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1.
Cell ; 153(1): 216-27, 2013 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-23540699

RESUMEN

Phospholipase Cε (PLCε) is a multifunctional enzyme implicated in cardiovascular, pancreatic, and inflammatory functions. Here we show that conditional deletion of PLCε in mouse cardiac myocytes protects from stress-induced pathological hypertrophy. PLCε small interfering RNA (siRNA) in ventricular myocytes decreases endothelin-1 (ET-1)-dependent elevation of nuclear calcium and activation of nuclear protein kinase D (PKD). PLCε scaffolded to muscle-specific A kinase-anchoring protein (mAKAP), along with PKCε and PKD, localizes these components at or near the nuclear envelope, and this complex is required for nuclear PKD activation. Phosphatidylinositol 4-phosphate (PI4P) is identified as a perinuclear substrate in the Golgi apparatus for mAKAP-scaffolded PLCε. We conclude that perinuclear PLCε, scaffolded to mAKAP in cardiac myocytes, responds to hypertrophic stimuli to generate diacylglycerol (DAG) from PI4P in the Golgi apparatus, in close proximity to the nuclear envelope, to regulate activation of nuclear PKD and hypertrophic signaling pathways.


Asunto(s)
Cardiomegalia/metabolismo , Cardiomegalia/patología , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoinositido Fosfolipasa C/metabolismo , Animales , Aorta/patología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Aparato de Golgi/metabolismo , Corazón , Ventrículos Cardíacos/citología , Masculino , Ratones , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Membrana Nuclear/metabolismo , Fosfoinositido Fosfolipasa C/genética , Ratas , Transducción de Señal
2.
Circ Res ; 131(12): 980-1000, 2022 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-36367103

RESUMEN

BACKGROUND: RBPs (RNA-binding proteins) perform indispensable functions in the post-transcriptional regulation of gene expression. Numerous RBPs have been implicated in cardiac development or physiology based on gene knockout studies and the identification of pathogenic RBP gene mutations in monogenic heart disorders. The discovery and characterization of additional RBPs performing indispensable functions in the heart will advance basic and translational cardiovascular research. METHODS: We performed a differential expression screen in zebrafish embryos to identify genes enriched in nkx2.5-positive cardiomyocytes or cardiopharyngeal progenitors compared to nkx2.5-negative cells from the same embryos. We investigated the myocardial-enriched gene RNA-binding protein with multiple splicing (variants) 2 [RBPMS2)] by generating and characterizing rbpms2 knockout zebrafish and human cardiomyocytes derived from RBPMS2-deficient induced pluripotent stem cells. RESULTS: We identified 1848 genes enriched in the nkx2.5-positive population. Among the most highly enriched genes, most with well-established functions in the heart, we discovered the ohnologs rbpms2a and rbpms2b, which encode an evolutionarily conserved RBP. Rbpms2 localizes selectively to cardiomyocytes during zebrafish heart development and strong cardiomyocyte expression persists into adulthood. Rbpms2-deficient embryos suffer from early cardiac dysfunction characterized by reduced ejection fraction. The functional deficit is accompanied by myofibril disarray, altered calcium handling, and differential alternative splicing events in mutant cardiomyocytes. These phenotypes are also observed in RBPMS2-deficient human cardiomyocytes, indicative of conserved molecular and cellular function. RNA-sequencing and comparative analysis of genes mis-spliced in RBPMS2-deficient zebrafish and human cardiomyocytes uncovered a conserved network of 29 ortholog pairs that require RBPMS2 for alternative splicing regulation, including RBFOX2, SLC8A1, and MYBPC3. CONCLUSIONS: Our study identifies RBPMS2 as a conserved regulator of alternative splicing, myofibrillar organization, and calcium handling in zebrafish and human cardiomyocytes.


Asunto(s)
Calcio , Miocardio , Proteínas de Unión al ARN , Proteínas de Pez Cebra , Animales , Humanos , Calcio/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Proteínas Represoras/metabolismo , Factores de Empalme de ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
3.
J Am Pharm Assoc (2003) ; 63(3): 939-945, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37024375

