RESUMO
This brief review summarizes the current state of the field for estrogen receptor actions in the cardiovascular system and the cardiovascular effects of hormone replacement therapy (HRT). It is organized into three parts: a short Introduction and overview of the current view of how estrogen works on blood vessels; a summary of the current status of clinical information regarding HRT and cardiovascular effects; and an update on state-of-the-art mouse models of estrogen action using estrogen receptor knockout mice.
Assuntos
Sistema Cardiovascular/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Terapia de Reposição de Estrogênios , Estrogênios/farmacologia , Receptores de Estrogênio/fisiologia , Animais , Fenômenos Fisiológicos Cardiovasculares/efeitos dos fármacos , Sistema Cardiovascular/metabolismo , Endotélio Vascular/metabolismo , Humanos , Camundongos , Camundongos Knockout , Óxido Nítrico/metabolismo , Receptores de Estrogênio/metabolismoRESUMO
Increased arterial stiffness and wave reflection have been identified as cardiovascular disease risk factors. In light of significant sex differences and the moderate heritability of vascular function measures, we hypothesized that variation in the genes coding for oestrogen receptors alpha (ESR1) and beta (ESR2) and aromatase (CYP19A1) is associated with aortic stiffness and pressure wave reflection as measured by non-invasive arterial tonometry. In all, 1261 unrelated Framingham Offspring Study participants who attended the seventh examination cycle (mean age 62+/-10 years, 52% women) and had arterial tonometry and genotyping data were included in the study. Analysis of covariance was used to assess the association of polymorphisms with forward wave amplitude, augmented pressure, augmentation index (AI), carotid-femoral pulse wave velocity and mean arterial pressure with adjustment for potential confounders. In the sex-pooled analysis, those homozygous for the minor allele at any of four ESR1 variants that were in strong linkage disequilibrium ((TA)(n), rs2077647, rs2234693 and rs9340799) had on an average 18% higher augmented pressure and 16% greater AI compared with carriers of one or two major alleles (P=0.0002-0.01). A similar magnitude of association was detected in those homozygous for the common allele at two ESR2 single-nucleotide polymorphisms (P=0.007-0.02). Our results are consistent with the hypothesis that variation in ESR1 and ESR2, but not CYP19A1, is associated with an increased wave reflection that may contribute to associations between these variants and adverse clinical events demonstrated earlier. Our findings will need to be replicated in additional cohorts.
Assuntos
Aromatase/genética , Artérias/fisiopatologia , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Doenças Vasculares Periféricas/genética , Polimorfismo de Nucleotídeo Único , Idoso , Artérias/diagnóstico por imagem , Pressão Sanguínea , Artéria Braquial/fisiopatologia , Artérias Carótidas/fisiopatologia , Elasticidade , Feminino , Artéria Femoral/fisiopatologia , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Homozigoto , Humanos , Desequilíbrio de Ligação , Masculino , Manometria , Pessoa de Meia-Idade , Doenças Vasculares Periféricas/diagnóstico por imagem , Doenças Vasculares Periféricas/fisiopatologia , Fenótipo , Fluxo Pulsátil , Ultrassonografia DopplerAssuntos
Doença das Coronárias/prevenção & controle , Terapia de Reposição de Estrogênios , Fatores Etários , Ensaios Clínicos Controlados como Assunto/estatística & dados numéricos , Interpretação Estatística de Dados , Estrogênios Conjugados (USP)/uso terapêutico , Feminino , Seguimentos , Humanos , Acetato de Medroxiprogesterona/uso terapêutico , Metanálise como Assunto , Pós-Menopausa/efeitos dos fármacos , Medição de Risco , Resultado do TratamentoRESUMO
Physiological effects of estrogen on myocardium are mediated by two intracellular estrogen receptors, ERalpha and ERbeta, that regulate transcription of target genes through binding to specific DNA target sequences. To define the role of ERbeta in the transcriptional activation of both endothelial (eNOS) and inducible nitric oxide synthase (iNOS) in cardiac myocytes, we used the complete ER-specific antagonist R,R-tetrahydrochrysene (R,R-THC). R,R-THC inhibited activation of iNOS/eNOS promoter-luciferase reporter constructs (iNOS/eNOS-Luc) in a dose-dependent fashion in COS7 cells selectively transfected with ERbeta, but failed to influence ERalpha-mediated increase of iNOS/ eNOS-Luc. In neonatal rat cardiomyocytes transfected with eNOS-Luc or iNOS-Luc, incubation with 17betaestradiol (E2, 10(-8) M) for 24 h stimulated expression of eNOS and iNOS. R,R-THC (10(-5) M) completely inhibited this effect. Furthermore, eNOS and iNOS protein expression in cardiac myocytes induced by E2 was completely blocked by R,R-THC as shown by immunoblot analysis. Taken together, these results show that ERbeta mediates transcriptional activation of eNOS and iNOS by E2.
