RESUMEN
Heart failure with preserved ejection fraction (HFpEF) is now the dominant form of heart failure and one for which no efficacious therapies exist. Obesity and lipid mishandling greatly contribute to HFpEF. However, molecular mechanism(s) governing metabolic alterations and perturbations in lipid homeostasis in HFpEF are largely unknown. Here, we report that cardiomyocyte steatosis in HFpEF is coupled with increases in the activity of the transcription factor FoxO1 (Forkhead box protein O1). FoxO1 depletion, as well as over-expression of the Xbp1s (spliced form of the X-box-binding protein 1) arm of the UPR (unfolded protein response) in cardiomyocytes each ameliorates the HFpEF phenotype in mice and reduces myocardial lipid accumulation. Mechanistically, forced expression of Xbp1s in cardiomyocytes triggers ubiquitination and proteasomal degradation of FoxO1 which occurs, in large part, through activation of the E3 ubiquitin ligase STUB1 (STIP1 homology and U-box-containing protein 1) a novel and direct transcriptional target of Xbp1s. Our findings uncover the Xbp1s-FoxO1 axis as a pivotal mechanism in the pathogenesis of cardiometabolic HFpEF and unveil previously unrecognized mechanisms whereby the UPR governs metabolic alterations in cardiomyocytes.
Asunto(s)
Proteína Forkhead Box O1/metabolismo , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Metabolismo de los Lípidos , Contracción Miocárdica , Volumen Sistólico , Proteína 1 de Unión a la X-Box/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Secuencia Conservada , Eliminación de Gen , Células HEK293 , Insuficiencia Cardíaca/genética , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Fenotipo , Estabilidad Proteica , Proteolisis , Transcripción Genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
BACKGROUND: BET (bromodomain and extraterminal) epigenetic reader proteins, in particular BRD4 (bromodomain-containing protein 4), have emerged as potential therapeutic targets in a number of pathological conditions, including cancer and cardiovascular disease. Small-molecule BET protein inhibitors such as JQ1 have demonstrated efficacy in reversing cardiac hypertrophy and heart failure in preclinical models. Yet, genetic studies elucidating the biology of BET proteins in the heart have not been conducted to validate pharmacological findings and to unveil potential pharmacological side effects. METHODS: By engineering a cardiomyocyte-specific BRD4 knockout mouse, we investigated the role of BRD4 in cardiac pathophysiology. We performed functional, transcriptomic, and mitochondrial analyses to evaluate BRD4 function in developing and mature hearts. RESULTS: Unlike pharmacological inhibition, loss of BRD4 protein triggered progressive declines in myocardial function, culminating in dilated cardiomyopathy. Transcriptome analysis of BRD4 knockout mouse heart tissue identified early and specific disruption of genes essential to mitochondrial energy production and homeostasis. Functional analysis of isolated mitochondria from these hearts confirmed that BRD4 ablation triggered significant changes in mitochondrial electron transport chain protein expression and activity. Computational analysis identified candidate transcription factors participating in the BRD4-regulated transcriptome. In particular, estrogen-related receptor α, a key nuclear receptor in metabolic gene regulation, was enriched in promoters of BRD4-regulated mitochondrial genes. CONCLUSIONS: In aggregate, we describe a previously unrecognized role for BRD4 in regulating cardiomyocyte mitochondrial homeostasis, observing that its function is indispensable to the maintenance of normal cardiac function.
