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Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11bDTR mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction.
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Sistema de Conducción Cardíaco , Macrófagos/fisiología , Animales , Conexina 43/metabolismo , Femenino , Atrios Cardíacos/citología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Miocitos Cardíacos/fisiologíaRESUMEN
Valvular heart disease is a common cause of morbidity and mortality worldwide and has no effective medical therapy. Severe disease is managed with valve replacement procedures, which entail high health care-related costs and postprocedural morbidity and mortality. Robust ongoing research programs have elucidated many important molecular pathways contributing to primary valvular heart disease. However, there remain several key challenges inherent in translating research on valvular heart disease to viable molecular targets that can progress through the clinical trials pathway and effectively prevent or modify the course of these common conditions. In this scientific statement, we review the basic cellular structures of the human heart valves and discuss how these structures change in primary valvular heart disease. We focus on the most common primary valvular heart diseases, including calcific aortic stenosis, bicuspid aortic valves, mitral valve prolapse, and rheumatic heart disease, and outline the fundamental molecular discoveries contributing to each. We further outline potential therapeutic molecular targets for primary valvular heart disease and discuss key knowledge gaps that might serve as future research priorities.
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American Heart Association , Enfermedades de las Válvulas Cardíacas , Humanos , Enfermedades de las Válvulas Cardíacas/tratamiento farmacológico , Enfermedades de las Válvulas Cardíacas/metabolismo , Estados Unidos , AnimalesRESUMEN
BACKGROUND: Clonal hematopoiesis of indeterminate potential (CHIP), a common age-associated phenomenon, associates with increased risk of both hematological malignancy and cardiovascular disease. Although CHIP is known to increase the risk of myocardial infarction and heart failure, the influence of CHIP in cardiac arrhythmias, such as atrial fibrillation (AF), is less explored. METHODS: CHIP prevalence was determined in the UK Biobank, and incident AF analysis was stratified by CHIP status and clone size using Cox proportional hazard models. Lethally irradiated mice were transplanted with hematopoietic-specific loss of Tet2, hematopoietic-specific loss of Tet2 and Nlrp3, or wild-type control and fed a Western diet, compounded with or without NLRP3 (NLR [NACHT, LRR {leucine rich repeat}] family pyrin domain containing protein 3) inhibitor, NP3-361, for 6 to 9 weeks. Mice underwent in vivo invasive electrophysiology studies and ex vivo optical mapping. Cardiomyocytes from Ldlr-/- mice with hematopoietic-specific loss of Tet2 or wild-type control and fed a Western diet were isolated to evaluate calcium signaling dynamics and analysis. Cocultures of pluripotent stem cell-derived atrial cardiomyocytes were incubated with Tet2-deficient bone marrow-derived macrophages, wild-type control, or cytokines IL-1ß (interleukin 1ß) or IL-6 (interleukin 6). RESULTS: Analysis of the UK Biobank showed individuals with CHIP, in particular TET2 CHIP, have increased incident AF. Hematopoietic-specific inactivation of Tet2 increases AF propensity in atherogenic and nonatherogenic mouse models and is associated with increased Nlrp3 expression and CaMKII (Ca2+/calmodulin-dependent protein kinase II) activation, with AF susceptibility prevented by inactivation of Nlrp3. Cardiomyocytes isolated from Ldlr-/- mice with hematopoietic inactivation of Tet2 and fed a Western diet have impaired calcium release from the sarcoplasmic reticulum into the cytosol, contributing to atrial arrhythmogenesis. Abnormal sarcoplasmic reticulum calcium release was recapitulated in cocultures of cardiomyocytes with the addition of Tet2-deficient macrophages or cytokines IL-1ß or IL-6. CONCLUSIONS: We identified a modest association between CHIP, particularly TET2 CHIP, and incident AF in the UK Biobank population. In a mouse model of AF resulting from hematopoietic-specific inactivation of Tet2, we propose altered calcium handling as an arrhythmogenic mechanism, dependent on Nlrp3 inflammasome activation. Our data are in keeping with previous studies of CHIP in cardiovascular disease, and further studies into the therapeutic potential of NLRP3 inhibition for individuals with TET2 CHIP may be warranted.