RESUMEN

BACKGROUND: Pharmacogenomics (PGx) is used as a medication management strategy by a small but growing number of institutions. PGx allows prescribers to individually treat patients concordant with their genes. Recent litigation for preventable PGx-mediated adverse events highlights the need to accelerate PGx implementation for patient safety. Genetic variations cause drug metabolism, transport, and target changes, affecting medication response and tolerability. PGx testing often consists of targeted testing aimed at specific gene-drug pairs or disease states. Conversely, expanded panel testing can evaluate all known actionable gene-drug interactions, enhancing proactive clarity regarding patient response. OBJECTIVES: Evaluate the divergence of targeted PGx testing with a single gene-drug pair test (cardiac), a two-gene panel, and a focused psychiatric panel compared to expanded PGx testing. METHODS: An expanded PGx panel (≥25 genes) was compared to a single gene-drug pair test of CYP2C19/clopidogrel, a dual gene test of CYP2C19/CYP2D6, a 7-gene psychiatric list, and a 14-gene psychiatric panel to inform specific depression and pain management drugs. The expanded panel provided a baseline to evaluate total PGx variations compared to those possibly missed by targeted testing. RESULTS: Targeted testing did not identify up to 95% of total PGx gene-drug interactions discovered. The expanded panel reported all gene-drug interactions for any medication with Clinical Pharmacogenomics Implementation Consortium (CPIC) guidance or U.S. Food and Drug Administration (FDA) labeling for that gene. Single gene CYP2C19/clopidogrel testing missed or did not report on ∼95% of total interactions, CYP2C19/CYP2D6 testing missed or did not report ∼89%, and the 14-gene panel missed or did not report on ∼73%. The 7-gene list missed ∼20% of discovered potential PGx interactions but was not designed to identify gene-drug interactions. CONCLUSIONS: Targeted PGx testing for limited genes or by specialty may miss or not report significant portions of PGx gene-drug interactions. This can lead to potential patient harm from the missed interactions and subsequent failed therapies and/or adverse reactions.


Asunto(s)
Farmacogenética , Humanos , Clopidogrel , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2D6/genética , Pruebas Genéticas
4.
J Am Pharm Assoc (2003) ; 63(1): 188-192, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36243653

RESUMEN

BACKGROUND: Pharmacogenomics (PGx) is an emerging field. Many drug-gene interactions are known but not yet routinely addressed in clinical practice. Therefore, there is a significant gap in care, necessitating development of implementation strategies. OBJECTIVE: The objective of the study was to assess the impact of implementing a PGx practice model which incorporates comprehensive pharmacogenomic risk evaluation, testing and medication optimization administered by 7 PGx-certified ambulatory care pharmacists embedded across 30 primary care clinic sites. METHODS: Pharmacogenomic services were implemented in 30 primary care clinics within the Cincinnati, Ohio area. Patients are identified for pharmacogenomic testing using a clinical decision support tool (CDST) that is fully integrated in the electronic medical record (EMR) or by provider designation (e.g., psychotropic drug failure). Pharmacogenomic testing is performed via buccal swab using standardized clinic processes. Discrete data results are returned directly into the EMR/CDST for review by PGx-certified ambulatory care pharmacists. Recommendations and prescriptive changes are then discussed and implemented as a collaborative effort between pharmacist, primary care provider, specialists, and patient. RESULTS: A total of 422 unique interactions were assessed by the embedded ambulatory care PGx pharmacists (N = 7) during this interim analysis. About half (213) were pharmacogenomic interactions, and of these, 124 were actionable. When an intervention was actionable, 82% of the time a change in medication was recommended. The underlying reasons for recommending therapy alterations were most commonly ineffective therapy (43%), adverse drug reaction prevented (34%), or adverse drug reaction observed (13%). CONCLUSION: Variations in drug metabolism, response, and tolerability can negatively impact patient outcomes across many disease states and treatment specialties. Incorporation of pharmacogenomic testing with accessible clinical decision support into the team-based care model allows for a truly comprehensive review and optimization of medications. Our initial analysis suggests that comprehensive PGx testing should be considered to enhance medication safety and efficacy in at-risk patients.


Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Farmacogenética , Humanos , Farmacogenética/métodos , Hospitales Comunitarios , Pruebas de Farmacogenómica , Atención Primaria de Salud
5.
Development ; 146(19)2019 10 09.
Artículo en Inglés | MEDLINE | ID: mdl-31427288

RESUMEN

Deciphering the genetic and epigenetic regulation of cardiomyocyte proliferation in organisms that are capable of robust cardiac renewal, such as zebrafish, represents an attractive inroad towards regenerating the human heart. Using integrated high-throughput transcriptional and chromatin analyses, we have identified a strong association between H3K27me3 deposition and reduced sarcomere and cytoskeletal gene expression in proliferative cardiomyocytes following cardiac injury in zebrafish. To move beyond an association, we generated an inducible transgenic strain expressing a mutant version of histone 3, H3.3K27M, that inhibits H3K27me3 catalysis in cardiomyocytes during the regenerative window. Hearts comprising H3.3K27M-expressing cardiomyocytes fail to regenerate, with wound edge cells showing heightened expression of structural genes and prominent sarcomeres. Although cell cycle re-entry was unperturbed, cytokinesis and wound invasion were significantly compromised. Collectively, our study identifies H3K27me3-mediated silencing of structural genes as requisite for zebrafish heart regeneration and suggests that repression of similar structural components in the border zone of an infarcted human heart might improve its regenerative capacity.


