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1.
Circulation ; 147(5): 409-424, 2023 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-36448446

RESUMO

BACKGROUND: Extensive evidence from single-center studies indicates that a subset of patients with chronic advanced heart failure (HF) undergoing left ventricular assist device (LVAD) support show significantly improved heart function and reverse structural remodeling (ie, termed "responders"). Furthermore, we recently published a multicenter prospective study, RESTAGE-HF (Remission from Stage D Heart Failure), demonstrating that LVAD support combined with standard HF medications induced remarkable cardiac structural and functional improvement, leading to high rates of LVAD weaning and excellent long-term outcomes. This intriguing phenomenon provides great translational and clinical promise, although the underlying molecular mechanisms driving this recovery are largely unknown. METHODS: To identify changes in signaling pathways operative in the normal and failing human heart and to molecularly characterize patients who respond favorably to LVAD unloading, we performed global RNA sequencing and phosphopeptide profiling of left ventricular tissue from 93 patients with HF undergoing LVAD implantation (25 responders and 68 nonresponders) and 12 nonfailing donor hearts. Patients were prospectively monitored through echocardiography to characterize their myocardial structure and function and identify responders and nonresponders. RESULTS: These analyses identified 1341 transcripts and 288 phosphopeptides that are differentially regulated in cardiac tissue from nonfailing control samples and patients with HF. In addition, these unbiased molecular profiles identified a unique signature of 29 transcripts and 93 phosphopeptides in patients with HF that distinguished responders after LVAD unloading. Further analyses of these macromolecules highlighted differential regulation in 2 key pathways: cell cycle regulation and extracellular matrix/focal adhesions. CONCLUSIONS: This is the first study to characterize changes in the nonfailing and failing human heart by integrating multiple -omics platforms to identify molecular indices defining patients capable of myocardial recovery. These findings may guide patient selection for advanced HF therapies and identify new HF therapeutic targets.


Assuntos
Insuficiência Cardíaca , Transplante de Coração , Coração Auxiliar , Humanos , Transcriptoma , Estudos Prospectivos , Fosfopeptídeos/metabolismo , Proteômica , Doadores de Tecidos , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/terapia , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo
2.
J Biol Chem ; 298(4): 101653, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35101445

RESUMO

PROteolysis TArgeting Chimeras (PROTACs) are hetero-bifunctional small molecules that can simultaneously recruit target proteins and E3 ligases to form a ternary complex, promoting target protein ubiquitination and degradation via the Ubiquitin-Proteasome System (UPS). PROTACs have gained increasing attention in recent years due to certain advantages over traditional therapeutic modalities and enabling targeting of previously "undruggable" proteins. To better understand the mechanism of PROTAC-induced Target Protein Degradation (TPD), several computational approaches have recently been developed to study and predict ternary complex formation. However, mounting evidence suggests that ubiquitination can also be a rate-limiting step in PROTAC-induced TPD. Here, we propose a structure-based computational approach to predict target protein ubiquitination induced by cereblon (CRBN)-based PROTACs by leveraging available structural information of the CRL4A ligase complex (CRBN/DDB1/CUL4A/Rbx1/NEDD8/E2/Ub). We generated ternary complex ensembles with Rosetta, modeled multiple CRL4A ligase complex conformations, and predicted ubiquitination efficiency by separating the ternary ensemble into productive and unproductive complexes based on the proximity of the ubiquitin to accessible lysines on the target protein. We validated our CRL4A ligase complex models with published ternary complex structures and additionally employed our modeling workflow to predict ubiquitination efficiencies and sites of a series of cyclin-dependent kinases (CDKs) after treatment with TL12-186, a pan-kinase PROTAC. Our predictions are consistent with CDK ubiquitination and site-directed mutagenesis of specific CDK lysine residues as measured using a NanoBRET ubiquitination assay in HEK293 cells. This work structurally links PROTAC-induced ternary formation and ubiquitination, representing an important step toward prediction of target "degradability."