Assuntos
Estradiol/farmacologia , Miocárdio/enzimologia , Óxido Nítrico Sintase/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Western Blotting , Células COS , Células Cultivadas , Crisenos/farmacologia , Receptor beta de Estrogênio , Coração/efeitos dos fármacos , Immunoblotting , Luciferases/metabolismo , Masculino , Miocárdio/química , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Regiões Promotoras Genéticas , Ratos , Ratos Wistar , Transfecção , Regulação para CimaRESUMO
The two known estrogen receptors, ERalpha and ERbeta, mediate the effects of estrogen in all target tissues, including blood vessels. We have shown previously that estrogen inhibits vascular injury response to the same extent in female wild-type (WT), ERalpha knockout (ERalphaKO(CH)), and ERbeta knockout (ERbetaKO(CH)) mice. We generated mice harboring disruptions of both ERalpha and ERbeta genes (ERalpha,betaKO(CH)) by breeding and studied the effect of 17beta-estradiol (E2) on vascular injury responses in ovariectomized female ERalpha,betaKO(CH) mice and WT littermates. E2 inhibited increases in vascular medial area following injury in the WT mice but not in the ERalpha,betaKO(CH) mice, demonstrating for the first time that the two known estrogen receptors are necessary and sufficient to mediate estrogen inhibition of a component of the vascular injury response. Surprisingly, as in WT littermates, E2 still significantly increased uterine weight and inhibited vascular smooth muscle cell (VSMC) proliferation following injury in the ERalpha,betaKO(CH) mice. These data support that the role of estrogen receptors differs for specific components of the vascular injury response in the ERalpha,betaKO(CH) mice. The results leave unresolved whether E2 inhibition of VSMC proliferation in ERalpha,betaKO(CH) mice is caused by a receptor-independent mechanism, an unidentified receptor responsive to estrogen, or residual activity of the ERalpha splice variant reported previously in the parental ERalphaKO(CH) mice. These possibilities may be resolved by studies of mice in which ERalpha has been fully disrupted (ERalphaKO(St)), which are in progress.
Assuntos
Lesões das Artérias Carótidas/tratamento farmacológico , Estrogênios/uso terapêutico , Receptores de Estrogênio/genética , Animais , Bromodesoxiuridina/metabolismo , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/genética , Divisão Celular/efeitos dos fármacos , Estradiol/uso terapêutico , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Feminino , Genótipo , Camundongos , Camundongos Knockout , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , OvariectomiaRESUMO
Clinical studies of hormone replacement therapy to prevent cardiovascular diseases have heightened interest in the cardiovascular effects of progestins. However, the role of the progesterone receptor (PR) in vascular biology has not been studied in vivo. We studied ovariectomized female PR knockout (PRKO) mice and their wild-type (WT) littermates using the mouse carotid artery injury model. Placebo-treated PRKO mice showed significantly greater vascular medial hypertrophy and vascular smooth muscle cell (VSMC) proliferation in response to vascular injury than did WT mice. Progesterone had no significant effect in the PRKO mice, but worsened the response to injury in WT mice. VSMCs cultured from PRKO mouse aortae were markedly hyperproliferative, and their growth was not affected by progesterone. In contrast to the in vivo findings, progesterone inhibited proliferation of WT-derived VSMCs. Furthermore, reintroduction of PR into PRKO-derived VSMCs using adenoviral methods restored progesterone-mediated inhibition of proliferation to these cells. This effect was reversed by the PR antagonist, RU 486. Thus, the effects of PR and progesterone differ markedly between cultured VSMCs and intact blood vessels. These data demonstrate a direct role for the PR in regulating the response to vascular injury and VSMC proliferation.
