RESUMO
Cardiac tolerance to ischaemia can be increased by dietary interventions such as fasting, which is associated with significant changes in myocardial gene expression. Among the possible mechanisms of how gene expression may be altered are epigenetic modifications of RNA - epitranscriptomics. N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are two of the most prevalent modifications in mRNA. These methylations are reversible and regulated by proteins called writers, erasers, readers, and m6A-repelled proteins. We analysed 33 of these epitranscriptomic regulators in rat hearts after cardioprotective 3-day fasting using RT-qPCR, Western blot, and targeted proteomic analysis. We found that the most of these regulators were changed on mRNA or protein levels in fasting hearts, including up-regulation of both demethylases - FTO and ALKBH5. In accordance, decreased methylation (m6A+m6Am) levels were detected in cardiac total RNA after fasting. We also identified altered methylation levels in Nox4 and Hdac1 transcripts, both of which play a role in the cytoprotective action of ketone bodies produced during fasting. Furthermore, we investigated the impact of inhibiting demethylases ALKBH5 and FTO in adult rat primary cardiomyocytes (AVCMs). Our findings indicate that inhibiting these demethylases reduced the hypoxic tolerance of AVCMs isolated from fasting rats. This study showed that the complex epitranscriptomic machinery around m6A and m6Am modifications is regulated in the fasting hearts and might play an important role in cardiac adaptation to fasting, a well-known cardioprotective intervention.
Assuntos
Adenosina , Proteômica , Animais , Ratos , Adenosina/genética , Adenosina/metabolismo , RNA/metabolismo , RNA Mensageiro/genética , JejumRESUMO
Macrophage fusion leading to the formation of multinucleated giant cells is a hallmark of chronic inflammation. Several membrane proteins have been implicated in mediating cell-cell attachment during fusion, but their binding partners remain unknown. Recently, we demonstrated that interleukin-4 (IL-4)-induced fusion of mouse macrophages depends on the integrin macrophage antigen 1 (Mac-1). Surprisingly, the genetic deficiency of intercellular adhesion molecule 1 (ICAM-1), an established ligand of Mac-1, did not impair macrophage fusion, suggesting the involvement of other counter-receptors. Here, using various approaches, including signal regulatory protein α (SIRPα) knockdown, recombinant proteins, adhesion and fusion assays, biolayer interferometry, and peptide libraries, we show that SIRPα, which, similar to ICAM-1, belongs to the Ig superfamily and has previously been implicated in cell fusion, interacts with Mac-1. The following results support the conclusion that SIRPα is a ligand of Mac-1: (a) recombinant ectodomain of SIRPα supports adhesion of Mac-1-expressing cells; (b) Mac-1-SIRPα interaction is mediated through the ligand-binding αMI-domain of Mac-1; (c) recognition of SIRPα by the αMI-domain conforms to general principles governing binding of Mac-1 to many of its ligands; (d) SIRPα reportedly binds CD47; however, anti-CD47 function-blocking mAb produced only a limited inhibition of macrophage adhesion to SIRPα; and (e) co-culturing of SIRPα- and Mac-1-expressing HEK293 cells resulted in the formation of multinucleated cells. Taken together, these results identify SIRPα as a counter-receptor for Mac-1 and suggest that the Mac-1-SIRPα interaction may be involved in macrophage fusion.
Assuntos
Antígenos de Diferenciação/metabolismo , Antígeno de Macrófago 1/metabolismo , Macrófagos/metabolismo , Receptores Imunológicos/metabolismo , Animais , Antígenos de Diferenciação/genética , Fusão Celular , Células HEK293 , Humanos , Antígeno de Macrófago 1/genética , Camundongos , Domínios Proteicos , Receptores Imunológicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The functional significance of having two nuclei in a cell is unknown. Having two stores of genetic material may be advantageous for cell growth. Nuclear protein import is at a critical juncture in the cell to modify cell growth. This study addressed the potential for differential nuclear protein import in two nuclei of the same cell. Isolated adult rat cardiomyocytes were microinjected with an exogenous fluorescent protein conjugated with nuclear localization amino acid sequences to facilitate nuclear import and its detection. Our results demonstrate the rate of nuclear protein import was not significantly different between the two nuclei in the same cell. These data demonstrate that the two nuclei are functionally similar in a binucleated cardiomyocyte, at least as far as nucleocytoplasmic transport is concerned.
