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1.
Cell ; 178(1): 242-260.e29, 2019 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-31155234

RESUMO

Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology.


Assuntos
Miocárdio/metabolismo , Biossíntese de Proteínas , Adolescente , Adulto , Idoso , Animais , Códon/genética , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fases de Leitura Aberta/genética , RNA Circular/genética , RNA Circular/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ribossomos/genética , Ribossomos/metabolismo , Adulto Jovem
2.
Biochem J ; 479(3): 401-424, 2022 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-35147166

RESUMO

The extracellular signal-regulated kinase 1/2 (ERK1/2) cascade promotes cardiomyocyte hypertrophy and is cardioprotective, with the three RAF kinases forming a node for signal integration. Our aims were to determine if BRAF is relevant for human heart failure, whether BRAF promotes cardiomyocyte hypertrophy, and if Type 1 RAF inhibitors developed for cancer (that paradoxically activate ERK1/2 at low concentrations: the 'RAF paradox') may have the same effect. BRAF was up-regulated in heart samples from patients with heart failure compared with normal controls. We assessed the effects of activated BRAF in the heart using mice with tamoxifen-activated Cre for cardiomyocyte-specific knock-in of the activating V600E mutation into the endogenous gene. We used echocardiography to measure cardiac dimensions/function. Cardiomyocyte BRAFV600E induced cardiac hypertrophy within 10 d, resulting in increased ejection fraction and fractional shortening over 6 weeks. This was associated with increased cardiomyocyte size without significant fibrosis, consistent with compensated hypertrophy. The experimental Type 1 RAF inhibitor, SB590885, and/or encorafenib (a RAF inhibitor used clinically) increased ERK1/2 phosphorylation in cardiomyocytes, and promoted hypertrophy, consistent with a 'RAF paradox' effect. Both promoted cardiac hypertrophy in mouse hearts in vivo, with increased cardiomyocyte size and no overt fibrosis. In conclusion, BRAF potentially plays an important role in human failing hearts, activation of BRAF is sufficient to induce hypertrophy, and Type 1 RAF inhibitors promote hypertrophy via the 'RAF paradox'. Cardiac hypertrophy resulting from these interventions was not associated with pathological features, suggesting that Type 1 RAF inhibitors may be useful to boost cardiomyocyte function.


Assuntos
Cardiomegalia/patologia , Sistema de Sinalização das MAP Quinases/fisiologia , Miócitos Cardíacos/patologia , Proteínas Proto-Oncogênicas B-raf/fisiologia , Animais , Carbamatos/farmacologia , Carbamatos/toxicidade , Cardiomegalia/metabolismo , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Dimerização , Técnicas de Introdução de Genes , Insuficiência Cardíaca/patologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Mutação Puntual , Conformação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/biossíntese , Ratos , Ratos Sprague-Dawley , Sulfonamidas/farmacologia , Sulfonamidas/toxicidade
3.
Circulation ; 141(5): 387-398, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31983221

RESUMO

BACKGROUND: Dilated cardiomyopathy (DCM) is genetically heterogeneous, with >100 purported disease genes tested in clinical laboratories. However, many genes were originally identified based on candidate-gene studies that did not adequately account for background population variation. Here we define the frequency of rare variation in 2538 patients with DCM across protein-coding regions of 56 commonly tested genes and compare this to both 912 confirmed healthy controls and a reference population of 60 706 individuals to identify clinically interpretable genes robustly associated with dominant monogenic DCM. METHODS: We used the TruSight Cardio sequencing panel to evaluate the burden of rare variants in 56 putative DCM genes in 1040 patients with DCM and 912 healthy volunteers processed with identical sequencing and bioinformatics pipelines. We further aggregated data from 1498 patients with DCM sequenced in diagnostic laboratories and the Exome Aggregation Consortium database for replication and meta-analysis. RESULTS: Truncating variants in TTN and DSP were associated with DCM in all comparisons. Variants in MYH7, LMNA, BAG3, TNNT2, TNNC1, PLN, ACTC1, NEXN, TPM1, and VCL were significantly enriched in specific patient subsets, with the last 2 genes potentially contributing primarily to early-onset forms of DCM. Overall, rare variants in these 12 genes potentially explained 17% of cases in the outpatient clinic cohort representing a broad range of adult patients with DCM and 26% of cases in the diagnostic referral cohort enriched in familial and early-onset DCM. Although the absence of a significant excess in other genes cannot preclude a limited role in disease, such genes have limited diagnostic value because novel variants will be uninterpretable and their diagnostic yield is minimal. CONCLUSIONS: In the largest sequenced DCM cohort yet described, we observe robust disease association with 12 genes, highlighting their importance in DCM and translating into high interpretability in diagnostic testing. The other genes analyzed here will need to be rigorously evaluated in ongoing curation efforts to determine their validity as Mendelian DCM genes but have limited value in diagnostic testing in DCM at present. This data will contribute to community gene curation efforts and will reduce erroneous and inconclusive findings in diagnostic testing.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Cardiomiopatia Dilatada/genética , Predisposição Genética para Doença , Testes Genéticos , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Adulto , Cardiomiopatia Dilatada/diagnóstico , Exoma/genética , Feminino , Heterogeneidade Genética , Humanos , Masculino , Adulto Jovem
4.
Circulation ; 140(11): 937-951, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31284728