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Fibrilación Atrial , Hematopoyesis Clonal , Proteínas de Unión al ADN , Dioxigenasas , Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas Proto-Oncogénicas , Animales , Dioxigenasas/metabolismo , Dioxigenasas/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Fibrilación Atrial/metabolismo , Fibrilación Atrial/etiología , Fibrilación Atrial/genética , Fibrilación Atrial/patología , Inflamasomas/metabolismo , Humanos , Ratones , Hematopoyesis Clonal/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Masculino , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Anciano , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Persona de Mediana Edad , Ratones Noqueados , Factores de RiesgoRESUMEN
BACKGROUND: ZFHX3 (zinc finger homeobox 3), a gene that encodes a large transcription factor, is at the second-most significantly associated locus with atrial fibrillation (AF), but its function in the heart is unknown. This study aims to identify causative genetic variation related to AF at the ZFHX3 locus and examine the impact of Zfhx3 loss on cardiac function in mice. METHODS: CRISPR-Cas9 genome editing, chromatin immunoprecipitation, and luciferase assays in pluripotent stem cell-derived cardiomyocytes were used to identify causative genetic variation related to AF at the ZFHX3 locus. Cardiac function was assessed by echocardiography, magnetic resonance imaging, electrophysiology studies, calcium imaging, and RNA sequencing in mice with heterozygous and homozygous cardiomyocyte-restricted Zfhx3 loss (Zfhx3 Het and knockout, respectively). Human cardiac single-nucleus ATAC (assay for transposase-accessible chromatin)-sequencing data was analyzed to determine which genes in atrial cardiomyocytes are directly regulated by ZFHX3. RESULTS: We found single-nucleotide polymorphism (SNP) rs12931021 modulates an enhancer regulating ZFHX3 expression, and the AF risk allele is associated with decreased ZFHX3 transcription. We observed a gene-dose response in AF susceptibility with Zfhx3 knockout mice having higher incidence, frequency, and burden of AF than Zfhx3 Het and wild-type mice, with alterations in conduction velocity, atrial action potential duration, calcium handling and the development of atrial enlargement and thrombus, and dilated cardiomyopathy. Zfhx3 loss results in atrial-specific differential effects on genes and signaling pathways involved in cardiac pathophysiology and AF. CONCLUSIONS: Our findings implicate ZFHX3 as the causative gene at the 16q22 locus for AF, and cardiac abnormalities caused by loss of cardiac Zfhx3 are due to atrial-specific dysregulation of pathways involved in AF susceptibility. Together, these data reveal a novel and important role for Zfhx3 in the control of cardiac genes and signaling pathways essential for normal atrial function.
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Fibrilación Atrial , Proteínas de Homeodominio , Animales , Humanos , Ratones , Fibrilación Atrial/genética , Calcio/metabolismo , Dilatación , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Miocitos Cardíacos/metabolismo , Factores de Transcripción/genéticaRESUMEN
While a multitude of studies have appeared touting the use of molecules as electronic components, the design of molecular switches is crucial for the next steps in molecular electronics. In this work, single-molecule devices incorporating spiropyrans, made using break junction techniques, are described. Linear spiropyrans with electrode-contacting groups linked by alkynyl spacers to both the indoline and chromenone moieties have previously provided very low conductance values, and removing the alkynyl spacer has resulted in a total loss of conductance. An orthogonal T-shaped approach to single-molecule junctions incorporating spiropyran moieties in which the conducting pathway lies orthogonal to the molecule backbone is described and characterized. This approach has provided singlemolecule conductance features with good correlation to molecular length. Additional higher conducting states are accessible using switching induced by UV light or protonation. Theoretical modeling demonstrates that upon (photo)chemical isomerization to the merocyanine, two cooperating phenomena increase conductance: release of steric hindrance allows the conductance pathway to become more planar (raising the mid-bandgap transmission) and a bound state introduces sharp interference near the Fermi level of the electrodes similarly responding to the change in state. This design step paves the way for future use of spiropyrans in single-molecule devices and electrosteric switches.
