RESUMO
BACKGROUND: Patients undergoing left atrial appendage (LAA) occlusion (LAAO) are multi-morbid, including mitral valve disease (MVD) which is associated with anatomic changes of the left atrium (LA). This study aims to identify how atrial myopathy in MVD influences outcomes in LAAO. METHODS: Atrial myopathy in MVD was defined as LA diameter > 45 mm (â) and > 48 mm (â) and existing MVD or history of surgical/interventional treatment. Patients were compared with controls from the prospective, multicentre LAArge registry of LAAO. RESULTS: A total of 528 patients (52 MVD, 476 no-MVD) were included. The MVD group was significantly more likely to be older (78.2 years vs 75.9 years, p = 0.036) and female (59.6% vs 37.8%, p = 0.002). Altered LA anatomy was observed in MVD with significantly larger LA diameter (53 mm vs. 48 mm, p < 0.001) and LAA Ostia [at 135° 23.0 mm (20.5, 26.0) vs 20.0 mm (18.0, 23.0), p = 0.002]. Implant success was high with 96.2% and 97.9%, respectively, without differences in severe complications (7.7% vs 4.6%, p = 0.31). One-year mortality (17.8% vs 11.5%, p = 0.19) and a combined outcome of death, stroke, and systemic embolism (20.3% vs 12.4%, p = 0.13) were not different. Independent predictors of the combined outcome were peripheral artery disease (HR 2.41, 95% CI 1.46-3.98, p < 0.001) and chronic kidney disease (HR 3.46, 95% CI 2.02-5.93, p < 0.001) but not MVD and atrial myopathy. CONCLUSION: Patients with MVD present with altered LA anatomy with increased LA and LAA diameter. However, procedural success and safety in LAAO are not compromised. One-year mortality is numerically higher in patients with MVD but driven by comorbidities.
Assuntos
Apêndice Atrial , Fibrilação Atrial , Doenças das Valvas Cardíacas , Doenças Musculares , Acidente Vascular Cerebral , Humanos , Feminino , Valva Mitral/diagnóstico por imagem , Valva Mitral/cirurgia , Apêndice Atrial/diagnóstico por imagem , Apêndice Atrial/cirurgia , Fibrilação Atrial/complicações , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/cirurgia , Estudos Prospectivos , Átrios do Coração/diagnóstico por imagem , Doenças das Valvas Cardíacas/complicações , Doenças das Valvas Cardíacas/diagnóstico , Doenças das Valvas Cardíacas/cirurgia , Acidente Vascular Cerebral/etiologia , Doenças Musculares/complicações , Resultado do TratamentoRESUMO
Heterotrimeric G proteins in physiological and pathological processes have been extensively studied so far. However, little is known about mechanisms regulating the cellular content and compartmentalization of G proteins. Here, we show that the association of nucleoside diphosphate kinase B (NDPK B) with the G protein betagamma dimer (Gbetagamma) is required for G protein function in vivo. In zebrafish embryos, morpholino-mediated knockdown of zebrafish NDPK B, but not NDPK A, results in a severe decrease in cardiac contractility. The depletion of NDPK B is associated with a drastic reduction in Gbeta(1)gamma(2) dimer expression. Moreover, the protein levels of the adenylyl cyclase (AC)-regulating Galpha(s) and Galpha(i) subunits as well as the caveolae scaffold proteins caveolin-1 and -3 are strongly reduced. In addition, the knockdown of the zebrafish Gbeta(1) orthologs, Gbeta(1) and Gbeta(1like), causes a cardiac phenotype very similar to that of NDPK B morphants. The loss of Gbeta(1)/Gbeta(1like) is associated with a down-regulation in caveolins, AC-regulating Galpha-subunits, and most important, NDPK B. A comparison of embryonic fibroblasts from wild-type and NDPK A/B knockout mice demonstrate a similar reduction of G protein, caveolin-1 and basal cAMP content in mammalian cells that can be rescued by re-expression of human NDPK B. Thus, our results suggest a role for the interaction of NDPK B with Gbetagamma dimers and caveolins in regulating membranous G protein content and maintaining normal G protein function in vivo.