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1.
Nat Commun ; 11(1): 138, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919361

RESUMO

In C. elegans, the conserved transcription factor DAF-16/FOXO is a powerful aging regulator, relaying dire conditions into expression of stress resistance and longevity promoting genes. For some of these functions, including low insulin/IGF signaling (IIS), DAF-16 depends on the protein SMK-1/SMEK, but how SMK-1 exerts this role has remained unknown. We show that SMK-1 functions as part of a specific Protein Phosphatase 4 complex (PP4SMK-1). Loss of PP4SMK-1 hinders transcriptional initiation at several DAF-16-activated genes, predominantly by impairing RNA polymerase II recruitment to their promoters. Search for the relevant substrate of PP4SMK-1 by phosphoproteomics identified the conserved transcriptional regulator SPT-5/SUPT5H, whose knockdown phenocopies the loss of PP4SMK-1. Phosphoregulation of SPT-5 is known to control transcriptional events such as elongation and termination. Here we also show that transcription initiating events are influenced by the phosphorylation status of SPT-5, particularly at DAF-16 target genes where transcriptional initiation appears rate limiting, rendering PP4SMK-1 crucial for many of DAF-16's physiological roles.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Envelhecimento/genética , Animais , Caenorhabditis elegans/genética , Proteínas Cromossômicas não Histona/genética , Longevidade/genética , Complexos Multiproteicos/metabolismo , Interferência de RNA , RNA Polimerase II/metabolismo , Estresse Fisiológico/genética , Transcrição Gênica/genética , Fatores de Elongação da Transcrição/genética
2.
JCI Insight ; 52019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31112528

RESUMO

The mechanisms regulating translation and splicing are not well understood. We provide insight into a new regulator of translation, OGFOD1 (2-oxoglutarate and iron dependent oxygenase domain-containing protein 1), which is a prolyl-hydroxylase that catalyzes the posttranslational hydroxylation of Pro-62 in the small ribosomal protein S23. We show that deletion of OGFOD1 in an in vitro model of human cardiomyocytes decreases translation of specific proteins (e.g., RNA-binding proteins) and alters splicing. RNA sequencing showed poor correlation between changes in mRNA and protein synthesis, suggesting that posttranscriptional regulation was the primary cause for the observed differences. We found that loss of OGFOD1 and the resultant alterations in protein translation modulates the cardiac proteome, shifting it towards higher protein amounts of sarcomeric proteins such as cardiac troponins, titin and cardiac myosin binding protein C. Furthermore, we found a decrease of OGFOD1 during cardiomyocyte differentiation. These results suggest that loss of OGFOD1 modulates protein translation and splicing, thereby leading to alterations in the cardiac proteome and highlight the role of altered translation and splicing in regulating the proteome..


Assuntos
Proteínas de Transporte/metabolismo , Diferenciação Celular/fisiologia , Miócitos Cardíacos/metabolismo , Proteínas Nucleares/metabolismo , Prolil Hidroxilases/metabolismo , Sequência de Bases , Proteínas de Transporte/genética , Linhagem Celular , Conectina , Técnicas de Inativação de Genes , Humanos , Proteínas Nucleares/genética , Prolil Hidroxilases/genética , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Transcriptoma , Troponina
3.
Stem Cell Reports ; 10(3): 834-847, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29503093

RESUMO

Energy metabolism is a key aspect of cardiomyocyte biology. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising tool for biomedical application, but they are immature and have not undergone metabolic maturation related to early postnatal development. To assess whether cultivation of hiPSC-CMs in 3D engineered heart tissue format leads to maturation of energy metabolism, we analyzed the mitochondrial and metabolic state of 3D hiPSC-CMs and compared it with 2D culture. 3D hiPSC-CMs showed increased mitochondrial mass, DNA content, and protein abundance (proteome). While hiPSC-CMs exhibited the principal ability to use glucose, lactate, and fatty acids as energy substrates irrespective of culture format, hiPSC-CMs in 3D performed more oxidation of glucose, lactate, and fatty acid and less anaerobic glycolysis. The increase in mitochondrial mass and DNA in 3D was diminished by pharmacological reduction of contractile force. In conclusion, contractile work contributes to metabolic maturation of hiPSC-CMs.