Asunto(s)
Silenciador del Gen , Corazón/fisiología , Histonas/metabolismo , Lisina/metabolismo , Regeneración/fisiología , Pez Cebra/genética , Pez Cebra/fisiología , Animales , Proliferación Celular , Citocinesis , Citoesqueleto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Metilación , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Sarcómeros/metabolismo
6.
Nature ; 534(7609): 700-4, 2016 06 30.
Artículo en Inglés | MEDLINE | ID: mdl-27357797

RESUMEN

Many organs are composed of complex tissue walls that are structurally organized to optimize organ function. In particular, the ventricular myocardial wall of the heart comprises an outer compact layer that concentrically encircles the ridge-like inner trabecular layer. Although disruption in the morphogenesis of this myocardial wall can lead to various forms of congenital heart disease and non-compaction cardiomyopathies, it remains unclear how embryonic cardiomyocytes assemble to form ventricular wall layers of appropriate spatial dimensions and myocardial mass. Here we use advanced genetic and imaging tools in zebrafish to reveal an interplay between myocardial Notch and Erbb2 signalling that directs the spatial allocation of myocardial cells to their proper morphological positions in the ventricular wall. Although previous studies have shown that endocardial Notch signalling non-cell-autonomously promotes myocardial trabeculation through Erbb2 and bone morphogenetic protein (BMP) signalling, we discover that distinct ventricular cardiomyocyte clusters exhibit myocardial Notch activity that cell-autonomously inhibits Erbb2 signalling and prevents cardiomyocyte sprouting and trabeculation. Myocardial-specific Notch inactivation leads to ventricles of reduced size and increased wall thickness because of excessive trabeculae, whereas widespread myocardial Notch activity results in ventricles of increased size with a single-cell-thick wall but no trabeculae. Notably, this myocardial Notch signalling is activated non-cell-autonomously by neighbouring Erbb2-activated cardiomyocytes that sprout and form nascent trabeculae. Thus, these findings support an interactive cellular feedback process that guides the assembly of cardiomyocytes to morphologically create the ventricular myocardial wall and more broadly provide insight into the cellular dynamics of how diverse cell lineages organize to create form.


Asunto(s)
Ventrículos Cardíacos/citología , Ventrículos Cardíacos/embriología , Morfogénesis , Miocitos Cardíacos/citología , Pez Cebra/embriología , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Linaje de la Célula , Retroalimentación Fisiológica , Ventrículos Cardíacos/anatomía & histología , Proteína Jagged-2 , Miocitos Cardíacos/metabolismo , Tamaño de los Órganos , Organogénesis , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/metabolismo , Receptores Notch/antagonistas & inhibidores , Receptores Notch/metabolismo , Transducción de Señal , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismo
7.
J Mol Cell Cardiol ; 156: 33-44, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33781820

RESUMEN

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N')-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C∆C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N'-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N'-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.


Asunto(s)
Proteínas Portadoras/genética , Contracción Miocárdica/genética , Miocardio/metabolismo , Dominios y Motivos de Interacción de Proteínas , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Corazón/diagnóstico por imagen , Imagen por Resonancia Magnética , Ratones , Ratones Transgénicos , Miocitos Cardíacos/metabolismo , Fosforilación , Sarcómeros/metabolismo
8.
Circulation ; 141(12): 1001-1026, 2020 03 24.
Artículo en Inglés | MEDLINE | ID: mdl-32202936

RESUMEN

Heart failure with preserved ejection fraction (HFpEF), a major public health problem that is rising in prevalence, is associated with high morbidity and mortality and is considered to be the greatest unmet need in cardiovascular medicine today because of a general lack of effective treatments. To address this challenging syndrome, the National Heart, Lung, and Blood Institute convened a working group made up of experts in HFpEF and novel research methodologies to discuss research gaps and to prioritize research directions over the next decade. Here, we summarize the discussion of the working group, followed by key recommendations for future research priorities. There was uniform recognition that HFpEF is a highly integrated, multiorgan, systemic disorder requiring a multipronged investigative approach in both humans and animal models to improve understanding of mechanisms and treatment of HFpEF. It was recognized that advances in the understanding of basic mechanisms and the roles of inflammation, macrovascular and microvascular dysfunction, fibrosis, and tissue remodeling are needed and ideally would be obtained from (1) improved animal models, including large animal models, which incorporate the effects of aging and associated comorbid conditions; (2) repositories of deeply phenotyped physiological data and human tissue, made accessible to researchers to enhance collaboration and research advances; and (3) novel research methods that take advantage of computational advances and multiscale modeling for the analysis of complex, high-density data across multiple domains. The working group emphasized the need for interactions among basic, translational, clinical, and epidemiological scientists and across organ systems and cell types, leveraging different areas or research focus, and between research centers. A network of collaborative centers to accelerate basic, translational, and clinical research of pathobiological mechanisms and treatment strategies in HFpEF was discussed as an example of a strategy to advance research progress. This resource would facilitate comprehensive, deep phenotyping of a multicenter HFpEF patient cohort with standardized protocols and a robust biorepository. The research priorities outlined in this document are meant to stimulate scientific advances in HFpEF by providing a road map for future collaborative investigations among a diverse group of scientists across multiple domains.