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Controlling the orientation of complex molecules in molecular junctions is crucial to their development into functional devices. To date, this has been achieved through the use of multipodal compounds (i.e., containing more than two anchoring groups), resulting in the formation of tri/tetrapodal compounds. While such compounds have greatly improved orientation control, this comes at the cost of lower surface coverage. In this study, we examine an alternative approach for generating multimodal compounds by binding multiple independent molecular wires together through metal coordination to form a molecular bundle. This was achieved by coordinating iron(II) and cobalt(II) to 5,5'-bis(methylthio)-2,2'-bipyridine (L1) and (methylenebis(4,1-phenylene))bis(1-(5-(methylthio)pyridin-2-yl)methanimine) (L2) to give two monometallic complexes, Fe-1 and Co-1, and two bimetallic helicates, Fe-2 and Co-2. Using XPS, all of the complexes were shown to bind to a gold surface in a fac fashion through three thiomethyl groups. Using single-molecule conductance and DFT calculations, each of the ligands was shown to conduct as an independent wire with no impact from the rest of the complex. These results suggest that this is a useful approach for controlling the geometry of junction formation without altering the conductance behavior of the individual molecular wires.
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BACKGROUND: Mutations in tafazzin (TAZ), a gene required for biogenesis of cardiolipin, the signature phospholipid of the inner mitochondrial membrane, causes Barth syndrome (BTHS). Cardiomyopathy and risk of sudden cardiac death are prominent features of BTHS, but the mechanisms by which impaired cardiolipin biogenesis causes cardiac muscle weakness and arrhythmia are poorly understood. METHODS: We performed in vivo electrophysiology to define arrhythmia vulnerability in cardiac-specific TAZ knockout mice. Using cardiomyocytes derived from human induced pluripotent stem cells and cardiac-specific TAZ knockout mice as model systems, we investigated the effect of TAZ inactivation on Ca2+ handling. Through genome editing and pharmacology, we defined a molecular link between TAZ mutation and abnormal Ca2+ handling and contractility. RESULTS: A subset of mice with cardiac-specific TAZ inactivation developed arrhythmias, including bidirectional ventricular tachycardia, atrial tachycardia, and complete atrioventricular block. Compared with wild-type controls, BTHS-induced pluripotent stem cell-derived cardiomyocytes had increased diastolic Ca2+ and decreased Ca2+ transient amplitude. BTHS-induced pluripotent stem cell-derived cardiomyocytes had higher levels of mitochondrial and cellular reactive oxygen species than wild-type controls, which activated CaMKII (Ca2+/calmodulin-dependent protein kinase II). Activated CaMKII phosphorylated the RYR2 (ryanodine receptor 2) on serine 2814, increasing Ca2+ leak through RYR2. Inhibition of this reactive oxygen species-CaMKII-RYR2 pathway through pharmacological inhibitors or genome editing normalized aberrant Ca2+ handling in BTHS-induced pluripotent stem cell-derived cardiomyocytes and improved their contractile function. Murine Taz knockout cardiomyocytes also exhibited elevated diastolic Ca2+ and decreased Ca2+ transient amplitude. These abnormalities were ameliorated by Ca2+/calmodulin-dependent protein kinase II or reactive oxygen species inhibition. CONCLUSIONS: This study identified a molecular pathway that links TAZ mutation with abnormal Ca2+ handling and decreased cardiomyocyte contractility. This pathway may offer therapeutic opportunities to treat BTHS and potentially other diseases with elevated mitochondrial reactive oxygen species production.
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Síndrome de Barth/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calcio/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Síndrome de Barth/fisiopatología , Humanos , Ratones , Ratones NoqueadosRESUMEN
This paper describes the syntheses of several functionalized dihydropyrene (DHP) molecular switches with different substitution patterns. Regioselective nucleophilic alkylation of a 5-substituted dimethyl isophthalate allowed the development of a workable synthetic protocol for the preparation of 2,7-alkyne-functionalized DHPs. Synthesis of DHPs with surface-anchoring groups in the 2,7- and 4,9-positions is described. The molecular structures of several intermediates and DHPs were elucidated by X-ray single-crystal diffraction. Molecular properties and switching capabilities of both types of DHPs were assessed by light irradiation experiments, spectroelectrochemistry, and cyclic voltammetry. Spectroelectrochemistry, in combination with density functional theory (DFT) calculations, shows reversible electrochemical switching from the DHP forms to the cyclophanediene (CPD) forms. Charge-transport behavior was assessed in single-molecule scanning tunneling microscope (STM) break junctions, combined with density functional theory-based quantum transport calculations. All DHPs with surface-contacting groups form stable molecular junctions. Experiments show that the molecular conductance depends on the substitution pattern of the DHP motif. The conductance was found to decrease with increasing applied bias.