Assuntos
Núcleo Celular/metabolismo , Miócitos Cardíacos/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Masculino , Microinjeções , Ratos Sprague-DawleyRESUMO
Although proteasome inhibitors (PIs) are modern targeted anticancer drugs, they have been associated with a certain risk of cardiotoxicity and heart failure (HF). Recently, PIs have been combined with anthracyclines (ANTs) to further boost their anticancer efficacy. However, this raised concerns regarding cardiac safety, which were further supported by several in vitro studies on immature cardiomyocytes. In the present study, we investigated the toxicity of clinically used PIs alone (bortezomib (BTZ), carfilzomib (CFZ)) as well as their combinations with an ANT (daunorubicin (DAU)) in both neonatal and adult ventricular cardiomyocytes (NVCMs and AVCMs) and in a chronic rabbit model of DAU-induced HF. Using NVCMs, we found significant cytotoxicity of both PIs around their maximum plasma concentration (cmax) as well as significant augmentation of DAU cytotoxicity. In AVCMs, BTZ did not induce significant cytotoxicity in therapeutic concentrations, whereas the toxicity of CFZ was significant and more profound. Importantly, neither PI significantly augmented the cardiotoxicity of DAU despite even more profound proteasome-inhibitory activity in AVCMs compared with NVCMs. Furthermore, in young adult rabbits, no significant augmentation of chronic ANT cardiotoxicity was noted with respect to any functional, morphological, biochemical or molecular parameter under study, despite significant inhibition of myocardial proteasome activity. Our experimental data show that combination of PIs with ANTs is not accompanied by an exaggerated risk of cardiotoxicity and HF in young adult animal cardiomyocytes and hearts.
Assuntos
Antraciclinas/toxicidade , Antineoplásicos/toxicidade , Cardiotoxicidade/etiologia , Inibidores de Proteassoma/toxicidade , Animais , Antraciclinas/administração & dosagem , Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Bortezomib/administração & dosagem , Bortezomib/toxicidade , Daunorrubicina/administração & dosagem , Daunorrubicina/toxicidade , Relação Dose-Resposta a Droga , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Oligopeptídeos/administração & dosagem , Oligopeptídeos/toxicidade , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/administração & dosagem , Coelhos , Ratos , Ratos WistarRESUMO
Adaptation to chronic hypoxia renders the heart more tolerant to ischemia/reperfusion injury. To evaluate changes in gene expression after adaptation to chronic hypoxia by RT-qPCR, it is essential to select suitable reference genes. In a chronically hypoxic rat model, no specific reference genes have been identified in the myocardium. This study aimed to select the best reference genes in the left (LV) and right (RV) ventricles of chronically hypoxic and normoxic rats. Sprague-Dawley rats were adapted to continuous normobaric hypoxia (CNH; 12% O2 or 10% O2) for 3 weeks. The expression levels of candidate genes were assessed by RT-qPCR. The stability of genes was evaluated by NormFinder, geNorm and BestKeeper algorithms. The best five reference genes in the LV were Top1, Nupl2, Rplp1, Ywhaz, Hprt1 for the milder CNH and Top1, Ywhaz, Sdha, Nupl2, Tomm22 for the stronger CNH. In the RV, the top five genes were Hprt1, Nupl2, Gapdh, Top1, Rplp1 for the milder CNH and Tomm22, Gapdh, Hprt1, Nupl2, Top1 for the stronger CNH. This study provides validation of reference genes in LV and RV of CNH rats and shows that suitable reference genes differ in the two ventricles and depend on experimental protocol.