Assuntos
Modelos Moleculares , Ubiquitina-Proteína Ligases , Ubiquitinação , Células HEK293 , Humanos , Estrutura Terciária de Proteína , Proteólise , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo
3.
J Proteome Res ; 20(5): 2780-2795, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33856812

RESUMO

Proteomic investigations of Alzheimer's and Parkinson's disease have provided valuable insights into neurodegenerative disorders. Thus far, these investigations have largely been restricted to bottom-up approaches, hindering the degree to which one can characterize a protein's "intact" state. Top-down proteomics (TDP) overcomes this limitation; however, it is typically limited to observing only the most abundant proteoforms and of a relatively small size. Therefore, fractionation techniques are commonly used to reduce sample complexity. Here, we investigate gas-phase fractionation through high-field asymmetric waveform ion mobility spectrometry (FAIMS) within TDP. Utilizing a high complexity sample derived from Alzheimer's disease (AD) brain tissue, we describe how the addition of FAIMS to TDP can robustly improve the depth of proteome coverage. For example, implementation of FAIMS with external compensation voltage (CV) stepping at -50, -40, and -30 CV could more than double the mean number of non-redundant proteoforms, genes, and proteome sequence coverage compared to without FAIMS. We also found that FAIMS can influence the transmission of proteoforms and their charge envelopes based on their size. Importantly, FAIMS enabled the identification of intact amyloid beta (Aß) proteoforms, including the aggregation-prone Aß1-42 variant which is strongly linked to AD. Raw data and associated files have been deposited to the ProteomeXchange Consortium via the MassIVE data repository with data set identifier PXD023607.


Assuntos
Espectrometria de Mobilidade Iônica , Proteômica , Peptídeos beta-Amiloides , Encéfalo , Química Encefálica , Proteoma
4.
Proc Natl Acad Sci U S A ; 115(33): E7871-E7880, 2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30061404

RESUMO

Smyd1, a muscle-specific histone methyltransferase, has established roles in skeletal and cardiac muscle development, but its role in the adult heart remains poorly understood. Our prior work demonstrated that cardiac-specific deletion of Smyd1 in adult mice (Smyd1-KO) leads to hypertrophy and heart failure. Here we show that down-regulation of mitochondrial energetics is an early event in these Smyd1-KO mice preceding the onset of structural abnormalities. This early impairment of mitochondrial energetics in Smyd1-KO mice is associated with a significant reduction in gene and protein expression of PGC-1α, PPARα, and RXRα, the master regulators of cardiac energetics. The effect of Smyd1 on PGC-1α was recapitulated in primary cultured rat ventricular myocytes, in which acute siRNA-mediated silencing of Smyd1 resulted in a greater than twofold decrease in PGC-1α expression without affecting that of PPARα or RXRα. In addition, enrichment of histone H3 lysine 4 trimethylation (a mark of gene activation) at the PGC-1α locus was markedly reduced in Smyd1-KO mice, and Smyd1-induced transcriptional activation of PGC-1α was confirmed by luciferase reporter assays. Functional confirmation of Smyd1's involvement showed an increase in mitochondrial respiration capacity induced by overexpression of Smyd1, which was abolished by siRNA-mediated PGC-1α knockdown. Conversely, overexpression of PGC-1α rescued transcript expression and mitochondrial respiration caused by silencing Smyd1 in cardiomyocytes. These findings provide functional evidence for a role of Smyd1, or any member of the Smyd family, in regulating cardiac energetics in the adult heart, which is mediated, at least in part, via modulating PGC-1α.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Metabolismo Energético/fisiologia , Histona-Lisina N-Metiltransferase/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/enzimologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/biossíntese , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/genética , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/genética , Proteínas Musculares/genética , PPAR alfa/biossíntese , PPAR alfa/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Receptor X Retinoide alfa/biossíntese , Receptor X Retinoide alfa/genética , Fatores de Transcrição/genética
5.
J Biol Chem ; 294(44): 16385-16399, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31530639

RESUMO

Bardet-Biedl syndrome (BBS) is a genetic disorder characterized by malfunctions in primary cilia resulting from mutations that disrupt the function of the BBSome, an 8-subunit complex that plays an important role in protein transport in primary cilia. To better understand the molecular basis of BBS, here we used an integrative structural modeling approach consisting of EM and chemical cross-linking coupled with MS analyses, to analyze the structure of a BBSome 2-7-9 subcomplex consisting of three homologous BBS proteins, BBS2, BBS7, and BBS9. The resulting molecular model revealed an overall structure that resembles a flattened triangle. We found that within this structure, BBS2 and BBS7 form a tight dimer through a coiled-coil interaction and that BBS9 associates with the dimer via an interaction with the α-helical domain of BBS2. Interestingly, a BBS-associated mutation of BBS2 (R632P) is located in its α-helical domain at the interface between BBS2 and BBS9, and binding experiments indicated that this mutation disrupts the BBS2-BBS9 interaction. This finding suggests that BBSome assembly is disrupted by the R632P substitution, providing molecular insights that may explain the etiology of BBS in individuals harboring this mutation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas/metabolismo , Síndrome de Bardet-Biedl/metabolismo , Cílios/metabolismo , Células HEK293 , Humanos , Espectrometria de Massas/métodos , Microscopia Eletrônica/métodos , Modelos Moleculares , Mutação
6.
Proc Natl Acad Sci U S A ; 112(8): 2413-8, 2015 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-25675501