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Sphingolipids are an important class of lipids found in mammalian cell membranes with important structural and signaling roles. They differ from another major group of lipids, the glycerophospholipids, in the connection of their hydrocarbon chains to their headgroups. In this study, a combination of electrochemical and structural methods has been used to elucidate the effect of this difference on sphingolipid behavior in an applied electric field. N-Palmitoyl sphingomyelin forms bilayers of similar coverage and thickness to its close analogue di-palmitoyl phosphatidylcholine. Grazing incidence diffraction data show slightly closer packing and a smaller chain tilt angle from the surface normal. Electrochemical IR results at low charge density show that the difference in tilt angle is retained on deposition to form bilayers. The bilayers respond differently to increasing electric field strength: chain tilt angles increase for both molecules, but sphingomyelin chains remain tilted as field strength is further increased. This behavior is correlated with disruption of the hydrogen-bonding network of small groups of sphingomyelin molecules, which may have significance for the behavior of molecules in lipid rafts in the presence of strong fields induced by ion gradients or asymmetric distribution of charged lipids.
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Membrana Dobles de Lípidos , Esfingomielinas , Animales , Esfingomielinas/química , Membrana Dobles de Lípidos/química , Fosfatidilcolinas , Membrana Celular , Microdominios de Membrana , MamíferosRESUMEN
Revealing how formation protocols influence the properties of the solid-electrolyte interphase (SEI) on Si electrodes is key to developing the next generation of Li-ion batteries. SEI understanding is, however, limited by the low-throughput nature of conventional characterisation techniques. Herein, correlative scanning electrochemical cell microscopy (SECCM) and shell-isolated nanoparticles for enhanced Raman spectroscopy (SHINERS) are used for combinatorial screening of the SEI formation under a broad experimental space (20 sets of different conditions with several repeats). This novel approach reveals the heterogeneous nature and dynamics of the SEI electrochemical properties and chemical composition on Si electrodes, which evolve in a characteristic manner as a function of cycle number. Correlative SECCM/SHINERS has the potential to screen thousands of candidate experiments on a variety of battery materials to accelerate the optimization of SEI formation methods, a key bottleneck in battery manufacturing.
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BACKGROUND: Ibrutinib is a Bruton tyrosine kinase inhibitor with remarkable efficacy against B-cell cancers. Ibrutinib also increases the risk of atrial fibrillation (AF), which remains poorly understood. METHODS: We performed electrophysiology studies on mice treated with ibrutinib to assess inducibility of AF. Chemoproteomic analysis of cardiac lysates identified candidate ibrutinib targets, which were further evaluated in genetic mouse models and additional pharmacological experiments. The pharmacovigilance database, VigiBase, was queried to determine whether drug inhibition of an identified candidate kinase was associated with increased reporting of AF. RESULTS: We demonstrate that treatment of mice with ibrutinib for 4 weeks results in inducible AF, left atrial enlargement, myocardial fibrosis, and inflammation. This effect was reproduced in mice lacking Bruton tyrosine kinase, but not in mice treated with 4 weeks of acalabrutinib, a more specific Bruton tyrosine kinase inhibitor, demonstrating that AF is an off-target side effect. Chemoproteomic profiling identified a short list of candidate kinases that was narrowed by additional experimentation leaving CSK (C-terminal Src kinase) as the strongest candidate for ibrutinib-induced AF. Cardiac-specific Csk knockout in mice led to increased AF, left atrial enlargement, fibrosis, and inflammation, phenocopying ibrutinib treatment. Disproportionality analyses in VigiBase confirmed increased reporting of AF associated with kinase inhibitors blocking Csk versus non-Csk inhibitors, with a reporting odds ratio of 8.0 (95% CI, 7.3-8.7; P<0.0001). CONCLUSIONS: These data identify Csk inhibition as the mechanism through which ibrutinib leads to AF. Registration: URL: https://ww.clinicaltrials.gov; Unique identifier: NCT03530215.