Assuntos
Metabolismo Energético/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Diferenciação Celular/fisiologia , Células Cultivadas , Ácidos Graxos/metabolismo , Glucose/metabolismo , Glicólise/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Ácido Láctico/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Contração Muscular/fisiologia , Miócitos Cardíacos/metabolismo
4.
J Am Heart Assoc ; 6(8)2017 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-28862954

RESUMO

BACKGROUND: Heart failure preceded by hypertrophy is a leading cause of death, and sex differences in hypertrophy are well known, although the basis for these sex differences is poorly understood. METHODS AND RESULTS: This study used a systems biology approach to investigate mechanisms underlying sex differences in cardiac hypertrophy. Male and female mice were treated for 2 and 3 weeks with angiotensin II to induce hypertrophy. Sex differences in cardiac hypertrophy were apparent after 3 weeks of treatment. RNA sequencing was performed on hearts, and sex differences in mRNA expression at baseline and following hypertrophy were observed, as well as within-sex differences between baseline and hypertrophy. Sex differences in mRNA were substantial at baseline and reduced somewhat with hypertrophy, as the mRNA differences induced by hypertrophy tended to overwhelm the sex differences. We performed an integrative analysis to identify mRNA networks that were differentially regulated in the 2 sexes by hypertrophy and obtained a network centered on PPARα (peroxisome proliferator-activated receptor α). Mouse experiments further showed that acute inhibition of PPARα blocked sex differences in the development of hypertrophy. CONCLUSIONS: The data in this study suggest that PPARα is involved in the sex-dimorphic regulation of cardiac hypertrophy.


Assuntos
Cardiomegalia/metabolismo , Miocárdio/metabolismo , PPAR alfa/metabolismo , Biologia de Sistemas/métodos , Angiotensina II , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/genética , Cardiomegalia/prevenção & controle , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Miocárdio/patologia , Oxazóis/farmacologia , PPAR alfa/antagonistas & inibidores , PPAR alfa/genética , Mapas de Interação de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Caracteres Sexuais , Fatores Sexuais , Transdução de Sinais , Fatores de Tempo , Tirosina/análogos & derivados , Tirosina/farmacologia
5.
Cardiovasc Res ; 110(3): 346-58, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27095734

RESUMO

AIMS: Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein-protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes. METHODS AND RESULTS: We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping. CONCLUSIONS: This study provides the first extensive characterization of the cardiac prolyl hydroxylome and demonstrates that inhibition of α-ketoglutarate hydroxylases alters protein stability, translation, and splicing.


Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/enzimologia , Miócitos Cardíacos/enzimologia , Prolina/química , Prolil Hidroxilases/metabolismo , Processamento de Proteína Pós-Traducional , Processamento Alternativo , Aminoácidos Dicarboxílicos/farmacologia , Linhagem Celular , Conectina/metabolismo , Humanos , Hidroxilação , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Fator de Processamento Associado a PTB/metabolismo , Inibidores de Prolil-Hidrolase/farmacologia , Biossíntese de Proteínas , Proteólise , Proteômica/métodos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo
6.
Tissue Eng Part A ; 22(3-4): 326-35, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26763667

RESUMO

Engineered heart tissue (EHT) from primary heart cells contains endothelial cells (ECs), but the extent to which ECs organize into vessel-like structures or even functional vessels remains unknown and is difficult to study by conventional methods. In this study, we generated fibrin-based mini-EHTs from a transgenic mouse line (Cdh5-CreERT2 × Rosa26-LacZ), in which ECs were specifically and inducibly labeled by applying tamoxifen (EC(iLacZ)). EHTs were generated from an unpurified cell mix of newborn mouse hearts and were cultured under standard serum-containing conditions. Cre expression in 15-day-old EHTs was induced by addition of o-hydroxytamoxifen to the culture medium for 48 h, and ECs were visualized by X-gal staining. EC(iLacZ) EHTs showed a dense X-gal-positive vessel-like network with distinct tubular structures. Immunofluorescence revealed that ECs were mainly associated with cardiomyocytes within the EHT. EC(iLacZ) EHT developed spontaneous and regular contractility with forces up to 0.1 mN. Coherent contractility and the presence of an extensive vessel-like network were both dependent on the presence of animal sera in the culture medium. Contractile EC(iLacZ) EHTs successfully served as grafts in implantation studies onto the hearts of immunodeficient mice. Four weeks after implantation, EHTs showed X-gal-positive lumen-forming vessel structures connected to the host myocardium circulation as they contained erythrocytes on a regular basis. Taken together, genetic labeling of ECs revealed the extensive formation of vessel-like structures in EHTs in vitro. The EC(iLacZ) EHT model could help simultaneously study biological effects of compounds on cardiomyocyte function and tissue vascularization.


