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Genomic experiments analyzing human papillomaviruses (HPVs) require a carefully selected list of sequences as a reference database to map millions of reads. The available sources, such as the Papillomavirus Episteme (PaVE), are organized based on variations in the L1 gene rather than the whole HPV sequence. Moreover, the PaVE process uses complex multiple sequence alignments containing hundreds or thousands of sequences. These issues complicate the generation of a reference database for genomics, leading to the generation of per-analysis-defined databases. Here, we propose a de novo strategy considering all HPV sequences reported in the NCBI database to define a subset of highly representative HPV sequences. The strategy is based on oligonucleotide frequency profiling of the whole sequence followed by hierarchical clustering. Using data from HPV capture experiments, we demonstrate that this strategy selects suitable sequences as a reference database to map most mappable reads unambiguously. We provide some recommendations to improve HPV mapping. The generated .fasta files can be accessed at https://github.com/vtrevino/HPV-Ref-Genomes .
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Alphapapillomavirus , Infecções por Papillomavirus , Mapeamento Cromossômico , Genômica , Humanos , Papillomaviridae/genéticaRESUMO
Heart failure in children is a complex clinical syndrome with multiple aetiologies. The underlying disorders that lead to heart failure in children differ significantly from those in adults. Some clinical biomarkers for heart failure status and prognosis appear to be useful in both age groups. This review outlines the use and the present status of biomarkers for heart failure in paediatric cardiology. Furthermore, clinical scenarios in which development of new biomarkers might address management or prognosis are discussed. Finally, strategies for proteomic discovery of novel biomarkers and application to practice are described.
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Biomarcadores/análise , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/terapia , Pediatria , Proteômica/métodos , Humanos , Prognóstico , Processamento de Proteína Pós-TraducionalRESUMO
In the United States alone, â¼14,000 children are hospitalised annually with acute heart failure. The science and art of caring for these patients continues to evolve. The International Pediatric Heart Failure Summit of Johns Hopkins All Children's Heart Institute was held on February 4 and 5, 2015. The 2015 International Pediatric Heart Failure Summit of Johns Hopkins All Children's Heart Institute was funded through the Andrews/Daicoff Cardiovascular Program Endowment, a philanthropic collaboration between All Children's Hospital and the Morsani College of Medicine at the University of South Florida (USF). Sponsored by All Children's Hospital Andrews/Daicoff Cardiovascular Program, the International Pediatric Heart Failure Summit assembled leaders in clinical and scientific disciplines related to paediatric heart failure and created a multi-disciplinary "think-tank". The purpose of this manuscript is to summarise the lessons from the 2015 International Pediatric Heart Failure Summit of Johns Hopkins All Children's Heart Institute, to describe the "state of the art" of the treatment of paediatric cardiac failure, and to discuss future directions for research in the domain of paediatric cardiac failure.
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Cardiopatias Congênitas/diagnóstico , Cardiopatias Congênitas/terapia , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/terapia , Pediatria/tendências , Congressos como Assunto , Cardiopatias Congênitas/epidemiologia , Insuficiência Cardíaca/epidemiologia , Hospitais Pediátricos , Humanos , Estados UnidosRESUMO
Sunlight exposure is a significant risk factor for UV-induced deteriorating transformations of epidermal homeostasis leading to skin carcinogenesis. The ability of UVB radiation to cause melanoma, as well as basal and squamous cell carcinomas, makes UVB the most harmful among the three known UV ranges. UVB-induced DNA mutations and dysregulation of signaling pathways contribute to skin cancer formation. Among various signaling pathways modulated by UVB, tyrosine phosphorylation signaling which is mediated by the action of protein tyrosine kinases (PTKs) on specific tyrosine residues is highly implicated in photocarcinogenesis. Following UVB irradiation, PTKs get activated and their downstream signaling pathways contribute to photocarcinogenesis by promoting the survival of damaged keratinocytes and increasing cell proliferation. While UVB activates oncogenic signaling pathways, it can also activate tumor suppressive signaling pathways as initial protective mechanisms to maintain epidermal homeostasis. Tyrosine dephosphorylation is one of the protective mechanisms and is mediated by the action of protein tyrosine phosphatases (PTPs). PTP can counteract UVB-mediated PTK activation and downregulate oncogenic signaling pathways. However, PTPs have not been studied extensively in photocarcinogenesis with previous studies regarding their inactivation induced by UVB. This current review will summarize the recent progress in the protective function of PTPs in epidermal photocarcinogenesis.
