here, we demonstrate the transcriptomic landscape of the postnatal mouse CCS at a single-cell resolution with spatial information. Integration of single-cell and spatial transcriptomics uncover region-specific markers and zonation patterns of expression. Network inference shows heterogeneous gene regulatory networks across the CCS. Notably, region-specific gene regulation is recapitulated in vitro using neonatal mouse atrial and ventricular myocytes overexpressing CCS-specific transcription factors, Tbx3 and/or Irx3. This finding is supported by ATAC-seq of different CCS regions, Tbx3 ChIP-seq, and Irx motifs. Overall, this study provides comprehensive molecular profiles of the postnatal CCS and elucidates gene regulatory mechanisms contributing to its heterogeneity.
Assuntos
Sistema de Condução Cardíaco , Proteínas de Homeodomínio , Miócitos Cardíacos , Proteínas com Domínio T , Animais , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Sistema de Condução Cardíaco/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Redes Reguladoras de Genes , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Regulação da Expressão Gênica , Animais Recém-Nascidos , Análise de Célula Única , Transcriptoma , Ramos Subendocárdicos/metabolismo , Ramos Subendocárdicos/fisiologia , Nó Atrioventricular/metabolismo , Nó Sinoatrial/metabolismo , Fascículo Atrioventricular/metabolismoRESUMO
Cardiac-specific overexpression of a constitutively active form of calcineurin A (CNA) leads directly to cardiac hypertrophy in the CNA mouse model. Because cardiac hypertrophy is a prominent characteristic of many cardiomyopathies, we deduced that delineating the proteomic profile of ventricular tissue from this model might identify novel, widely applicable therapeutic targets. Proteomic analysis was carried out by subjecting fractionated cardiac samples from CNA mice and their WT littermates to gel-free liquid chromatography linked to shotgun tandem mass spectrometry. We identified 1,918 proteins with high confidence, of which 290 were differentially expressed. Microarray analysis of the same tissue provided us with alterations in the ventricular transcriptome. Because bioinformatic analyses of both the proteome and transcriptome demonstrated the up-regulation of endoplasmic reticulum stress, we validated its occurrence in adult CNA hearts through a series of immunoblots and RT-PCR analyses. Endoplasmic reticulum stress often leads to increased apoptosis, but apoptosis was minimal in CNA hearts, suggesting that activated calcineurin might protect against apoptosis. Indeed, the viability of cultured neonatal mouse cardiomyocytes (NCMs) from CNA mice was higher than WT after serum starvation, an apoptotic trigger. Proteomic data identified α-crystallin B (Cryab) as a potential mediator of this protective effect and we showed that silencing of Cryab via lentivector-mediated transduction of shRNAs in NCMs led to a significant reduction in NCM viability and loss of protection against apoptosis. The identification of Cryab as a downstream effector of calcineurin-induced protection against apoptosis will permit elucidation of its role in cardiac apoptosis and its potential as a therapeutic target.
Assuntos
Calcineurina/metabolismo , Retículo Endoplasmático/metabolismo , Miocárdio/metabolismo , Cadeia B de alfa-Cristalina/metabolismo , Animais , Apoptose/fisiologia , Calcineurina/genética , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Expressão Gênica , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Transgênicos , Miocárdio/citologia , Análise Serial de Proteínas , Proteômica , RNA Interferente Pequeno/genética , Estresse Fisiológico , Cadeia B de alfa-Cristalina/antagonistas & inibidores , Cadeia B de alfa-Cristalina/genéticaRESUMO
Cardiac metabolism is deranged in heart failure, but underlying mechanisms remain unclear. Here, we show that lysine demethylase 8 (Kdm8) maintains an active mitochondrial gene network by repressing Tbx15, thus preventing dilated cardiomyopathy leading to lethal heart failure. Deletion of Kdm8 in mouse cardiomyocytes increased H3K36me2 with activation of Tbx15 and repression of target genes in the NAD+ pathway before dilated cardiomyopathy initiated. NAD+ supplementation prevented dilated cardiomyopathy in Kdm8 mutant mice, and TBX15 overexpression blunted NAD+-activated cardiomyocyte respiration. Furthermore, KDM8 was downregulated in human hearts affected by dilated cardiomyopathy, and higher TBX15 expression defines a subgroup of affected hearts with the strongest downregulation of genes encoding mitochondrial proteins. Thus, KDM8 represses TBX15 to maintain cardiac metabolism. Our results suggest that epigenetic dysregulation of metabolic gene networks initiates myocardium deterioration toward heart failure and could underlie heterogeneity of dilated cardiomyopathy.