RESUMO

G-protein signaling depends on the ability of the individual subunits of the G-protein heterotrimer to assemble into a functional complex. Formation of the G-protein ßγ (Gßγ) dimer is particularly challenging because it is an obligate dimer in which the individual subunits are unstable on their own. Recent studies have revealed an intricate chaperone system that brings Gß and Gγ together. This system includes cytosolic chaperonin containing TCP-1 (CCT; also called TRiC) and its cochaperone phosducin-like protein 1 (PhLP1). Two key intermediates in the Gßγ assembly process, the Gß-CCT and the PhLP1-Gß-CCT complexes, were isolated and analyzed by a hybrid structural approach using cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and unnatural amino acid cross-linking. The structures show that Gß interacts with CCT in a near-native state through interactions of the Gγ-binding region of Gß with the CCTγ subunit. PhLP1 binding stabilizes the Gß fold, disrupting interactions with CCT and releasing a PhLP1-Gß dimer for assembly with Gγ. This view provides unique insight into the interplay between CCT and a cochaperone to orchestrate the folding of a protein substrate.


Assuntos
Proteínas de Transporte/química , Chaperonina com TCP-1/química , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/química , Proteínas do Tecido Nervoso/química , Multimerização Proteica , Aminoácidos/metabolismo , Animais , Benzofenonas , Proteínas de Transporte/ultraestrutura , Chaperonina com TCP-1/ultraestrutura , Reagentes de Ligações Cruzadas/metabolismo , Microscopia Crioeletrônica , Subunidades beta da Proteína de Ligação ao GTP/ultraestrutura , Subunidades gama da Proteína de Ligação ao GTP/ultraestrutura , Humanos , Espectrometria de Massas , Modelos Moleculares , Proteínas do Tecido Nervoso/ultraestrutura , Fenilalanina/análogos & derivados , Estrutura Secundária de Proteína
7.
J Virol ; 86(15): 7907-17, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22593163

RESUMO

Halophage CW02 infects a Salinivibrio costicola-like bacterium, SA50, isolated from the Great Salt Lake. Following isolation, cultivation, and purification, CW02 was characterized by DNA sequencing, mass spectrometry, and electron microscopy. A conserved module of structural genes places CW02 in the T7 supergroup, members of which are found in diverse aquatic environments, including marine and freshwater ecosystems. CW02 has morphological similarities to viruses of the Podoviridae family. The structure of CW02, solved by cryogenic electron microscopy and three-dimensional reconstruction, enabled the fitting of a portion of the bacteriophage HK97 capsid protein into CW02 capsid density, thereby providing additional evidence that capsid proteins of tailed double-stranded DNA phages have a conserved fold. The CW02 capsid consists of bacteriophage lambda gpD-like densities that likely contribute to particle stability. Turret-like densities were found on icosahedral vertices and may represent a unique adaptation similar to what has been seen in other extremophilic viruses that infect archaea, such as Sulfolobus turreted icosahedral virus and halophage SH1.


Assuntos
Capsídeo , DNA Viral , Ecossistema , Podoviridae , Vibrionaceae/virologia , Capsídeo/metabolismo , Capsídeo/ultraestrutura , DNA Viral/genética , DNA Viral/metabolismo , Água Doce/virologia , Podoviridae/genética , Podoviridae/metabolismo , Podoviridae/ultraestrutura , Análise de Sequência de DNA
8.
PNAS Nexus ; 1(3): pgac124, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-36003074