Assuntos
Lesões das Artérias Carótidas , Receptores de Progesterona/fisiologia , Animais , Artéria Carótida Primitiva/patologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Feminino , Antagonistas de Hormônios/farmacologia , Hiperplasia , Camundongos , Camundongos Knockout , Mifepristona/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Ovariectomia , Progesterona/antagonistas & inibidores , Progesterona/farmacologia , Receptores de Progesterona/deficiência , Receptores de Progesterona/efeitos dos fármacos , Receptores de Progesterona/genética , Proteínas Recombinantes de Fusão/fisiologia , TransfecçãoRESUMO
Atherosclerosis is the primary cause of cardiovascular disease, and the risk for atherosclerosis is inversely proportional to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL is atheroprotective are complex and not well understood. Here we show that HDL stimulates endothelial nitric oxide synthase (eNOS) in cultured endothelial cells. In contrast, eNOS is not activated by purified forms of the major HDL apolipoproteins ApoA-I and ApoA-II or by low-density lipoprotein. Heterologous expression experiments in Chinese hamster ovary cells reveal that scavenger receptor-BI (SR-BI) mediates the effects of HDL on the enzyme. HDL activation of eNOS is demonstrable in isolated endothelial-cell caveolae where SR-BI and eNOS are colocalized, and the response in isolated plasma membranes is blocked by antibodies to ApoA-I and SR-BI, but not by antibody to ApoA-II. HDL also enhances endothelium- and nitric-oxide-dependent relaxation in aortae from wild-type mice, but not in aortae from homozygous null SR-BI knockout mice. Thus, HDL activates eNOS via SR-BI through a process that requires ApoA-I binding. The resulting increase in nitric-oxide production might be critical to the atheroprotective properties of HDL and ApoA-I.
Assuntos
Antígenos CD36/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de Membrana , Óxido Nítrico Sintase/metabolismo , Receptores Imunológicos , Receptores de Lipoproteínas , Animais , Sequência de Bases , Antígenos CD36/genética , Antígenos CD36/fisiologia , Células CHO , Linhagem Celular Transformada , Cricetinae , Primers do DNA , Ativação Enzimática , Óxido Nítrico Sintase Tipo III , Ligação Proteica , Receptores Depuradores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptores Depuradores Classe B , OvinosRESUMO
Estrogen causes rapid endothelial nitric oxide (NO) production because of the activation of plasma membrane-associated estrogen receptors (ER) coupled to endothelial NO synthase (eNOS). In the present study, we determined the role of G proteins in eNOS activation by estrogen. Estradiol-17beta (E(2), 10(-8) m) and acetylcholine (10(-5) m) caused comparable increases in NOS activity (15 min) in intact endothelial cells that were fully blocked by pertussis toxin (Ptox). In addition, exogenous guanosine 5'-O-(2- thiodiphosphate) inhibited E(2)-mediated eNOS stimulation in isolated endothelial plasma membranes, and Ptox prevented enzyme activation by E(2) in COS-7 cells expressing ERalpha and eNOS. Coimmunoprecipitation studies of plasma membranes from COS-7 cells transfected with ERalpha and specific Galpha proteins demonstrated E(2)-stimulated interaction between ERalpha and Galpha(i) but not between ERalpha and either Galpha(q) or Galpha(s); the observed ERalpha-Galpha(i) interaction was blocked by the ER antagonist ICI 182,780 and by Ptox. E(2)-stimulated ERalpha-Galpha(i) interaction was also demonstrable in endothelial cell plasma membranes. Cotransfection of Galpha(i) into COS-7 cells expressing ERalpha and eNOS yielded a 3-fold increase in E(2)-mediated eNOS stimulation, whereas cotransfection with a protein regulator of G protein signaling, RGS4, inhibited the E(2) response. These findings indicate that eNOS stimulation by E(2) requires plasma membrane ERalpha coupling to Galpha(i) and that activated Galpha(i) mediates the requisite downstream signaling events. Thus, novel G protein coupling enables a subpopulation of ERalpha to initiate signal transduction at the cell surface. Similar mechanisms may underly the nongenomic actions of other steroid hormones.