RESUMO
Elevated circulating dipeptidyl peptidase-4 (DPP4) is a biomarker for liver disease, but its involvement in gluconeogenesis and metabolic associated fatty liver disease progression remains unclear. Here, we identified that DPP4 in hepatocytes but not TEK receptor tyrosine kinase-positive endothelial cells regulates the local bioactivity of incretin hormones and gluconeogenesis. However, the complete absence of DPP4 (Dpp4-/-) in aged mice with metabolic syndrome accelerates liver fibrosis without altering dyslipidemia and steatosis. Analysis of transcripts from the livers of Dpp4-/- mice displayed enrichment for inflammasome, p53, and senescence programs compared with littermate controls. High-fat, high-cholesterol feeding decreased Dpp4 expression in F4/80+ cells, with only minor changes in immune signaling. Moreover, in a lean mouse model of severe nonalcoholic fatty liver disease, phosphatidylethanolamine N-methyltransferase mice, we observed a 4-fold increase in circulating DPP4, in contrast with previous findings connecting DPP4 release and obesity. Last, we evaluated DPP4 levels in patients with hepatitis C infection with dysglycemia (Homeostatic Model Assessment of Insulin Resistance > 2) who underwent direct antiviral treatment (with/without ribavirin). DPP4 protein levels decreased with viral clearance; DPP4 activity levels were reduced at long-term follow-up in ribavirin-treated patients; but metabolic factors did not improve. These data suggest elevations in DPP4 during hepatitis C infection are not primarily regulated by metabolic disturbances.
Assuntos
Hepatite C , Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , Hepatopatia Gordurosa não Alcoólica/metabolismo , Glucose/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Dipeptidil Peptidase 4/metabolismo , Células Endoteliais/metabolismo , Ribavirina/metabolismo , Hepatócitos/metabolismoRESUMO
Heart disease is the leading cause of morbidity and mortality worldwide. Due to their low cost, ease of handling, and abundance of transgenic strains, rodents have become essential models for cardiovascular research. However, spontaneous lethal cardiac arrhythmias that often cause mortality in heart disease patients are rare in rodent models of heart disease. This is primarily due to the species differences in cardiac electrical properties between human and rodents and poses a challenge to the study of cardiac arrhythmias using rodents. This protocol describes an approach to enable efficient transgene expression in mouse and rat ventricular myocardium using echocardiography-guided intramuscular injections of recombinant virus (adenovirus and adeno-associated virus). This work also outlines a method to enable reliable assessment of cardiac susceptibility to arrhythmias using isolated, Langendorff-perfused mouse and rat hearts with both adrenergic and programmed electrical stimulations. These techniques are critical for studying heart rhythm disorders associated with adverse cardiac remodeling after injuries, such as myocardial infarction.