Assuntos
Regulação da Expressão Gênica , Hipóxia/genética , Miocárdio/metabolismo , Miocárdio/patologia , Animais , Doença Crônica , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Masculino , Ratos Sprague-Dawley , Padrões de ReferênciaRESUMO
Epoxyeicosatrienoic acids (EETs) decrease cardiac ischemia-reperfusion injury; however, the mechanism of their protective effect remains elusive. Here, we investigated the cardioprotective action of a novel EET analog, EET-B, in reperfusion and the role of hypoxia-inducible factor (HIF)-1α in such action of EET-B. Adult male rats were subjected to 30 min of left coronary artery occlusion followed by 2 h of reperfusion. Administration of 14,15-EET (2.5 mg/kg) or EET-B (2.5 mg/kg) 5 min before reperfusion reduced infarct size expressed as a percentage of the area at risk from 64.3 ± 1.3% in control to 42.6 ± 1.9% and 46.0 ± 1.6%, respectively, and their coadministration did not provide any stronger effect. The 14,15-EET antagonist 14,15-epoxyeicosa-5( Z)-enoic acid (2.5 mg/kg) inhibited the infarct size-limiting effect of EET-B (62.5 ± 1.1%). Similarly, the HIF-1α inhibitors 2-methoxyestradiol (2.5 mg/kg) and acriflavine (2 mg/kg) completely abolished the cardioprotective effect of EET-B. In a separate set of experiments, the immunoreactivity of HIF-1α and its degrading enzyme prolyl hydroxylase domain protein 3 (PHD3) were analyzed in the ischemic areas and nonischemic septa. At the end of ischemia, the HIF-1α immunogenic signal markedly increased in the ischemic area compared with the septum (10.31 ± 0.78% vs. 0.34 ± 0.08%). After 20 min and 2 h of reperfusion, HIF-1α immunoreactivity decreased to 2.40 ± 0.48% and 1.85 ± 0.43%, respectively, in the controls. EET-B blunted the decrease of HIF-1α immunoreactivity (7.80 ± 0.69% and 6.44 ± 1.37%, respectively) and significantly reduced PHD3 immunogenic signal in ischemic tissue after reperfusion. In conclusion, EET-B provides an infarct size-limiting effect at reperfusion that is mediated by HIF-1α and downregulation of its degrading enzyme PHD3. NEW & NOTEWORTHY The present study shows that EET-B is an effective agonistic 14,15-epoxyeicosatrienoic acid analog, and its administration before reperfusion markedly reduced myocardial infarction in rats. Most importantly, we demonstrate that increased hypoxia-inducible factor-1α levels play a role in cardioprotection mediated by EET-B in reperfusion likely by mechanisms including downregulation of the hypoxia-inducible factor -1α-degrading enzyme prolyl hydroxylase domain protein 3.
Assuntos
Ácido 8,11,14-Eicosatrienoico/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/enzimologia , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/uso terapêutico , Animais , Modelos Animais de Doenças , Regulação para Baixo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Masculino , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/patologia , Proteólise , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
The heart is characterized by a remarkable degree of heterogeneity. Since different cardiac pathologies affect different cardiac regions, it is important to understand molecular mechanisms by which these parts respond to pathological stimuli. In addition to already described left ventricular (LV)/right ventricular (RV) and transmural differences, possible baso-apical heterogeneity has to be taken into consideration. The aim of our study has been, therefore, to compare proteomes in the apical and basal parts of the rat RV and LV. Two-dimensional electrophoresis was used for the proteomic analysis. The major result of this study has revealed for the first time significant baso-apical differences in concentration of several proteins, both in the LV and RV. As far as the LV is concerned, five proteins had higher concentration in the apical compared to basal part of the ventricle. Three of them are mitochondrial and belong to the "metabolism and energy pathways" (myofibrillar creatine kinase M-type, L-lactate dehydrogenase, dihydrolipoamide dehydrogenase). Myosin light chain 3 is a contractile protein and HSP60 belongs to heat shock proteins. In the RV, higher concentration in the apical part was observed in two mitochondrial proteins (creatine kinase S-type and proton pumping NADH:ubiquinone oxidoreductase). The described changes were more pronounced in the LV, which is subjected to higher workload. However, in both chambers was the concentration of proteins markedly higher in the apical than that in basal part, which corresponds to the higher energetic demand and contractile activity of these segments of both ventricles.