Asunto(s)
Insuficiencia Cardíaca/epidemiología , Investigación/normas , Humanos , National Heart, Lung, and Blood Institute (U.S.) , Volumen Sistólico , Estados Unidos
9.
Anal Chem ; 93(26): 9041-9048, 2021 07 06.
Artículo en Inglés | MEDLINE | ID: mdl-34165299

RESUMEN

Measurements of protein higher order structure (HOS) provide important information on stability, potency, efficacy, immunogenicity, and biosimilarity of biopharmaceuticals, with a significant number of techniques and methods available to perform these measurements. The comparison of the analytical performance of HOS methods and the standardization of the results is, however, not a trivial task, due to the lack of reference protocols and reference measurement procedures. Here, we developed a protocol to structurally alter and compare samples of somatropin, a recombinant biotherapeutic, and describe the results obtained by using a number of techniques, methods and in different laboratories. This, with the final aim to provide tools and generate a pool of data to compare and benchmark analytical platforms and define method sensitivity to structural changes. Changes in somatropin HOS, induced by the presence of zinc at increasing concentrations, were observed, both globally and at more localized resolution, across many of the methods utilized in this study and with different sensitivities, suggesting the suitability of the protocol to improve understanding of inter- and cross-platform measurement comparability and assess analytical performance as appropriate.


Asunto(s)
Laboratorios , Estándares de Referencia
10.
J Appl Microbiol ; 130(3): 971-981, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-32743931

RESUMEN

AIMS: This study compared the bag-mediated filtration system (BMFS) and standard WHO two-phase separation methods for poliovirus (PV) environmental surveillance, examined factors impacting PV detection and monitored Sabin-like (SL) PV type 2 presence with withdrawal of oral polio vaccine type 2 (OPV2) in April 2016. METHODS AND RESULTS: Environmental samples were collected in Nairobi, Kenya (Sept 2015-Feb 2017), concentrated via BMFS and two-phase separation methods, then assayed using the WHO PV isolation algorithm and intratypic differentiation diagnostic screening kit. SL1, SL2 and SL3 were detected at higher rates in BMFS than two-phase samples (P < 0·05). In BMFS samples, SL PV detection did not significantly differ with volume filtered, filtration time or filter shipment time (P > 0·05), while SL3 was detected less frequently with higher shipment temperatures (P = 0·027). SL2 was detected more frequently before OPV2 withdrawal in BMFS and two-phase samples (P < 1 × 10-5 ). CONCLUSIONS: Poliovirus was detected at higher rates with the BMFS, a method that includes a secondary concentration step, than using the standard WHO two-phase method. SL2 disappearance from the environment was commensurate with OPV2 withdrawal. SIGNIFICANCE AND IMPACT OF THE STUDY: The BMFS offers comparable or improved PV detection under the conditions in this study, relative to the two-phase method.


Asunto(s)
Monitoreo del Ambiente/métodos , Filtración/métodos , Poliovirus/aislamiento & purificación , Filtración/normas , Humanos , Kenia/epidemiología , Poliomielitis/epidemiología , Poliomielitis/virología , Vacuna Antipolio Oral/aislamiento & purificación , Serogrupo , Aguas del Alcantarillado/virología
11.
Nucleic Acids Res ; 47(21): e138, 2019 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-31529053

RESUMEN

To understand the molecular pathogenesis of human disease, precision analyses to define alterations within and between disease-associated cell populations are desperately needed. Single-cell genomics represents an ideal platform to enable the identification and comparison of normal and diseased transcriptional cell populations. We created cellHarmony, an integrated solution for the unsupervised analysis, classification, and comparison of cell types from diverse single-cell RNA-Seq datasets. cellHarmony efficiently and accurately matches single-cell transcriptomes using a community-clustering and alignment strategy to compute differences in cell-type specific gene expression over potentially dozens of cell populations. Such transcriptional differences are used to automatically identify distinct and shared gene programs among cell-types and identify impacted pathways and transcriptional regulatory networks to understand the impact of perturbations at a systems level. cellHarmony is implemented as a python package and as an integrated workflow within the software AltAnalyze. We demonstrate that cellHarmony has improved or equivalent performance to alternative label projection methods, is able to identify the likely cellular origins of malignant states, stratify patients into clinical disease subtypes from identified gene programs, resolve discrete disease networks impacting specific cell-types, and illuminate therapeutic mechanisms. Thus, this approach holds tremendous promise in revealing the molecular and cellular origins of complex disease.