Asunto(s)
Cardiomiopatía Dilatada/metabolismo , Núcleo Celular/metabolismo , Metabolismo Energético , Mitocondrias Cardíacas/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Transcriptoma , Disfunción Ventricular Izquierda/metabolismo , Función Ventricular Izquierda , Animales , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/patología , Cardiomiopatía Dilatada/fisiopatología , Núcleo Celular/genética , Núcleo Celular/patología , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Metabolismo Energético/genética , Epigénesis Genética , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Perfilación de la Expresión Génica , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Ratones Noqueados , Mitocondrias Cardíacas/genética , Mitocondrias Cardíacas/patología , Miocitos Cardíacos/patología , Proteínas Nucleares/genética , Factores de Transcripción/genética , Disfunción Ventricular Izquierda/genética , Disfunción Ventricular Izquierda/patología , Disfunción Ventricular Izquierda/fisiopatología , Función Ventricular Izquierda/genéticaRESUMEN
BACKGROUND: Polycystin-1 (PC1) is a transmembrane protein originally identified in autosomal dominant polycystic kidney disease where it regulates the calcium-permeant cation channel polycystin-2. Autosomal dominant polycystic kidney disease patients develop renal failure, hypertension, left ventricular hypertrophy, and diastolic dysfunction, among other cardiovascular disorders. These individuals harbor PC1 loss-of-function mutations in their cardiomyocytes, but the functional consequences are unknown. PC1 is ubiquitously expressed, and its experimental ablation in cardiomyocyte-specific knockout mice reduces contractile function. Here, we set out to determine the pathophysiological role of PC1 in cardiomyocytes. METHODS: Wild-type and cardiomyocyte-specific PC1 knockout mice were analyzed by echocardiography. Excitation-contraction coupling was assessed in isolated cardiomyocytes and human embryonic stem cell-derived cardiomyocytes, and functional consequences were explored in heterologous expression systems. Protein-protein interactions were analyzed biochemically and by means of ab initio calculations. RESULTS: PC1 ablation reduced action potential duration in cardiomyocytes, decreased Ca2+ transients, and myocyte contractility. PC1-deficient cardiomyocytes manifested a reduction in sarcoendoplasmic reticulum Ca2+ stores attributable to a reduced action potential duration and sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) activity. An increase in outward K+ currents decreased action potential duration in cardiomyocytes lacking PC1. Overexpression of full-length PC1 in HEK293 cells significantly reduced the current density of heterologously expressed Kv4.3, Kv1.5 and Kv2.1 potassium channels. PC1 C terminus inhibited Kv4.3 currents to the same degree as full-length PC1. Additionally, PC1 coimmunoprecipitated with Kv4.3, and a modeled PC1 C-terminal structure suggested the existence of 2 docking sites for PC1 within the N terminus of Kv4.3, supporting a physical interaction. Finally, a naturally occurring human mutant PC1R4228X manifested no suppressive effects on Kv4.3 channel activity. CONCLUSIONS: Our findings uncover a role for PC1 in regulating multiple Kv channels, governing membrane repolarization and alterations in SERCA activity that reduce cardiomyocyte contractility.
Asunto(s)
Potenciales de Acción/fisiología , Miocitos Cardíacos/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Canales Catiónicos TRPP/deficiencia , Animales , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Canales Catiónicos TRPP/genéticaRESUMEN
Heart failure with preserved ejection fraction (HFpEF) is a common syndrome with high morbidity and mortality for which there are no evidence-based therapies. Here we report that concomitant metabolic and hypertensive stress in mice-elicited by a combination of high-fat diet and inhibition of constitutive nitric oxide synthase using Nω-nitro-L-arginine methyl ester (L-NAME)-recapitulates the numerous systemic and cardiovascular features of HFpEF in humans. Expression of one of the unfolded protein response effectors, the spliced form of X-box-binding protein 1 (XBP1s), was reduced in the myocardium of our rodent model and in humans with HFpEF. Mechanistically, the decrease in XBP1s resulted from increased activity of inducible nitric oxide synthase (iNOS) and S-nitrosylation of the endonuclease inositol-requiring protein 1α (IRE1α), culminating in defective XBP1 splicing. Pharmacological or genetic suppression of iNOS, or cardiomyocyte-restricted overexpression of XBP1s, each ameliorated the HFpEF phenotype. We report that iNOS-driven dysregulation of the IRE1α-XBP1 pathway is a crucial mechanism of cardiomyocyte dysfunction in HFpEF.