Assuntos
Ventrículos do Coração/metabolismo , Proteínas Musculares/metabolismo , Proteômica , Animais , Chaperonina 60/metabolismo , Cromatografia Líquida , Creatina Quinase Forma MM/metabolismo , Di-Hidrolipoamida Desidrogenase/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Eletroforese em Gel Bidimensional , Metabolismo Energético , Ventrículos do Coração/enzimologia , L-Lactato Desidrogenase/metabolismo , Masculino , Proteínas Mitocondriais/metabolismo , Proteínas Musculares/isolamento & purificação , Cadeias Leves de Miosina/metabolismo , Ratos Wistar , Espectrometria de Massas em TandemRESUMO
Mitochondria play an essential role in improved cardiac ischaemic tolerance conferred by adaptation to chronic hypoxia. In the present study, we analysed the effects of continuous normobaric hypoxia (CNH) on mitochondrial functions, including the sensitivity of the mitochondrial permeability transition pore (MPTP) to opening, and infarct size (IS) in hearts of spontaneously hypertensive rats (SHR) and the conplastic SHR-mtBN strain, characterized by the selective replacement of the mitochondrial genome of SHR with that of the more ischaemia-resistant brown Norway (BN) strain. Rats were adapted to CNH (10% O2, 3 weeks) or kept at room air as normoxic controls. In the left ventricular mitochondria, respiration and cytochrome c oxidase (COX) activity were measured using an Oxygraph-2k and the sensitivity of MPTP opening was assessed spectrophotometrically as Ca2+-induced swelling. Myocardial infarction was analysed in anaesthetized open-chest rats subjected to 20 min of coronary artery occlusion and 3 h of reperfusion. The IS reached 68±3.0% and 65±5% of the area at risk in normoxic SHR and SHR-mtBN strains, respectively. CNH significantly decreased myocardial infarction to 46±3% in SHR. In hypoxic SHR-mtBN strain, IS reached 33±2% and was significantly smaller compared with hypoxic SHR. Mitochondria isolated from hypoxic hearts of both strains had increased detergent-stimulated COX activity and were less sensitive to MPTP opening. The maximum swelling rate was significantly lower in hypoxic SHR-mtBN strain compared with hypoxic SHR, and positively correlated with myocardial infarction in all experimental groups. In conclusion, the mitochondrial genome of SHR modulates the IS-limiting effect of adaptation to CNH by affecting mitochondrial energetics and MPTP sensitivity to opening.
Assuntos
DNA Mitocondrial/genética , Hipóxia , Mitocôndrias Cardíacas/genética , Animais , Western Blotting , Doença Crônica , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Genoma Mitocondrial/genética , Masculino , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos SHR , Ratos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Adaptation to chronic intermittent hypoxia (CIH) is associated with reactive oxygen species (ROS) generation implicated in the improved cardiac tolerance against acute ischemia-reperfusion injury. Phospholipases A2 (PLA2s) play an important role in cardiomyocyte phospholipid metabolism influencing membrane homeostasis. Here we aimed to determine the effect of CIH (7000 m, 8 h/day, 5 weeks) on the expression of cytosolic PLA2 (cPLA2α), its phosphorylated form (p-cPLA2α), calcium-independent (iPLA2), and secretory (sPLA2IIA) at protein and mRNA levels, as well as fatty acids (FA) profile in left ventricular myocardium of adult male Wistar rats. Chronic administration of antioxidant tempol was used to verify the ROS involvement in CIH effect on PLA2s expression and phospholipid FA remodeling. While CIH did not affect PLA2s mRNA levels, it increased the total cPLA2α protein in cytosol and membranes (by 191% and 38%, respectively) and p-cPLA2α (by 23%) in membranes. On the contrary, both iPLA2 and sPLA2IIA were downregulated by CIH. CIH further decreased phospholipid n-6 polyunsaturated FA (PUFA) and increased n-3 PUFA proportion. Tempol treatment prevented only CIH-induced cPLA2α up-regulation and its phosphorylation on Ser505. Our results show that CIH diversely affect myocardial PLA2s and suggest that ROS are responsible for the activation of cPLA2α under these conditions.