Asunto(s)
Algoritmos , Perfilación de la Expresión Génica/métodos , Genómica/métodos , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Transcriptoma/genética , Bases de Datos Genéticas , Conjuntos de Datos como Asunto , Humanos
12.
Development ; 144(24): 4616-4624, 2017 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-29061637

RESUMEN

During mammalian embryogenesis, cardiac progenitor cells constituting the second heart field (SHF) give rise to the right ventricle and primitive outflow tract (OFT). In zebrafish, previous lineage-tracing and mutant analyses suggested that SHF ventricular and OFT progenitors co-migrate to the arterial pole of the zebrafish heart tube soon after their specification in the nkx2.5+ field of anterior lateral plate mesoderm (ALPM). Using additional prospective lineage tracing, we demonstrate that while SHF ventricular progenitors migrate directly to the arterial pole, OFT progenitors become temporarily sequestered in the mesodermal cores of pharyngeal arch 2 (PA2), where they downregulate nkx2.5 expression. While there, they intermingle with precursors for PA2-derived head muscles (HMs) and hypobranchial artery endothelium, which we demonstrate are co-specified with SHF progenitors in the nkx2.5+ ALPM. Soon after their sequestration in PA2, OFT progenitors migrate to the arterial pole of the heart and differentiate into OFT lineages. Lastly, we demonstrate that SHF ventricular and OFT progenitors exhibit unique sensitivities to a mutation in fgf8a Our data highlight novel aspects of SHF, OFT and HM development in zebrafish that will inform mechanistic interpretations of cardiopharyngeal phenotypes in zebrafish models of human congenital disorders.


Asunto(s)
Cardiopatías Congénitas/embriología , Ventrículos Cardíacos/embriología , Células Madre/citología , Pez Cebra/embriología , Animales , Región Branquial/metabolismo , Linaje de la Célula , Movimiento Celular/fisiología , Factores de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Ventrículos Cardíacos/metabolismo , Proteína Homeótica Nkx-2.5/biosíntesis , Mesodermo/metabolismo , Miocardio/citología , Miocardio/metabolismo , Transducción de Señal/genética , Pez Cebra/genética , Proteínas de Pez Cebra/biosíntesis , Proteínas de Pez Cebra/genética
13.
Circ Res ; 123(12): 1285-1297, 2018 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-30566042

RESUMEN

RATIONALE: Hypertrophic cardiomyopathy occurs with a frequency of about 1 in 500 people. Approximately 30% of those affected carry mutations within the gene encoding cMyBP-C (cardiac myosin binding protein C). Cardiac stress, as well as cMyBP-C mutations, can trigger production of a 40kDa truncated fragment derived from the amino terminus of cMyBP-C (Mybpc340kDa). Expression of the 40kDa fragment in mouse cardiomyocytes leads to hypertrophy, fibrosis, and heart failure. Here we use genetic approaches to establish a causal role for excessive myofibroblast activation in a slow, progressive genetic cardiomyopathy-one that is driven by a cardiomyocyte-intrinsic genetic perturbation that models an important human disease. OBJECTIVE: TGFß (transforming growth factor-ß) signaling is implicated in a variety of fibrotic processes, and the goal of this study was to define the role of myofibroblast TGFß signaling during chronic Mybpc340kDa expression. METHODS AND RESULTS: To specifically block TGFß signaling only in the activated myofibroblasts in Mybpc340kDa transgenic mice and quadruple compound mutant mice were generated, in which the TGFß receptor II (TßRII) alleles ( Tgfbr2) were ablated using the periostin ( Postn) allele, myofibroblast-specific, tamoxifen-inducible Cre ( Postnmcm) gene-targeted line. Tgfbr2 was ablated either early or late during pathological fibrosis. Early myofibroblast-specific Tgfbr2 ablation during the fibrotic response reduced cardiac fibrosis, alleviated cardiac hypertrophy, preserved cardiac function, and increased lifespan of the Mybpc340kDa transgenic mice. Tgfbr2 ablation late in the pathological process reduced cardiac fibrosis, preserved cardiac function, and prolonged Mybpc340kDa mouse survival but failed to reverse cardiac hypertrophy. CONCLUSIONS: Fibrosis and cardiac dysfunction induced by cardiomyocyte-specific expression of Mybpc340kDa were significantly decreased by Tgfbr2 ablation in the myofibroblast. Surprisingly, preexisting fibrosis was partially reversed if the gene was ablated subsequent to fibrotic deposition, suggesting that continued TGFß signaling through the myofibroblasts was needed to maintain the heart fibrotic response to a chronic, disease-causing cardiomyocyte-only stimulus.