Asunto(s)
Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Estrés Nitrosativo , Volumen Sistólico , Animales , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Endorribonucleasas/metabolismo , Insuficiencia Cardíaca/prevención & control , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/deficiencia , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fenotipo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteína 1 de Unión a la X-Box/genética , Proteína 1 de Unión a la X-Box/metabolismoRESUMEN
BACKGROUND: The primary cilium is a singular cellular structure that extends from the surface of many cell types and plays crucial roles in vertebrate development, including that of the heart. Whereas ciliated cells have been described in developing heart, a role for primary cilia in adult heart has not been reported. This, coupled with the fact that mutations in genes coding for multiple ciliary proteins underlie polycystic kidney disease, a disorder with numerous cardiovascular manifestations, prompted us to identify cells in adult heart harboring a primary cilium and to determine whether primary cilia play a role in disease-related remodeling. METHODS: Histological analysis of cardiac tissues from C57BL/6 mouse embryos, neonatal mice, and adult mice was performed to evaluate for primary cilia. Three injury models (apical resection, ischemia/reperfusion, and myocardial infarction) were used to identify the location and cell type of ciliated cells with the use of antibodies specific for cilia (acetylated tubulin, γ-tubulin, polycystin [PC] 1, PC2, and KIF3A), fibroblasts (vimentin, α-smooth muscle actin, and fibroblast-specific protein-1), and cardiomyocytes (α-actinin and troponin I). A similar approach was used to assess for primary cilia in infarcted human myocardial tissue. We studied mice silenced exclusively in myofibroblasts for PC1 and evaluated the role of PC1 in fibrogenesis in adult rat fibroblasts and myofibroblasts. RESULTS: We identified primary cilia in mouse, rat, and human heart, specifically and exclusively in cardiac fibroblasts. Ciliated fibroblasts are enriched in areas of myocardial injury. Transforming growth factor ß-1 signaling and SMAD3 activation were impaired in fibroblasts depleted of the primary cilium. Extracellular matrix protein levels and contractile function were also impaired. In vivo, depletion of PC1 in activated fibroblasts after myocardial infarction impaired the remodeling response. CONCLUSIONS: Fibroblasts in the neonatal and adult heart harbor a primary cilium. This organelle and its requisite signaling protein, PC1, are required for critical elements of fibrogenesis, including transforming growth factor ß-1-SMAD3 activation, production of extracellular matrix proteins, and cell contractility. Together, these findings point to a pivotal role of this organelle, and PC1, in disease-related pathological cardiac remodeling and suggest that some of the cardiovascular manifestations of autosomal dominant polycystic kidney disease derive directly from myocardium-autonomous abnormalities.
Asunto(s)
Fibroblastos/ultraestructura , Miocardio/patología , Riñón Poliquístico Autosómico Dominante/patología , Células 3T3/ultraestructura , Animales , Animales Recién Nacidos , Remodelación Atrial , Cilios , Corazón Fetal/citología , Fibrosis , Lesiones Cardíacas/patología , Humanos , Cinesinas/deficiencia , Cinesinas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/patología , Riñón Poliquístico Autosómico Dominante/genética , Ratas , Transducción de Señal , Proteína smad3/fisiología , Canales Catiónicos TRPP/deficiencia , Canales Catiónicos TRPP/fisiología , Factor de Crecimiento Transformador beta1/fisiología , Remodelación VentricularRESUMEN
Cardiac progenitor cells (CPCs) have rapidly advanced to clinical trials, yet little is known regarding their interaction with the microenvironment. Signaling cues present in the microenvironment change with development and disease. This work aims to assess the influence of two distinct signaling moieties on CPCs: cyclic biaxial strain and extracellular matrix. We evaluate four endpoints for improving CPC therapy: paracrine signaling, proliferation, connexin43 expression, and alignment. Vascular endothelial growth factor A (about 900 pg/mL) was secreted by CPCs cultured on fibronectin and collagen I. The application of mechanical strain increased vascular endothelial growth factor A secretion 2-4-fold for CPCs cultured on poly-L-lysine, laminin, or a naturally derived cardiac extracellular matrix. CPC proliferation was at least 25% higher on fibronectin than that on other matrices, especially for lower strain magnitudes. At 5% strain, connexin43 expression was highest on fibronectin. With increasing strain magnitude, connexin43 expression decreased by as much as 60% in CPCs cultured on collagen I and a naturally derived cardiac extracellular matrix. Cyclic mechanical strain induced the strongest CPC alignment when cultured on fibronectin or collagen I. This study demonstrates that culturing CPCs on fibronectin with 5% strain magnitude is optimal for their vascular endothelial growth factor A secretion, proliferation, connexin43 expression, and alignment.
