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1.
Nature ; 608(7924): 766-777, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35948637

RESUMEN

Myocardial infarction is a leading cause of death worldwide1. Although advances have been made in acute treatment, an incomplete understanding of remodelling processes has limited the effectiveness of therapies to reduce late-stage mortality2. Here we generate an integrative high-resolution map of human cardiac remodelling after myocardial infarction using single-cell gene expression, chromatin accessibility and spatial transcriptomic profiling of multiple physiological zones at distinct time points in myocardium from patients with myocardial infarction and controls. Multi-modal data integration enabled us to evaluate cardiac cell-type compositions at increased resolution, yielding insights into changes of the cardiac transcriptome and epigenome through the identification of distinct tissue structures of injury, repair and remodelling. We identified and validated disease-specific cardiac cell states of major cell types and analysed them in their spatial context, evaluating their dependency on other cell types. Our data elucidate the molecular principles of human myocardial tissue organization, recapitulating a gradual cardiomyocyte and myeloid continuum following ischaemic injury. In sum, our study provides an integrative molecular map of human myocardial infarction, represents an essential reference for the field and paves the way for advanced mechanistic and therapeutic studies of cardiac disease.


Asunto(s)
Remodelación Atrial , Ensamble y Desensamble de Cromatina , Perfilación de la Expresión Génica , Infarto del Miocardio , Análisis de la Célula Individual , Remodelación Ventricular , Remodelación Atrial/genética , Estudios de Casos y Controles , Cromatina/genética , Epigenoma , Humanos , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Factores de Tiempo , Remodelación Ventricular/genética
2.
Hum Mutat ; 41(11): 1931-1943, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32840935

RESUMEN

Mutations in RBM20 encoding the RNA-binding motif protein 20 (RBM20) are associated with an early onset and clinically severe forms of cardiomyopathies. Transcriptome analyses revealed RBM20 as an important regulator of cardiac alternative splicing. RBM20 mutations are especially localized in exons 9 and 11 including the highly conserved arginine and serine-rich domain (RS domain). Here, we investigated in several cardiomyopathy patients, the previously described RBM20-mutation p.Pro638Leu localized within the RS domain. In addition, we identified in a patient the novel mutation p.Val914Ala localized in the (glutamate-rich) Glu-rich domain of RBM20 encoded by exon 11. Its impact on the disease was investigated with a novel TTN- and RYR2-splicing assay based on the patients' cardiac messenger RNA. Furthermore, we showed in cell culture and in human cardiac tissue that mutant RBM20-p.Pro638Leu is not localized in the nuclei but causes an abnormal cytoplasmic localization of the protein. In contrast the splicing deficient RBM20-p.Val914Ala has no influence on the intracellular localization. These results indicate that disease-associated variants in RBM20 lead to aberrant splicing through different pathomechanisms dependent on the localization of the mutation. This might have an impact on the future development of therapeutic strategies for the treatment of RBM20-induced cardiomyopathies.


Asunto(s)
Cardiomiopatías/genética , Mutación , Proteínas de Unión al ARN/genética , Adulto , Empalme Alternativo , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Linaje
3.
J Mol Cell Cardiol ; 129: 303-313, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30885746

RESUMEN

Arrhythmogenic right ventricular cardiomyopathy is a heritable cardiac disease causing severe ventricular arrhythmias, heart failure and sudden cardiac death. It is mainly caused by mutations in genes encoding several structural proteins of the cardiac desmosomes including the DSG2 gene encoding the desmosomal cadherin desmoglein-2. Although the molecular structure of the extracellular domain of desmoglein-2 is known, it remains an open question, how mutations in DSG2 contribute to the pathogenesis of arrhythmogenic right ventricular cardiomyopathy. In the present study, we analyzed the impact of different DSG2 mutations on the glycosylation pattern using de-glycosylation assays, lectin blot analysis and genetic inhibition studies. Remarkably, wildtype and mutant desmoglein-2 displayed different glycosylation patterns, although the investigated DSG2 mutations do not directly affect the consensus sequences of the N-glycosylation sites. Our study reveals complex molecular interactions between DSG2 mutations and N-glycosylations of desmoglein-2, which may contribute to the molecular understanding of the patho-mechanisms associated with arrhythmogenic right ventricular cardiomyopathy.