Asunto(s)
Cardiomiopatía Hipertrófica/metabolismo , Proteínas Portadoras/genética , Miocitos Cardíacos/metabolismo , Miofibroblastos/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Transducción de Señal , Animales , Cardiomiopatía Hipertrófica/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Ratones , Mutación , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética
14.
Circulation ; 137(20): 2152-2165, 2018 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-29348261

RESUMEN

BACKGROUND: Defining conserved molecular pathways in animal models of successful cardiac regeneration could yield insight into why adult mammals have inadequate cardiac regeneration after injury. Insight into the transcriptomic landscape of early cardiac regeneration from model organisms will shed light on evolutionarily conserved pathways in successful cardiac regeneration. METHODS: Here we describe a cross-species transcriptomic screen in 3 model organisms for cardiac regeneration: axolotl, neonatal mice, and zebrafish. Apical resection to remove ≈10% to 20% of ventricular mass was carried out in these model organisms. RNA-sequencing analysis was performed on the hearts harvested at 3 time points: 12, 24, and 48 hours after resection. Sham surgery was used as internal control. RESULTS: Genes associated with inflammatory processes were found to be upregulated in a conserved manner. Complement receptors (activated by complement components, part of the innate immune system) were found to be highly upregulated in all 3 species. This approach revealed induction of gene expression for complement 5a receptor 1 in the regenerating hearts of zebrafish, axolotls, and mice. Inhibition of complement 5a receptor 1 significantly attenuated the cardiomyocyte proliferative response to heart injury in all 3 species. Furthermore, after left ventricular apical resection, the cardiomyocyte proliferative response was diminished in mice with genetic deletion of complement 5a receptor 1. CONCLUSIONS: These data reveal that activation of complement 5a receptor 1 mediates an evolutionarily conserved response that promotes cardiomyocyte proliferation after cardiac injury and identify complement pathway activation as a common pathway of successful heart regeneration.


Asunto(s)
Evolución Molecular , Corazón/fisiología , Receptor de Anafilatoxina C5a/metabolismo , Regeneración/fisiología , Ambystoma mexicanum , Animales , Animales Recién Nacidos , Proliferación Celular , Perfilación de la Expresión Génica , Ontología de Genes , Ratones , Miocardio/patología , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Péptidos Cíclicos/farmacología , ARN/química , ARN/aislamiento & purificación , ARN/metabolismo , Receptor de Anafilatoxina C5a/antagonistas & inhibidores , Receptor de Anafilatoxina C5a/genética , Análisis de Secuencia de ARN , Troponina T/análisis , Pez Cebra
15.
Circulation ; 138(12): 1236-1252, 2018 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-29653926

RESUMEN

BACKGROUND: Fibronectin (FN) polymerization is necessary for collagen matrix deposition and is a key contributor to increased abundance of cardiac myofibroblasts (MFs) after cardiac injury. We hypothesized that interfering with FN polymerization or its genetic ablation in fibroblasts would attenuate MF and fibrosis and improve cardiac function after ischemia/reperfusion (I/R) injury. METHODS: Mouse and human MFs were used to assess the impact of the FN polymerization inhibitor (pUR4) in attenuating pathological cellular features such as proliferation, migration, extracellular matrix deposition, and associated mechanisms. To evaluate the therapeutic potential of inhibiting FN polymerization in vivo, wild-type mice received daily intraperitoneal injections of either pUR4 or control peptide (III-11C) immediately after cardiac surgery for 7 consecutive days. Mice were analyzed 7 days after I/R to assess MF markers and inflammatory cell infiltration or 4 weeks after I/R to evaluate long-term effects of FN inhibition on cardiac function and fibrosis. Furthermore, inducible, fibroblast-restricted, FN gene-ablated (Tcf21MerCreMer; Fnflox) mice were used to evaluate cell specificity of FN expression and polymerization in the heart. RESULTS: pUR4 administration on activated MFs reduced FN and collagen deposition into the extracellular matrix and attenuated cell proliferation, likely mediated through decreased c-myc signaling. pUR4 also ameliorated fibroblast migration accompanied by increased ß1 integrin internalization and reduced levels of phosphorylated focal adhesion kinase protein. In vivo, daily administration of pUR4 for 7 days after I/R significantly reduced MF markers and neutrophil infiltration. This treatment regimen also significantly attenuated myocardial dysfunction, pathological cardiac remodeling, and fibrosis up to 4 weeks after I/R. Last, inducible ablation of FN in fibroblasts after I/R resulted in significant functional cardioprotection with reduced hypertrophy and fibrosis. The addition of pUR4 to the FN-ablated mice did not confer further cardioprotection, suggesting that the salutary effects of inhibiting FN polymerization may be mediated largely through effects on FN secreted from the cardiac fibroblast lineage. CONCLUSIONS: Inhibiting FN polymerization or cardiac fibroblast gene expression attenuates pathological properties of MFs in vitro and ameliorates adverse cardiac remodeling and fibrosis in an in vivo model of heart failure. Interfering with FN polymerization may be a new therapeutic strategy for treating cardiac fibrosis and heart failure.