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana/metabolismo , Óxido Nítrico Sintase/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Linhagem Celular Transformada , Membrana Celular/metabolismo , Ativação Enzimática , Óxido Nítrico Sintase Tipo III , Testes de Precipitina , Transdução de SinaisRESUMO
OBJECTIVES: The goal of this study was to examine the effect of heart failure etiology on peripheral vascular endothelial function in cardiac transplant recipients. BACKGROUND: Peripheral vascular endothelial dysfunction occurs in patients with heart failure of either ischemic or nonischemic etiology. The effect of heart failure etiology on peripheral endothelial function after cardiac transplantation is unknown. METHODS: Using brachial artery ultrasound, endothelium-dependent, flow-mediated dilation (FMD) was assessed in patients with heart failure with either nonischemic cardiomyopathy (n = 10) or ischemic cardiomyopathy (n = 7), cardiac transplant recipients with prior nonischemic cardiomyopathy (n = 10) or prior ischemic cardiomyopathy (n = 10) and normal controls (n = 10). RESULTS: Patients with heart failure with either ischemic cardiomyopathy or nonischemic cardiomyopathy had impaired FMD (3.6 +/- 1.0% and 5.1 +/- 1.2%, respectively, p = NS) compared with normal subjects (13.9 +/- 1.3%, p < 0.01 compared with either heart failure group). In transplant recipients with antecedent nonischemic cardiomyopathy, FMD was markedly higher than that of heart failure patients with nonischemic cardiomyopathy (13.0 +/- 2.4%, p < 0.001) and similar to that of normal subjects (p = NS). However, FMD remained impaired in transplant recipients with prior ischemic cardiomyopathy (5.5 +/- 1.5%, p = 0.001 compared with normal, p = 0.002 vs. transplant recipients with previous nonischemic cardiomyopathy). CONCLUSIONS: Peripheral vascular endothelial function is normal in cardiac transplant recipients with antecedent nonischemic cardiomyopathy, but remains impaired in those with prior ischemic cardiomyopathy. In contrast, endothelial function is uniformly abnormal for patients with heart failure, regardless of etiology. These findings indicate that cardiac transplantation corrects peripheral endothelial function for patients without ischemic heart disease, but not in those with prior atherosclerotic coronary disease.
Assuntos
Endotélio Vascular/fisiopatologia , Insuficiência Cardíaca/etiologia , Transplante de Coração/fisiologia , Complicações Pós-Operatórias/etiologia , Vasodilatação/fisiologia , Adulto , Artéria Braquial/fisiopatologia , Feminino , Insuficiência Cardíaca/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Isquemia Miocárdica/complicações , Isquemia Miocárdica/fisiopatologia , Isquemia Miocárdica/cirurgia , Complicações Pós-Operatórias/fisiopatologia , Fatores de RiscoRESUMO
The administration of estrogens in animals tends to inhibit the development of atherosclerosis. [Therefore], attempts are being made in human males to prevent further progress of the disease in cases of angina or previous coronary thrombosis by long-range estrogen therapy. This work is in its infancy. It is hoped that, with further research, compounds might be found that eliminate the undesirable action of such hormones and yet retain its beneficial effects on arteries (Samuel A. Levine, 1958).