RESUMO

Human leukocyte antigen class I (HLA-I) molecules bind and present peptides at the cell surface to facilitate the induction of appropriate CD8+ T cell-mediated immune responses to pathogen- and self-derived proteins. The HLA-I peptide-binding cleft contains dominant anchor sites in the B and F pockets that interact primarily with amino acids at peptide position 2 and the C-terminus, respectively. Nonpocket peptide-HLA interactions also contribute to peptide binding and stability, but these secondary interactions are thought to be unique to individual HLA allotypes or to specific peptide antigens. Here, we show that two positively charged residues located near the top of peptide-binding cleft facilitate interactions with negatively charged residues at position 4 of presented peptides, which occur at elevated frequencies across most HLA-I allotypes. Loss of these interactions was shown to impair HLA-I/peptide binding and complex stability, as demonstrated by both in vitro and in silico experiments. Furthermore, mutation of these Arginine-65 (R65) and/or Lysine-66 (K66) residues in HLA-A*02:01 and A*24:02 significantly reduced HLA-I cell surface expression while also reducing the diversity of the presented peptide repertoire by up to 5-fold. The impact of the R65 mutation demonstrates that nonpocket HLA-I/peptide interactions can constitute anchor motifs that exert an unexpectedly broad influence on HLA-I-mediated antigen presentation. These findings provide fundamental insights into peptide antigen binding that could broadly inform epitope discovery in the context of viral vaccine development and cancer immunotherapy.

9.
Nat Commun ; 12(1): 2529, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953175

RESUMO

In the past decade, many long noncoding RNAs (lncRNAs) have been identified and their in vitro functions defined, although in some cases their functions in vivo remain less clear. Moreover, unlike nuclear lncRNAs, the roles of cytoplasmic lncRNAs are less defined. Here, using a gene trapping approach in mouse embryonic stem cells, we identify Caren (short for cardiomyocyte-enriched noncoding transcript), a cytoplasmic lncRNA abundantly expressed in cardiomyocytes. Caren maintains cardiac function under pathological stress by inactivating the ataxia telangiectasia mutated (ATM)-DNA damage response (DDR) pathway and activating mitochondrial bioenergetics. The presence of Caren transcripts does not alter expression of nearby (cis) genes but rather decreases translation of an mRNA transcribed from a distant gene encoding histidine triad nucleotide-binding protein 1 (Hint1), which activates the ATM-DDR pathway and reduces mitochondrial respiratory capacity in cardiomyocytes. Therefore, the cytoplasmic lncRNA Caren functions in cardioprotection by regulating translation of a distant gene and maintaining cardiomyocyte homeostasis.


Assuntos
Dano ao DNA/fisiologia , Insuficiência Cardíaca/metabolismo , Biogênese de Organelas , RNA Longo não Codificante/metabolismo , Animais , Núcleo Celular , Metabolismo Energético , Fibroblastos , Insuficiência Cardíaca/patologia , Homeostase , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/metabolismo , Células-Tronco Embrionárias Murinas , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Longo não Codificante/genética , RNA Mensageiro/metabolismo
10.
Cell Rep ; 29(2): 270-282.e5, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31597091

RESUMO

Accumulation of visceral (VIS) is a predictor of metabolic disorders and insulin resistance. This is due in part to the limited capacity of VIS fat to buffer lipids allowing them to deposit in insulin-sensitive tissues. Mechanisms underlying selective hypertrophic growth and tissue remodeling properties of VIS fat are not well understood. We identified subsets of adipose progenitors (APs) unique to VIS fat with differential Cd34 expression and adipogenic capacity. VIS low (Cd34 low) APs are adipogenic, whereas VIS high (Cd34 high) APs are not. Furthermore, VIS high APs inhibit adipogenic differentiation of SUB and VIS low APs in vitro through the secretion of soluble inhibitory factor(s). The number of VIS high APs increased with adipose tissue expansion, and their abundance in vivo caused hypertrophic growth, fibrosis, inflammation, and metabolic dysfunction. This study unveils the presence of APs unique to VIS fat involved in the paracrine regulation of adipogenesis and tissue remodeling.


Assuntos
Antígenos CD34/metabolismo , Gordura Intra-Abdominal/citologia , Comunicação Parácrina , Transdução de Sinais , Células-Tronco/metabolismo , Adipogenia/efeitos dos fármacos , Tecido Adiposo Branco/citologia , Animais , Proteína Morfogenética Óssea 4/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Resistência à Insulina , Gordura Intra-Abdominal/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Comunicação Parácrina/efeitos dos fármacos , Fenótipo , Rosiglitazona/farmacologia , Transdução de Sinais/efeitos dos fármacos , Solubilidade , Células-Tronco/efeitos dos fármacos , Aumento de Peso/efeitos dos fármacos
11.
Nat Commun ; 10(1): 2865, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253771

RESUMO

The mechanistic target of rapamycin (mTOR) kinase forms two multi-protein signaling complexes, mTORC1 and mTORC2, which are master regulators of cell growth, metabolism, survival and autophagy. Two of the subunits of these complexes are mLST8 and Raptor, ß-propeller proteins that stabilize the mTOR kinase and recruit substrates, respectively. Here we report that the eukaryotic chaperonin CCT plays a key role in mTORC assembly and signaling by folding both mLST8 and Raptor. A high resolution (4.0 Å) cryo-EM structure of the human mLST8-CCT intermediate isolated directly from cells shows mLST8 in a near-native state bound to CCT deep within the folding chamber between the two CCT rings, and interacting mainly with the disordered N- and C-termini of specific CCT subunits of both rings. These findings describe a unique function of CCT in mTORC assembly and a distinct binding site in CCT for mLST8, far from those found for similar ß-propeller proteins.


