RESUMO
During atherosclerotic plaque formation, smooth muscle cells (SMCs) switch from a contractile/differentiated to a synthetic/dedifferentiated phenotype. We previously isolated differentiated spindle-shaped (S) and dedifferentiated rhomboid (R) SMCs from porcine coronary artery. R-SMCs express S100A4, a calcium-binding protein. We investigated the role of apelin in this phenotypic conversion, as well as its relationship with S100A4. We found that apelin was highly expressed in R-SMCs compared with S-SMCs. We observed a nuclear expression of apelin in SMCs within experimentally-induced intimal thickening of the porcine coronary artery and rat aorta. Plasmids targeting apelin to the nucleus (N. Ap) and to the secretory vesicles (S. Ap) were transfected into S-SMCs where apelin was barely detectable. Both plasmids induced the SMC transition towards a R-phenotype. Overexpression of N. Ap, and to a lesser degree S. Ap, led to a nuclear localization of S100A4. Stimulation of S-SMCs with platelet-derived growth factor-BB, known to induce the transition toward the R-phenotype, yielded the direct interaction and nuclear expression of both apelin and S100A4. In conclusion, apelin induces a SMC phenotypic transition towards the synthetic phenotype. These results suggest that apelin acts via nuclear re-localization of S100A4, raising the possibility of a new pro-atherogenic relationship between apelin and S100A4.
Assuntos
Aterosclerose , Animais , Ratos , Apelina/genética , Apelina/metabolismo , Aterosclerose/metabolismo , Movimento Celular , Células Cultivadas , Miócitos de Músculo Liso/metabolismo , Fenótipo , SuínosRESUMO
OBJECTIVES: The aim of this study was to test whether adenosine triphosphate-sensitive K(+) (KATP) channel expression relates to mechanical and hypoxic stress within the left human heart. BACKGROUND: The KATP channels play a vital role in preserving the metabolic integrity of the stressed heart. However, the mechanisms that govern the expression of their subunits (e.g., potassium inward rectifier [Kir] 6.2) in adult pathologies are mostly unknown. METHODS: We collected biopsies from the 4 cardiac chambers and 50 clinical parameters from 30 surgical patients with severe mitral dysfunction. Proteins and messenger ribonucleic acids (mRNAs) of KATP pore subunits and mRNAs of their known transcriptional regulators (forkhead box [FOX] F2, FOXO1, FOXO3, and hypoxia inducible factor [HIF]-1α) were measured respectively by Western blotting, immunohistochemistry, and quantitative real-time polymerase chain reaction, and submitted to statistical analysis. RESULTS: In all heart chambers, Kir6.2 mRNA correlated with HIF-1α mRNA. Neither Kir6.1 nor Kir6.2 proteins positively correlated with their respective mRNAs. The HIF-1α mRNA related in the left ventricle to aortic pressure, in the left atrium to left atrial pressure, and in all heart chambers to a decreased Kir6.2 protein/mRNA ratio. Interestingly, in the left heart, Kir6.2 protein and its immunohistochemical detection in myocytes were maximal at low venous PO(2). In the left ventricle, the Kir6.2 protein/mRNA ratio was also significantly higher at low venous PO(2), suggesting that tissue hypoxia might stabilize the Kir6.2 protein. CONCLUSIONS: Results suggest that post-transcriptional events determine Kir6.2 protein expression in the left ventricle of patients with severe mitral dysfunction and low venous PO(2). Mechanical stress mainly affects transcription of HIF-1α and Kir6.2. This study implies that new therapies could aim at the proteasome for stabilizing the left ventricular Kir6.2 protein.