Assuntos
Antioxidantes/farmacologia , Óxidos N-Cíclicos/farmacologia , Fosfolipases A2 do Grupo IV/metabolismo , Hipóxia/enzimologia , Animais , Doença Crônica , Ácidos Graxos/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fosfolipases A2 do Grupo IV/genética , Hipóxia/metabolismo , Masculino , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Marcadores de SpinRESUMO
Cardiac resistance against acute ischemia/reperfusion (I/R) injury can be enhanced by adaptation to chronic intermittent hypoxia (CIH), but the changes at the molecular level associated with this adaptation are still not fully explored. Phospholipase A2 (PLA2) plays an important role in phospholipid metabolism and may contribute to membrane destruction under conditions of energy deprivation during I/R. The aim of this study was to determine the effect of CIH (7000 m, 8 h/day, 5 weeks) on the expression of cytosolic PLA2α (cPLA2α) and its phosphorylated form (p-cPLA2α), as well as other related signaling proteins in the left ventricular myocardium of adult male Wistar rats. Adaptation to CIH increased the total content of cPLA2α by 14 % in myocardial homogenate, and enhanced the association of p-cPLA2α with the nuclear membrane by 85 %. The total number of ß-adrenoceptors (ß-ARs) did not change but the ß2/ß1 ratio markedly increased due to the elevation of ß2-ARs and drop in ß1-ARs. In parallel, the amount of adenylyl cyclase decreased by 49 % and Giα proteins increased by about 50 %. Besides that, cyclooxygenase 2 (COX-2) and prostaglandin E2 (PGE2) increased by 36 and 84 %, respectively. In parallel, we detected increased phosphorylation of protein kinase Cα, ERK1/2 and p38 (by 12, 48 and 19 %, respectively). These data suggest that adaptive changes induced in the myocardium by CIH may include activation of cPLA2α and COX-2 via ß2-AR/Gi-mediated stimulation of the ERK/p38 pathway.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Fosfolipases A2 do Grupo IV/metabolismo , Sistema de Sinalização das MAP Quinases , Isquemia Miocárdica/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Doença Crônica , Masculino , Isquemia Miocárdica/patologia , Ratos , Ratos WistarRESUMO
Pulmonary hypertension is a cardiovascular disease with a low survival rate. The protein galectin-3 (Gal-3) binding ß-galactosides of cellular glycoproteins plays an important role in the onset and development of this disease. Carbohydrate-based drugs that target Gal-3 represent a new therapeutic strategy in the treatment of pulmonary hypertension. Here, we present the synthesis of novel hydrophilic glycopolymer inhibitors of Gal-3 based on a polyoxazoline chain decorated with carbohydrate ligands. Biolayer interferometry revealed a high binding affinity of these glycopolymers to Gal-3 in the subnanomolar range. In the cell cultures of cardiac fibroblasts and pulmonary artery smooth muscle cells, the most potent glycopolymer 18 (Lac-high) caused a decrease in the expression of markers of tissue remodeling in pulmonary hypertension. The glycopolymers were shown to penetrate into the cells. In a biodistribution and pharmacokinetics study in rats, the glycopolymers accumulated in heart and lung tissues, which are most affected by pulmonary hypertension.
Assuntos
Galectina 3 , Hipertensão Pulmonar , Animais , Galectina 3/antagonistas & inibidores , Galectina 3/metabolismo , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/metabolismo , Ratos , Humanos , Distribuição Tecidual , Masculino , Biomarcadores , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Polímeros/química , Polímeros/farmacologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismoRESUMO
The rapidly developing research field of epitranscriptomics has recently emerged into the spotlight of researchers due to its vast regulatory effects on gene expression and thereby cellular physiology and pathophysiology. N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are among the most prevalent and well-characterized modified nucleosides in eukaryotic RNA. Both of these modifications are dynamically regulated by a complex set of epitranscriptomic regulators called writers, readers, and erasers. Altered levels of m6A and also several regulatory proteins were already associated with diabetic tissues. This review summarizes the current knowledge and gaps about m6A and m6Am modifications and their respective regulators in the pathophysiology of diabetes mellitus. It focuses mainly on the more prevalent type 2 diabetes mellitus (T2DM) and its treatment by metformin, the first-line antidiabetic agent. A better understanding of epitranscriptomic modifications in this highly prevalent disease deserves further investigation and might reveal clinically relevant discoveries in the future.
Assuntos
Diabetes Mellitus Tipo 2 , Humanos , RNA Mensageiro/metabolismo , Diabetes Mellitus Tipo 2/genética , Adenosina/metabolismo , RNA/genética , RNA/metabolismo , Processamento Pós-Transcricional do RNARESUMO
Epitranscriptomic modifications have recently emerged into the spotlight of researchers due to their vast regulatory effects on gene expression and thereby cellular physiology and pathophysiology. N6,2'-O-dimethyladenosine (m6Am) is one of the most prevalent chemical marks on RNA and is dynamically regulated by writers (PCIF1, METTL4) and erasers (FTO). The presence or absence of m6Am in RNA affects mRNA stability, regulates transcription, and modulates pre-mRNA splicing. Nevertheless, its functions in the heart are poorly known. This review summarizes the current knowledge and gaps about m6Am modification and its regulators in cardiac biology. It also points out technical challenges and lists the currently available techniques to measure m6Am. A better understanding of epitranscriptomic modifications is needed to improve our knowledge of the molecular regulations in the heart which may lead to novel cardioprotective strategies.