Assuntos
Arritmias Cardíacas , Transgenes , Animais , Animais Geneticamente Modificados , Arritmias Cardíacas/fisiopatologia , Modelos Animais de Doenças , Coração , Humanos , Camundongos , Miocárdio/metabolismo , RatosRESUMO
OBJECTIVE: Aberrant ketogenesis is correlated with the degree of steatosis in non-alcoholic fatty liver disease (NAFLD) patients, and an inborn error of ketogenesis (mitochondrial HMG-CoA synthase deficiency) is commonly associated with the development of the fatty liver. Here we aimed to determine the impact of Hmgcs2-mediated ketogenesis and its modulations on the development and treatment of fatty liver disease. METHODS: Loss- and gain-of-ketogenic function models, achieved by Hmgcs2 knockout and overexpression, respectively, were utilized to investigate the role of ketogenesis in the hepatic lipid accumulation during postnatal development and in a high-fat diet-induced NAFLD mouse model. RESULTS: Ketogenic function was decreased in NAFLD mice with a reduction in Hmgcs2 expression. Mice lacking Hmgcs2 developed spontaneous fatty liver phenotype during postnatal development, which was rescued by a shift to a low-fat dietary composition via early weaning. Hmgcs2 heterozygous adult mice, which exhibited lower ketogenic activity, were more susceptible to diet-induced NAFLD development, whereas HMGCS2 overexpression in NAFLD mice improved hepatosteatosis and glucose homeostasis. CONCLUSIONS: Our study adds new knowledge to the field of ketone body metabolism and shows that Hmgcs2-mediated ketogenesis modulates hepatic lipid regulation under a fat-enriched nutritional environment. The regulation of hepatic ketogenesis may be a viable therapeutic strategy in the prevention and treatment of hepatosteatosis.
Assuntos
Dieta Hiperlipídica , Hidroximetilglutaril-CoA Sintase , Cetose , Hepatopatia Gordurosa não Alcoólica , Animais , Dieta Hiperlipídica/efeitos adversos , Humanos , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Corpos Cetônicos/genética , Corpos Cetônicos/metabolismo , Cetose/genética , Cetose/metabolismo , Lipídeos , Camundongos , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
Voltage-activated outward K(+) currents (I (Kv)) are essential for cardiac repolarization and are major factors in the electrophysiological remodeling and arrhythmias seen in heart disease. Mouse models have been useful for understanding cardiac electrophysiology. However, previous methods for separating and quantifying the components of I (Kv) in mouse myocardium have yielded inconsistencies. In this study, we developed a statistically rigorous method to uniquely quantify various I (Kv) in adult mouse ventricular myocytes, and concluded that tri-exponential functions combined with depolarizing pulses of duration greater than 20 s are essential to adequately separate the different I (Kv) components. This method enabled us to reliably dissect the kinetic components of the decay phase of I (Kv) into fast (I (to)), intermediate (K(V)1.5-encoded I (K,slow1)) and slow (K(V)2-encoded I (K,slow2)) components. The most rapid kinetic phase, I (to), can be further dissected into fast (K(V)4-encoded I (to,f)) and slow (K(V)1.4-encoded I (to,s)) components by measuring recovery from inactivation, voltage-dependence of activation and sensitivity to HpTx-2 and 4-AP. The applicability of our dissection method was validated using transgenic mice over-expressing dominant-negative K(V)1.1 transgene which largely abolished the 4-AP-sensitive portion of I (to) (i.e., I (to,s)) and the I (K,slow1) component. We also applied our method to Irx5-deficient mice and verified selective elevations of I (to) in endocardial myocytes. Our method should prove useful in future electrophysiological studies using mouse.
Assuntos
Técnicas Eletrofisiológicas Cardíacas , Miócitos Cardíacos/metabolismo , Potássio/metabolismo , Animais , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Cardiovasculares , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismoRESUMO
Obesity is mainly due to excessive food intake. IRX3 and IRX5 have been suggested as determinants of obesity in connection with the intronic variants of FTO, but how these genes contribute to obesity via changes in food intake remains unclear. Here, we show that mice doubly heterozygous for Irx3 and Irx5 mutations exhibit lower food intake with enhanced hypothalamic leptin response. By lineage tracing and single-cell RNA sequencing using the Ins2-Cre system, we identify a previously unreported radial glia-like neural stem cell population with high Irx3 and Irx5 expression in early postnatal hypothalamus and demonstrate that reduced dosage of Irx3 and Irx5 promotes neurogenesis in postnatal hypothalamus leading to elevated numbers of leptin-sensing arcuate neurons. Furthermore, we find that mice with deletion of Irx3 in these cells also exhibit a similar food intake and hypothalamic phenotype. Our results illustrate that Irx3 and Irx5 play a regulatory role in hypothalamic postnatal neurogenesis and leptin response.