Assuntos
Hipóxia/metabolismo , Canais KATP/metabolismo , Insuficiência da Valva Mitral/metabolismo , Miocárdio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Estresse Mecânico , Idoso , Animais , Gasometria , Pressão Sanguínea , Ecocardiografia , Feminino , Regulação da Expressão Gênica , Ventrículos do Coração/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Oxigênio/fisiologia , Pressão Parcial , Estabilidade Proteica , RNA Mensageiro/metabolismoRESUMO
Connexins are a large family of proteins that form gap junction channels allowing exchange of ions and small metabolites between neighboring cells. They have been implicated in pathological processes such as tumourigenesis in which they may act as tumour suppressors. A polymorphism in the human connexin37 (Cx37) gene (C1019T), resulting in a non-conservative amino acid change in the regulatory C-terminus (CT) of the Cx37 protein (P319S) has been suggested to be implicated in predisposition to angiosarcomas. In this study, we have used communication-deficient HeLa and SK-HEP-1 cells transfected with Cx37-319S, Cx37-319P or empty vector. We showed that the expression of Cx37-319P limited proliferation of HeLa and SK-HEP-1 cells, whereas Cx37-319S expression was without effect. Using an in vitro kinase assay, we demonstrated phosphorylation of Cx37 CT by glycogen synthase kinase-3 (GSK-3), a kinase known to be implicated in cell proliferation and cancer. GSK-3-induced phosphorylation was associated with reduced gap junctional intercellular communication (GJIC) as measured by microinjection of the tracer neurobiotin. Inhibition of GSK-3 by LiCl or SB415286 reduced phosphorylation of Cx37-319P and increased GJIC. This latter effect on GJIC involved the beta and not the alpha isoform of GSK-3. In contrast, GSK-3 inhibitors were without effect on HeLa cells expressing Cx37-319S. In conclusion, our data indicate functional effects of the Cx37 C1019T polymorphism on GJIC that might contribute to tumour cell growth.
Assuntos
Proliferação de Células , Conexinas/genética , Neoplasias/genética , Neoplasias/patologia , Polimorfismo Genético/fisiologia , Western Blotting , Comunicação Celular , Ciclo Celular , Conexinas/metabolismo , Imunofluorescência , Junções Comunicantes , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Células HeLa , Humanos , Fosforilação , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína alfa-4 de Junções ComunicantesRESUMO
ATP-sensitive potassium channels couple cell excitability to energy metabolism, thereby providing life-saving protection of stressed cardiomyocytes. The signaling for ATP-sensitive potassium channel expression is still unknown. We tested involvement of biochemical and biophysical parameters and potential transcription factors Forkhead box (FOX) and hypoxia-inducible factor (HIF-1alpha). Right atrial tissues were obtained during surgery from 28 children with heart disease. Expression of K(+)-inward-rectifier subunits Kir6.1/Kir6.2; sulfonyl urea receptors (SURs) SUR1A/B and SUR2A/B; and FOX class O (FOXO) 1, FOXO3, FOXF2, and HIF-1alpha were related to 31 parameters, including personal data, blood chemistry, and echocardiography. Venous hypoxemia (but not other ischemia indicators, such as venous hypercapnia or low glucose) predicts increased Kir6.1 (P<0.003) and Kir6.2 (P<0.03) protein. Kir6.1 associates with SUR2A/B mRNA (P<0.05) and correlates with FOXOs (P<0.002). FOXOs correlate with HIF-1alpha (P<0.01) and HIF-1alpha with venous hypoxemia (P<0.003). Electrophoretic mobility-shift assays suggest causal links among hypoxia, HIF-1alpha, FOXO1, and Kir6.1. To mimic mild ischemia encountered in some patients, cultured rat atrial myocytes were tested in hypoxia, hypercapnia, or low glucose, with normal conditions serving as the control. Mild hypoxia (24-hour) increases expression of HIF-1alpha, FOXO1, and SUR2A/B/Kir6.1 in culture (P<0.01), whereas hypercapnia and low glucose have no or opposite effects. Gene knockdown of HIF-1alpha or FOXO1 by small-interfering RNAs abolishes hypoxia-induced expression of FOXO1 and SUR2A/B/Kir6.1. These results suggest that low tissue oxygen determines increased expression of the atrial SUR2A/B/Kir6.1 gene via activation of HIF-1alpha-FOXO1. Because increased SUR2A/B/Kir6.1 has known survival benefits, this pathway offers novel therapeutic targets for children with heart disease.