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , AMP Cíclico/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Contração Miocárdica/genética , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Nucleosídeo NM23 Difosfato Quinases/genética , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Heterotrimeric G proteins are pivotal regulators of myocardial contractility. In addition to the receptor-induced GDP/GTP exchange, G protein alpha subunits can be activated by a phosphate transfer via a plasma membrane-associated complex of nucleoside diphosphate kinase B (NDPK B) and G protein betagamma-dimers (Gbetagamma). To investigate the physiological role of this phosphate transfer in cardiomyocytes, we generated a Gbeta1gamma2-dimer carrying a single amino acid exchange at the intermediately phosphorylated His-266 in the beta1 subunit (Gbeta1H266Lgamma2). Recombinantly expressed Gbeta1H266Lgamma2 were integrated into heterotrimeric G proteins in rat cardiomyocytes but were deficient in intermediate Gbeta phosphorylation. Compared with wild-type Gbeta1gamma2 (Gbeta1WTgamma2), overexpression of Gbeta1H266Lgamma2 suppressed basal cAMP formation up to 55%. A similar decrease in basal cAMP production occurred when the formation of NDPK B/Gbetagamma complexes was attenuated by siRNA-mediated NDPK B knockdown. In adult rat cardiomyocytes expressing Gbeta1H266Lgamma2, the basal contractility was suppressed by approximately 50% which correlated to similarly reduced basal cAMP levels and reduced Ser16-phosphorylation of phospholamban. In the presence of the beta-adrenoceptor agonist isoproterenol, the total cAMP formation and contractility were significantly lower in Gbeta1H266Lgamma2 than in Gbeta1WTgamma2 expressing cardiomyocytes. However, the relative isoproterenol-induced increased was not affected by Gbeta1H266Lgamma2. We conclude that the receptor-independent activation of G proteins via NDPK B/Gbetagamma complexes requires the intermediate phosphorylation of G protein beta subunits at His-266. Our results highlight the histidine kinase activity of NDPK B for Gbeta and demonstrate its contribution to the receptor-independent regulation of cAMP synthesis and contractility in intact cardiomyocytes.
Assuntos
AMP Cíclico/biossíntese , Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Núcleosídeo-Difosfato Quinase/biossíntese , Subunidades Proteicas/fisiologia , Animais , Linhagem Celular Transformada , AMP Cíclico/genética , AMP Cíclico/fisiologia , Dimerização , Proteínas Heterotriméricas de Ligação ao GTP/biossíntese , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Contração Miocárdica/genética , Nucleosídeo NM23 Difosfato Quinases , Núcleosídeo-Difosfato Quinase/genética , Núcleosídeo-Difosfato Quinase/fisiologia , Subunidades Proteicas/biossíntese , Subunidades Proteicas/genética , RatosRESUMO
Iatrogenic aortic dissection (AD) is quite a rare complication during percutaneous coronary intervention (PCI). The exact mechanism of iatrogenic AD during PCI is unknown. A standard of care in the management of iatrogenic AD is still lacking. We describe a case of an 83-year-old man, with an aortocoronary bypass graft, who underwent complex PCI with rotational atherectomy catheterization with a radial approach for a chronic right coronary artery stenosis, complicated by AD. According to our experience, retrograde dissection into the aorta during PCI can be sealed with a coronary stent that covers the coronary ostium and should be treated conservatively in most circumstances, unless complication like extension of dissection into other vessels or pericardial effusion or hemodynamic instability occurs.