Assuntos
Adenosina , Metilação de DNA , RNA Mensageiro/genética , Adenosina/metabolismo , RNA/metabolismo , BiologiaRESUMO
The heart is capable of extensive adaptive growth in response to the demands of the body. When the heart is confronted with an increased workload over a prolonged period, it tends to cope with the situation by increasing its muscle mass. The adaptive growth response of the cardiac muscle changes significantly during phylogenetic and ontogenetic development. Cold-blooded animals maintain the ability for cardiomyocyte proliferation even in adults. On the other hand, the extent of proliferation during ontogenetic development in warm-blooded species shows significant temporal limitations: whereas fetal and neonatal cardiac myocytes express proliferative potential (hyperplasia), after birth proliferation declines and the heart grows almost exclusively by hypertrophy. It is, therefore, understandable that the regulation of the cardiac growth response to the increased workload also differs significantly during development. The pressure overload (aortic constriction) induced in animals before the switch from hyperplastic to hypertrophic growth leads to a specific type of left ventricular hypertrophy which, in contrast with the same stimulus applied in adulthood, is characterized by hyperplasia of cardiomyocytes, capillary angiogenesis and biogenesis of collagenous structures, proportional to the growth of myocytes. These studies suggest that timing may be of crucial importance in neonatal cardiac interventions in humans: early definitive repairs of selected congenital heart disease may be more beneficial for the long-term results of surgical treatment.
RESUMO
The adaptation to chronic hypoxia confers long-lasting cardiac protection against acute ischemia-reperfusion injury. Protein kinase C (PKC) appears to play a role in the cardioprotective mechanism but the involvement of individual PKC isoforms remains unclear. The aim of this study was to examine the effects of chronic intermittent hypoxia (CIH; 7,000 m, 8 h/day) and acute administration of PKC-δ inhibitor (rottlerin, 0.3 mg/kg) on the expression and subcellular distribution of PKC-δ and PKC-ε in the left ventricular myocardium of adult male Wistar rats by Western blot and quantitative immunofluorescence microscopy. CIH decreased the total level of PKC-ε in homogenate without affecting the level of phosphorylated PKC-ε (Ser729). In contrast, CIH up-regulated the total level of PKC-δ as well as the level of phosphorylated PKC-δ (Ser643) in homogenate. Rottlerin partially reversed the hypoxia-induced increase in PKC-δ in the mitochondrial fraction. Immunofluorescent staining of ventricular cryo-sections revealed increased co-localization of PKC-δ with mitochondrial and sarcolemmal membranes in CIH hearts that was suppressed by rottlerin. The formation of nitrotyrosine as a marker of oxidative stress was enhanced in CIH myocardium, particularly in mitochondria. The expression of total oxidative phosphorylation complexes was slightly decreased by CIH mainly due to complex II decline. In conclusion, up-regulated PKC-δ in CIH hearts is mainly localized to mitochondrial and sarcolemmal membranes. The inhibitory effects of rottlerin on PKC-δ subcellular redistribution and cardioprotection (as shown previously) support the view that this isoform plays a role in the mechanism of CIH-induced ischemic tolerance.