Assuntos
Pressão Venosa Central/fisiologia , Átrios do Coração/metabolismo , Cardiopatias Congênitas/genética , Cardiopatias/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Hipóxia/etiologia , Canais de Potássio/genética , Animais , Criança , Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Cardiopatias Congênitas/sangue , Cardiopatias Congênitas/fisiopatologia , Cardiopatias/fisiopatologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Células Musculares/fisiologia , Oxigênio/sangue , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologiaRESUMO
Coordinate adaptation of myocyte metabolism and function is fundamental to survival of the stressed heart, but the mechanisms for this coordination remain unclear. Bioinformatics led us to discover that Foxs are key transcription factors involved. We performed experiments on the mouse atrial cell line HL-1, neonate rat heart myocytes, and an adult rat model of myocardial infarction. In electrophoretic mobility-shift assays, FoxO1 binds to the FoxO concensus site of the KATP channel subunit KIR6.1 promoter. In primary atrial culture, targeting FoxO1 and FoxO3 with siRNA specifically reduces mRNA expression of FoxO1 and -O3 and KIR6.1. Western blots, confocal immunofluorescence, and quantitative RT-PCR was applied for measuring expression of 10 Fox, 6 KATP channel subunits, and 12 metabolic genes. FoxF2, -O1, and -O3 strongly associate with expression of KATP channel subunits (in particular, KIR6.1, SUR1A and SUR2B) in different heart tissues and in the periinfarct zone of the left ventricle. Patch-clamp recordings demonstrate that molecular plasticity of these channels is matched by pharmacological plasticity and increased sensitivity to a metabolic challenge mimicked by the protonophore CCCP. A balance of FoxF2 and FoxO also regulates expression of at least 9 metabolic genes involved in setting the balance of glycolysis and beta-oxidation. Bioinformatics shows that the transcriptional mechanisms are highly conserved among chicken, mouse, rat, and human, and Fox are intimately linked to other metabolic sensors. Thus, FoxF2 and -O are key transcription factors coordinating expression of KATP channels and energy metabolism.
Assuntos
Metabolismo Energético , Fatores de Transcrição Forkhead/fisiologia , Canais KATP/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Animais , Células Cultivadas , Biologia Computacional , Proteína Forkhead Box O3 , Regulação da Expressão Gênica , Células Musculares , Infarto do Miocárdio , Proteínas do Tecido Nervoso , RatosRESUMO
Secretory polypeptides are vital for nervous system function, sleep, reproduction, growth, and metabolism. Ribosomes scanning the 5'-end of mRNA usually detect the first AUG site for initiating translation. The nascent propeptide chain is then directed via a signal-peptide into the endoplasmic reticulum, processed through the Golgi stacks, and packaged into secretory vesicles. By expressing prepropeptide-EGFP fusion proteins, we observed unusual destinations, mitochondria, nucleus, and cytoplasm, of neuropeptide Y (NPY), atrial natriuretic peptide, and growth hormone in living murine cardiac cells and hypothalamic slices. Subcellular expression was modulated by Zn++ or mutations of N-terminal prohormone sequences but was not due to overexpression in the trans-Golgi network. Mitochondrial targeting of NPY also occurred without the EGFP tag, was enhanced by site-directed mutagenesis of the first AUG initiation site, and abolished by mutation of the second AUG. Immunological methods indicated the presence of N-terminal truncated NPY in mitochondria. Imaging studies showed depolarization of NPY-containing mitochondria. P-SORT software correctly predicted the secondary intracellular destinations and suggested such destinations for many neuropeptides and peptide hormones known. Thus, mammalian cells may retarget secretory peptides from extracellular to intracellular sites by skipping the first translation-initiation codon and thereby alter mitochondrial function, gene expression, and secretion.