Assuntos
Aneurisma Aórtico/terapia , Dissecção Aórtica/terapia , Aterectomia Coronária/efeitos adversos , Aneurisma Coronário/terapia , Estenose Coronária/cirurgia , Complicações Pós-Operatórias/terapia , Idoso de 80 Anos ou mais , Dissecção Aórtica/etiologia , Angioplastia Coronária com Balão/efeitos adversos , Aneurisma Aórtico/etiologia , Calcinose/cirurgia , Aneurisma Coronário/etiologia , Angiografia Coronária , Humanos , Doença Iatrogênica , MasculinoRESUMO
BACKGROUND: A high intake of dietary flavonoids, which are abundant in fruits, vegetables, tea, and wine, is known to reduce cardiovascular mortality. The effects of flavonoids on cardiac electrophysiology, which theoretically may have both antiarrhythmic and proarrhythmic consequences, have not been studied systematically to date. METHODS AND RESULTS: We screened a broad spectrum of flavonoids for their inhibitory activity on HERG channels by using heterologous expression in Xenopus oocytes. At a concentration of 1 mmol/L, 10 compounds caused a significant inhibition of HERG currents, whereas 11 other flavonoids had no effect. The IC50 value for HERG block by naringenin, the most potent inhibitor, was 102.3 micromol/L in Xenopus oocytes and 36.5 micromol/L in HEK cells. To demonstrate the physiological relevance of these findings, we studied the effects of pink grapefruit juice, which contains large amounts of naringenin glycosides (>1000 micromol/L), in human volunteers. In 10 persons, we observed a peak QTc prolongation of 12.5+/-4.2 ms 5 hours after oral ingestion of 1 L of grapefruit juice. This effect was significant (P=0.02). CONCLUSIONS: We found a significant QTc prolongation by grapefruit juice in healthy volunteers, probably caused by block of HERG channels by flavonoids. These findings reveal new perspectives on the potential for dietary modification of cardiac electrophysiology.
Assuntos
Proteínas de Transporte de Cátions/antagonistas & inibidores , Citrus paradisi/química , Eletrocardiografia , Flavonoides/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Adulto , Animais , Antiarrítmicos , Bebidas , Canal de Potássio ERG1 , Eletrofisiologia , Canais de Potássio Éter-A-Go-Go , Feminino , Flavanonas/farmacologia , Flavonoides/administração & dosagem , Humanos , Concentração Inibidora 50 , Masculino , Oócitos , Transdução Genética , XenopusRESUMO
Coronary artery ectasia (CAE) is found in 0.3-5% of patients undergoing coronary angiography. Atherosclerosis is the main cause, followed by Kawasaki disease and infectious emboli. The exact pathogenesis has not been diagnosed as yet, but an inflammatory process is underlying. Symptoms, if present, are usually related to myocardial ischemia. Angiography is the mainstay for diagnosis. The prognosis is generally favorable. Thromboembolic complications are rare with antiplatelet therapy, and spontaneous rupture generally is rare but occurs more commonly in Kawasaki disease. Management varies from antithrombotic therapy to surgical ligation. Controlling coronary heart disease risk factors sharply affects the prognosis in patients with CAE.
Assuntos
Aneurisma Coronário , Aterosclerose , Biomarcadores/metabolismo , Aneurisma Coronário/diagnóstico , Aneurisma Coronário/etiologia , Aneurisma Coronário/mortalidade , Aneurisma Coronário/terapia , Angiografia Coronária , Diagnóstico Diferencial , Dilatação Patológica , Humanos , Mediadores da Inflamação/metabolismo , Prognóstico , Fatores de RiscoRESUMO
Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to receptor independent G protein activation. Apparently, the NDPK B isoform forms complexes with Gbetagamma dimers and thereby phosphorylates His266 in Gbeta1 subunits. Phosphorylated His266 mediates G protein activation by a transfer of the high energetic phosphate onto GDP, thus leading to de novo synthesis of GTP. Moreover, it has been demonstrated that the sarcolemmal content of NDPK isoforms is increased in hearts with terminal congestive heart failure leading to enhanced G protein activation. Similar data were reported in a rat model for beta-adrenoceptor-induced cardiac hypertrophy. We therefore describe in this chapter several methods which can be used for analysis of NDPK mediated G protein activation: (1) The quantification of NDPK isoforms in highly purified cardiac sarcolemmal membranes, (2) the enrichment of the NDPK B/Gbetagamma-complex from preparations of the retinal G protein transducin, (3) the analysis of the enhanced NDPK activated and high energy phosphate transfer in a neonatal rat cardiac myocyte derived cell line stably overexpressing NDPK (H10 cells), and (4) the increased activation of adenylyl cyclase by the enhanced receptor-independent activation of the stimulatory G protein alpha subunit in these cells.