RESUMEN
UNLABELLED: Children with congenital heart diseases have increased morbidity and mortality, despite various surgical treatments, therefore warranting better treatment strategies. Here we investigate the role of age of human pediatric cardiac progenitor cells (hCPCs) on ventricular remodeling in a model of juvenile heart failure. hCPCs isolated from children undergoing reconstructive surgeries were divided into 3 groups based on age: neonate (1 day to 1 month), infant (1 month to 1 year), and child (1 to 5 years). Adolescent athymic rats were subjected to sham or pulmonary artery banding surgery to generate a model of right ventricular (RV) heart failure. Two weeks after surgery, hCPCs were injected in RV musculature noninvasively. Analysis of cardiac function 4 weeks post-transplantation demonstrated significantly increased tricuspid annular plane systolic excursion and RV ejection fraction and significantly decreased wall thickness and fibrosis in rats transplanted with neonatal hCPCs compared with saline-injected rats. Computational modeling and systems biology analysis were performed on arrays and gave insights into potential mechanisms at the microRNA and gene level. Mechanisms including migration and proliferation assays, as suggested by computational modeling, showed improved chemotactic and proliferative capacity of neonatal hCPCs compared with infant/child hCPCs. In vivo immunostaining further suggested increased recruitment of stem cell antigen 1-positive cells in the right ventricle. This is the first study to assess the role of hCPC age in juvenile RV heart failure. Interestingly, the reparative potential of hCPCs is age-dependent, with neonatal hCPCs exerting the maximum beneficial effect compared with infant and child hCPCs. SIGNIFICANCE: Stem cell therapy for children with congenital heart defects is moving forward, with several completed and ongoing clinical trials. Although there are studies showing how children differ from adults, few focus on the differences among children. This study using human cardiac progenitor cells shows age-related changes in the reparative ability of cells in a model of pediatric heart failure and uses computational and systems biology to elucidate potential mechanisms.
Asunto(s)
Envejecimiento/fisiología , Cardiopatías Congénitas/terapia , Insuficiencia Cardíaca/terapia , Miocardio/citología , Trasplante de Células Madre , Células Madre/citología , Adulto , Animales , Proliferación Celular , Células Cultivadas , Preescolar , Cardiopatías Congénitas/patología , Insuficiencia Cardíaca/patología , Humanos , Lactante , Recién Nacido , Ratas , Ratas Desnudas , Ratas Transgénicas , Remodelación VentricularRESUMEN
Cardiovascular disease, including myocardial infarction, is the number one cause of death. Current treatments are palliative and slow the progression toward heart failure, but to not regenerate healthy tissue. Self-assembling peptides are biomimietic, readily produced, non-immunogenic and non-cytotoxic. They do not assemble into hydrogels until triggered, allowing them to be injected into the myocardium and providing opportunities for minimally invasive therapies. The ability to tune the mechanical and bioactive properties of self-assembling peptides will continue to make them readily adaptable for mimicking natural microenvironments. To date, a variety of growth factors and signaling moieties have been incorporated into self-assembling peptide hydrogels, enhancing cell behavior and tissue function. Furthermore, the hydrogels serve as delivery vehicles for cells in vivo and platforms for improved cell culture. In addition to a brief review of self-assembling peptides, we will discuss a variety of their approaches for myocardial infarction therapy. Moreover, we will assess approaches taken in other tissue and discuss how these could benefit therapies for myocardial infarction.