Assuntos
Vasos Coronários/crescimento & desenvolvimento , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Neovascularização Fisiológica , Engenharia Tecidual/métodos , Animais , Vasos Coronários/citologia , Camundongos , Camundongos SCID , Camundongos Transgênicos , Miocárdio/citologia , Miócitos Cardíacos/citologia
7.
Nat Commun ; 5: 5515, 2014 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-25463264

RESUMO

Homozygous or compound heterozygous frameshift mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C) cause neonatal hypertrophic cardiomyopathy (HCM), which rapidly evolves into systolic heart failure and death within the first year of life. Here we show successful long-term Mybpc3 gene therapy in homozygous Mybpc3-targeted knock-in (KI) mice, which genetically mimic these human neonatal cardiomyopathies. A single systemic administration of adeno-associated virus (AAV9)-Mybpc3 in 1-day-old KI mice prevents the development of cardiac hypertrophy and dysfunction for the observation period of 34 weeks and increases Mybpc3 messenger RNA (mRNA) and cMyBP-C protein levels in a dose-dependent manner. Importantly, Mybpc3 gene therapy unexpectedly also suppresses accumulation of mutant mRNAs. This study reports the first successful long-term gene therapy of HCM with correction of both haploinsufficiency and production of poison peptides. In the absence of alternative treatment options except heart transplantation, gene therapy could become a realistic treatment option for severe neonatal HCM.


Assuntos
Cardiomiopatia Hipertrófica Familiar/genética , Proteínas de Transporte/genética , Terapia Genética/métodos , RNA Mensageiro/metabolismo , Animais , Cardiomiopatia Hipertrófica Familiar/metabolismo , Cardiomiopatia Hipertrófica Familiar/terapia , Proteínas de Transporte/metabolismo , Dependovirus , Técnicas de Introdução de Genes , Homozigoto , Camundongos
8.
J Mol Cell Cardiol ; 74: 151-61, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24852842

RESUMO

Spontaneously beating engineered heart tissue (EHT) represents an advanced in vitro model for drug testing and disease modeling, but cardiomyocytes in EHTs are less mature and generate lower forces than in the adult heart. We devised a novel pacing system integrated in a setup for videooptical recording of EHT contractile function over time and investigated whether sustained electrical field stimulation improved EHT properties. EHTs were generated from neonatal rat heart cells (rEHT, n=96) or human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hEHT, n=19). Pacing with biphasic pulses was initiated on day 4 of culture. REHT continuously paced for 16-18 days at 0.5Hz developed 2.2× higher forces than nonstimulated rEHT. This was reflected by higher cardiomyocyte density in the center of EHTs, increased connexin-43 abundance as investigated by two-photon microscopy and remarkably improved sarcomere ultrastructure including regular M-bands. Further signs of tissue maturation include a rightward shift (to more physiological values) of the Ca(2+)-response curve, increased force response to isoprenaline and decreased spontaneous beating activity. Human EHTs stimulated at 2Hz in the first week and 1.5Hz thereafter developed 1.5× higher forces than nonstimulated hEHT on day 14, an ameliorated muscular network of longitudinally oriented cardiomyocytes and a higher cytoplasm-to-nucleus ratio. Taken together, continuous pacing improved structural and functional properties of rEHTs and hEHTs to an unprecedented level. Electrical stimulation appears to be an important step toward the generation of fully mature EHT.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Miocárdio/citologia , Miócitos Cardíacos/citologia , Técnicas de Cultura de Tecidos/métodos , Engenharia Tecidual/métodos , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Cálcio/metabolismo , Diferenciação Celular , Núcleo Celular/fisiologia , Núcleo Celular/ultraestrutura , Conexina 43/metabolismo , Citoplasma/fisiologia , Citoplasma/ultraestrutura , Estimulação Elétrica , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Isoproterenol/farmacologia , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos , Sarcômeros/fisiologia , Sarcômeros/ultraestrutura
9.
Nature ; 509(7502): 641-4, 2014 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-24747400