Asunto(s)
Fibronectinas/antagonistas & inhibidores , Insuficiencia Cardíaca/tratamiento farmacológico , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Miofibroblastos/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Función Ventricular Izquierda/efectos de los fármacos , Remodelación Ventricular/efectos de los fármacos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colágeno/metabolismo , Modelos Animales de Enfermedad , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrosis , Quinasa 1 de Adhesión Focal/metabolismo , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Humanos , Integrina beta1/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Miofibroblastos/metabolismo , Miofibroblastos/patología , Infiltración Neutrófila/efectos de los fármacos , Fosforilación , Polimerizacion , Transducción de Señal/efectos de los fármacos
16.
Am J Physiol Renal Physiol ; 316(6): F1293-F1298, 2019 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-31017009

RESUMEN

Fibrosis is a common feature of chronic kidney disease; however, no clinical therapies effectively target the progression of fibrosis. Inhibition of fibronectin polymerization with the small peptide pUR4 attenuates fibrosis in the liver and heart. Here, we show that pUR4 decreases renal fibrosis and tissue remodeling using a clinically relevant model of kidney injury, unilateral ischemia-reperfusion. This work highlights the benefits of inhibiting matrix polymerization, alone or in conjunction with cell-based therapies, as a novel approach to diminish the maladaptive responses to ischemic kidney injury that lead to chronic renal failure.


Asunto(s)
Lesión Renal Aguda/prevención & control , Matriz Extracelular/efectos de los fármacos , Fibronectinas/metabolismo , Riñón/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Daño por Reperfusión/prevención & control , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Animales , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Fibrosis , Riñón/metabolismo , Riñón/patología , Masculino , Ratones Endogámicos C57BL , Polimerizacion , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología
17.
Development ; 143(1): 113-22, 2016 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-26732840

RESUMEN

The vertebrate heart forms through successive phases of cardiomyocyte differentiation. Initially, cardiomyocytes derived from first heart field (FHF) progenitors assemble the linear heart tube. Thereafter, second heart field (SHF) progenitors differentiate into cardiomyocytes that are accreted to the poles of the heart tube over a well-defined developmental window. Although heart tube elongation deficiencies lead to life-threatening congenital heart defects, the variables controlling the initiation, rate and duration of myocardial accretion remain obscure. Here, we demonstrate that the AP-1 transcription factor, Fos-like antigen 2 (Fosl2), potentiates the rate of myocardial accretion from the zebrafish SHF. fosl2 mutants initiate accretion appropriately, but cardiomyocyte production is sluggish, resulting in a ventricular deficit coupled with an accumulation of SHF progenitors. Surprisingly, mutant embryos eventually correct the myocardial deficit by extending the accretion window. Overexpression of Fosl2 also compromises production of SHF-derived ventricular cardiomyocytes, a phenotype that is consistent with precocious depletion of the progenitor pool. Our data implicate Fosl2 in promoting the progenitor to cardiomyocyte transition and uncover the existence of regulatory mechanisms to ensure appropriate SHF-mediated cardiomyocyte contribution irrespective of embryonic stage.


Asunto(s)
Diferenciación Celular/fisiología , Antígeno 2 Relacionado con Fos/metabolismo , Corazón/embriología , Miocitos Cardíacos/citología , Factor de Transcripción AP-1/metabolismo , Pez Cebra/embriología , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Proliferación Celular/genética , Antígeno 2 Relacionado con Fos/biosíntesis , Antígeno 2 Relacionado con Fos/genética , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Cardiopatías Congénitas/genética , Miocardio/citología , Análisis de Secuencia de Proteína , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
18.
Pediatr Radiol ; 49(3): 332-341, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30607435