RESUMO

BACKGROUND: Fibrosis is a common pathology in many cardiac disorders and is driven by the activation of resident fibroblasts. The global posttranscriptional mechanisms underlying fibroblast-to-myofibroblast conversion in the heart have not been explored. METHODS: Genome-wide changes of RNA transcription and translation during human cardiac fibroblast activation were monitored with RNA sequencing and ribosome profiling. We then used RNA-binding protein-based analyses to identify translational regulators of fibrogenic genes. The integration with cardiac ribosome occupancy levels of 30 dilated cardiomyopathy patients demonstrates that these posttranscriptional mechanisms are also active in the diseased fibrotic human heart. RESULTS: We generated nucleotide-resolution translatome data during the transforming growth factor ß1-driven cellular transition of human cardiac fibroblasts to myofibroblasts. This identified dynamic changes of RNA transcription and translation at several time points during the fibrotic response, revealing transient and early-responder genes. Remarkably, about one-third of all changes in gene expression in activated fibroblasts are subject to translational regulation, and dynamic variation in ribosome occupancy affects protein abundance independent of RNA levels. Targets of RNA-binding proteins were strongly enriched in posttranscriptionally regulated genes, suggesting genes such as MBNL2 can act as translational activators or repressors. Ribosome occupancy in the hearts of patients with dilated cardiomyopathy suggested the same posttranscriptional regulatory network was underlying cardiac fibrosis. Key network hubs include RNA-binding proteins such as Pumilio RNA binding family member 2 (PUM2) and Quaking (QKI) that work in concert to regulate the translation of target transcripts in human diseased hearts. Furthermore, silencing of both PUM2 and QKI inhibits the transition of fibroblasts toward profibrotic myofibroblasts in response to transforming growth factor ß1. CONCLUSIONS: We reveal widespread translational effects of transforming growth factor ß1 and define novel posttranscriptional regulatory networks that control the fibroblast-to-myofibroblast transition. These networks are active in human heart disease, and silencing of hub genes limits fibroblast activation. Our findings show the central importance of translational control in fibrosis and highlight novel pathogenic mechanisms in heart failure.


Assuntos
Cardiopatias/genética , Cardiopatias/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Biossíntese de Proteínas/genética , Proteínas de Ligação a RNA/genética , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Perfilação da Expressão Gênica/métodos , Cardiopatias/patologia , Humanos , Análise de Sequência de RNA/métodos , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
5.
Genet Med ; 21(1): 133-143, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29892087

RESUMO

PURPOSE: We evaluated strategies for identifying disease-causing variants in genetic testing for dilated cardiomyopathy (DCM). METHODS: Cardiomyopathy gene panel testing was performed in 532 DCM patients and 527 healthy control subjects. Rare variants in 41 genes were stratified using variant-level and gene-level characteristics. RESULTS: A majority of DCM cases and controls carried rare protein-altering cardiomyopathy gene variants. Variant-level characteristics alone had limited discriminative value. Differentiation between groups was substantially improved by addition of gene-level information that incorporated ranking of genes based on literature evidence for disease association. The odds of DCM were increased to nearly 9-fold for truncating variants or high-impact missense variants in the subset of 14 genes that had the strongest biological links to DCM (P <0.0001). For some of these genes, DCM-associated variants appeared to be clustered in key protein functional domains. Multiple rare variants were present in many family probands, however, there was generally only one "driver" pathogenic variant that cosegregated with disease. CONCLUSION: Rare variants in cardiomyopathy genes can be effectively stratified by combining variant-level and gene-level information. Prioritization of genes based on their a priori likelihood of disease causation is a key factor in identifying clinically actionable variants in cardiac genetic testing.