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Isoenzimas/metabolismo , Miocárdio , Núcleosídeo-Difosfato Quinase/metabolismo , 5'-Nucleotidase/metabolismo , Animais , Fracionamento Celular , AMP Cíclico/metabolismo , Ativação Enzimática , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/isolamento & purificação , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Humanos , Isoenzimas/isolamento & purificação , Substâncias Macromoleculares , Miocárdio/citologia , Miocárdio/enzimologia , Núcleosídeo-Difosfato Quinase/genética , Núcleosídeo-Difosfato Quinase/isolamento & purificação , Fosforilação , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Transporte Proteico , Ratos , Segmento Externo da Célula Bastonete/química , Sarcolema/química , Sarcolema/enzimologia , Transducina/química , Transducina/metabolismoRESUMO
1. Receptor-independent activation of heterotrimeric G proteins by plasma membrane-associated nucleoside diphosphate kinase (NDPK) has been demonstrated in vivo, and elevated levels of NDPK were found in purified sarcolemmal membranes of patients with end-stage heart failure. 2. Among 22 consecutive patients with chronic heart failure who underwent cardiac transplantation, those treated with a beta-blocker (n=8) had a 65% lower NDPK content and activity in the cardiac sarcolemma, compared to patients with similar base line characteristics who had no beta-blocker therapy (n=14). 3. The lower NDPK was associated with a reduced NDPK-dependent, Gi-mediated inhibition of adenylyl cyclase activity, as assessed by in vitro measurement of adenylyl cyclase activity in the presence of GDP or its kinase-resistant analog guanosine 5'-O-(2-thio)diphosphate (GDPbetaS). 4. We further tested whether treatment with a beta-adrenergic agonist would induce an increase in sarcolemmal NDPK. Rats treated with isoproterenol developed myocardial hypertrophy, and NDPK in the sarcolemma rose by 60% during 14 days of treatment. The beta-blocker propranolol prevented both effects. When hypertrophy was induced with thyroid hormone, NDPK did not increase. 5. In conclusion, chronic activation of beta-adrenergic receptors increases the binding of NDPK to cardiac sarcolemma, where it may activate heterotrimeric G proteins.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Miocárdio/enzimologia , Núcleosídeo-Difosfato Quinase/metabolismo , Sarcolema/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/uso terapêutico , Adulto , Idoso , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Cardiomiopatia Dilatada/tratamento farmacológico , Cardiopatias Congênitas/tratamento farmacológico , Humanos , Isoproterenol/farmacologia , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Miocárdio/patologia , Ratos , Ratos Wistar , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores Adrenérgicos beta/metabolismo , Sarcolema/enzimologia , Hormônios Tireóideos/farmacologia , Fatores de TempoRESUMO
Several studies have shown an activation of adenylyl cyclase by the G protein-inactivating guanine nucleotides, GDP and its phosphate transfer-resistant analog, guanosine 5'- O-(2-thiodiphosphate; GDPbetaS). Here, we studied the mechanism underlying this unconventional activation. Adenylyl cyclase activity in sarcolemmal membranes from failing human ventricular myocardium, at a low Mg2+ concentration, decreased rapidly during incubation at 37 degrees C. This decrease in enzyme activity was paralleled by a rapid release of GDP from Gi proteins, amounting to 75% release of total Gi-bound GDP within 10 min at 37 degrees C. In contrast, no GDP release was observed at 4 degrees C, and adenylyl cyclase activity remained stable for up to 20 min at 4 degrees C. GDPbetaS did not alter the initial rates of cyclic AMP formation by the adenylyl cyclase, at either 37 degrees C or 4 degrees C, but almost fully prevented the decrease in enzyme activity occurring at 37 degrees C. Hence, after 10 min of incubation at 37 degrees C, adenylyl cyclase activity in the presence of GDPbetaS was increased by 150%, while no difference in activity was observed at 4 degrees C. Under conditions where adenylyl cyclase is uncoupled from regulation by the inhibitory Gi proteins, i.e., at high concentrations of Mg2+ or Mn2+, adenylyl cyclase activity remained stable even at 37 degrees C and GDPbetaS did not stimulate activity. In conclusion, Gi proteins in sarcolemmal membranes from failing human hearts rapidly release bound GDP. The data, furthermore, suggest that this process results in adenylyl cyclase inhibition by the empty but apparently active Gi proteins.