Assuntos
Retículo Endoplasmático/enzimologia , Óxido Nítrico Sintase/metabolismo , Animais , Doenças Cardiovasculares/enzimologia , Doenças Cardiovasculares/prevenção & controle , Ativação Enzimática/fisiologia , Estrogênios/uso terapêutico , Feminino , Proteínas de Choque Térmico HSP90/metabolismo , Terapia de Reposição Hormonal , Humanos , Masculino , Óxido Nítrico Sintase Tipo III , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Subunidades Proteicas , Receptores de Estrogênio/metabolismo , Transdução de SinaisRESUMO
Estrogen causes nitric oxide (NO)-dependent vasodilation due to estrogen receptor (ER) alpha-mediated, nongenomic activation of endothelial NO synthase (eNOS). The subcellular site of interaction between ERalpha and eNOS was determined in studies of isolated endothelial cell plasma membranes. Estradiol (E(2), 10(-8) mol/L) caused an increase in eNOS activity in plasma membranes in the absence of added calcium, calmodulin, or eNOS cofactors, which was blocked by ICI 182,780 and ERalpha antibody. Immunoidentification studies detected the same 67-kDa protein in endothelial cell nucleus, cytosol, and plasma membrane. Plasma membranes from COS-7 cells expressing eNOS and ERalpha displayed ER-mediated eNOS stimulation, whereas membranes from cells expressing eNOS alone or ERalpha plus a myristoylation-deficient mutant eNOS were insensitive. Fractionation of endothelial cell plasma membranes revealed ERalpha protein in caveolae, and E(2) caused stimulation of eNOS in isolated caveolae that was ER-dependent; noncaveolae membranes were insensitive. Acetylcholine and bradykinin also activated eNOS in isolated caveolae. Furthermore, the effect of E(2) on eNOS in caveolae was prevented by calcium chelation. Thus, a subpopulation of ERalpha is localized to endothelial cell caveolae where they are coupled to eNOS in a functional signaling module that may regulate the local calcium environment. The full text of this article is available at http://www.circresaha.org.
Assuntos
Cavéolas/metabolismo , Óxido Nítrico Sintase/metabolismo , Receptores de Estrogênio/metabolismo , Transdução de Sinais , Acetilcolina/farmacologia , Animais , Células COS , Cálcio/metabolismo , Calmodulina/metabolismo , Caveolina 1 , Caveolinas/metabolismo , Membrana Celular/enzimologia , Células Cultivadas , Quelantes , Colinérgicos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Estradiol/farmacologia , Receptor alfa de Estrogênio , Immunoblotting , Óxido Nítrico Sintase Tipo III , Ovinos , Transdução de Sinais/efeitos dos fármacosRESUMO
Estrogen receptors (ERs) are ligand-activated transcription factors that regulate gene expression and cell growth. Two ERs now have been identified: ERalpha and the more recently discovered ERbeta. The physiological function of ERbeta remains unclear, but evidence from vascular injury studies and from ERbeta knockout mice suggests that ERbeta may be involved in the regulation of cellular proliferation. Here we show a direct and specific interaction between ERbeta and the cell cycle mitotic spindle assembly checkpoint protein, MAD2 (mitosis arrest-deficient 2). The ERbeta-MAD2 interaction was identified by screening of a yeast two-hybrid system vascular endothelial cell library with ERbeta and confirmed with glutathione S-transferase-fusion protein interaction studies. In contrast, ERalpha did not interact with MAD2 in either the two-hybrid system or in the protein-protein interaction experiments. Amino acids 173-208 in the hinge region of ERbeta were sufficient to mediate the interaction with MAD2 in the two-hybrid system and in glutathione S-transferase-fusion protein studies. These data identify a link between ERbeta and MAD2 of potential importance to regulation of the cell cycle and support a function of ERbeta distinct from the established role of ERs as transcription factors.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte , Proteínas Fúngicas/metabolismo , Receptores de Estrogênio/metabolismo , Sequência de Aminoácidos , Animais , Ciclo Celular , Proteínas de Ciclo Celular , Endotélio Vascular/metabolismo , Receptor beta de Estrogênio , Glutationa Transferase/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Nucleares , Ligação Proteica , Artéria Pulmonar/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Ovinos , Técnicas do Sistema de Duplo-HíbridoAssuntos
Sistema Cardiovascular/efeitos dos fármacos , Estrogênios/farmacologia , Animais , Sistema Cardiovascular/citologia , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/patologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Deleção de Genes , Humanos , Camundongos , Camundongos Knockout , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miocárdio/metabolismo , Receptores de Estrogênio/deficiência , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Fatores de TempoRESUMO
The protective effects of estrogen in the cardiovascular system result from both systemic effects and direct actions of the hormone on the vasculature. Two estrogen receptors have been identified, ERalpha and ERbeta. We demonstrated previously that estrogen inhibits the response to vascular injury in both wild-type and ERalpha-deficient mice, and that ERbeta is expressed in the blood vessels of each, suggesting a role for ERbeta in the vascular protective effects of estrogen. In the present study, we examined the effect of estrogen administration on mouse carotid arterial injury in ERbeta-deficient mice. Surprisingly, in ovariectomized female wild-type and ERbeta knockout mice, 17beta-estradiol markedly and equally inhibited the increase in vascular medial area and the proliferation of vascular smooth muscle cells after vascular injury. These data demonstrate that ERbeta is not required for estrogen-mediated inhibition of the response to vascular injury, and suggest that either of the two known estrogen receptors is sufficient to protect against vascular injury, or that another unidentified estrogen receptor mediates the vascular protective effects of estrogen.