Assuntos
Cardiomiopatia Dilatada/genética , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Doenças Raras/genética , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Dilatada/patologia , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Linhagem , Doenças Raras/diagnóstico , Doenças Raras/patologia
6.
Eur Heart J ; 38(46): 3461-3468, 2017 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-28082330

RESUMO

AIM: Hypertrophic cardiomyopathy (HCM) exhibits genetic heterogeneity that is dominated by variation in eight sarcomeric genes. Genetic variation in a large number of non-sarcomeric genes has also been implicated in HCM but not formally assessed. Here we used very large case and control cohorts to determine the extent to which variation in non-sarcomeric genes contributes to HCM. METHODS AND RESULTS: We sequenced known and putative HCM genes in a new large prospective HCM cohort (n = 804) and analysed data alongside the largest published series of clinically genotyped HCM patients (n = 6179), previously published HCM cohorts and reference population samples from the exome aggregation consortium (ExAC, n = 60 706) to assess variation in 31 genes implicated in HCM. We found no significant excess of rare (minor allele frequency < 1:10 000 in ExAC) protein-altering variants over controls for most genes tested and conclude that novel variants in these genes are rarely interpretable, even for genes with previous evidence of co-segregation (e.g. ACTN2). To provide an aid for variant interpretation, we integrated HCM gene sequence data with aggregated pedigree and functional data and suggest a means of assessing gene pathogenicity in HCM using this evidence. CONCLUSION: We show that genetic variation in the majority of non-sarcomeric genes implicated in HCM is not associated with the condition, reinforce the fact that the sarcomeric gene variation is the primary cause of HCM known to date and underscore that the aetiology of HCM is unknown in the majority of patients.


Assuntos
Cardiomiopatia Hipertrófica/genética , Genes/genética , Sarcômeros/genética , Estudos de Casos e Controles , Feminino , Variação Genética , Humanos , Masculino , Mutação/genética , Estudos Prospectivos
7.
Nature ; 478(7367): 114-8, 2011 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-21979051

RESUMO

Left ventricular mass (LVM) is a highly heritable trait and an independent risk factor for all-cause mortality. So far, genome-wide association studies have not identified the genetic factors that underlie LVM variation, and the regulatory mechanisms for blood-pressure-independent cardiac hypertrophy remain poorly understood. Unbiased systems genetics approaches in the rat now provide a powerful complementary tool to genome-wide association studies, and we applied integrative genomics to dissect a highly replicated, blood-pressure-independent LVM locus on rat chromosome 3p. Here we identified endonuclease G (Endog), which previously was implicated in apoptosis but not hypertrophy, as the gene at the locus, and we found a loss-of-function mutation in Endog that is associated with increased LVM and impaired cardiac function. Inhibition of Endog in cultured cardiomyocytes resulted in an increase in cell size and hypertrophic biomarkers in the absence of pro-hypertrophic stimulation. Genome-wide network analysis unexpectedly implicated ENDOG in fundamental mitochondrial processes that are unrelated to apoptosis. We showed direct regulation of ENDOG by ERR-α and PGC1α (which are master regulators of mitochondrial and cardiac function), interaction of ENDOG with the mitochondrial genome and ENDOG-mediated regulation of mitochondrial mass. At baseline, the Endog-deleted mouse heart had depleted mitochondria, mitochondrial dysfunction and elevated levels of reactive oxygen species, which were associated with enlarged and steatotic cardiomyocytes. Our study has further established the link between mitochondrial dysfunction, reactive oxygen species and heart disease and has uncovered a role for Endog in maladaptive cardiac hypertrophy.


Assuntos
Cardiomegalia/enzimologia , Cardiomegalia/patologia , Endodesoxirribonucleases/metabolismo , Mitocôndrias/metabolismo , Animais , Apoptose , Peso Corporal/genética , Cardiomegalia/genética , Cardiomegalia/fisiopatologia , Respiração Celular , Cromossomos de Mamíferos/genética , Cruzamentos Genéticos , Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/genética , Feminino , Regulação da Expressão Gênica , Genes Mitocondriais/genética , Hipertrofia Ventricular Esquerda/enzimologia , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Metabolismo dos Lipídeos , Masculino , Mitocôndrias/genética , Mitocôndrias/patologia , Tamanho do Órgão/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Locos de Características Quantitativas/genética , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Endogâmicos , Espécies Reativas de Oxigênio/metabolismo , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/metabolismo , Receptor ERRalfa Relacionado ao Estrogênio
8.
Circ Res ; 109(7): 758-69, 2011 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-21799151