Assuntos
Fator Natriurético Atrial/metabolismo , Encéfalo/metabolismo , Hormônio do Crescimento/metabolismo , Miocárdio/metabolismo , Neuropeptídeo Y/metabolismo , Animais , Fator Natriurético Atrial/química , Fator Natriurético Atrial/genética , Células Cultivadas , Regulação da Expressão Gênica , Hormônio do Crescimento/química , Hormônio do Crescimento/genética , Humanos , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Neuropeptídeo Y/química , Neuropeptídeo Y/genética , Transporte Proteico , Ratos , Ratos Sprague-DawleyRESUMO
ANP (atrial natriuretic peptide) is widely recognized as an important vasorelaxant, diuretic, and cardioprotective hormone. Little is known, however, about how ANP-secretory vesicles form within the atrial myocytes. Secretory vesicles were visualized by fluorescence microscope imaging in live rat atrial myocytes expressing proANP-enhanced green fluorescent protein (EGFP), or N-terminal-mutated fusion proteins thought to suppress the calcium-dependent aggregation of proANP. Results showed the following: (1) aggregates of proANP and coexpressed proANP-EGFP recruited peptidylglycine alpha-amidating monooxygenase (PAM)-1, an abundant atrial integral vesicle membrane protein; (2) coexpressed N-terminal-mutated (Glu23,24-->Gln23,24) and N-terminal-deleted proANP-EGFP inhibited recruitment of PAM-1 by up to 60%; (3) 4-phenyl-3-butenoic acid (PBA) (10 mumol/L), a pharmacological inhibitor of the lumenal peptidylglycine alpha-hydroxylating monooxygenase domain of PAM proteins, inhibited recruitment of endogenous PAM-1 and of coexpressed pro-EGFP-PAM-1; (4) PBA had no effect on exocytosis of the potassium inward rectifier KIR2.1; (5) PBA induced a deformation of the secretory vesicles but did not inhibit docking. These findings suggest that recruitment of PAM-1 to secretory vesicles depends on intact N-terminal proANP and on the lumenal domain of PAM-1. Conversely, PAM-1 participates in shaping the proANP-secretory vesicles. The full text of this article is available online at http://circres.ahajournals.org.
Assuntos
Apêndice Atrial/metabolismo , Fator Natriurético Atrial/fisiologia , Ácidos Graxos Monoinsaturados/farmacologia , Oxigenases de Função Mista/fisiologia , Complexos Multienzimáticos/fisiologia , Vesículas Secretórias/fisiologia , Animais , Apêndice Atrial/ultraestrutura , Sítios de Ligação , Eletroporação , Exocitose/efeitos dos fármacos , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Oxigenases de Função Mista/antagonistas & inibidores , Complexos Multienzimáticos/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/fisiologia , Vesículas Secretórias/enzimologia , Vesículas Secretórias/ultraestrutura , Deleção de Sequência , Relação Estrutura-AtividadeRESUMO
Cardiomyocytes express mRNA for all major subunits of ATP-sensitive potassium (K(ATP)) channels: KIR6.1, KIR6.2, SUR1A, SUR2A, and SUR2B. It has remained controversial as to whether KIR6.1 may associate with KIR6.2 to form the tetrameric pore of K(ATP) channels in cardiomyocytes. To explore this possibility, cultured rat cardiomyocytes were examined for an inhibition of K(ATP) current by overexpression of pore loop-mutated (inactive) KIR6.x. Bicistronic plasmids were constructed encoding loop-mutated (AFA or SFG for GFG) rat KIR6.x followed by EGFP. In ventricular myocytes, the overexpression of KIR6.1SFG-pIRES(2)-EGFP or KIR6.2AFA-pIRES(2)-EGFP DNA caused, after 72 h, a major decrease of K(ATP) current density of 85.8% and 82.7%, respectively (P < 0.01), relative to EGFP controls (59 +/- 9 pA/pF). In atrial myocytes, overexpression of these pore-mutated KIR6.x by 6.0-fold and 10.6-fold, as assessed by quantitative immunohistochemistry, caused a decrease of K(ATP) current density of 73.7% and 58.5%, respectively (P < 0.01). Expression of wild-type rat KIR6.2 increased the ventricular and atrial K(ATP) current density by 58.3% and 42.9%, respectively (P < 0.01), relative to corresponding EGFP controls, indicating a reserve of SUR to accommodate increased KIR6.x trafficking to the sarcolemma. The results favor the view that KIR6.1 may associate with KIR6.2 to form heterotetrameric pores of native K(ATP) channels in cardiomyocytes.