Assuntos
Lesões das Artérias Carótidas/tratamento farmacológico , Estradiol/uso terapêutico , Músculo Liso Vascular/efeitos dos fármacos , Receptores de Estrogênio/genética , Animais , Divisão Celular/efeitos dos fármacos , Receptor beta de Estrogênio , Feminino , Camundongos , Camundongos Knockout , Músculo Liso Vascular/citologia , OvariectomiaRESUMO
Contraction and relaxation of smooth muscle are regulated by myosin light-chain kinase and myosin phosphatase through phosphorylation and dephosphorylation of myosin light chains. Cyclic guanosine monophosphate (cGMP)-dependent protein kinase Ialpha (cGKIalpha) mediates physiologic relaxation of vascular smooth muscle in response to nitric oxide and cGMP. It is shown here that cGKIalpha is targeted to the smooth muscle cell contractile apparatus by a leucine zipper interaction with the myosin-binding subunit (MBS) of myosin phosphatase. Uncoupling of the cGKIalpha-MBS interaction prevents cGMP-dependent dephosphorylation of myosin light chain, demonstrating that this interaction is essential to the regulation of vascular smooth muscle cell tone.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Músculo Liso Vascular/enzimologia , Cadeias Leves de Miosina/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Células Cultivadas , Proteína Quinase Dependente de GMP Cíclico Tipo I , Proteínas Quinases Dependentes de GMP Cíclico/química , Proteínas Quinases Dependentes de GMP Cíclico/genética , Histonas/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Zíper de Leucina , Contração Muscular , Relaxamento Muscular , Músculo Liso Vascular/fisiologia , Mutagênese Sítio-Dirigida , Fosfatase de Miosina-de-Cadeia-Leve , Fosfoproteínas Fosfatases/química , Fosforilação , Testes de Precipitina , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
INTRODUCTION: A mouse strain lacking functional myotonic dystrophy protein kinase (DMPK) has recently been developed. DMPK-/- mice exhibit muscular and conduction abnormalities consistent with the disease; however, the site of abnormal cardiac conduction is unknown. METHODS AND RESULTS: Nine homozygous DMPK-/- mice and seven age matched wild-type (WT) controls underwent in vivo electrophysiologic studies using an endocardial 2-French catheter. Baseline intervals as well as Wenckebach and 2:1 cycle lengths were measured to assess AV and ventriculoatrial (VA) conduction. Effective refractory periods (ERP) and functional refractory periods were determined during atrial and ventricular premature stimulation. His-bundle recordings were obtained on all the studied animals (16/16). DMPK-/- mice had significantly prolonged PR (48.1 +/- 5.5 vs 40.9 +/- 3.9 msec, P = 0.010) and AH (36.7 +/- 4.0 vs 31.6 +/- 4.8 msec, P = 0.037) intervals compared to WT controls. HV intervals were very significantly prolonged as well (14.7 +/- 2.0 vs 10.3 +/- 0.8 msec; P < 0.0001). Three of 9 DMPK-/- and 1 of 7 WT mice exhibited VA block. Atrial ERP was reached before AV node ERP in 2 (22%) of 9 of the knockout mice and 5 (71%) of 7 of the controls (P = 0.06). Only one mouse (DMPK-/-) exhibited infra-Hisian block on premature atrial stimulation. CONCLUSION: In this mouse model of myotonic dystrophy, AV conduction abnormalities were localized to the supra-Hisian and infra-Hisian conduction tissues, with a higher predilection to the latter, a finding similar to the human form of the disease.