RESUMO

RATIONALE: Telethonin (also known as titin-cap or t-cap) is a 19-kDa Z-disk protein with a unique ß-sheet structure, hypothesized to assemble in a palindromic way with the N-terminal portion of titin and to constitute a signalosome participating in the process of cardiomechanosensing. In addition, a variety of telethonin mutations are associated with the development of several different diseases; however, little is known about the underlying molecular mechanisms and telethonin's in vivo function. OBJECTIVE: Here we aim to investigate the role of telethonin in vivo and to identify molecular mechanisms underlying disease as a result of its mutation. METHODS AND RESULTS: By using a variety of different genetically altered animal models and biophysical experiments we show that contrary to previous views, telethonin is not an indispensable component of the titin-anchoring system, nor is deletion of the gene or cardiac specific overexpression associated with a spontaneous cardiac phenotype. Rather, additional titin-anchorage sites, such as actin-titin cross-links via α-actinin, are sufficient to maintain Z-disk stability despite the loss of telethonin. We demonstrate that a main novel function of telethonin is to modulate the turnover of the proapoptotic tumor suppressor p53 after biomechanical stress in the nuclear compartment, thus linking telethonin, a protein well known to be present at the Z-disk, directly to apoptosis ("mechanoptosis"). In addition, loss of telethonin mRNA and nuclear accumulation of this protein is associated with human heart failure, an effect that may contribute to enhanced rates of apoptosis found in these hearts. CONCLUSIONS: Telethonin knockout mice do not reveal defective heart development or heart function under basal conditions, but develop heart failure following biomechanical stress, owing at least in part to apoptosis of cardiomyocytes, an effect that may also play a role in human heart failure.


Assuntos
Insuficiência Cardíaca/metabolismo , Coração/fisiopatologia , Mecanotransdução Celular , Proteínas Musculares/deficiência , Miocárdio/metabolismo , Adaptação Fisiológica , Animais , Animais Geneticamente Modificados , Apoptose , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Conectina , Modelos Animais de Doenças , Ecocardiografia , Fibrose , Genótipo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Miocárdio/patologia , Fenótipo , Interferência de RNA , Ratos , Sarcômeros/metabolismo , Estresse Mecânico , Transfecção , Proteína Supressora de Tumor p53/metabolismo
9.
Circulation ; 123(24): 2838-47, 2011 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-21632490

RESUMO

BACKGROUND: Calcineurin is a calcium-regulated phosphatase that plays a major role in cardiac hypertrophy. We previously described that alternative splicing of the calcineurin Aß (CnAß) gene generates the CnAß1 isoform, with a unique C-terminal region that is different from the autoinhibitory domain present in all other CnA isoforms. In skeletal muscle, CnAß1 is necessary for myoblast proliferation and stimulates regeneration, reducing fibrosis and accelerating the resolution of inflammation. Its role in the heart is currently unknown. METHODS AND RESULTS: We generated transgenic mice overexpressing CnAß1 in postnatal cardiomyocytes under the control of the α-myosin heavy chain promoter. In contrast to previous studies using an artificially truncated calcineurin, CnAß1 overexpression did not induce cardiac hypertrophy. Moreover, transgenic mice showed improved cardiac function and reduced scar formation after myocardial infarction, with reduced neutrophil and macrophage infiltration and decreased expression of proinflammatory cytokines. Immunoprecipitation and Western blot analysis showed interaction of CnAß1 with the mTOR complex 2 and activation of the Akt/SGK cardioprotective pathway in a PI3K-independent manner. In addition, gene expression profiling revealed that CnAß1 activated the transcription factor ATF4 downstream of the Akt/mTOR pathway to promote the amino acid biosynthesis program, to reduce protein catabolism, and to induce the antifibrotic and antiinflammatory factor growth differentiation factor 15, which protects the heart through Akt activation. CONCLUSIONS: Calcineurin Aß1 shows a unique mode of action that improves cardiac function after myocardial infarction, activating different cardioprotective pathways without inducing maladaptive hypertrophy. These features make CnAß1 an attractive candidate for the development of future therapeutic approaches.


Assuntos
Calcineurina/genética , Coração/fisiologia , Contração Miocárdica/fisiologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/fisiopatologia , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Calcineurina/metabolismo , Cardiomegalia/genética , Cardiomegalia/fisiopatologia , Fibrose , Perfilação da Expressão Gênica , Camundongos , Camundongos Transgênicos , Infarto do Miocárdio/metabolismo , Miocardite/genética , Miocardite/metabolismo , Miocardite/fisiopatologia , Miocárdio/metabolismo , Miocárdio/patologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Isoformas de Proteínas/genética , Transdução de Sinais/fisiologia
10.
Science ; 377(6606): eabo1984, 2022 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-35926050

RESUMO

Pathogenic variants in genes that cause dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM) convey high risks for the development of heart failure through unknown mechanisms. Using single-nucleus RNA sequencing, we characterized the transcriptome of 880,000 nuclei from 18 control and 61 failing, nonischemic human hearts with pathogenic variants in DCM and ACM genes or idiopathic disease. We performed genotype-stratified analyses of the ventricular cell lineages and transcriptional states. The resultant DCM and ACM ventricular cell atlas demonstrated distinct right and left ventricular responses, highlighting genotype-associated pathways, intercellular interactions, and differential gene expression at single-cell resolution. Together, these data illuminate both shared and distinct cellular and molecular architectures of human heart failure and suggest candidate therapeutic targets.