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Neoplasias Cutâneas , Raios Ultravioleta , Humanos , Fosforilação , Queratinócitos/efeitos da radiação , Proteínas Tirosina Fosfatases/metabolismo , Carcinogênese , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/metabolismo , Tirosina/metabolismoRESUMO
Neutralizing antibodies (NAs) are key immunological markers and are part of the humoral response of the adaptive immune system. NA assays determine the presence of functional antibodies to prevent SARS-CoV-2 infection. We performed a real-world evidence study to detect NAs that confer protection against SARS-CoV-2 after the application of five vaccines (Pfizer/BioNTech, AstraZeneca, Sinovac, Moderna, and CanSino) in the Mexican population. Side effects of COVID-19 vaccines and clinical and demographic factors associated with low immunogenicity were also evaluated. A total of 242 SARS-CoV-2-vaccinated subjects were recruited. Pfizer/BioNTech and Moderna proved the highest percentage of inhibition in a mono-vaccine scheme. Muscular pain, headache, and fatigue were the most common adverse events. None of the patients reported severe adverse events. We found an estimated contagion-free time of 207 (IQR: 182-231) and 187 (IQR: 184-189) days for Pfizer/BioNTech and CanSino in 12 cases in each group. On the basis of our results, we consider that the emerging vaccination strategy in Mexico is effective and safe.
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Alterations of serine/threonine phosphorylation of the cardiac proteome are a hallmark of heart failure. However, the contribution of tyrosine phosphorylation (pTyr) to the pathogenesis of cardiac hypertrophy remains unclear. We use global mapping to discover and quantify site-specific pTyr in two cardiac hypertrophic mouse models, i.e., cardiac overexpression of ErbB2 (TgErbB2) and α myosin heavy chain R403Q (R403Q-αMyHC Tg), compared to control hearts. From this, there are significant phosphoproteomic alterations in TgErbB2 mice in right ventricular cardiomyopathy, hypertrophic cardiomyopathy (HCM), and dilated cardiomyopathy (DCM) pathways. On the other hand, R403Q-αMyHC Tg mice indicated that the EGFR1 pathway is central for cardiac hypertrophy, along with angiopoietin, ErbB, growth hormone, and chemokine signaling pathways activation. Surprisingly, most myofilament proteins have downregulation of pTyr rather than upregulation. Kinase-substrate enrichment analysis (KSEA) shows a marked downregulation of MAPK pathway activity downstream of k-Ras in TgErbB2 mice and activation of EGFR, focal adhesion, PDGFR, and actin cytoskeleton pathways. In vivo ErbB2 inhibition by AG-825 decreases cardiomyocyte disarray. Serine/threonine and tyrosine phosphoproteome confirm the above-described pathways and the effectiveness of AG-825 Treatment. Thus, altered pTyr may play a regulatory role in cardiac hypertrophic models.
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Cardiomiopatia Hipertrófica , Proteoma , Camundongos , Animais , Proteoma/metabolismo , Fosforilação , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/patologia , Cardiomegalia , Serina/metabolismo , Treonina/metabolismo , Tirosina/metabolismoRESUMO
An early detection tool for latent COVID-19 infections in oncology staff and patients is essential to prevent outbreaks in a cancer center. (1) Background: In this study, we developed and implemented two early detection tools for the radiotherapy area to identify COVID-19 cases opportunely. (2) Methods: Staff and patients answered a questionnaire (electronic and paper surveys, respectively) with clinical and epidemiological information. The data were collected through two online survey tools: Real-Time Tracking (R-Track) and Summary of Factors (S-Facts). Cut-off values were established according to the algorithm models. SARS-CoV-2 qRT-PCR tests confirmed the positive algorithms individuals. (3) Results: Oncology staff members (n = 142) were tested, and 14% (n = 20) were positives for the R-Track algorithm; 75% (n = 15) were qRT-PCR positive. The S-Facts Algorithm identified 7.75% (n = 11) positive oncology staff members, and 81.82% (n = 9) were qRT-PCR positive. Oncology patients (n = 369) were evaluated, and 1.36% (n = 5) were positive for the Algorithm used. The five patients (100%) were confirmed by qRT-PCR. (4) Conclusions: The proposed early detection tools have proved to be a low-cost and efficient tool in a country where qRT-PCR tests and vaccines are insufficient for the population.