Asunto(s)
Displasia Ventricular Derecha Arritmogénica/genética , Desmogleína 2/genética , Desmogleína 2/metabolismo , Mutación/genética , Adhesión Celular , Línea Celular Tumoral , Membrana Celular/metabolismo , Desmogleína 2/química , Glicosilación , Humanos , Lectinas/metabolismo , Proteínas Mutantes/metabolismo , Unión Proteica , Dominios Proteicos , Proteínas Recombinantes/metabolismo
4.
Am J Pathol ; 187(4): 752-766, 2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-28183531

RESUMEN

Arrhythmogenic cardiomyopathy (AC) is a hereditary disease leading to sudden cardiac death or heart failure. AC pathology is characterized by cardiomyocyte loss and replacement fibrosis. Our goal was to determine whether cardiomyocytes respond to AC progression by pathological hypertrophy. To this end, we examined tissue samples from AC patients with end-stage heart failure and tissue samples that were collected at different disease stages from desmoglein 2-mutant mice, a well characterized AC model. We find that cardiomyocyte diameters are significantly increased in right ventricles of AC patients. Increased mRNA expression of the cardiac stress marker natriuretic peptide B is also observed in the right ventricle of AC patients. Elevated myosin heavy chain 7 mRNA expression is detected in left ventricles. In desmoglein 2-mutant mice, cardiomyocyte diameters are normal during the concealed disease phase but increase significantly after acute disease onset on cardiomyocyte death and fibrotic myocardial remodeling. Hypertrophy progresses further during the chronic disease stage. In parallel, mRNA expression of myosin heavy chain 7 and natriuretic peptide B is up-regulated in both ventricles with right ventricular preference. Calcineurin/nuclear factor of activated T cells (Nfat) signaling, which is linked to pathological hypertrophy, is observed during AC progression, as evidenced by Nfatc2 and Nfatc3 mRNA in cardiomyocytes and increased mRNA of the Nfat target regulator of calcineurin 1. Taken together, we demonstrate that pathological hypertrophy occurs in AC and is secondary to cardiomyocyte loss and cardiac remodeling.


Asunto(s)
Arritmias Cardíacas/complicaciones , Cardiomegalia/complicaciones , Cardiomiopatías/complicaciones , Miocitos Cardíacos/patología , Animales , Arritmias Cardíacas/sangre , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatología , Señalización del Calcio/genética , Cardiomegalia/sangre , Cardiomegalia/genética , Cardiomegalia/fisiopatología , Cardiomiopatías/sangre , Cardiomiopatías/genética , Cardiomiopatías/fisiopatología , Tamaño de la Célula , Desmogleína 2/metabolismo , Dilatación , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Insuficiencia Cardíaca/patología , Pruebas de Función Cardíaca , Ventrículos Cardíacos/patología , Humanos , Inmunoglobulina G/sangre , Ratones , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Factores de Transcripción NFATC/metabolismo , Necrosis , Tamaño de los Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal
5.
J Card Surg ; 33(10): 693-702, 2018 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-30199919

RESUMEN

BACKGROUND: The associations between mechanical circulatory support (MCS), acquired von Willebrand syndrome (AvWS), and clinical outcome are incompletely understood. METHODS: In 128 heart failure patients with pulsatile MCS implants (65 total artificial heart or biventricular assist device implants, 63 left ventricular assist device [LVAD] implants) and 76 patients with continuous flow LVAD implants, we analyzed the von Willebrand factor (vWF) profile before (≤24 h) and 17.5 (standard deviation: 5.1) days after device implant. We determined vWF concentrations, vWF activity, and vWF collagen binding capacity and calculated ratios of vWF activity/binding capacity with vWF concentration. The relation of the vWF profile with clinical outcomes such as stroke, gastrointestinal bleeding, and survival was also evaluated. Events were assessed up to 1 year of device implant. RESULTS: All entities of vWF were already significantly elevated preoperatively and remained high after MCS implantation. The ratios of vWF activity/concentration (vWF:RCo/Ag) and collagen binding capacity/concentration (vWF:CBA/Ag) were significantly reduced preoperatively and remained low postoperatively, indicating AvWS. The preoperative alterations in the vWF profile were already present in patients without intra-aortic balloon pump and/or extracorporeal circulatory membrane oxygenation implants. The vWF profile was unrelated to postoperative stroke. However, a higher postoperative ratio of vWF:CBA/Ag was independently associated with increased gastrointestinal bleeding. In addition, a postoperative increase in vWF concentrations and activity were independent predictors of increased 1-year mortality. CONCLUSIONS: Our data indicate that AvWS is present in heart failure patients before device implantation, and is independently associated with clinical outcomes, especially with 1-year mortality.


Asunto(s)
Insuficiencia Cardíaca/terapia , Corazón Auxiliar , Factor de von Willebrand/metabolismo , Anciano , Estudios de Cohortes , Colágeno/metabolismo , Femenino , Hemorragia Gastrointestinal , Insuficiencia Cardíaca/mortalidad , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Unión Proteica , Accidente Cerebrovascular , Tasa de Supervivencia , Factores de Tiempo , Resultado del Tratamiento
6.
Eur Heart J ; 36(14): 872-81, 2015 Apr 07.
Artículo en Inglés | MEDLINE | ID: mdl-24598986