Assuntos
Displasia Arritmogênica Ventricular Direita , Cardiomiopatia Dilatada , Insuficiência Cardíaca , Análise de Célula Única , Transcriptoma , Displasia Arritmogênica Ventricular Direita/genética , Atlas como Assunto , Cardiomiopatia Dilatada/genética , Núcleo Celular/genética , Insuficiência Cardíaca/genética , Ventrículos do Coração , Humanos , RNA-Seq
11.
Sci Transl Med ; 13(597)2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108253

RESUMO

Acetaminophen (N-acetyl-p-aminophenol; APAP) toxicity is a common cause of liver damage. In the mouse model of APAP-induced liver injury (AILI), interleukin 11 (IL11) is highly up-regulated and administration of recombinant human IL11 (rhIL11) has been shown to be protective. Here, we demonstrate that the beneficial effect of rhIL11 in the mouse model of AILI is due to its inhibition of endogenous mouse IL11 activity. Our results show that species-matched IL11 behaves like a hepatotoxin. IL11 secreted from APAP-damaged human and mouse hepatocytes triggered an autocrine loop of NADPH oxidase 4 (NOX4)-dependent cell death, which occurred downstream of APAP-initiated mitochondrial dysfunction. Hepatocyte-specific deletion of Il11 receptor subunit alpha chain 1 (Il11ra1) in adult mice protected against AILI despite normal APAP metabolism and glutathione (GSH) depletion. Mice with germline deletion of Il11 were also protected from AILI, and deletion of Il1ra1 or Il11 was associated with reduced c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activation and quickly restored GSH concentrations. Administration of a neutralizing IL11RA antibody reduced AILI in mice across genetic backgrounds and promoted survival when administered up to 10 hours after APAP. Inhibition of IL11 signaling was associated with the up-regulation of markers of liver regenerations: cyclins and proliferating cell nuclear antigen (PCNA) as well as with phosphorylation of retinoblastoma protein (RB) 24 hours after AILI. Our data suggest that species-matched IL11 is a hepatotoxin and that IL11 signaling might be an effective therapeutic target for APAP-induced liver damage.


Assuntos
Doença Hepática Crônica Induzida por Substâncias e Drogas , Doença Hepática Induzida por Substâncias e Drogas , Acetaminofen/toxicidade , Animais , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Hepatócitos , Interleucina-11 , Subunidade alfa de Receptor de Interleucina-11 , Fígado , Camundongos , Camundongos Endogâmicos C57BL
12.
J Mol Cell Cardiol ; 47(1): 133-41, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19376125

RESUMO

Elevated levels of the cardiac transcription factor Hand1 have been reported in several adult cardiac diseases but it is unclear whether this change is itself maladaptive with respect to heart function. To test this possibility, we have developed a novel, inducible transgenic system, and used it to overexpress Hand1 in adult mouse hearts. Overexpression of Hand1 in the adult mouse heart leads to mild cardiac hypertrophy and a reduction in life expectancy. Treated mice show no significant fibrosis, myocyte disarray or congestive heart failure, but have a greatly reduced threshold for induced ventricular tachycardia, indicating a predisposition to cardiac arrhythmia. Within 48 h, they show a significant loss of connexin43 protein from cardiac intercalated discs, with increased intercalated disc beta-catenin expression at protein and RNA levels. These changes are sustained during prolonged Hand1 overexpression. We propose that cardiac overexpression of Hand1 offers a useful mouse model of arrhythmogenesis and elevated HAND1 may provide one of the molecular links between the failing heart and arrhythmia.


Assuntos
Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cardiomegalia/genética , Cardiomegalia/metabolismo , Eletrofisiologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Cardiovasc Res ; 77(4): 695-706, 2008 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-18178572