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Prolactin (PRL) is a hormone expressed in lactotrophs cells of the pituitary gland in primates. Extra pituitary expression of PRL has been reported, including the eye; however, expression in the developing eye of primates is limited. The aim of the study was determining the expression of PRL and PRL receptor (PRLR) (mRNAs and proteins) in adult and fetal baboon (Papio hamadryas) ocular tissues. METHODS: We analyzed PRL and PRLR in baboon eyes tissues by immunofluorescence. The mRNAs of PRL and PRLR were detected by RT-PCR, cDNA was cloned, and sequenced. Furthermore, we performed a phylogenetic analysis to identify the evolutionary forces that underlie the divergence of PRL and PRLR primate genes. RESULTS: We observed the expression of PRL and PRLR (mRNAs and proteins) in all retinal cell lineages of fetal and adult baboon. PRL and PRLR fit the hypothesis of evolutionary purifying gene selection. CONCLUSIONS: mRNA and protein of PRL and PRLR are expressed in fetal and adult baboon retinal tissue. PRL may trigger autocrine and paracrine-specific actions in retinal cell lines.
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Breast cancer (BC) has one of the highest incidences and mortality worldwide. Single nucleotide polymorphisms (SNPs) in TOX3 rs3803662 and MMP7 rs1943779 have been associated with susceptibility to BC. In this case-control study, we evaluated the association of rs3803662 (TOX3)/rs1943779 (MMP7) SNPs with clinical features, immunohistochemical reactivity, and risk association with BC in women from northeastern Mexico. We compared 212 BC cases and 212 controls. DNA was isolated from peripheral blood to perform the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. We calculated genotype frequencies, odds ratios, and 95% confidence intervals. We found that CT (Cytocine-Thymine) and TT (Thymine -Thymine) genotypes, and T alleles of TOX3 rs3803662, were associated with BC risk (p = 0.034, p = 0.011, respectively). SNP TOX3 rs3803662 was associated with positive progesterone receptors (PR) and triple-negative BC (TNBC) but not with estrogen receptor (ER) or HER2 reactivity. CT and TT genotypes (p = 0.006) and T alleles (p = 0.002) of SNP MMP7 rs1943779 were associated with risk of BC. We found that T alleles of TOX3 rs3803662 and MMP7 rs1943779 SNPs are associated with BC risk. These findings contribute to personalized medicine in Mexican women.
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Proteínas Reguladoras de Apoptose/genética , Neoplasias da Mama , Transativadores/genética , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/genética , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Humanos , Metaloproteinase 7 da Matriz/genética , México , Polimorfismo de Nucleotídeo Único/genética , Receptores de Progesterona/genéticaRESUMO
The sarcomeres form the molecular motor of the cardiomyocyte and consist of a complex multi-protein of thick and thin filaments which are anchored to the cytoskeleton. The thick filament, composed of myosin and associated proteins, and the thin filament composed of actin, tropomyosin and the troponins develop actinmyosin crossbridges which cycle in response to calcium resulting in sliding of the filaments and contraction. The thin filament in fixed to the cardiomyocyte cytoskeleton at the Z-disc, a complex of structural and regulatory proteins. A giant protein, titin, provides an external scaffold and regulates passive force in diastole. Both genetic disorders and acquired conditions may affect proteins of the sarcomere. Genetic disorders of the thick and thin filament proteins are the predominant cause of hypertrophic cardiomyopathy. These mutations lead to abnormal sarcomere function, often an enhanced sensitivity to calcium, and impaired relaxation. This may result in secondary changes in calcium cycling and amplification of hypertrophic signaling cascades. Correcting the abnormal function of the sarcomere as well as intervening in later stages of the pathophysiologic cascades may ameliorate disease. In dilated cardiomyopathy genetic abnormalities in the sarcomere, Z-disc, calcium regulatory and cytoskeletal proteins as well as the dystrophin complex may be causal for disease. In dilated cardiomyopathy, disturbances in post-translational modifications of the sarcomere my also play a prominent role. Experimental models indicate that altered phosphorylation of sarcomeric proteins may impair systolic and diastolic function as well as the response to heart rate and afterload. Thus correcting these post-translational changes are legitimate targets for future therapeutic strategies for dilated cardiomyopathy.