Assuntos
Inibidores de Adenilil Ciclases , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/metabolismo , Miocárdio/enzimologia , Miocárdio/metabolismo , Sarcolema/enzimologia , Sarcolema/metabolismo , Guanosina Difosfato/farmacologia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/enzimologia , Humanos , Sarcolema/efeitos dos fármacos , Tionucleotídeos/farmacologiaRESUMO
Heterotrimeric G proteins are key regulators of signaling pathways in mammalian cells. Beyond G protein-coupled receptors, the amount and mutual ratio of specific G protein α, ß, and γ subunits determine the G protein signaling. However, little is known about mechanisms that regulate the concentration and composition of G protein subunits at the plasma membrane. Here, we show a novel cross-talk between stimulatory and inhibitory G protein α subunits (Gα) that is mediated by G protein ßγ dimers and controls the abundance of specific Gα subunits at the plasma membrane. Firstly, we observed in heart tissue from constitutively Gαi2- and Gαi3-deficient mice that the loss of Gαi2 and Gαi3 was accompanied by a slight increase in the protein content of the nontargeted Gαi isoform. Therefore, we analyzed whether overexpression of selected Gα subunits conversely impairs endogenous G protein α and ß subunit levels in cardiomyocytes. Integration of overexpressed Gαi2 subunits into heterotrimeric G proteins was verified by co-immunoprecipitation. Adenoviral expression of increasing amounts of Gαi2 led to a reduction of Gαi3 (up to 90 %) and Gαs (up to 75 %) protein levels. Likewise, increasing amounts of adenovirally expressed Gαs resulted in a linear 75 % decrease in both Gαi2 and Gαi3 protein levels. In contrast, overexpression of either Gαi or Gαs isoform did not influence the amount of Gαo and Gαq, both of which are not involved in the regulation of adenylyl cyclase activity. The mRNA expression of the disappearing endogenous Gα subunits was not affected, indicating a posttranslational mechanism. Interestingly, the amount of endogenous G protein ßγ dimers was not altered by any Gα overexpression. However, the increase of Gßγ level by adenoviral expression prevented the loss of endogenous Gαs and Gαi3 in Gαi2 overexpressing cardiomyocytes. Thus, our results provide evidence for a novel mechanism cross-regulating adenylyl cyclase-modulating Gαi isoforms and Gαs proteins. The Gα subunits apparently compete for a limited amount of Gßγ dimers, which are required for G protein heterotrimer formation at the plasma membrane.
Assuntos
Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Adenoviridae/genética , Adenilil Ciclases/metabolismo , Animais , Membrana Celular/metabolismo , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Ratos , Receptor Cross-Talk , Transdução de Sinais/fisiologiaRESUMO
Increasing evidence suggests that reversible phosphorylation of histidine residues in proteins is important for signaling cascades in eukaryotic cells. Recently, the first eukaryotic protein histidine phosphatase (PHP) was identified. The beta1-subunit of heterotrimeric G proteins (Gbeta) undergoes phosphorylation on His266 which is apparently involved in receptor-independent G protein activation. We studied whether phosphorylated Gbeta-subunits are substrates of PHP. Phosphorylated Gbetagamma dimers of the retinal G protein transducin and Gbeta in membrane preparations of H10 cells (neonatal rat cardiomyocytes) were dephosphorylated by PHP. Overexpression of PHP in H10 cells showed that PHP and Gbeta also interfere within cells. In membranes of cells overexpressing PHP, the amount of phosphorylated Gbeta was largely reduced. Both our in vitro and cell studies indicate that phosphorylated Gbeta-subunits of heterotrimeric G proteins are substrates of PHP. Therefore, PHP might play a role in the regulation of signal transduction via heterotrimeric G proteins.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Miócitos Cardíacos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Animais Recém-Nascidos , Sítios de Ligação , Linhagem Celular , Feminino , Especificidade de Órgãos , Ligação Proteica , Subunidades Proteicas , Ratos , Ratos Endogâmicos F344 , Distribuição TecidualRESUMO