RESUMEN

AIMS: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare genetic condition caused predominantly by mutations within desmosomal genes. The mutation leading to ARVC-5 was recently identified on the island of Newfoundland and caused by the fully penetrant missense mutation p.S358L in TMEM43. Although TMEM43-p.S358L mutation carriers were also found in the USA, Germany, and Denmark, the genetic relationship between North American and European patients and the disease mechanism of this mutation remained to be clarified. METHODS AND RESULTS: We screened 22 unrelated ARVC patients without mutations in desmosomal genes and identified the TMEM43-p.S358L mutation in a German ARVC family. We excluded TMEM43-p.S358L in 22 unrelated patients with dilated cardiomyopathy. The German family shares a common haplotype with those from Newfoundland, USA, and Denmark, suggesting that the mutation originated from a common founder. Examination of 40 control chromosomes revealed an estimated age of 1300-1500 years for the mutation, which proves the European origin of the Newfoundland mutation. Skin fibroblasts from a female and two male mutation carriers were analysed in cell culture using atomic force microscopy and revealed that the cell nuclei exhibit an increased stiffness compared with TMEM43 wild-type controls. CONCLUSION: The German family is not affected by a de novo TMEM43 mutation. It is therefore expected that an unknown number of European families may be affected by the TMEM43-p.S358L founder mutation. Due to its deleterious clinical phenotype, this mutation should be checked in any case of ARVC-related genotyping. It appears that the increased stiffness of the cell nucleus might be related to the massive loss of cardiomyocytes, which is typically found in ventricles of ARVC hearts.


Asunto(s)
Displasia Ventricular Derecha Arritmogénica/genética , Núcleo Celular/fisiología , Proteínas de la Membrana/genética , Mutación Missense/genética , Adulto , Anciano , Displasia Ventricular Derecha Arritmogénica/etnología , Estudios de Cohortes , Femenino , Fibroblastos/fisiología , Efecto Fundador , Alemania/etnología , Haplotipos , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Terranova y Labrador/etnología , Linaje , Piel
7.
Physiol Genomics ; 44(1): 99-109, 2012 Jan 18.
Artículo en Inglés | MEDLINE | ID: mdl-22085907

RESUMEN

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy primarily of the right ventricle characterized through fibrofatty replacement of cardiomyocytes. The genetic etiology in ARVC patients is most commonly caused by dominant inheritance and high genetic heterogeneity. Though histological examinations of ARVC-affected human myocardium reveals fibrolipomatous replacement, the molecular mechanisms leading to loss of cardiomyocytes are largely unknown. We therefore analyzed the transcriptomes of six ARVC hearts and compared our findings to six nonfailing donor hearts (NF). To characterize the ARVC-specific transcriptome, we compared our findings to samples from seven patients with idiopathic dilated cardiomyopathy (DCM). The myocardial DCM and ARVC samples were prepared from hearts explanted during an orthotopic heart transplantation representing myocardium from end-stage heart failure patients (NYHA IV). From each heart, left (LV) and right ventricular (RV) myocardial samples were analyzed by Affymetrix HG-U133 Plus 2.0 arrays, adding up to six sample groups. Unsupervised cluster analyses of the groups revealed a clear separation of NF and cardiomyopathy samples. However, in contrast to the other samples, the analyses revealed no distinct expression pattern in LV and RV of myocardial ARVC samples. We further identified differentially expressed transcripts using t-tests and found transcripts separating diseased and NF ventricular myocardium. Of note, in failing myocardium only ~15-16% of the genes are commonly regulated compared with NF samples. In addition both cardiomyopathies are clearly distinct on the transcriptome level. Comparison of the expression patterns between the failing RV and LV using a paired t-test revealed a lack of major differences between LV and RV gene expression in ARVC hearts. Our study is the first analysis of specific ARVC-related RV and LV gene expression patterns in terminal failing human hearts.


Asunto(s)
Displasia Ventricular Derecha Arritmogénica/genética , Miocardio/metabolismo , Transcriptoma , Adolescente , Adulto , Anciano , Displasia Ventricular Derecha Arritmogénica/metabolismo , Displasia Ventricular Derecha Arritmogénica/patología , Estudios de Casos y Controles , Análisis por Conglomerados , Estudios de Cohortes , Femenino , Perfilación de la Expresión Génica , Estudios de Asociación Genética , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Humanos , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Miocardio/patología , Transcriptoma/genética , Adulto Joven
8.
Eur J Heart Fail ; 23(2): 324-334, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33038287