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Familial adenomatous polyposis (FAP) is an autosomal-dominant condition characterized by the presence of multiple colorectal adenomas, caused by germline variants in the adenomatous polyposis coli (APC) gene. More than 300 germline variants have been characterized. The detection of novel variants is important to understand the mechanisms of pathophysiology. We identified a novel pathogenic germline variant using next-generation sequencing (NGS) in a proband patient. The variant is a complex rearrangement (c.422+1123_532-577 del ins 423-1933_423-1687 inv) that generates a complete deletion of exon 5 of the APC gene. To study the variant in other family members, we designed an endpoint PCR method followed by Sanger sequencing. The variant was identified in the proband patient's mother, one daughter, her brother, two cousins, a niece, and a second nephew. In patients where the variant was identified, we found atypical clinical symptoms, including mandibular, ovarian, breast, pancreatic, and gastric cancer. Genetic counseling and cancer prevention strategies were provided for the family. According to the American College of Medical Genetics (ACMG) guidelines, this novel variant is considered a PVS1 variant (very strong evidence of pathogenicity), and it can be useful in association with clinical data for early surveillance and suitable treatment.
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Transgenic models with pseudo phosphorylation mutants of troponin I, PKA sites at Ser 22 and 23 (cTnIDD(22,23) mice) or PKC sites at Ser 42 and 44 (cTnIAD(22,23)DD(42,44)) displayed differential force-frequency relationships and afterload relaxation delay in vivo. We hypothesized that cTnI PKA and PKC phosphomimics impact cardiac muscle rate-related developed twitch force and relaxation kinetics in opposite directions. cTnIDD(22,23) transgenic mice produce a force frequency relationship (FFR) equivalent to control NTG albeit at lower peak [Ca(2+)](i), while cTnIAD(22,23)DD(42,44) TG mice had a flat FFR with normal peak systolic [Ca(2+)](i), thus suggestive of diminished responsiveness to [Ca(2+)](i) at higher frequencies. Force-[Ca(2+)](i) hysteresis analysis revealed that cTnIDD(22,23) mice have a combined enhanced myofilament calcium peak response with an enhanced slope of force development and decline per unit of [Ca(2+)](i), whereas cTnIAD(22,23)DD(42,44) transgenic mice showed the opposite. The computational ECME model predicts that the TG lines may be distinct from each other due to different rate constants for association/dissociation of Ca(2+) at the regulatory site of cTnC. Our data indicate that cTnI phosphorylation at PKA sites plays a critical role in the FFR by increasing relative myofilament responsiveness, and results in a distinctive transition between activation and relaxation, as displayed by force-[Ca(2+)](i) hysteresis loops. These findings may have important implications for understanding the specific contribution of cTnI to beta-adrenergic inotropy and lusitropy and to adverse contractile effects of PKC activation, which is relevant during heart failure development.