RESUMO

AIMS: Combined left ventricular assist device (LVAD) and pharmacological therapy has been proposed to favour myocardial recovery in patients with end-stage heart failure (HF). Clenbuterol (Clen), a beta(2)-adrenoceptor (beta(2)-AR) agonist, has been used as a part of this strategy. In this study, we investigated the direct effects of clenbuterol on unloaded myocardium in HF. METHODS AND RESULTS: Left coronary artery ligation or sham operation was performed in male Lewis rats. After 4-6 weeks, heterotopic abdominal transplantation of the failing hearts into normal recipients was performed to induce LV unloading (UN). Recipient rats were treated with saline (Sal) or clenbuterol (2 mg/kg/day) via osmotic minipumps (HF + UN + Sal or HF + UN + Clen) for 7 days. Non-transplanted HF animals were treated with Sal (Sham + Sal, HF + Sal) or clenbuterol (HF + Clen). LV myocytes were isolated and studied using optical, fluorescence, and electrophysiological techniques. Clenbuterol treatment improved in vivo LV function measured with echocardiography (LVEF (%): HF 35.9 +/- 2 [16], HF + Clen 52.1 +/- 1.4 [16]; P < 0.001; mean +/- SEM [n]). In combination with unloading, clenbuterol increased sarcomere shortening (amplitude (microm): HF + UN + Clen 0.1 +/- 0.01 [50], HF + UN + Sal 0.07 +/- 0.01 [38]; P < 0.001) by normalizing the depressed myofilament sensitivity to Ca(2+) (slope of the linear relationship between Ca(2+) transient and sarcomere shortening hysteresis loop during relaxation (microm/ratio unit): HF + UN + Clen 2.13 +/- 0.2 [52], HF + UN + Sal 1.42 +/- 0.13 [38]; P < 0.05). CONCLUSION: Clenbuterol treatment of failing rat hearts, alone or in combination with mechanical unloading, improves LV function at the whole-heart and cellular levels by affecting cell morphology, excitation-contraction coupling, and myofilament sensitivity to calcium. This study supports the use of this drug in the strategy to enhance recovery in HF patients treated with LVADs and also begins to elucidate some of the possible cellular mechanisms responsible for the improvement in LV function.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2 , Agonistas Adrenérgicos beta/farmacologia , Clembuterol/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Transplante de Coração , Miocárdio/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Potenciais de Ação , Agonistas Adrenérgicos beta/administração & dosagem , Animais , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Tamanho Celular , Clembuterol/administração & dosagem , Modelos Animais de Doenças , Insuficiência Cardíaca/diagnóstico por imagem , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Bombas de Infusão Implantáveis , Masculino , Camundongos , Contração Miocárdica/efeitos dos fármacos , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Isoformas de Proteínas/metabolismo , Ratos , Ratos Endogâmicos Lew , Receptores Adrenérgicos beta 2/metabolismo , Sarcômeros/metabolismo , Trocador de Sódio e Cálcio/efeitos dos fármacos , Trocador de Sódio e Cálcio/metabolismo , Ultrassonografia
15.
Nat Commun ; 10(1): 3616, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399586

RESUMO

Cardiac fibrosis is a final common pathology in inherited and acquired heart diseases that causes cardiac electrical and pump failure. Here, we use systems genetics to identify a pro-fibrotic gene network in the diseased heart and show that this network is regulated by the E3 ubiquitin ligase WWP2, specifically by the WWP2-N terminal isoform. Importantly, the WWP2-regulated pro-fibrotic gene network is conserved across different cardiac diseases characterized by fibrosis: human and murine dilated cardiomyopathy and repaired tetralogy of Fallot. Transgenic mice lacking the N-terminal region of the WWP2 protein show improved cardiac function and reduced myocardial fibrosis in response to pressure overload or myocardial infarction. In primary cardiac fibroblasts, WWP2 positively regulates the expression of pro-fibrotic markers and extracellular matrix genes. TGFß1 stimulation promotes nuclear translocation of the WWP2 isoforms containing the N-terminal region and their interaction with SMAD2. WWP2 mediates the TGFß1-induced nucleocytoplasmic shuttling and transcriptional activity of SMAD2.


Assuntos
Fibrose/metabolismo , Redes Reguladoras de Genes , Predisposição Genética para Doença , Proteína Smad2/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adolescente , Adulto , Idoso , Animais , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibrose/genética , Regulação da Expressão Gênica , Predisposição Genética para Doença/genética , Cardiopatias/genética , Cardiopatias/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Isoformas de Proteínas , Proteína Smad2/genética , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/genética , Adulto Jovem
16.
Circulation ; 115(17): 2254-61, 2007 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-17438152