RESUMEN

AIMS: Mechanical unloading by ventricular assist devices (VADs) has become increasingly important for the therapy of end-stage heart failure during the last decade. However, VAD support was claimed to be associated with partial reverse remodelling. Unfortunately, the literature describes the contradictory effects of VAD systems on cardiac fibrosis, a hallmark of cardiac remodelling. To clarify these inconsistent results, the effects on cardiac fibrosis before and after mechanical unloading in 125 patients were examined. METHODS AND RESULTS: Left ventricular myocardial tissue from ischaemic or non-ischaemic cardiomyopathy patients undergoing VAD implantation and subsequent cardiac transplantation and non-failing hearts of the control group were analysed for 4-hydroxyproline (4OH-P) content as a marker for collagen protein. In addition, collagen cross-linking and mRNAs of collagens I and III and transforming growth factor beta-1 were measured. 4OH-P content was significantly increased in failing hearts compared with the control group and increased (P < 0.05) after mechanical unloading (nmol/mg tissue, mean ± standard deviation: 16.74 ± 9.68 vs. 7.75 ± 2.39 and 18.57 ± 9.19). However, plotting of the 4OH-P ratios (post/pre-VAD) against the collagen content pre-VAD could be fitted by non-linear regression. Collagen cross-linking correlated strongly with the total collagen content in pre- and post-VAD myocardium (r2  = 0.73 and 0.71, respectively). In contrast to the total collagen content, all three mRNAs of fibrotic genes were significantly down-regulated during VAD support when compared to pre-VAD. CONCLUSIONS: This investigation of a comparably large patient cohort revealed that cardiac fibrosis was strongly increased in heart failure and increased even after mechanical unloading. The mRNAs of collagens I and III are independently regulated from the collagen protein.


Asunto(s)
Cardiomiopatías , Insuficiencia Cardíaca , Corazón Auxiliar , Fibrosis , Insuficiencia Cardíaca/patología , Humanos , Miocardio/patología
9.
Genes (Basel) ; 12(6)2021 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-34201072

RESUMEN

A major cause of heart failure is cardiomyopathies, with dilated cardiomyopathy (DCM) as the most common form. Over 40 genes are linked to DCM, among them TTN and RBM20. Next Generation Sequencing in clinical DCM cohorts revealed truncating variants in TTN (TTNtv), accounting for up to 25% of familial DCM cases. Mutations in the cardiac splicing factor RNA binding motif protein 20 (RBM20) are also known to be associated with severe cardiomyopathies. TTN is one of the major RBM20 splicing targets. Most of the pathogenic RBM20 mutations are localized in the highly conserved arginine serine rich domain (RS), leading to a cytoplasmic mislocalization of mutant RBM20. Here, we present a patient with an early onset DCM carrying a combination of (likely) pathogenic TTN and RBM20 mutations. We show that the splicing of RBM20 target genes is affected in the mutation carrier. Furthermore, we reveal RBM20 haploinsufficiency presumably caused by the frameshift mutation in RBM20.


Asunto(s)
Cardiomiopatía Dilatada/genética , Conectina/genética , Proteínas de Unión al ARN/genética , Adulto , Animales , Cardiomiopatía Dilatada/patología , Línea Celular , Femenino , Haploinsuficiencia , Humanos , Masculino , Ratones , Mutación , Linaje , Fenotipo , Dominios Proteicos , Transporte de Proteínas , Empalme del ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo
10.
Pharmaceuticals (Basel) ; 14(8)2021 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-34451836

RESUMEN

Thrombus formation and thromboembolic events play important roles in various cardiovascular pathologies. The key receptor involved in platelet aggregation is the fibrinogen receptor glycoprotein IIb/IIIa. [18F]GP1, a derivative of the GPIIb/IIIa antagonist elarofiban, is a specific 18F-labeled small-molecule radiotracer that binds with high affinity to GPIIb/IIIa receptors of activated platelets. An improved, robust and fully automated radiosynthesis of [18F]GP1 has been developed. [18F]GP1 has been synthesized with decay corrected radiochemical yields of 38 ± 6%, with a radiochemical concentration up to 1900 MBq/mL, molar activities of 952-9428 GBq/µmol and a radio-chemical purity >98%. After determination of the optimal reaction conditions, in particular for HPLC separation, adaption of the reaction conditions to PET center requirements, validation of the manufacturing process and the quality control methods, the synthesis of [18F]GP1 was successfully implemented to GMP standards and was available for clinical application. We describe the GMP-compliant synthesis of the novel radiotracer [18F]GP1. Moreover, we provide some proof-of-concept examples for clinical application in the cardiovascular field. PET/CT with the novel small-molecular radiotracer [18F]GP1 may serve as a novel highly sensitive tool for visualizing active platelet aggregation at the molecular level.