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Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteína Quinase C/metabolismo , Troponina I/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Simulação por Computador , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocárdio/metabolismo , FosforilaçãoRESUMO
In addition to O-phosphorylation, O-linked modifications of serine and threonine by beta-N-acetyl-D-glucosamine (GlcNAc) may regulate muscle contractile function. This study assessed the potential role of O-GlcNAcylation in cardiac muscle contractile activation. To identify specific sites of O-GlcNAcylation in cardiac myofilament proteins, a recently developed methodology based on GalNAz-biotin labeling followed by dithiothreitol replacement and light chromatography/tandem mass spectrometry site mapping was adopted. Thirty-two O-GlcNAcylated peptides from cardiac myofilaments were identified on cardiac myosin heavy chain, actin, myosin light chains, and troponin I. To assess the potential physiological role of the GlcNAc, force-[Ca(2+)] relationships were studied in skinned rat trabeculae. Exposure to GlcNAc significantly decreased calcium sensitivity (pCa50), whereas maximal force (F(max)) and Hill coefficient (n) were not modified. Using a pan-specific O-GlcNAc antibody, it was determined that acute exposure of myofilaments to GlcNAc induced a significant increase in actin O-GlcNAcylation. This study provides the first identification of O-GlcNAcylation sites in cardiac myofilament proteins and demonstrates their potential role in regulating myocardial contractile function.
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Acetilglucosamina/fisiologia , Citoesqueleto de Actina/fisiologia , Contração Miocárdica/fisiologia , Miocárdio , Acetilglucosamina/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Acilação , Animais , Configuração de Carboidratos , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica/efeitos dos fármacos , RatosRESUMO
Modification of electrical conduction would be a useful principle to recruit in preventing or treating certain arrhythmias, notably ventricular tachycardia (VT). Here we pursue a novel gene transfer approach to modulate electrical conduction by reducing gap junctional intercellular communication (GJIC) and hence potentially modify the arrhythmia substrate. The ultimate goal is to develop a nondestructive approach to uncouple zones of slow conduction by focal gene transfer. Lentiviral vectors encoding connexin43 (Cx43) internal loop mutants were produced and studied in vitro. Transduction of neonatal rat ventricular myocytes (NRVMs) revealed the expected subcellular localization of the mutant gene product. Fluorescent dye transfer studies showed a significant reduction of GJIC in NRVMs that had been genetically modified. Additionally, adjacent mutant gene-modified NRVMs displayed delayed calcium transients, indicative of electrical uncoupling. Multi-site optical mapping of action potential (AP) propagation in gene-modified NRVM monolayers revealed a 3-fold slowing of conduction velocity (CV) relative to nontransduced NRVMs. In conclusion, lentiviral vector-mediated gene transfer of Cx43 mutants reduced GJIC in NRVMs. Electrical charge transfer was also reduced as evidenced by delayed calcium transients in adjacent NRVMs and reduced CV in NRVM monolayers. These data validate a molecular tool that opens the prospect for gene transfer targeting gap junctions as an approach to modulate cardiac conduction.
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Comunicação Celular/fisiologia , Conexina 43/fisiologia , Junções Comunicantes/fisiologia , Sistema de Condução Cardíaco/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Animais Recém-Nascidos , Sinalização do Cálcio/genética , Comunicação Celular/genética , Células Cultivadas , Técnicas de Cocultura , Conexina 43/biossíntese , Conexina 43/genética , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacocinética , Junções Comunicantes/genética , Técnicas de Transferência de Genes , Genes Dominantes , Terapia Genética/métodos , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Ventrículos do Coração/citologia , Humanos , Lentivirus/genética , Mutagênese Sítio-Dirigida , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Deleção de SequênciaRESUMO
Calcium cycling figures prominently in excitation-contraction coupling and in various signaling cascades involved in the development of left ventricular hypertrophy. We hypothesized that genetic suppression of the L-type calcium channel accessory beta-subunit would modulate calcium current and suppress cardiac hypertrophy. A short hairpin RNA template sequence capable of mediating the knockdown of the L-type calcium channel accessory beta-subunit gene was incorporated into a lentiviral vector (PPT.CG.H1.beta(2)). Transduction of ventricular myocytes in vivo with the active short hairpin RNA partially inhibited the L-type calcium current. In neonatal rat cardiomyocytes, L-type calcium channel accessory beta-subunit gene knockdown reduced calcium transient amplitude. Similarly, [(3)H]leucine incorporation was attenuated in PPT.CG.H1.beta(2)-transduced neonatal rat cardiomyocytes compared with nonsilencing controls in a phenylephrine-induced hypertrophy model. In vivo gene transfer attenuated the hypertrophic response in an aortic-banded rat model of left ventricular hypertrophy, with reduced left ventricular wall thickness and heart weight/body weight ratios in PPT.CG.H1.beta(2)-injected rats at four weeks post transduction. Fractional shortening was preserved in rats treated with PPT.CG.H1.beta(2). These findings indicate that knockdown of L-type calcium channel accessory beta-subunit is capable of attenuating the hypertrophic response both in vitro and in vivo without compromising systolic performance. Suppression of the calcium channel beta subunit may represent a novel and useful therapeutic strategy for left ventricular hypertrophy.