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Adenina/análogos & derivados , Antineoplásicos/toxicidad , Fibrilación Atrial/inducido químicamente , Función del Atrio Izquierdo/efectos de los fármacos , Proteína Tirosina Quinasa CSK/antagonistas & inhibidores , Atrios Cardíacos/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Piperidinas/toxicidad , Inhibidores de Proteínas Quinasas/toxicidad , Potenciales de Acción/efectos de los fármacos , Adenina/toxicidad , Agammaglobulinemia Tirosina Quinasa/deficiencia , Agammaglobulinemia Tirosina Quinasa/genética , Animales , Fibrilación Atrial/enzimología , Fibrilación Atrial/fisiopatología , Proteína Tirosina Quinasa CSK/genética , Proteína Tirosina Quinasa CSK/metabolismo , Bases de Datos Genéticas , Atrios Cardíacos/enzimología , Atrios Cardíacos/fisiopatología , Humanos , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Medición de Riesgo , Factores de RiesgoRESUMEN
The most probable single-molecule conductance of each member of a series of 12 conjugated molecular wires, 6 of which contain either a ruthenium or platinum center centrally placed within the backbone, has been determined. The measurement of a small, positive Seebeck coefficient has established that transmission through these molecules takes place by tunneling through the tail of the HOMO resonance near the middle of the HOMO-LUMO gap in each case. Despite the general similarities in the molecular lengths and frontier-orbital compositions, experimental and computationally determined trends in molecular conductance values across this series cannot be satisfactorily explained in terms of commonly discussed "single-parameter" models of junction conductance. Rather, the trends in molecular conductance are better rationalized from consideration of the complete molecular junction, with conductance values well described by transport calculations carried out at the DFT level of theory, on the basis of the Landauer-Büttiker model.
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Lipid bilayers form the basis of biological cell membranes, selective and responsive barriers vital to the function of the cell. The structure and function of the bilayer are controlled by interactions between the constituent molecules and so vary with the composition of the membrane. These interactions also influence how a membrane behaves in the presence of electric fields they frequently experience in nature. In this study, we characterize the electrochemical phase behavior of dipalmitoylphosphatidylcholine (DPPC), a glycerophospholipid prevalent in nature and often used in model systems and healthcare applications. DPPC bilayers were formed on Au(111) electrodes using Langmuir-Blodgett and Langmuir-Schaefer deposition and studied with electrochemical methods, atomic force microscopy (AFM) and in situ polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). The coverage of the substrate determined with AFM is in accord with that estimated from differential capacitance measurements, and the bilayer thickness is slightly higher than for bilayers of the similar but shorter-chained lipid, dimyristoylphosphatidylcholine (DMPC). DPPC bilayers exhibit similar electrochemical response to DMPC bilayers, but the organization of molecules differs, particularly at negative charge densities. Infrared spectra show that DPPC chains tilt as the charge density on the metal is increased in the negative direction, but, unlike in DMPC, the chains then return to their original tilt angle at the most negative potentials. The onset of the increase in the chain tilt angle coincides with a decrease in solvation around the ester carbonyl groups, and the conformation around the acyl chain linkage differs from that in DMPC. We interpret the differences in behavior between bilayers formed from these structurally similar lipids in terms of stronger dispersion forces between DPPC chains and conclude that relatively subtle changes in molecular structure may have a significant impact on a membrane's response to its environment.