RESUMO

BACKGROUND: Therapeutic efficacy of bone marrow (BM) cell injection for treating ischemic chronic heart failure has not been established. In addition, experimental data are lacking on arrhythmia occurrence after BM cell injection. We hypothesized that therapeutic efficacy and arrhythmia occurrence induced by BM cell injection may be affected by the cell delivery route. METHODS AND RESULTS: Three weeks after left coronary artery ligation, wild-type female rats were injected with 1x10(7) mononuclear BM cells derived from green fluorescent protein-transgenic male rats through either a direct intramyocardial or a retrograde intracoronary route. Both intramyocardial and intracoronary injection of BM cells demonstrated similar improvement in left ventricular ejection fraction measured by echocardiography and a similar graft size analyzed by real-time polymerase chain reaction for the Y chromosome-specific Sry gene. Noticeably, intramyocardial injection of BM cells induced frequent ventricular premature contractions (108+/-73 per hour at 7 days after BM cell injection), including multiform, consecutive ventricular premature contractions and ventricular tachycardia for the initial 14 days; intracoronary injection of BM cells and intramyocardial injection of phosphate-buffered saline rarely induced arrhythmias. Immunohistochemistry demonstrated that intramyocardial BM cell injection formed distinct cell clusters containing donor-derived cells and accumulated host-derived inflammatory cells in the infarct border zone, whereas intracoronary BM cell injection provided more homogeneous donor cell dissemination with less inflammation and without disrupting the native myocardial structure. CONCLUSIONS: BM cell injection is able to improve cardiac function in ischemic chronic heart failure but has a risk of arrhythmia occurrence when the intramyocardial route is used. Such arrhythmias may be prevented by using the intracoronary route.


Assuntos
Transplante de Medula Óssea/efeitos adversos , Transplante de Medula Óssea/métodos , Insuficiência Cardíaca/terapia , Isquemia Miocárdica/terapia , Taquicardia Ventricular/etiologia , Animais , Animais Geneticamente Modificados , Doença Crônica , Modelos Animais de Doenças , Feminino , Sobrevivência de Enxerto , Insuficiência Cardíaca/patologia , Injeções , Masculino , Isquemia Miocárdica/patologia , Miocárdio/patologia , Ratos , Ratos Sprague-Dawley , Taquicardia Ventricular/mortalidade
17.
Endocrinology ; 149(11): 5822-7, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18617621

RESUMO

Follistatins play roles in diverse biological processes including cell proliferation, wound healing, inflammation, and skeletal muscle growth, yet their role in the heart is currently unknown. We have investigated the myocardial expression profile and cellular distribution of follistatin (FST) and the FST-like genes FSTL1 and FSTL3 in the normal and failing heart. Expression was further analyzed in the novel setting of recovery from heart failure in myocardium obtained from patients who received combined mechanical (left ventricular assist device) and pharmacological therapy. Real-time PCR revealed that FSTL1 and FSTL3 expression was elevated in heart failure but returned to normal after recovery. FSTL3 expression levels correlated with molecular markers of disease severity and FSTL1 with the endothelial cell marker CD31, suggesting a potential link with vascularization. FSTL1 levels before treatment correlated with cardiac function after recovery, suggesting initial levels may influence long-term outcome. Immunohistochemistry revealed that FST was primarily localized to fibroblasts and vascular endothelium within the heart, whereas FSTL1 was localized to myocytes, endothelium, and smooth muscle cells and FSLT3 to myocytes and endothelium. Microarray analysis revealed that FST and FSTL1 were associated with extracellular matrix-related and calcium-binding proteins, whereas FSTL3 was associated mainly with cell signaling and transcription. These data show for the first time that elevated myocardial expression of FST-like genes is a feature of heart failure and may be linked to both disease severity and mechanisms underlying recovery, revealing new insight into the pathogenesis of heart failure and offering novel therapeutic targets.


Assuntos
Proteínas Relacionadas à Folistatina/genética , Insuficiência Cardíaca/genética , Animais , Estudos de Coortes , Proteínas Relacionadas à Folistatina/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/cirurgia , Coração Auxiliar , Humanos , Miocárdio/metabolismo , Miocárdio/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Índice de Gravidade de Doença , Distribuição Tecidual , Pressão Ventricular , Remodelação Ventricular/genética
18.
Biochem Biophys Res Commun ; 371(4): 615-20, 2008 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-18413147

RESUMO

Side population cells have been found in various types of adult tissue including heart and are presumed to be tissue-specific stem/progenitor cells. In the present study, we confirmed the presence of cardiac side population (cSP) cells, which showed both the Hoechst 33342 efflux ability and ABCG2 expression, in adult murine heart. Flow cytometric analysis showed that more than half of cSP cells expressed the endothelial marker VE-cadherin or the smooth muscle markers, alpha-smooth muscle actin and desmin. In addition, immunohistochemical analysis demonstrated that ABCG2(+) cells were mainly localized within vascular walls. Quantitative RT-PCR analysis demonstrated that VE-cadherin(-) cSP cells progressively expressed Nkx2.5 and cardiac troponin T with time in culture. VE-cadherin(-) cSP cells also expressed mesodermal-mesenchymal-associated markers and differentiated into osteocytes and adipocytes. These results highlight the heterogeneic nature of cSP cells, consisting of vascular endothelial cells, smooth muscle cells, and mesenchymal stem/progenitor cells including potential cardiomyogenic cells.