Asunto(s)
Materiales Biomiméticos/química , Sistemas de Liberación de Medicamentos/métodos , Péptidos y Proteínas de Señalización Intercelular/uso terapéutico , Infarto del Miocardio/tratamiento farmacológico , Péptidos/química , Ingeniería de Tejidos/métodos , Animales , Materiales Biomiméticos/administración & dosificación , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/química , Humanos , Hidrogeles/administración & dosificación , Hidrogeles/química , Péptidos y Proteínas de Señalización Intercelular/administración & dosificación , Infarto del Miocardio/terapia , Miocitos Cardíacos/citología , Miocitos Cardíacos/trasplante , Nanofibras/administración & dosificación , Nanofibras/química , Péptidos/administración & dosificación , Conformación Proteica , Trasplante de Células Madre , Células Madre/citologíaRESUMEN
RATIONALE: Myocardial infarction is a leading cause of death in developed nations, and there remains a need for cardiac therapeutic systems that mitigate tissue damage. Cardiac progenitor cells (CPCs) and other stem cell types are attractive candidates for treatment of myocardial infarction; however, the benefit of these cells may be as a result of paracrine effects. OBJECTIVE: We tested the hypothesis that CPCs secrete proregenerative exosomes in response to hypoxic conditions. METHODS AND RESULTS: The angiogenic and antifibrotic potential of secreted exosomes on cardiac endothelial cells and cardiac fibroblasts were assessed. We found that CPC exosomes secreted in response to hypoxia enhanced tube formation of endothelial cells and decreased profibrotic gene expression in TGF-ß-stimulated fibroblasts, indicating that these exosomes possess therapeutic potential. Microarray analysis of exosomes secreted by hypoxic CPCs identified 11 miRNAs that were upregulated compared with exosomes secreted by CPCs grown under normoxic conditions. Principle component analysis was performed to identify miRNAs that were coregulated in response to distinct exosome-generating conditions. To investigate the cue-signal-response relationships of these miRNA clusters with a physiological outcome of tube formation or fibrotic gene expression, partial least squares regression analysis was applied. The importance of each up- or downregulated miRNA on physiological outcomes was determined. Finally, to validate the model, we delivered exosomes after ischemia-reperfusion injury. Exosomes from hypoxic CPCs improved cardiac function and reduced fibrosis. CONCLUSIONS: These data provide a foundation for subsequent research of the use of exosomal miRNA and systems biology as therapeutic strategies for the damaged heart.
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Exosomas/fisiología , MicroARNs/fisiología , Miocitos Cardíacos/fisiología , Células Madre/fisiología , Biología de Sistemas/métodos , Animales , Animales Recién Nacidos , Hipoxia de la Célula/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Cell therapy techniques are a promising option for tissue regeneration; especially in cases such as heart failure where transplantation is limited by donor availability. Multiple cell types have been examined for myocardial regeneration, including mesenchymal stem cells (and other bone marrow-derived cells), induced pluripotent stem cells, embryonic stem cells, cardiosphere-derived cells, and cardiac progenitor cells (CPCs). CPCs are multipotent and clonogenic, can be harvested from mature tissue, and have the distinct advantages of autologous transplant and lack of tumor formation in a clinical setting. Here we focus on the isolation, expansion, and myocardial differentiation of rat CPCs. Brief adaptations of the protocol for isolation from mouse and human tissue are also provided.
Asunto(s)
Separación Celular/métodos , Separación Inmunomagnética/métodos , Mioblastos Cardíacos/citología , Mioblastos Cardíacos/metabolismo , Miocitos Cardíacos/citología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Animales , Azacitidina/farmacología , Diferenciación Celular/efectos de los fármacos , Dexametasona/farmacología , Humanos , Ratones , Microesferas , Mioblastos Cardíacos/efectos de los fármacos , RatasRESUMEN
Myocardial infarction (MI) produces a collagen scar, altering the local microenvironment and impeding cardiac function. Cell therapy is a promising therapeutic option to replace the billions of myocytes lost following MI. Despite early successes, chronic function remains impaired and is likely a result of poor cellular retention, proliferation, and differentiation/maturation. While some efforts to deliver cells with scaffolds have attempted to address these shortcomings, they lack the natural cues required for optimal cell function. The goal of this study was to determine whether a naturally derived cardiac extracellular matrix (cECM) could enhance cardiac progenitor cell (CPC) function in vitro. CPCs were isolated via magnetic sorting of c-kit(+) cells and were grown on plates coated with either cECM or collagen I (Col). Our results show an increase in early cardiomyocyte markers on cECM compared with Col, as well as corresponding protein expression at a later time. CPCs show stronger serum-induced proliferation on cECM compared with Col, as well as increased resistance to apoptosis following serum starvation. Finally, a microfluidic adhesion assay demonstrated stronger adhesion of CPCs to cECM compared with Col. These data suggest that cECM may be optimal for CPC therapeutic delivery, as well as providing potential mechanisms to overcome the shortcomings of naked cell therapy.