Long-term treatment with angiotensin-converting enzyme (ACE) inhibitors as well as angiotensin II type 1 (AT(1)) receptor antagonists and statins reduces cardiovascular mortality in patients with coronary artery disease as well as chronic heart failure. Little is known about the acute effects of these compounds on vascular reactivity of coronary resistance vessels. Coronary arterioles were obtained from patients undergoing coronary bypass operation (atherosclerosis group) or valve replacement (control group). Responses to endothelium-dependent agonists (histamine, serotonin, and acetylcholine) as well as to the endothelium-independent agonist sodium nitroprusside (SNP) were investigated under baseline conditions and after incubation (15 min) with lisinopril (ACE inhibitor), candesartan (AT(1) receptor antagonist), or fluvastatin. In atherosclerotic vessels, vasorelaxation was significantly reduced to all endothelium-dependent agonists but not, however, to SNP (77 +/- 8, -24 +/- 16, -46 +/- 24, and 98 +/- 8% relaxation for histamine, serotonin, acetylcholine, and SNP, respectively). Lisinopril and fluvastatin but not candesartan significantly improved the responses to the endothelium-dependent agonists (lisinopril: 94 +/- 4, 17 +/- 22, and -20 +/- 13%; fluvastatin: 96 +/- 8, 23 +/- 21, and -25 +/- 18% relaxation for histamine, serotonin, and acetylcholine, respectively). The effect of lisinopril was prevented by pretreatment with a bradykinin antagonist (HOE-130) and dichloroisocoumarine, an inhibitor of kinine-forming enzymes. Pretreatment with a nitric oxide (NO) synthase inhibitor abolished the improvement of endothelial function by lisinopril and fluvastatin. Vascular reactivity in the control group was not influenced by any of the pharmacological interventions. The data demonstrate that in atherosclerosis, endothelium-dependent relaxation of coronary resistance arteries is severely compromised. The impairment can acutely be reversed by ACE inhibitors and statins via increasing the availability of NO.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Vasos Coronários/fisiopatologia , Endotélio Vascular/fisiopatologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Doenças Vasculares/tratamento farmacológico , Acetilcolina/farmacologia , Idoso , Bloqueadores do Receptor Tipo 1 de Angiotensina II , Arteríolas/efeitos dos fármacos , Arteriosclerose/tratamento farmacológico , Arteriosclerose/fisiopatologia , Benzimidazóis/uso terapêutico , Compostos de Bifenilo , Doença das Coronárias/tratamento farmacológico , Doença das Coronárias/fisiopatologia , Ácidos Graxos Monoinsaturados/uso terapêutico , Feminino , Fluvastatina , Histamina/farmacologia , Humanos , Técnicas In Vitro , Indóis/uso terapêutico , Lisinopril/uso terapêutico , Masculino , Pessoa de Meia-Idade , Contração Muscular/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I , Nitroprussiato/farmacologia , Serotonina/farmacologia , Tetrazóis/uso terapêutico , Doenças Vasculares/fisiopatologia , Resistência Vascular/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
A reduced availability of tetrahydrobiopterin (BH4), an essential cofactor for NO-synthesis, is causally involved in the development of endothelial dysfunction associated with ischemia/reperfusion. We, therefore, investigated the effect of sepiapterin, a substrate for BH4 synthesis, on postischemic injury in myocardial infarction and myocardial stunning. In rats, myocardial stunning was induced by repetitive ischemia (5 x 10-min ligature of the left coronary artery, 5 x 20-min reperfusion) and myocardial infarction by 50-min ligature and 60-min reperfusion. Myocardial blood flow was determined by H2-clearance, regional myocardial function by pulsed Doppler and infarct size by tetrazolium staining. Myeloperoxidase (MPO) activity was measured as a marker of neutrophil extravasation. cGMP was determined in rat serum as an indicator of increased NO synthesis. In animals treated with sepiapterin, regional myocardial function was significantly improved in both myocardial stunning and infarction and infarct size was significantly reduced. MPO activity decreased with sepiapterin treatment in both models. The systemic level of cGMP was reduced both following myocardial stunning and myocardial infarction in the control group. Pretreatment with sepiapterin induced a significant increase of cGMP level at the end of the protocol in both models. Substitution of sepiapterin reduces postischemic injury both in myocardial stunning and infarction apparently by ameliorating the availability of NO, thereby attenuating the activation of neutrophils in ischemia/reperfusion.