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Canais de Cálcio Tipo L/deficiência , Canais de Cálcio Tipo L/metabolismo , Inativação Gênica , Terapia Genética , Hipertrofia Ventricular Esquerda/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Humanos , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Hipertrofia Ventricular Esquerda/terapia , Lentivirus , Miócitos Cardíacos/patologia , Tamanho do Órgão/genética , Subunidades Proteicas/deficiência , Subunidades Proteicas/metabolismo , Ratos , Transdução GenéticaRESUMO
INTRODUCTION: Obesity and psychosocial stress (PS) co-exist in individuals of Western society. Nevertheless, how PS impacts cardiac and hippocampal phenotype in obese subjects is still unknown. Nor is it clear whether changes in local brain-derived neurotrophic factor (BDNF) account, at least in part, for myocardial and behavioral abnormalities in obese experiencing PS. METHODS: In adult male WT mice, obesity was induced via a high-fat diet (HFD). The resident-intruder paradigm was superimposed to trigger PS. In vivo left ventricular (LV) performance was evaluated by echocardiography and pressure-volume loops. Behaviour was indagated by elevated plus maze (EPM) and Y-maze. LV myocardium was assayed for apoptosis, fibrosis, vessel density and oxidative stress. Hippocampus was analyzed for volume, neurogenesis, GABAergic markers and astrogliosis. Cardiac and hippocampal BDNF and TrkB levels were measured by ELISA and WB. We investigated the pathogenetic role played by BDNF signaling in additional cardiac-selective TrkB (cTrkB) KO mice. FINDINGS: When combined, obesity and PS jeopardized LV performance, causing prominent apoptosis, fibrosis, oxidative stress and remodeling of the larger coronary branches, along with lower BDNF and TrkB levels. HFD/PS weakened LV function similarly in WT and cTrkB KO mice. The latter exhibited elevated LV ROS emission already at baseline. Obesity/PS augmented anxiety-like behaviour and impaired spatial memory. These changes were coupled to reduced hippocampal volume, neurogenesis, local BDNF and TrkB content and augmented astrogliosis. INTERPRETATION: PS and obesity synergistically deteriorate myocardial structure and function by depleting cardiac BDNF/TrkB content, leading to augmented oxidative stress. This comorbidity triggers behavioral deficits and induces hippocampal remodeling, potentially via lower BDNF and TrkB levels. FUND: J.A. was in part supported by Rotary Foundation Global Study Scholarship. G.K. was supported by T32 National Institute of Health (NIH) training grant under award number 1T32AG058527. S.C. was funded by American Heart Association Career Development Award (19CDA34760185). G.A.R.C. was funded by NIH (K01HL133368-01). APB was funded by a Grant from the Friuli Venezia Giulia Region entitled: "Heart failure as the Alzheimer disease of the heart; therapeutic and diagnostic opportunities". M.C. was supported by PRONAT project (CNR). N.P. was funded by NIH (R01 HL136918) and by the Magic-That-Matters fund (JHU). V.L. was in part supported by institutional funds from Scuola Superiore Sant'Anna (Pisa, Italy), by the TIM-Telecom Italia (WHITE Lab, Pisa, Italy), by a research grant from Pastificio Attilio Mastromauro Granoro s.r.l. (Corato, Italy) and in part by ETHERNA project (Prog. n. 161/16, Fondazione Pisa, Italy). Funding source had no such involvement in study design, in the collection, analysis, interpretation of data, in the writing of the report; and in the decision to submit the paper for publication.