11.
Sci Transl Med ; 13(618): eabd3079, 2021 11 03.
Artículo en Inglés | MEDLINE | ID: mdl-34731013

RESUMEN

Heterozygous truncating variants in TTN (TTNtv), the gene coding for titin, cause dilated cardiomyopathy (DCM), but the underlying pathomechanisms are unclear and disease management remains uncertain. Truncated titin proteins have not yet been considered as a contributor to disease development. Here, we studied myocardial tissues from nonfailing donor hearts and 113 patients with end-stage DCM for titin expression and identified a TTNtv in 22 patients with DCM (19.5%). We directly demonstrate titin haploinsufficiency in TTNtv-DCM hearts and the absence of compensatory changes in the alternative titin isoform Cronos. Twenty-one TTNtv-DCM hearts in our cohort showed stable expression of truncated titin proteins. Expression was variable, up to half of the total titin protein pool, and negatively correlated with patient age at heart transplantation. Truncated titin proteins were not detected in sarcomeres but were present in intracellular aggregates, with deregulated ubiquitin-dependent protein quality control. We produced human induced pluripotent stem cell­derived cardiomyocytes (hiPSC-CMs), comparing wild-type controls to cells with a patient-derived, prototypical A-band-TTNtv or a CRISPR-Cas9­generated M-band-TTNtv. TTNtv-hiPSC-CMs showed reduced wild-type titin expression and contained truncated titin proteins whose proportion increased upon inhibition of proteasomal activity. In engineered heart muscle generated from hiPSC-CMs, depressed contractility caused by TTNtv could be reversed by correction of the mutation using CRISPR-Cas9, eliminating truncated titin proteins and raising wild-type titin content. Functional improvement also occurred when wild-type titin protein content was increased by proteasome inhibition. Our findings reveal the major pathomechanisms of TTNtv-DCM and can be exploited for new therapies to treat TTNtv-related cardiomyopathies.


Asunto(s)
Cardiomiopatías , Conectina , Trasplante de Corazón , Células Madre Pluripotentes Inducidas , Cardiomiopatías/genética , Conectina/genética , Conectina/metabolismo , Haploinsuficiencia , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Miocitos Cardíacos/metabolismo , Donantes de Tejidos
12.
Physiol Genomics ; 42(3): 397-405, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20460602

RESUMEN

Mechanical unloading by ventricular assist devices (VAD) leads to significant gene expression changes often summarized as reverse remodeling. However, little is known on individual transcriptome changes during VAD support and its relationship to nonfailing hearts (NF). In addition no data are available for the transcriptome regulation during nonpulsatile VAD support. Therefore we analyzed the gene expression patterns of 30 paired samples from VAD-supported (including 8 nonpulsatile VADs) and 8 nonfailing control hearts (NF) using the first total human genome array available. Transmural myocardial samples were collected for RNA isolation. RNA was isolated by commercial methods and processed according to chip-manufacturer recommendations. cRNA were hybridized on Affymetrix HG-U133 Plus 2.0 arrays, providing coverage of the whole human genome Array. Data were analyzed using Microarray Analysis Suite 5.0 (Affymetrix) and clustered by Expressionist software (Genedata). We found 352 transcripts were differentially regulated between samples from VAD implantation and NF, whereas 510 were significantly regulated between VAD transplantation and NF (paired t-test P < 0.001, fold change >or=1.6). Remarkably, only a minor fraction of 111 transcripts was regulated in heart failure (HF) and during VAD support. Unsupervised hierarchical clustering of paired VAD and NF samples revealed separation of HF and NF samples; however, individual differentiation of VAD implantation and VAD transplantation was not accomplished. Clustering of pulsatile and nonpulsatile VAD did not lead to robust separation of gene expression patterns. During VAD support myocardial gene expression changes do not indicate reversal of the HF phenotype but reveal a distinct HF-related pattern. Transcriptome analysis of pulsatile and nonpulsatile VAD-supported hearts did not provide evidence for a pump mode-specific transcriptome pattern.


Asunto(s)
Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/terapia , Corazón Auxiliar , Miocardio/metabolismo , Adulto , Estudios de Casos y Controles , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/fisiología , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Humanos , Masculino , Persona de Mediana Edad , Miocardio/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Flujo Pulsátil
13.
Physiol Genomics ; 38(1): 7-15, 2009 Jun 10.
Artículo en Inglés | MEDLINE | ID: mdl-19293330

RESUMEN

Cardiomyocytes derived from pluripotent embryonic stem cells (ESC) have the advantage of providing a source for standardized cell cultures. However, little is known on the regulation of the genome during differentiation of ESC to cardiomyocytes. Here, we characterize the transcriptome of the mouse ESC line CM7/1 during differentiation into beating cardiomyocytes and compare the gene expression profiles with those from primary adult murine cardiomyocytes and left ventricular myocardium. We observe that the cardiac gene expression pattern of fully differentiated CM7/1-ESC is highly similar to adult primary cardiomyocytes and murine myocardium, respectively. This finding is underlined by demonstrating pharmacological effects of catecholamines and endothelin-1 on ESC-derived cardiomyocytes. Furthermore, we monitor the temporal changes in gene expression pattern during ESC differentiation with a special focus on transcription factors involved in cardiomyocyte differentiation. Thus, CM7/1-ESC-derived cardiomyocytes are a promising new tool for functional studies of cardiomyocytes in vitro and for the analysis of the transcription factor network regulating pluripotency and differentiation to cardiomyocytes.