Assuntos
Diferenciação Celular , Coração , Mioblastos Cardíacos/citologia , Mioblastos Cardíacos/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/análise , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Actinas/análise , Actinas/genética , Actinas/metabolismo , Animais , Benzimidazóis/metabolismo , Caderinas/análise , Caderinas/genética , Caderinas/metabolismo , Separação Celular , Células Cultivadas , Desmina/análise , Desmina/genética , Desmina/metabolismo , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/análise , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mioblastos Cardíacos/química , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Troponina T/análise , Troponina T/genética , Troponina T/metabolismo
19.
Circulation ; 114(1 Suppl): I16-20, 2006 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-16820567

RESUMO

BACKGROUND: Combination therapy consisting of mechanical unloading using a left ventricular assist device (LVAD) and pharmacological intervention can promote recovery from end-stage heart failure, but the mechanism is unknown. Preliminary microarray analysis revealed a significant and unexpected decrease in myocardial arginine:glycine amidinotransferase (AGAT) gene expression during recovery in these patients. The aim of this study was to evaluate the expression and role of AGAT expression in heart failure and recovery. METHODS AND RESULTS: We used quantitative real time (TaqMan) polymerase chain reaction to examine myocardial AGAT mRNA expression in implant and explant samples from recovering patients after combination therapy (n=12), end-stage heart failure (ESHF) samples from stable patients undergoing transplantation without LVAD support (n=10), and donor hearts with normal hemodynamic function (n=8). AGAT mRNA expression was significantly elevated in all heart failure patients relative to donors (4.3-fold [P<0.001] and 2.7-fold [P<0.005] in LVAD and ESHF relative to donors, respectively) and returned to normal levels after recovery. AGAT enzyme activity was detectable in both human and rat myocardia and was elevated in heart failure. CONCLUSIONS: Our data highlight local and potentially regulated expression of AGAT activity in the myocardium and suggest a specific response to heart failure involving elevated local creatine synthesis. These findings have implications both for the management of recovery patients undergoing combination therapy and for heart failure in general.


Assuntos
Amidinotransferases/biossíntese , Insuficiência Cardíaca/enzimologia , Miocárdio/enzimologia , Adolescente , Adulto , Amidinotransferases/genética , Animais , Criança , Sistemas Computacionais , Convalescença , Creatina/metabolismo , Metabolismo Energético , Indução Enzimática , Feminino , Perfilação da Expressão Gênica , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/cirurgia , Transplante de Coração , Coração Auxiliar , Humanos , Rim/enzimologia , Fígado/enzimologia , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/enzimologia , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Ratos
20.
Vascul Pharmacol ; 46(3): 181-7, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17126612

RESUMO

Alternatively spliced endothelin (ET-1) receptor transcripts have been identified, but their significance to the functional effects of ET-1 has not been established. We have investigated the presence and influence of alternatively spliced ET(A) receptor transcripts on ET-1 mediated contraction of segments of human saphenous vein. The expression of ET(A) receptor transcripts was examined with quantitative reverse transcription-polymerase chain reaction (qPCR) studies, while the response of veins to ET-1 was tested with in vitro organ bath techniques. The expression of four different transcripts for the ET(A) receptor, in which either exon 3 is spliced out (Delta3), exon 4 is spliced out (Delta4), both 3 and 4 spliced out (Delta3,4) and when both exons 2 and 4 (Delta2,4) are spliced out were identified. Functional studies showed that a lack of efficacy and potency of ET-1 is associated with a significantly lower expression of the Delta3,4 transcript. ET(A) receptor antagonism was insurmountable in samples that had lower levels of the Delta3,4 transcript, while samples from patients with higher expression of the Delta3,4 showed surmountable antagonism with BQ123. These results suggest that there is a genetic basis for the variability between individuals for the contractile effect of ET-1 at ET(A) receptors.


Assuntos
Processamento Alternativo , Endotélio Vascular/fisiologia , Receptor de Endotelina A/genética , Veia Safena/fisiologia , Adulto , Idoso , Antagonistas do Receptor de Endotelina A , Endotelina-1/farmacologia , Endotélio Vascular/efeitos dos fármacos , Éxons , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Contração Muscular , Técnicas de Cultura de Órgãos/métodos , Peptídeos Cíclicos/farmacologia , Reação em Cadeia da Polimerase/métodos , Receptor de Endotelina A/metabolismo , Veia Safena/efeitos dos fármacos
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