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Fator Neurotrófico Derivado do Encéfalo/metabolismo , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Miocárdio/metabolismo , Estresse Psicológico , Animais , Apoptose , Comportamento Animal , Biomarcadores , Comorbidade , Dieta Hiperlipídica , Ecocardiografia , Fibrose , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Camundongos Obesos , Neurogênese , Estresse Oxidativo , Proteínas Tirosina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Troponin I (TnI) variant Pro82Ser (cTnIP82S) was initially considered a disease-causing mutation; however, later studies suggested the contrary. We tested the hypothesis of whether a causal link exists between cTnIP82S and cardiac structural and functional remodeling, such as during aging or chronic pressure overload. A cardiac-specific transgenic (Tg) mouse model of cTnIP82S was created to test this hypothesis. During aging, Tg cTnIP82S displayed diastolic dysfunction, characterized by longer isovolumetric relaxation time, and impaired ejection and relaxation time. In young, Tg mice in vivo pressure-volume loops and intact trabecular preparations revealed normal cardiac contractility at baseline. However, upon ß-adrenergic stimulation, a blunted contractile reserve and no hastening in left ventricle relaxation were evident in vivo, whereas, in isolated muscles, Ca(2+) transient amplitude isoproterenol dose-response was blunted. In addition, when exposed to chronic pressure overload, Tg mice show exacerbated hypertrophy and decreased contractility compared with age-matched non-Tg littermates. At the molecular level, this mutation significantly impairs myofilament cooperative activation. Importantly, this occurs in the absence of alterations in TnI or myosin-binding protein C phosphorylation. The cTnIP82S variant occurs near a region of interactions with troponin T; therefore, structural changes in this region could explain its meaningful effects on myofilament cooperativity. Our data indicate that cTnIP82S mutation modifies age-dependent diastolic dysfunction and impairs overall contractility after ß-adrenergic stimulation or chronic pressure overload. Thus cTnIP82S variant should be regarded as a disease-modifying factor for dysfunction and adverse remodeling with aging and chronic pressure overload.
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Envelhecimento/fisiologia , Cardiopatias/genética , Coração/fisiologia , Miocárdio/metabolismo , Receptores Adrenérgicos beta/metabolismo , Troponina I/genética , Substituição de Aminoácidos , Animais , Diástole , Feminino , Cardiopatias/metabolismo , Hipertrofia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Transgênicos , Miocárdio/patologia , Estresse FisiológicoRESUMO
Contractile dysfunction and increased deposition of O-linked ß-N-acetyl-d-glucosamine (O-GlcNAc) in cardiac proteins are a hallmark of the diabetic heart. However, whether and how this posttranslational alteration contributes to lower cardiac function remains unclear. Using a refined ß-elimination/Michael addition with tandem mass tags (TMT)-labeling proteomic technique, we show that CpOGA, a bacterial analog of O-GlcNAcase (OGA) that cleaves O-GlcNAc in vivo, removes site-specific O-GlcNAcylation from myofilaments, restoring Ca(2+) sensitivity in streptozotocin (STZ) diabetic cardiac muscles. We report that in control rat hearts, O-GlcNAc and O-GlcNAc transferase (OGT) are mainly localized at the Z-line, whereas OGA is at the A-band. Conversely, in diabetic hearts O-GlcNAc levels are increased and OGT and OGA delocalized. Consistent changes were found in human diabetic hearts. STZ diabetic hearts display increased physical interactions of OGA with α-actin, tropomyosin, and myosin light chain 1, along with reduced OGT and increased OGA activities. Our study is the first to reveal that specific removal of O-GlcNAcylation restores myofilament response to Ca(2+) in diabetic hearts and that altered O-GlcNAcylation is due to the subcellular redistribution of OGT and OGA rather than to changes in their overall activities. Thus, preventing sarcomeric OGT and OGA displacement represents a new possible strategy for treating diabetic cardiomyopathy.