Assuntos
Hipóxia/enzimologia , Miocárdio/patologia , Proteína Quinase C-delta/fisiologia , Regulação para Cima , Animais , Doença Crônica , Mitocôndrias/metabolismo , Miocárdio/metabolismo , Fosforilação , Substâncias Protetoras , Proteína Quinase C-delta/genética , Proteína Quinase C-delta/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar , Sarcolema/metabolismo , Distribuição TecidualRESUMO
The effects of dietary supplementation with fat of different fatty acid profile and chronic intermittent hypoxia (CIH) on the fatty acid composition of serum and heart lipids were analysed. Adult male Wistar rats were fed a standard non-fat diet enriched with 10 % of lard, fish oil (n-3 PUFA) or maize oil (n-6 PUFA) for 10 weeks. After 4 weeks on the diets, each group was divided in two subgroups, either exposed to CIH in a barochamber (7000 m, twenty-five exposures) or kept at normoxia. In normoxic rats, the fish oil diet increased the level of conjugated dienes. The n-6:n-3 PUFA ratio in serum TAG, phospholipids (PL), cholesteryl esters (CE) and heart TAG, PL and diacylglycerols (DAG) followed the ratio in the fed diet (in the sequence maize oil>lard>fish oil). In heart TAG, PL and DAG, 20 : 4n-6 and 18 : 2n-6 were replaced by 22 : 6n-3 in the fish oil group. The main fatty acid in CE was 20 : 4n-6 in the lard and maize oil groups whereas in the fish oil group, half of 20 : 4n-6 was replaced by 20 : 5n-3. CIH further increased 20 : 5n-3 in CE in the fish oil group. CIH decreased the n-6:n-3 PUFA ratio in serum CE, heart TAG, PL and DAG in all dietary groups and stimulated the activity of catalase in the maize and fish oil groups. In conclusion, PUFA diets and CIH, both interventions considered to be cardioprotective, distinctly modified the fatty acid profile in serum and heart lipids with specific effects on conjugated diene production and catalase activity.
Assuntos
Gorduras Insaturadas na Dieta/farmacologia , Ácidos Graxos Insaturados/farmacologia , Hipóxia/fisiopatologia , Peroxidação de Lipídeos/efeitos dos fármacos , Miocárdio/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/fisiologia , Animais , Cardiotônicos/farmacologia , Catalase/metabolismo , Doença Crônica , Óleo de Milho/farmacologia , Gorduras na Dieta/farmacologia , Ácidos Graxos Ômega-3/farmacologia , Ácidos Graxos Ômega-6/farmacologia , Ácidos Graxos Insaturados/análise , Óleos de Peixe/farmacologia , Lipídeos/sangue , Masculino , Ratos , Ratos WistarRESUMO
Cardiovascular disease (CVD) remains the leading cause of death worldwide and, despite continuous advances, better diagnostic and prognostic tools, as well as therapy, are needed. The human transcriptome, which is the set of all RNA produced in a cell, is much more complex than previously thought and the lack of dialogue between researchers and industrials and consensus on guidelines to generate data make it harder to compare and reproduce results. This European Cooperation in Science and Technology (COST) Action aims to accelerate the understanding of transcriptomics in CVD and further the translation of experimental data into usable applications to improve personalized medicine in this field by creating an interdisciplinary network. It aims to provide opportunities for collaboration between stakeholders from complementary backgrounds, allowing the functions of different RNAs and their interactions to be more rapidly deciphered in the cardiovascular context for translation into the clinic, thus fostering personalized medicine and meeting a current public health challenge. Thus, this Action will advance studies on cardiovascular transcriptomics, generate innovative projects, and consolidate the leadership of European research groups in the field.COST (European Cooperation in Science and Technology) is a funding organization for research and innovation networks (www.cost.eu).
RESUMO
Remodeling of the cellular distribution of gap junctions formed mainly by connexin-43 (Cx43) can be related to the increased incidence of cardiac arrhythmias. It has been shown that adaptation to chronic intermittent hypobaric hypoxia (IHH) attenuates the incidence and severity of ischemic and reperfusion ventricular arrhythmias and increases the proportion of anti-arrhythmic n-3 polyunsaturated fatty acids (n-3 PUFA) in heart phospholipids. Wistar rats were exposed to simulated IHH (7,000 m, 8-h/day, 35 exposures) and compared with normoxic controls (N). Cx43 expression, phosphorylation, localization and n-3 PUFA proportion were analyzed in left ventricular myocardium. Compared to N, IHH led to higher expression of total Cx43, its variant phosphorylated at Ser368 [p-Cx43(Ser368)], which maintains "end to end" communication, as well as p-Cx43(Ser364/365), which facilitates conductivity. By contrast, expression of non-phosphorylated Cx43 and p-Cx43(Ser278/289), attenuating intercellular communication, was lower in IHH than in N. IHH also resulted in increased expression of protein kinase A and protein kinase G while casein kinase 1 did not change compared to N. In IHH group, which exhibited reduced incidence of ischemic ventricular arrhythmias, Cx43 and p-Cx43(Ser368) were more abundant at "end to end" gap junctions than in N group and this difference was preserved after acute regional ischemia (10 min). We further confirmed higher n-3 PUFA proportion in heart phospholipids after adaptation to IHH, which was even further increased by ischemia. Our results suggest that adaptation to IHH alters expression, phosphorylation and distribution of Cx43 as well as cardioprotective n-3PUFA proportion suggesting that the anti-arrhythmic phenotype elicited by IHH can be at least partly related to the stabilization of the "end to end" conductivity between cardiomyocytes during brief ischemia.