Assuntos
Trifosfato de Adenosina/metabolismo , Mutação , Miócitos Cardíacos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , DNA , Condutividade Elétrica , Eletroporação , Átrios do Coração , Ventrículos do Coração , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: The aim is to compare the activation of ATP-sensitive potassium channels (K(ATP) channels) in intact and metabolically impaired atrial and ventricular myocytes. METHODS: The K(ATP) channel current is measured by whole cell and gramicidin-perforated patch clamp recordings in 164 cultured neonate rat cardiomyocytes. RESULTS: In whole cell recordings with 84 micromol/l ADP in pipette, spontaneous activity is significantly higher in atrium than ventricle, and EC(50) for the K(ATP) channel opener diazoxide is 0.13 micromol/l (atrium) versus 3.1 micromol/l (ventricle). With an ATP-regenerating system in pipette, EC(50) for diazoxide is 19.7 micromol/l (atrium) versus 54.9 micromol/l (ventricle). In gramicidin-perforated patch recordings, atrial myocytes respond significantly to 100 nmol/l of the mitochondrial protonophore CCCP, while ventricular myocytes do not. EC(50) for diazoxide is 129 micromol/l (atrium) versus >2500 micromol/l (ventricle) for myocytes exposed to CCCP, and 676 versus >2500 micromol/l, respectively, without CCCP. CONCLUSIONS: (1) K(ATP) channels are significantly more sensitive to metabolic inhibition in atrial than ventricular myocytes. (2) Sensitivity of atrium versus ventricle to the channel opener diazoxide increases from 3:1 to > or = 24:1 with ADP or metabolic inhibition. If extended to intact hearts, the results would predict a higher atrial sensitivity to ischemia, and a high sensitivity of the ischemic atrium to K(ATP) channel openers.
Assuntos
Trifosfato de Adenosina/fisiologia , Miócitos Cardíacos/metabolismo , Canais de Potássio/metabolismo , Animais , Animais Recém-Nascidos , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Células Cultivadas , Diazóxido/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Gramicidina/farmacologia , Átrios do Coração/citologia , Átrios do Coração/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Ionóforos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacos , Ratos , VasodilatadoresRESUMO
The relative role of plasmalemmal and mitochondrial ATP-sensitive K(+) (K(ATP)) channels in calcium homeostasis of the atrium is little understood. Electrically triggered (1 Hz) cytoplasmic calcium transients were measured by 340-to-380-nm wavelength fura 2 emission ratios in cultured rat atrial myocytes. CCCP, a mitochondrial protonophore (100-400 nmol/l), dose dependently reduced the transient amplitude by up to 85%, caused a slow rise in baseline calcium, and reduced the recovery time constant of the transient from 143 to 91 ms (P < 0.05). However, neither 5-hydroxydecanoate, a mitochondrial K(ATP) channel blocker, nor diazoxide (500 micromol/l) affected the amplitude, baseline, or time constant in CCCP-treated cells. HMR-1098 (30 micromol/l), a plasmalemmal K(ATP) channel blocker, and glibenclamide (1 micromol/l) increased the amplitude in CCCP-treated myocytes by 69-82%, sharply elevated the calcium baseline, and prolonged the recovery time constant to 181-193 ms (P < 0.01). Thus opening of plasmalemmal but not mitochondrial K(ATP) channels reduces the calcium overload in metabolically compromised but otherwise intact atrial myocytes. Mitochondrial K(ATP) channels probably operate through a different mechanism to afford ischemic protection.