Assuntos
Proteínas de Homeodomínio/genética , Hipotálamo/metabolismo , Insulina/genética , Leptina/metabolismo , Neurogênese/genética , Fatores de Transcrição/genética , Animais , Comportamento Alimentar , Imunofluorescência , Regulação da Expressão Gênica , Estudos de Associação Genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco Neurais , Neurônios/metabolismo , Fenótipo , RNA Citoplasmático Pequeno/genética , Fatores de Transcrição/metabolismoRESUMO
The paraventricular nucleus of the hypothalamus (PVH) contains a heterogeneous cluster of Sim1-expressing neurons critical for feeding regulation. Sim1 haploinsufficiency results in hyperphagic obesity with disruption of PVH neurons, yet the molecular profiles of PVH neurons and the mechanism underlying the defects of Sim1 haploinsufficiency are not well understood. By single-cell RNA sequencing, we identified two major populations of Sim1+ PVH neurons, which are differentially affected by Sim1 haploinsufficiency. The Iroquois homeobox genes Irx3 and Irx5 have been implicated in the hypothalamic control of energy homeostasis. We found that Irx3 and Irx5 are ectopically expressed in the Sim1+ PVH cells of Sim1+/− mice. By reducing their dosage and PVH-specific deletion of Irx3, we demonstrate that misexpression of Irx3 and Irx5 contributes to the defects of Sim1+/− mice. Our results illustrate abnormal hypothalamic activities of Irx3 and Irx5 as a central mechanism disrupting PVH development and feeding regulation in Sim1 haploinsufficiency.
RESUMO
The global prevalence of metabolic disorders, such as obesity, diabetes and fatty liver disease, is dramatically increasing. Both genetic and environmental factors are well-known contributors to the development of these diseases and therefore, the study of epigenetics can provide additional mechanistic insight. Dietary interventions, including caloric restriction, intermittent fasting or time-restricted feeding, have shown promising improvements in patients' overall metabolic profiles (i.e., reduced body weight, improved glucose homeostasis), and an increasing number of studies have associated these beneficial effects with epigenetic alterations. In this article, we review epigenetic changes involved in both metabolic diseases and dietary interventions in primary metabolic tissues (i.e., adipose, liver, and pancreas) in hopes of elucidating potential biomarkers and therapeutic targets for disease prevention and treatment.
RESUMO
Atrial Fibrillation (AF) is the most common supraventricular tachyarrhythmia that is typically associated with cardiovascular disease (CVD) and poor cardiovascular health. Paradoxically, endurance athletes are also at risk for AF. While it is well-established that persistent AF is associated with atrial fibrosis, hypertrophy and inflammation, intensely exercised mice showed similar adverse atrial changes and increased AF vulnerability, which required tumor necrosis factor (TNF) signaling, even though ventricular structure and function improved. To identify some of the molecular factors underlying the chamber-specific and TNF-dependent atrial changes induced by exercise, we performed transcriptome analyses of hearts from wild-type and TNF-knockout mice following exercise for 2 days, 2 or 6 weeks of exercise. Consistent with the central role of atrial stretch arising from elevated venous pressure in AF promotion, all 3 time points were associated with differential regulation of genes in atria linked to mechanosensing (focal adhesion kinase, integrins and cell-cell communications), extracellular matrix (ECM) and TNF pathways, with TNF appearing to play a permissive, rather than causal, role in gene changes. Importantly, mechanosensing/ECM genes were only enriched, along with tubulin- and hypertrophy-related genes after 2 days of exercise while being downregulated at 2 and 6 weeks, suggesting that early reactive strain-dependent remodeling with exercise yields to compensatory adjustments. Moreover, at the later time points, there was also downregulation of both collagen genes and genes involved in collagen turnover, a pattern mirroring aging-related fibrosis. By comparison, twofold fewer genes were differentially regulated in ventricles vs. atria, independently of TNF. Our findings reveal that exercise promotes TNF-dependent atrial transcriptome remodeling of ECM/mechanosensing pathways, consistent with increased preload and atrial stretch seen with exercise. We propose that similar preload-dependent mechanisms are responsible for atrial changes and AF in both CVD patients and athletes.