Assuntos
Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Pterinas/uso terapêutico , Animais , GMP Cíclico/metabolismo , Feminino , Isquemia Miocárdica/tratamento farmacológico , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Ratos , Ratos Endogâmicos WFRESUMO
Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to G protein activation in vitro. To study the effect of NDPK on G protein activity in living cells, the NDPK isoforms A and B were stably expressed in H10 cells, a cell line derived from neonatal rat cardiomyocytes. Overexpression of either NDPK isoform had no effect on cellular GTP and ATP levels, basal cAMP levels, basal adenylyl cyclase activity, and the expression of G(s)alpha and G(i)alpha proteins. However, co-expression of G(s)alpha led to an increase in cAMP synthesis that was largely enhanced by the expression of NDPK B, but not NDPK A, and that was confirmed by direct measurement of adenylyl cyclase activity. Cells expressing an inactive NDPK B mutant (H118N) exhibited a decreased cAMP formation in response to G(s)alpha. Co-immunoprecipitation studies demonstrated a complex formation of the NDPK with Gbetagamma dimers. The overexpression of NDPK B, but not its inactive mutant or NDPK A, increased the phosphorylation of Gbeta subunits. In summary, our data demonstrate a specific NDPK B-mediated activation of a G protein in intact cells, which is apparently caused by formation of NDPK B.Gbetagamma complexes and which appears to contribute to the receptor-independent activation of heterotrimeric G proteins.
Assuntos
Proteínas de Ligação ao GTP/química , Núcleosídeo-Difosfato Quinase/metabolismo , Fosfatos/química , Trifosfato de Adenosina/metabolismo , Adenoviridae/genética , Animais , Animais Recém-Nascidos , Western Blotting , Catálise , Membrana Celular/metabolismo , Clonagem Molecular , AMP Cíclico/metabolismo , DNA Complementar/metabolismo , Dimerização , Relação Dose-Resposta a Droga , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Modelos Biológicos , Mutação , Miocárdio/citologia , Fosforilação , Testes de Precipitina , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Ratos , Temperatura , Fatores de Tempo , TransfecçãoRESUMO
G protein betagamma dimers can be phosphorylated in membranes from various tissues by GTP at a histidine residue in the beta subunit. The phosphate is high energetic and can be transferred onto GDP leading to formation of GTP. Purified Gbetagamma dimers do not display autophosphorylation, indicating the involvement of a separate protein kinase. We therefore enriched the Gbeta-phosphorylating activity present in preparations of the retinal G protein transducin and in partially purified G(i/o) proteins from bovine brain. Immunoblots, autophosphorylation, and enzymatic activity measurements demonstrated enriched nucleoside diphosphate kinase (NDPK) B in both preparations, together with residual Gbetagamma dimers. In the retinal NDPK B-enriched fractions, a Gbeta-specific antiserum co-precipitated phosphorylated NDPK B, and an antiserum against the human NDPK co-precipitated phosphorylated Gbetagamma. In addition, the NDPK-containing fractions from bovine brain reconstituted the phosphorylation of purified Gbetagamma. For identification of the phosphorylated histidine residue, bovine brain Gbetagamma and G(t)betagamma were thiophosphorylated with guanosine 5'-O-(3-[(35)S]thio)triphosphate, followed by digestion with endoproteinase Glu-C and trypsin, separation of the resulting peptides by gel electrophoresis and high pressure liquid chromatography, respectively, and sequencing of the radioactive peptides. The sequence information produced by both methods identified specific labeled fragments of bovine Gbeta(1) that overlapped in the heptapeptide, Leu-Met-Thr-Tyr-Ser-His-Asp (amino acids 261-267). We conclude that NDPK B forms complexes with Gbetagamma dimers and contributes to G protein activation by increasing the high energetic phosphate transfer onto GDP via intermediately phosphorylated His-266 in Gbeta(1) subunits.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Núcleosídeo-Difosfato Quinase/metabolismo , Fosfatos/metabolismo , Animais , Western Blotting , Encéfalo/metabolismo , Bovinos , Membrana Celular/metabolismo , Dietil Pirocarbonato/farmacologia , Dimerização , Relação Dose-Resposta a Droga , Durapatita/farmacologia , Humanos , Immunoblotting , Modelos Moleculares , Peptídeos/química , Fosfatos/química , Fosforilação , Testes de Precipitina , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Retina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Serina Endopeptidases/metabolismo , Tripsina/farmacologiaRESUMO