RESUMO
AIM: Heat shock protein 60 (Hsp60) is a mediator of stress-induced vascular smooth muscle cell (VSMC) proliferation. This study will determine, first, if the mitochondrial or cytoplasmic localization of Hsp60 is critical to VSMC proliferation and, second, the mechanism of Hsp60 induction of VSMC proliferation with a focus on modification of nucleocytoplasmic trafficking. METHODS AND RESULTS: Hsp60 was overexpressed in primary rabbit VSMCs with or without a mitochondrial targeting sequence (AdHsp60mito-). Both interventions induced an increase in VSMC PCNA expression and proliferation. The increase in VSMC PCNA expression and growth was not observed after siRNA-mediated knockdown of Hsp60 expression. Nuclear protein import in VSMC was measured by fluorescent microscopy using a microinjected fluorescent import substrate. Nuclear protein import was stimulated by both AdHsp60 and AdHsp60mito- treatments. AdHsp60 treatment also induced increases in nucleoporin (Nup) 62, Nup153, importin-α, importin-ß and Ran expression as well as cellular ATP levels compared to control. AdHsp60mito- treatment induced an up-regulation in importin-α, importin-ß and Ran expression compared to control. Hsp60 knockdown did not change nuclear protein import nor the expression of any nuclear transport receptors or nucleoporins. Both heat shock treatment and Hsp60 overexpression promoted the interaction of Ran with Hsp60. CONCLUSIONS: VSMC proliferation can be modulated via an Hsp60 dependent, cytosol localized mechanism that in part involves a stimulation of nuclear protein import through an interaction with Ran. This novel cellular signaling role for Hsp60 may be important in growth-based vascular pathologies like atherosclerosis and hypertension.
Assuntos
Proliferação de Células , Chaperonina 60/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Chaperonina 60/antagonistas & inibidores , Chaperonina 60/genética , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Coelhos , Temperatura , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismoRESUMO
We examined the influence of dietary fatty acid (FA) classes on the expression of protein kinase C (PKC) delta and epsilon in relation to the cardioprotective effects of chronic intermittent hypoxia (CIH). Adult male Wistar rats were fed a nonfat diet enriched with 10% lard (saturated FA [SFA]), fish oil (n-3 polyunsaturated FA [n-3 PUFA]), or corn oil (n-6 PUFA) for 10 weeks. After 4 weeks on the diet, each group was divided into two subgroups that were either exposed to CIH in a barochamber (7000 m, 8 hrs/ day) or kept at normoxia for an additional 5-6 weeks. A FA phospholipid profile and Western blot analysis of PKC were performed in left ventricles. Infarct size was assessed in anesthetized animals subjected to 20-min coronary artery occlusion and 3-hr reperfusion. CIH decreased the n-6/n-3 PUFA ratio in all groups by 23% independently of the initial value set by various diets. The combination of n-3 diet and CIH had a stronger antiarrhythmic effect during reperfusion than the n-3 diet alone; this effect was less pronounced in rats fed the n-6 diet. The normoxic n-6 group exhibited smaller infarctions (by 22%) than the n-3 group. CIH decreased the infarct size in n-3 and SFA groups (by 20% and 23%, respectively) but not in the n-6 group. Unlike PKC epsilon, the abundance of PKC delta in the myocardial particulate fraction was increased by CIH except for the n-6 group. Myocardial infarct size was negatively correlated (r=- 0.79) with the abundance of PKC delta in the particulate fraction. We conclude that lipid diets modify the infarct size-limiting effect of CIH by a mechanism that involves the PKC delta-dependent pathway.