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Oro , Fosfolípidos , 1,2-Dipalmitoilfosfatidilcolina , Dimiristoilfosfatidilcolina , Membrana Dobles de Lípidos , Estructura Molecular , Espectrofotometría InfrarrojaRESUMEN
Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery. Despite a clear heritable component, the genetic aetiology leading to non-syndromic MVP has remained elusive. Four affected individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of the linked interval on chromosome 11. We report a missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds), that segregates with MVP in the family. Morpholino knockdown of the zebrafish homologue dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 messenger RNA with the familial mutation. Further genetic studies identified two additional families in which a second deleterious DCHS1 mutation segregates with MVP. Both DCHS1 mutations reduce protein stability as demonstrated in zebrafish, cultured cells and, notably, in mitral valve interstitial cells (MVICs) obtained during mitral valve repair surgery of a proband. Dchs1(+/-) mice had prolapse of thickened mitral leaflets, which could be traced back to developmental errors in valve morphogenesis. DCHS1 deficiency in MVP patient MVICs, as well as in Dchs1(+/-) mouse MVICs, result in altered migration and cellular patterning, supporting these processes as aetiological underpinnings for the disease. Understanding the role of DCHS1 in mitral valve development and MVP pathogenesis holds potential for therapeutic insights for this very common disease.
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Cadherinas/genética , Cadherinas/metabolismo , Prolapso de la Válvula Mitral/genética , Prolapso de la Válvula Mitral/patología , Mutación/genética , Animales , Tipificación del Cuerpo/genética , Proteínas Relacionadas con las Cadherinas , Cadherinas/deficiencia , Movimiento Celular/genética , Cromosomas Humanos Par 11/genética , Femenino , Humanos , Masculino , Ratones , Válvula Mitral/anomalías , Válvula Mitral/embriología , Válvula Mitral/patología , Válvula Mitral/cirugía , Linaje , Fenotipo , Estabilidad Proteica , ARN Mensajero/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
RATIONALE: Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are a readily available, robustly reproducible, and physiologically appropriate human cell source for cardiac disease modeling, drug discovery, and toxicity screenings in vitro. However, unlike adult myocardial cells in vivo, hPSC-CMs cultured in vitro maintain an immature metabolic phenotype, where majority of ATP is produced through aerobic glycolysis instead of oxidative phosphorylation in the mitochondria. Little is known about the underlying signaling pathways controlling hPSC-CMs' metabolic and functional maturation. OBJECTIVE: To define the molecular pathways controlling cardiomyocytes' metabolic pathway selections and improve cardiomyocyte metabolic and functional maturation. METHODS AND RESULTS: We cultured hPSC-CMs in different media compositions including glucose-containing media, glucose-containing media supplemented with fatty acids, and glucose-free media with fatty acids as the primary carbon source. We found that cardiomyocytes cultured in the presence of glucose used primarily aerobic glycolysis and aberrantly upregulated HIF1α (hypoxia-inducible factor 1α) and its downstream target lactate dehydrogenase A. Conversely, glucose deprivation promoted oxidative phosphorylation and repressed HIF1α. Small molecule inhibition of HIF1α or lactate dehydrogenase A resulted in a switch from aerobic glycolysis to oxidative phosphorylation. Likewise, siRNA inhibition of HIF1α stimulated oxidative phosphorylation while inhibiting aerobic glycolysis. This metabolic shift was accompanied by an increase in mitochondrial content and cellular ATP levels. Furthermore, functional gene expressions, sarcomere length, and contractility were improved by HIF1α/lactate dehydrogenase A inhibition. CONCLUSIONS: We show that under standard culture conditions, the HIF1α-lactate dehydrogenase A axis is aberrantly upregulated in hPSC-CMs, preventing their metabolic maturation. Chemical or siRNA inhibition of this pathway results in an appropriate metabolic shift from aerobic glycolysis to oxidative phosphorylation. This in turn improves metabolic and functional maturation of hPSC-CMs. These findings provide key insight into molecular control of hPSC-CMs' metabolism and may be used to generate more physiologically mature cardiomyocytes for drug screening, disease modeling, and therapeutic purposes.