At present, patients with documented sustained VT or resuscitated cardiac arrest (CA) are treated with ICDs. The aim of this study was to retrospectively evaluate if a routine electrophysiological study should be recommended prior to ICD implantation. In 462 patients referred for ICD implantation because of supposedly documented VT (n = 223) or CA (n = 239), electrophysiological study was routinely performed. In 48% of the patients with CA, sustained VT or VF was inducible. Electrophysiological study suggested conduction abnormalities (n = 11) or supraventricular tachyarrhythmias (n = 3) in conjunction with severely impaired left ventricular function to have been the most likely cause of CA in 14 (5.9%) of 239 patients. Likewise, sustained VT was only inducible in 48% of patients with supposedly documented VT. Of these inducible VTs, nine were diagnosed as right ventricular outflow tract tachycardia or as bundle branch reentry tachycardia. Supraventricular tachyarrhythmias judged to represent the clinical event were the only inducible arrhythmia in 35 (16%) patients (AV nodal reentrant tachycardia [n = 7], AV reentry tachycardia [n = 4], atrial flutter [n = 19], and atrial tachycardia [n = 5]). Based on findings from the electrophysiological study, ICD implantation was withheld in 14 (5.9%) of 239 patients with CA and in 44 (19.7%) of 223 patients with supposedly documented VT. During electrophysiological study, VT or VF was only reproducible in about 50% of patients with supposedly documented VT or CA. Electrophysiological study revealed other, potentially curable causes for CA or supposedly documented VT in 12.6% (58/462) of all patients, indicating that ICD implantation can potentially be avoided or at least postponed in some of these patients. Based on these retrospective data, routine electrophysiological study prior to ICD implantation seems to be advisable.
Assuntos
Desfibriladores Implantáveis , Técnicas Eletrofisiológicas Cardíacas , Parada Cardíaca/terapia , Taquicardia Ventricular/terapia , Distribuição de Qui-Quadrado , Feminino , Parada Cardíaca/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Estatísticas não Paramétricas , Taquicardia Ventricular/fisiopatologiaRESUMO
BACKGROUND: The prognostic role of asymptomatic nonsustained ventricular tachycardia (NSVT) and programmed ventricular stimulation (PVS) in patients with idiopathic dilated cardiomyopathy (IDC) remains controversial. METHODS: The prognostic significance of ventricular arrhythmias, ejection fraction, NYHA class, atrial fibrillation and age for overall and sudden death mortality was prospectively studied in 157 patients with IDC (group 1) free of documented sustained ventricular arrhythmia and syncope. In 99 patients with asymptomatic NSVT (group 2), PVS with 2 - 3 extrastimuli was performed. Non-inducible patients were discharged without specific antiarrhythmic therapy, whereas those with inducible monomorphic ventricular tachycardia were implanted with an ICD. RESULTS: In group 1, 48% of patients had NSVT. Overall and sudden death mortality were significantly higher in patients with NSVT (34.2 vs. 9.8%, p = 0.0001 and 15.8 vs. 3.7%, p = 0.0037; follow-up 22 +/- 14 months). Multivariate analysis revealed that NSVT independently predicts both overall and sudden death mortality (p = 0.0021 and.0221, respectively; adjusted for EF, NYHA class and age). In group 2, inducibility of sustained ventricular tachyarrhythmia was 7%, but sustained monomorphic VT occurred in 3% only. Two of 7 inducible patients experienced arrhythmic events during a follow-up of 25 +/- 21 months (positive predictive value 29%). Overall and sudden death mortality were 29% and 0% in the inducible group vs. 17 and 4% in the non-inducible group. Both overall and sudden death mortality were significantly lower in non-inducible patients from group 2 as compared to patients from group 1 with NSVT (p = 0.0043 and 0.0048), most likely due to a more common use of betablockers and a higher EF in the former group (p < 0.001, respectively). CONCLUSIONS: In patients with IDC, NSVT independently predicts both overall and sudden death mortality. Due to a low inducibility rate and a poor positive predictive value, PVS seems inappropriate for further arrhythmia risk assessment. However, in spite of documented NSVT, the incidence of SCD in patients on optimized medical treatment including betablockers seems to be very low, questioning the need for specific arrhythmia risk stratification.