RESUMO

Despite the introduction of antiproliferative drug-eluting stents, coronary heart disease remains the leading cause of death in the United States. In-stent restenosis and bypass graft failure are characterized by excessive smooth muscle cell (SMC) proliferation and concomitant myointima formation with luminal obliteration. Here we show that during the development of myointimal hyperplasia in human arteries, SMCs show hyperpolarization of their mitochondrial membrane potential (ΔΨm) and acquire a temporary state with a high proliferative rate and resistance to apoptosis. Pyruvate dehydrogenase kinase isoform 2 (PDK2) was identified as a key regulatory protein, and its activation proved necessary for relevant myointima formation. Pharmacologic PDK2 blockade with dichloroacetate or lentiviral PDK2 knockdown prevented ΔΨm hyperpolarization, facilitated apoptosis and reduced myointima formation in injured human mammary and coronary arteries, rat aortas, rabbit iliac arteries and swine (pig) coronary arteries. In contrast to several commonly used antiproliferative drugs, dichloroacetate did not prevent vessel re-endothelialization. Targeting myointimal ΔΨm and alleviating apoptosis resistance is a novel strategy for the prevention of proliferative vascular diseases.


Assuntos
Aorta/lesões , Artérias/lesões , Constrição Patológica/prevenção & controle , Ácido Dicloroacético/farmacologia , Ácido Dicloroacético/uso terapêutico , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/patologia , Angioplastia com Balão/efeitos adversos , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Apoptose/efeitos dos fármacos , Artérias/efeitos dos fármacos , Artérias/patologia , Proliferação de Células/efeitos dos fármacos , Constrição Patológica/patologia , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/lesões , Vasos Coronários/patologia , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Hiperplasia/tratamento farmacológico , Hiperplasia/patologia , Artéria Ilíaca/efeitos dos fármacos , Artéria Ilíaca/lesões , Artéria Ilíaca/patologia , Artéria Torácica Interna/efeitos dos fármacos , Artéria Torácica Interna/lesões , Artéria Torácica Interna/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil , Coelhos , Ratos , Prevenção Secundária , Stents/efeitos adversos , Suínos , Túnica Íntima/lesões
10.
Am J Physiol Heart Circ Physiol ; 306(9): H1353-63, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24585781

RESUMO

Contraction and relaxation are fundamental aspects of cardiomyocyte functional biology. They reflect the response of the contractile machinery to the systolic increase and diastolic decrease of the cytoplasmic Ca(2+) concentration. The analysis of contractile function and Ca(2+) transients is therefore important to discriminate between myofilament responsiveness and changes in Ca(2+) homeostasis. This article describes an automated technology to perform sequential analysis of contractile force and Ca(2+) transients in up to 11 strip-format, fibrin-based rat, mouse, and human fura-2-loaded engineered heart tissues (EHTs) under perfusion and electrical stimulation. Measurements in EHTs under increasing concentrations of extracellular Ca(2+) and responses to isoprenaline and carbachol demonstrate that EHTs recapitulate basic principles of heart tissue functional biology. Ca(2+) concentration-response curves in rat, mouse, and human EHTs indicated different maximal twitch forces (0.22, 0.05, and 0.08 mN in rat, mouse, and human, respectively; P < 0.001) and different sensitivity to external Ca(2+) (EC50: 0.15, 0.39, and 1.05 mM Ca(2+) in rat, mouse, and human, respectively; P < 0.001) in the three groups. In contrast, no difference in myofilament Ca(2+) sensitivity was detected between skinned rat and human EHTs, suggesting that the difference in sensitivity to external Ca(2+) concentration is due to changes in Ca(2+) handling proteins. Finally, this study confirms that fura-2 has Ca(2+) buffering effects and is thereby changing the force response to extracellular Ca(2+).


Assuntos
Sinalização do Cálcio , Microscopia de Fluorescência/métodos , Contração Miocárdica , Miocárdio/citologia , Miócitos Cardíacos/metabolismo , Engenharia Tecidual/métodos , Animais , Automação Laboratorial , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/instrumentação , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Ratos , Ratos Endogâmicos Lew , Ratos Wistar
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