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Aminoquinolinas/farmacología , Diferenciación Celular/efectos de los fármacos , Disulfuros/farmacología , Metabolismo Energético/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Alcaloides Indólicos/farmacología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Mitocondrias Cardíacas/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Sulfonamidas/farmacología , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Glucólisis/efectos de los fármacos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Células Madre Pluripotentes Inducidas/enzimología , L-Lactato Deshidrogenasa/metabolismo , Masculino , Ratones Endogámicos C57BL , Mitocondrias Cardíacas/enzimología , Mitocondrias Cardíacas/genética , Miocitos Cardíacos/enzimología , Fosforilación Oxidativa/efectos de los fármacos , Fenotipo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
RATIONALE: Cardiac pacing is a critical technology for the treatment of arrhythmia and heart failure. The impact of specific pacing strategies on myocardial function is an area of intense research and high clinical significance. Mouse models have proven extremely useful for probing mechanisms of heart disease, but there is currently no reliable technology for long-term pacing in the mouse. OBJECTIVE: We sought to develop a device for long-term pacing studies in mice. We evaluated the device for (1) treating third-degree atrioventricular block after macrophage depletion, (2) ventricular pacing-induced cardiomyopathy, and (3) high-rate atrial pacing. METHODS AND RESULTS: We developed a mouse pacemaker by refashioning a 26 mm×6.7 mm clinical device powered by a miniaturized, highly efficient battery. The electrode was fitted with a single flexible lead, and custom software extended the pacing rate to up to 1200 bpm. The wirelessly programmable device was implanted in the dorsal subcutaneous space of 39 mice. The tunneled lead was passed through a left thoracotomy incision and attached to the epicardial surface of the apex (for ventricular pacing) or the left atrium (for atrial pacing). Mice tolerated the implantation and both long-term atrial and ventricular pacing over weeks. We then validated the pacemaker's suitability for the treatment of atrioventricular block after macrophage depletion in Cd11b DTR mice. Ventricular pacing increased the heart rate from 313±59 to 550 bpm ( P<0.05). In addition, we characterized tachypacing-induced cardiomyopathy in mice. Four weeks of ventricular pacing resulted in reduced left ventricular function, fibrosis, and an increased number of cardiac leukocytes and endothelial activation. Finally, we demonstrated the feasibility of chronic atrial pacing at 1200 bpm. CONCLUSIONS: Long-term pacing with a fully implantable, programmable, and battery-powered device enables previously impossible investigations of arrhythmia and heart failure in the mouse.
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Arritmias Cardíacas/fisiopatología , Marcapaso Artificial , Telemetría/métodos , Animales , Electrodos Implantados , Diseño de Equipo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Miniaturización , Programas Informáticos , TiempoRESUMEN
Fibromuscular dysplasia (FMD) is a nonatherosclerotic vascular disease leading to stenosis, dissection and aneurysm affecting mainly the renal and cerebrovascular arteries. FMD is often an underdiagnosed cause of hypertension and stroke, has higher prevalence in females (~80%) but its pathophysiology is unclear. We analyzed ~26K common variants (MAF>0.05) generated by exome-chip arrays in 249 FMD patients and 689 controls. We replicated 13 loci (P<10-4) in 402 cases and 2,537 controls and confirmed an association between FMD and a variant in the phosphatase and actin regulator 1 gene (PHACTR1). Three additional case control cohorts including 512 cases and 669 replicated this result and overall reached the genomic level of significance (OR = 1.39, P = 7.4×10-10, 1,154 cases and 3,895 controls). The top variant, rs9349379, is intronic to PHACTR1, a risk locus for coronary artery disease, migraine, and cervical artery dissection. The analyses of geometrical parameters of carotids from ~2,500 healthy volunteers indicate higher intima media thickness (P = 1.97×10-4) and wall to lumen ratio (P = 0.002) in rs9349379-A carriers, suggesting indices of carotid hypertrophy previously described in carotids of FMD patients. Immunohistochemistry detected PHACTR1 in endothelium and smooth muscle cells of FMD and normal human carotids. The expression of PHACTR1 by genotypes in primary human fibroblasts showed higher expression in rs9349379-A carriers (N = 86, P = 0.003). Phactr1 knockdown in zebrafish resulted in dilated vessels indicating subtle impaired vascular development. We report the first susceptibility locus for FMD and provide evidence for a complex genetic pattern of inheritance and indices of shared pathophysiology between FMD and other cardiovascular and neurovascular diseases.