Assuntos
Chaperonina com TCP-1/fisiologia , Proteína Regulatória Associada a mTOR/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Homólogo LST8 da Proteína Associada a mTOR/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Microscopia Crioeletrônica , Regulação da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Células HEK293 , Células Hep G2 , Humanos , Espectrometria de Massas , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Proteína Regulatória Associada a mTOR/genética , Serina-Treonina Quinases TOR/genética , Homólogo LST8 da Proteína Associada a mTOR/genética
12.
Curr Opin Physiol ; 1: 140-152, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29435515

RESUMO

Protein methylation plays a pivotal role in the regulation of various cellular processes including chromatin remodeling and gene expression. SET and MYND domain-containing proteins (Smyd) are a special class of lysine methyltransferases whose catalytic SET domain is split by an MYND domain. The hallmark feature of this family was thought to be the methylation of histone H3 (on lysine 4). However, several studies suggest that the role of the Smyd family is dynamic, targeting unique histone residues associated with both transcriptional activation and repression. Smyd proteins also methylate several non-histone proteins to regulate various cellular processes. Although we are only beginning to understand their specific molecular functions and role in chromatin remodeling, recent studies have advanced our understanding of this relatively uncharacterized family, highlighting their involvement in development, cell growth and differentiation and during disease in various animal models. This review summarizes our current knowledge of the structure, function and methylation targets of the Smyd family and provides a compilation of data emphasizing their prominent role in cardiac and skeletal muscle physiology and pathology.

13.
PLoS One ; 10(3): e0118974, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25790351

RESUMO

Heart failure (HF) is accompanied by complex alterations in myocardial energy metabolism. Up to 40% of HF patients have dyssynchronous ventricular contraction, which is an independent indicator of mortality. We hypothesized that electromechanical dyssynchrony significantly affects metabolic remodeling in the course of HF. We used a canine model of tachypacing-induced HF. Animals were paced at 200 bpm for 6 weeks either in the right atrium (synchronous HF, SHF) or in the right ventricle (dyssynchronous HF, DHF). We collected biopsies from left ventricular apex and performed comprehensive metabolic pathway analysis using multi-platform metabolomics (GC/MS; MS/MS; HPLC) and LC-MS/MS label-free proteomics. We found important differences in metabolic remodeling between SHF and DHF. As compared to Control, ATP, phosphocreatine (PCr), creatine, and PCr/ATP (prognostic indicator of mortality in HF patients) were all significantly reduced in DHF, but not SHF. In addition, the myocardial levels of carnitine (mitochondrial fatty acid carrier) and fatty acids (12:0, 14:0) were significantly reduced in DHF, but not SHF. Carnitine parmitoyltransferase I, a key regulatory enzyme of fatty acid ß-oxidation, was significantly upregulated in SHF but was not different in DHF, as compared to Control. Both SHF and DHF exhibited a reduction, but to a different degree, in creatine and the intermediates of glycolysis and the TCA cycle. In contrast to this, the enzymes of creatine kinase shuttle were upregulated, and the enzymes of glycolysis and the TCA cycle were predominantly upregulated or unchanged in both SHF and DHF. These data suggest a systemic mismatch between substrate supply and demand in pacing-induced HF. The energy deficit observed in DHF, but not in SHF, may be associated with a critical decrease in fatty acid delivery to the ß-oxidation pipeline, primarily due to a reduction in myocardial carnitine content.


Assuntos
Metabolismo Energético/fisiologia , Insuficiência Cardíaca/metabolismo , Redes e Vias Metabólicas/fisiologia , Metabolômica/métodos , Miocárdio/metabolismo , Disfunção Ventricular/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Ciclo do Ácido Cítrico/fisiologia , Creatina/metabolismo , Creatina Quinase/metabolismo , Cães , Cromatografia Gasosa-Espectrometria de Massas , Glicólise , Proteômica/métodos , Espectrometria de Massas em Tandem , Fatores de Tempo , Disfunção Ventricular/fisiopatologia
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