Asunto(s)
Células Madre Embrionarias/metabolismo , Perfilación de la Expresión Génica , Miocardio/metabolismo , Recombinación Genética , Factores de Transcripción/genética , Animales , Diferenciación Celular , Línea Celular , Células Madre Embrionarias/citología , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
14.
Circulation ; 113(23): 2724-32, 2006 Jun 13.
Artículo en Inglés | MEDLINE | ID: mdl-16754798

RESUMEN

BACKGROUND: In heart failure (HF), ventricular myocardium expresses brain natriuretic peptide (BNP). Despite the association of elevated serum levels with poor prognosis, BNP release is considered beneficial because of its antihypertrophic, vasodilating, and diuretic properties. However, there is evidence that BNP-mediated signaling may adversely influence cardiac remodeling, with further impairment of calcium homeostasis. METHODS AND RESULTS: We studied the effects of BNP on preload-dependent myocardial sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) expression. In rabbit isolated muscle strips stretched to high preload and shortening isotonically over 6 hours, the SERCA/glyceraldehyde phosphate dehydrogenase mRNA ratio was enhanced by 168% (n=8) compared with unloaded preparations (n=8; P<0.001). Recombinant human BNP at a concentration typically found in end-stage HF patients (350 pg/mL) abolished SERCA upregulation by stretch (n=9; P<0.0001 versus BNP free). Inhibition of cyclic guanosine 3',5' monophosphate (cGMP)-phosphodiesterase-5 mimicked this effect, whereas inhibition of cGMP-dependent protein kinase restored preload-dependent SERCA upregulation in the presence of recombinant human BNP. Furthermore, in myocardium from human end-stage HF patients undergoing cardiac transplantation (n=15), BNP expression was inversely correlated with SERCA levels. Moreover, among 23 patients treated with left ventricular assist devices, significant SERCA2a recovery occurred in those downregulating BNP. CONCLUSIONS: Our data indicate that preload stimulates SERCA expression. BNP antagonizes this mechanism via guanylyl cyclase-A, cGMP, and cGMP-dependent protein kinase. This novel action of BNP to uncouple preload-dependent SERCA expression may adversely affect contractility in patients with HF.


Asunto(s)
ATPasas Transportadoras de Calcio/biosíntesis , Insuficiencia Cardíaca/fisiopatología , Péptido Natriurético Encefálico/fisiología , Retículo Sarcoplasmático/enzimología , 3',5'-GMP Cíclico Fosfodiesterasas/fisiología , Adulto , Animales , Calcineurina/fisiología , Señalización del Calcio , ATPasas Transportadoras de Calcio/genética , Cardiomiopatía Dilatada/complicaciones , Estudios de Cohortes , GMP Cíclico/fisiología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5 , Inducción Enzimática/efectos de los fármacos , Femenino , Guanilato Ciclasa/fisiología , Insuficiencia Cardíaca/enzimología , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/terapia , Corazón Auxiliar , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Isquemia Miocárdica/complicaciones , Miocardio/enzimología , Factores de Transcripción NFATC/fisiología , Péptido Natriurético Encefálico/genética , Péptido Natriurético Encefálico/farmacología , ARN Mensajero/biosíntesis , Receptores del Factor Natriurético Atrial/fisiología , Proteínas Recombinantes de Fusión/farmacología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Estrés Mecánico
15.
PLoS One ; 12(1): e0169896, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28095452

RESUMEN

BACKGROUND: In terminal failing hearts ventricular assist devices (VAD) are implanted as a bridge to transplantation. Endothelin receptor (ETR) antagonists are used for treatment of secondary pulmonary hypertension in VAD patients. However, the cardiac ETR regulation in human heart failure and during VAD support is incompletely understood. METHODS: In paired left ventricular samples of 12 dilated cardiomyopathy patients we investigated the density of endothelin A (ETA) and B (ETB) receptors before VAD implantation and after device removal. Left ventricular samples of 12 non-failing donor hearts served as control. Receptor quantification was performed by binding of [125I]-ET-1 in the presence of nonselective and ETA selective ETR ligands as competitors. Additionally, the ETR mRNA expression was analyzed using quantitative real-time-PCR. RESULTS: The mRNA of ETA but not ETB receptors was significantly elevated in heart failure, whereas total ETR density analyzed by radioligand binding was significantly reduced due to ETB receptor down regulation. ETA and ETB receptor density showed poor correlation to mRNA data (spearman correlation factor: 0.43 and 0.31, respectively). VAD support had no significant impact on the density of both receptors and on mRNA expression of ETA whereas ETB mRNA increased during VAD. A meta-analysis reveals that the ETA receptor regulation in human heart failure appears to depend on non-failing hearts. CONCLUSIONS: In deteriorating hearts of patients suffering from dilated cardiomyopathy the ETA receptor density is not changed whereas the ETB receptor is down regulated. The mRNA and the proteins of ETA and ETB show a weak correlation. Non-failing hearts might influence the interpretation of ETA receptor regulation. Mechanical unloading of the failing hearts has no impact on the myocardial ETR density.