Asunto(s)
Displasia Fibromuscular/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Proteínas de Microfilamentos/genética , Animales , Arterias/metabolismo , Arterias/patología , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Grosor Intima-Media Carotídeo , Modelos Animales de Enfermedad , Exoma/genética , Femenino , Displasia Fibromuscular/patología , Regulación de la Expresión Génica , Genotipo , Humanos , Hipertensión/genética , Hipertensión/patología , Masculino , Proteínas de Microfilamentos/biosíntesis , Miocitos del Músculo Liso , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/patología , Pez Cebra/genéticaRESUMEN
Aims: Filamin-A (FLNA) was identified as the first gene of non-syndromic mitral valve dystrophy (FLNA-MVD). We aimed to assess the phenotype of FLNA-MVD and its impact on prognosis. Methods and results: We investigated the disease in 246 subjects (72 mutated) from four FLNA-MVD families harbouring three different FLNA mutations. Phenotype was characterized by a comprehensive echocardiography focusing on mitral valve apparatus in comparison with control relatives. In this X-linked disease valves lesions were severe in men and moderate in women. Most men had classical features of mitral valve prolapse (MVP), but without chordal rupture. By contrast to regular MVP, mitral leaflet motion was clearly restricted in diastole and papillary muscles position was closer to mitral annulus. Valvular abnormalities were similar in the four families, in adults and young patients from early childhood suggestive of a developmental disease. In addition, mitral valve lesions worsened over time as encountered in degenerative conditions. Polyvalvular involvement was frequent in males and non-diagnostic forms frequent in females. Overall survival was moderately impaired in men (P = 0.011). Cardiac surgery rate (mainly valvular) was increased (33.3 ± 9.8 vs. 5.0 ± 4.9%, P < 0.0001; hazard ratio 10.5 [95% confidence interval: 2.9-37.9]) owing mainly to a lifetime increased risk in men (76.8 ± 14.1 vs. 9.1 ± 8.7%, P < 0.0001). Conclusion: FLNA-MVD is a developmental and degenerative disease with complex phenotypic expression which can influence patient management. FLNA-MVD has unique features with both MVP and paradoxical restricted motion in diastole, sub-valvular mitral apparatus impairment and polyvalvular lesions in males. FLNA-MVD conveys a substantial lifetime risk of valve surgery in men.
Asunto(s)
Filaminas/genética , Prolapso de la Válvula Mitral/genética , Prolapso de la Válvula Mitral/patología , Válvula Mitral/patología , Adolescente , Adulto , Ecocardiografía , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Válvula Mitral/diagnóstico por imagen , Mutación/genética , Fenotipo , Pronóstico , Estudios Retrospectivos , Factores de Riesgo , Adulto JovenRESUMEN
RATIONALE: More than 25 million individuals have heart failure worldwide, with ≈4000 patients currently awaiting heart transplantation in the United States. Donor organ shortage and allograft rejection remain major limitations with only ≈2500 hearts transplanted each year. As a theoretical alternative to allotransplantation, patient-derived bioartificial myocardium could provide functional support and ultimately impact the treatment of heart failure. OBJECTIVE: The objective of this study is to translate previous work to human scale and clinically relevant cells for the bioengineering of functional myocardial tissue based on the combination of human cardiac matrix and human induced pluripotent stem cell-derived cardiomyocytes. METHODS AND RESULTS: To provide a clinically relevant tissue scaffold, we translated perfusion-decellularization to human scale and obtained biocompatible human acellular cardiac scaffolds with preserved extracellular matrix composition, architecture, and perfusable coronary vasculature. We then repopulated this native human cardiac matrix with cardiomyocytes derived from nontransgenic human induced pluripotent stem cells and generated tissues of increasing 3-dimensional complexity. We maintained such cardiac tissue constructs in culture for 120 days to demonstrate definitive sarcomeric structure, cell and matrix deformation, contractile force, and electrical conduction. To show that functional myocardial tissue of human scale can be built on this platform, we then partially recellularized human whole-heart scaffolds with human induced pluripotent stem cell-derived cardiomyocytes. Under biomimetic culture, the seeded constructs developed force-generating human myocardial tissue and showed electrical conductivity, left ventricular pressure development, and metabolic function. CONCLUSIONS: Native cardiac extracellular matrix scaffolds maintain matrix components and structure to support the seeding and engraftment of human induced pluripotent stem cell-derived cardiomyocytes and enable the bioengineering of functional human myocardial-like tissue of multiple complexities.