RESUMEN

BACKGROUND: Primary sclerosing cholangitis, autoimmune hepatitis and autoimmune sclerosing cholangitis are forms of chronic, progressive autoimmune liver disease (AILD) that can affect the pediatric population. OBJECTIVE: To determine whether quantitative MRI- and laboratory-based biomarkers are associated with conventional imaging findings of portal hypertension (radiologic portal hypertension) in children and young adults with AILD. MATERIALS AND METHODS: Forty-four patients with AILD enrolled in an institutional registry underwent a research abdominal MRI examination at 1.5 tesla (T). Five quantitative MRI techniques were performed: liver MR elastography, spleen MR elastography, liver iron-corrected T1 mapping, liver T2 mapping, and liver diffusion-weighted imaging (DWI, quantified as apparent diffusion coefficients). Two anatomical sequences were used to document splenomegaly, varices and ascites. We calculated aspartate aminotransferase (AST)-to-platelet ratio index (APRI) and fibrosis-4 (FIB-4) scores - laboratory-based biomarkers of liver fibrosis. We used receiver operating characteristic (ROC) curve analyses to establish the diagnostic performance of quantitative MRI and laboratory biomarkers for indicating the presence of radiologic portal hypertension. RESULTS: Twenty-three (52%) patients were male; mean age was 15.2±4.0 years. Thirteen (30%) patients had radiologic portal hypertension. Liver and spleen stiffness demonstrated the greatest diagnostic performance for indicating the presence of portal hypertension (area-under-the-ROC-curve [AUROC]=0.98 and 0.96, respectively). The APRI and FIB-4 scores also demonstrated good diagnostic performance (AUROC=0.87 and 0.88, respectively). CONCLUSION: MRI-derived measures of liver and spleen stiffness as well as laboratory-based APRI and FIB-4 scores are highly associated with imaging findings of portal hypertension in children and young adults with AILD and thus might be useful for predicting portal hypertension impending onset and directing personalized patient management.


Asunto(s)
Colangitis Esclerosante/diagnóstico por imagen , Hepatitis Autoinmune/diagnóstico por imagen , Hipertensión Portal/diagnóstico por imagen , Hipertensión Portal/inmunología , Imagen por Resonancia Magnética/métodos , Adolescente , Aspartato Aminotransferasas/análisis , Biomarcadores/análisis , Niño , Estudios Transversales , Imagen de Difusión por Resonancia Magnética , Diagnóstico por Imagen de Elasticidad , Femenino , Humanos , Interpretación de Imagen Asistida por Computador , Masculino , Valor Predictivo de las Pruebas , Sistema de Registros , Adulto Joven
19.
Proc Natl Acad Sci U S A ; 113(51): 14645-14650, 2016 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-27930313

RESUMEN

The existence of charge-density-wave (CDW) correlations in cuprate superconductors has now been established. However, the nature of the CDW ground state has remained uncertain because disorder and the presence of superconductivity typically limit the CDW correlation lengths to only a dozen unit cells or less. Here we explore the field-induced 3D CDW correlations in extremely pure detwinned crystals of YBa2Cu3O2 (YBCO) ortho-II and ortho-VIII at magnetic fields in excess of the resistive upper critical field ([Formula: see text]) where superconductivity is heavily suppressed. We observe that the 3D CDW is unidirectional and possesses a long in-plane correlation length as well as significant correlations between neighboring CuO2 planes. It is significant that we observe only a single sharply defined transition at a critical field proportional to [Formula: see text], given that the field range used in this investigation overlaps with other high-field experiments including quantum oscillation measurements. The correlation volume is at least two to three orders of magnitude larger than that of the zero-field CDW. This is by far the largest CDW correlation volume observed in any cuprate crystal and so is presumably representative of the high-field ground state of an "ideal" disorder-free cuprate.

20.
Pediatr Cardiol ; 40(7): 1410-1418, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31399780

RESUMEN

Numerous RNA-binding proteins (RBPs) are expressed in the heart, and mutations in several RBPs have been implicated in cardiovascular disease through genetic associations, animal modeling, and mechanistic studies. However, the functions of many more cardiac RBPs, and their relevance to disease states, remain to be elucidated. Recently, we have initiated studies to characterize the functions of the RBPs RBPMS and RBPMS2 in regulating myocardial biology in zebrafish and higher vertebrate species. These studies began when we learned, using an unbiased gene discovery approach, that rbpms2a and rbpms2b in zebrafish are robust markers of embryonic myocardium. This observation, which is consistent with published data, suggests that the encoded proteins are likely to be performing critical functions in regulating one or more aspects of cardiomyocyte differentiation, proliferation, survival, and/or contractility. This notion is supported by recent reports demonstrating that zebrafish embryos with disrupted Rbpms2 function exhibit gross signs of cardiac distress. Interestingly, a 20-year-old study determined that myocardial tissue from the frog, chick, and mouse also express high levels of Rbpms and/or Rbpms2, which is suggestive of evolutionary conservation of function. In this review, we will provide a historical account of how RBPMS and RBPMS2 genes were discovered, attempt to clarify some potentially confusing nomenclature, and summarize published observations that inform our ongoing studies.


Asunto(s)
Miocardio/citología , Proteínas de Unión al ARN/metabolismo , Animales , Diferenciación Celular , Humanos , Ratones , Proteínas de Unión al ARN/genética , Pez Cebra
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