Asunto(s)
Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/cirugía , Ventrículos Cardíacos/metabolismo , Corazón Auxiliar , Miocardio/metabolismo , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Western Blotting , Estudios de Casos y Controles , Humanos , Técnicas para Inmunoenzimas , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor de Endotelina A/genética , Receptor de Endotelina B/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Función Ventricular Izquierda/fisiología
16.
PLoS One ; 12(12): e0189489, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29253866

RESUMEN

Cardiomyopathies might lead to end-stage heart disease with the requirement of drastic treatments like bridging up to transplant or heart transplantation. A not precisely known proportion of these diseases are genetically determined. We genotyped 43 index-patients (30 DCM, 10 ARVC, 3 RCM) with advanced or end stage cardiomyopathy using a gene panel which covered 46 known cardiomyopathy disease genes. Fifty-three variants with possible impact on disease in 33 patients were identified. Of these 27 (51%) were classified as likely pathogenic or pathogenic in the MYH7, MYL2, MYL3, NEXN, TNNC1, TNNI3, DES, LMNA, PKP2, PLN, RBM20, TTN, and CRYAB genes. Fifty-six percent (n = 24) of index-patients carried a likely pathogenic or pathogenic mutation. Of these 75% (n = 18) were familial and 25% (n = 6) sporadic cases. However, severe cardiomyopathy seemed to be not characterized by a specific mutation profile. Remarkably, we identified a novel homozygous PKP2-missense variant in a large consanguineous family with sudden death in early childhood and several members with heart transplantation in adolescent age.


Asunto(s)
Cardiomiopatías/diagnóstico , Cardiomiopatías/genética , Mutación , Placofilinas/genética , Adolescente , Adulto , Anciano , Niño , Estudios de Cohortes , Salud de la Familia , Femenino , Genotipo , Insuficiencia Cardíaca/genética , Trasplante de Corazón , Secuenciación de Nucleótidos de Alto Rendimiento , Homocigoto , Humanos , Recién Nacido , Masculino , Persona de Mediana Edad , Mutación Missense , Adulto Joven
17.
Circulation ; 112(8): 1136-44, 2005 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-16103240

RESUMEN

BACKGROUND: Alterations in the balance of matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs) are involved in left ventricular (LV) remodeling. Whether their expression is related to interstitial fibrosis or LV dysfunction in patients with chronic pressure overload-induced LV hypertrophy, however, is unknown. METHODS AND RESULTS: Therefore, cardiac biopsies were taken in 36 patients with isolated aortic stenosis (AS) and in 29 control patients without LV hypertrophy. Microarray analysis revealed significantly increased mRNA expression of collagen types I, III, and IV and transcripts involved in collagen synthesis, including procollagen endopeptidase and lysine and proline hydroxylases, in AS compared with control patients. Collagen deposition was greater in AS than in control patients and was most pronounced in AS patients with severe diastolic dysfunction. Cardiac mRNA expression of TIMP-1 and TIMP-2 was significantly increased in AS compared with control patients (mRNA transcript levels normalized to GAPDH: TIMP-1, 0.67+/-0.1 in AS versus 0.37+/-0.08 in control patients; TIMP-2, 9.5+/-2.6 in AS versus 1.6+/-0.4 in control patients; P<0.05 for both) but did not differ significantly for MMP-1, -2, or -9. Cardiac TIMP-1 and -2 transcripts were significantly related to the degree of interstitial fibrosis and proportional to diastolic dysfunction in AS patients. CONCLUSIONS: Cardiac expression of TIMP-1 and TIMP-2 is significantly increased in chronic pressure-overloaded human hearts compared with controls and is related to the degree of interstitial fibrosis.


Asunto(s)
Hipertensión/fisiopatología , Hipertrofia Ventricular Izquierda/fisiopatología , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-2/genética , Presión Ventricular , Estenosis de la Válvula Aórtica/patología , Estenosis de la Válvula Aórtica/fisiopatología , Biopsia , Enfermedad Crónica , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Colágeno Tipo III/genética , Puente de Arteria Coronaria , Fibrosis , Expresión Génica , Humanos , Hipertensión/patología , Hipertrofia Ventricular Izquierda/patología , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Miocardio/patología , ARN Mensajero/metabolismo
18.
Eur J Heart Fail ; 8(3): 278-83, 2006 May.
Artículo en Inglés | MEDLINE | ID: mdl-16309954

RESUMEN

BACKGROUND: Whether adverse structural changes in the myocardium due to remodelling can be reversed by ventricular assist device (VAD) support in patients with end-stage heart failure is controversial. AIMS: To investigate the effect of VAD support on the extra-cellular matrix. METHODS: We analysed the collagen content in terminal failing ventricles of VAD-patients and donor hearts using 4-hydroxyproline for total collagen and real time RT-PCR for fibronectin (FN), collagen I alpha 1 (Col1A1), III alpha 1 (Col3A1) and TGF beta 1 analysis. RESULTS: Compared to donor hearts we found similar increases in Col1A1 and TGF beta1 but not Col3A1 and FN mRNAs, which were similar in the myocardium from patients receiving a VAD or heart transplant. However, patients receiving ACE-I during VAD-support had lower Col1A1 mRNA content at transplantation. The total collagen content was not influenced by mechanical unloading or by ACE-I medication. CONCLUSION: Mechanical unloading by VAD does not reduce the collagen content of the terminal failing ventricle possibly due to increased TGF beta1 levels. However, Col1A1 production may be reduced by ACE-I medication during VAD support.


Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Proteínas de la Matriz Extracelular/genética , Trasplante de Corazón , Corazón Auxiliar , Miocardio/metabolismo , Adulto , Anciano , Niño , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Humanos , Persona de Mediana Edad , Contracción Miocárdica , ARN Mensajero/análisis , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta1
19.
J Heart Lung Transplant ; 22(11): 1209-16, 2003 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-14585382

RESUMEN

BACKGROUND: Myocardial recovery is observed in some end-stage heart failure patients after mechanical circulatory support. The sarcoplasmic reticulum Ca(2+)-adenosine triphosphatase (Ca2+-ATPase) activity is down-regulated in failing myocardium and contributes to heart failure-associated contraction/relaxation abnormalities. Regulation of Ca(2+)-ATPase after mechanical support was shown to be heterogeneous. Thus, we analyzed Ca(2+)-ATPase activity and protein expression in the paired myocardial samples of 21 patients supported by ventricular assist devices to identify factors that influence restoration of the Ca(2+)-transient after ventricular assist device support. METHODS: We measured Ca(2+)-ATPase activity using a reduced nicotinamide-adenine dinucleotide-coupled reaction, determined sarcoplasmic reticulum Ca(2+)-dependent ATPase protein using Western blotting, and determined 4-hydroxyproline using amino-acid analysis. RESULTS: The mean Ca(2+)-ATPase activity decreased at assist-device implantation and slightly increased at transplantation, but remained significantly lower than in non-failing donor hearts. However, individual responses were heterogeneous. Patients with older age, increased left ventricular diameter, and increased 4-hydroxyproline content showed down-regulation of Ca(2+)-ATPase activity, whereas we found up-regulation in patients with low values for these parameters after assist-device support. CONCLUSIONS: Sarcoplasmic reticulum Ca(2+)-ATPase activity, which influences the myocardial Ca(2+)-transient, generally is not restored to normal values in assist-device-supported hearts, but depends on a combined score of the left ventricular end-diastolic diameter, degree of ventricular fibrosis, and age of the patient at the time of assist-device implantation.


Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Corazón Auxiliar , Miocardio/enzimología , Adulto , Anciano , Western Blotting , Niño , Ventrículos Cardíacos , Humanos , Hidroxiprolina/metabolismo , Persona de Mediana Edad , Miocardio/ultraestructura , Curva ROC , Retículo Sarcoplasmático/enzimología
20.
J Heart Lung Transplant ; 23(4): 396-404, 2004 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15063398

RESUMEN

BACKGROUND: Chronic heart failure is a multifactorial, progressive disease of many causes and is associated with complex ventricular remodeling. Deposition of extracellular matrix proteins and sarcomeric disarray of the myocytes occur in end-stage heart failure. Ventricular assist devices (VAD), implanted as bridge to transplantation, may reverse ventricular remodeling. Although successfully weaning patients from VAD support has been reported, it is not clear to what degree reversal of remodeling occurs in unloaded failing hearts. Because collagen deposition and ultrastructural disarray are hallmarks of myocardial remodeling, we analyzed the myocardial ultrastructure and collagen content of VAD-supported hearts before and after mechanical unloading. METHODS: We used amino acid analysis to measure collagen content (4-hydroxyproline content) in 24 transplant candidates receiving VAD support. We used transmission electron microscopy to examine the ultrastructure in 6 patients receiving VAD support. RESULTS: The 4-hydroxyproline content increased significantly at VAD implantation and was not altered by mechanical unloading. The ultrastructure showed signs of persisting cardiomyopathy. CONCLUSION: Mechanical unloading does not alter the total collagen content of the supported, failing heart. Thus, structural reversal of the remodeling process associated with heart failure is not a general phenomenon in mechanically unloaded hearts.


Asunto(s)
Insuficiencia Cardíaca/terapia , Corazón Auxiliar , Hidroxiprolina/metabolismo , Miocardio/metabolismo , Miocardio/ultraestructura , Remodelación Ventricular/fisiología , Adolescente , Adulto , Anciano , Niño , Colágeno/metabolismo , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Trasplante de Corazón , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/ultraestructura , Humanos , Microscopía Electrónica , Persona de Mediana Edad
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