Assuntos
Eletrocardiografia , Sistema de Condução Cardíaco , Coração/fisiopatologia , Distrofia Miotônica/fisiopatologia , Animais , Humanos , Camundongos , Camundongos Knockout , Distrofia Miotônica/genética , Miotonina Proteína Quinase , Proteínas Serina-Treonina Quinases/genéticaRESUMO
The investigation of fetal pulmonary endothelial cell gene expression and function has been limited by the requirement for primary cells. In an effort to establish an immortalized cell line, ovine fetal pulmonary artery endothelial cells (PAECs; passage 5) were permanently transfected with the E6 and E7 open reading frames of human papillomavirus type 16, and phenotypes related to nitric oxide (NO) production were evaluated up to passage 28. Acetylated low-density lipoprotein uptake, endothelial NO synthase (eNOS) expression, and proliferation rates were unaltered by immortalization. Acetylcholine-stimulated eNOS activity was 218-255% above basal levels in immortalized cells, and this was comparable to the 250% increase seen in primary PAECs (passage 6). eNOS was also acutely activated by estradiol to levels 197-309% above basal, paralleling the stimulation obtained in primary cells. In addition, the expression of estrogen receptor-alpha, which has recently been shown to mediate the acute response in primary PAECs, was conserved. Thus fetal PAECs transfected with E6 and E7 show no signs of senescence with passage, and mechanisms of NO production, including those mediated by estradiol, are conserved. Immortalized PAECs will provide an excellent model for further studies of eNOS gene expression and function in fetal pulmonary endothelium.
Assuntos
Linhagem Celular Transformada , Endotélio Vascular/embriologia , Artéria Pulmonar/embriologia , Animais , Divisão Celular/fisiologia , Transformação Celular Viral/fisiologia , Ativação Enzimática/fisiologia , Receptor alfa de Estrogênio , Feto/citologia , Feto/enzimologia , Óxido Nítrico Sintase/metabolismo , Fases de Leitura Aberta/fisiologia , Papillomaviridae/genética , Receptores de Estrogênio/metabolismo , Ovinos/embriologia , TransfecçãoAssuntos
Fenômenos Fisiológicos Cardiovasculares , Estrogênios/fisiologia , Vasos Sanguíneos/fisiologia , Doenças Cardiovasculares/prevenção & controle , Fenômenos Fisiológicos Cardiovasculares/efeitos dos fármacos , Estradiol/metabolismo , Estradiol/farmacologia , Estradiol/fisiologia , Estrogênios/uso terapêutico , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Receptores de Estrogênio/química , Receptores de Estrogênio/fisiologiaRESUMO
Myotonic dystrophy (DM) is the most common form of muscular dystrophy and is caused by expansion of a CTG trinucleotide repeat on human chromosome 19. Patients with DM develop atrioventricular conduction disturbances, the principal cardiac manifestation of this disease. The etiology of the pathophysiological changes observed in DM has yet to be resolved. Haploinsufficiency of myotonic dystrophy protein kinase (DMPK), DM locus-associated homeodomain protein (DMAHP) and/or titration of RNA-binding proteins by expanded CUG sequences have been hypothesized to underlie the multi-system defects observed in DM. Using an in vivo murine electrophysiology study, we show that cardiac conduction is exquisitely sensitive to DMPK gene dosage. DMPK-/- mice develop cardiac conduction defects which include first-, second-, and third-degree atrioventricular (A-V) block. Our results demonstrate that the A-V node and the His-Purkinje regions of the conduction system are specifically compromised by DMPK loss. Importantly, DMPK+/- mice develop first-degree heart block, a conduction defect strikingly similar to that observed in DM patients. These results demonstrate that DMPK dosage is a critical element modulating cardiac conduction integrity and conclusively link haploinsufficiency of DMPK with cardiac disease in myotonic dystrophy.