RESUMEN
OBJECTIVES: To elucidate signal transduction pathways regulating expression of myocardial gap junction channel proteins (connexins) and to determine whether mediators of cardiac hypertrophy might promote remodeling of gap junctions, we characterized the effects of angiotensin II on expression of the major cardiac gap junction protein connexin43 (Cx43) in cultured neonatal rat ventricular myocytes. BACKGROUND: Remodeling of the distribution of myocardial gap junctions appears to be an important feature of anatomic substrates of ventricular arrhythmias in patients with heart disease. Remodeling of intercellular connections may be initiated by changes in connexin expression caused by chemical mediators of the hypertrophic response. METHODS: Cultures were exposed to 0.1 micromol/liter angiotensin II for 6 or 24 h, and Cx43 expression was characterized by immunoblotting, confocal microscopy and electron microscopy. RESULTS: Immunoblot analysis revealed a twofold increase in Cx43 content in cells treated for 24 h with angiotensin II (n=4, p < 0.05). This response was inhibited by the presence of 1.0 micromol/liter losartan, an AT1-receptor blocker. Confocal and electron microscopy demonstrated enhanced Cx43 immunoreactivity and increases in the number and size of gap junction profiles in cells exposed to angiotensin II for 24 h. These effects were also blocked by losartan. Immunoprecipitation of Cx43 from cells metabolically labeled with [35S]methionine demonstrated 2.4- and 2.9-fold increases in Cx43 radioactivity after 6 and 24 h exposure to angiotensin II, respectively (p < 0.03 at each time point). CONCLUSIONS: Angiotensin II up-regulates gap junctions in cultured neonatal rat ventricular myocytes by increasing Cx43 synthesis. Signal transduction pathways activated by angiotensin II under pathophysiologic conditions could initiate remodeling of conduction pathways, leading to the development of anatomic substrates of arrhythmias.
Asunto(s)
Angiotensina II/fisiología , Cardiomegalia/fisiopatología , Conexina 43/fisiología , Ventrículos Cardíacos/fisiopatología , Transducción de Señal/fisiología , Animales , Animales Recién Nacidos , Cardiomegalia/patología , Técnicas de Cultivo , Uniones Comunicantes/fisiología , Uniones Comunicantes/ultraestructura , Ventrículos Cardíacos/patología , Microscopía Confocal , Microscopía Electrónica , Microscopía Fluorescente , Ratas , Regulación hacia Arriba/fisiologíaRESUMEN
Remodeling of conduction pathways in the hypertrophic response to myocardial injury is a potential mechanism leading to the development of anatomic substrates of lethal arrhythmias. To delineate the responsible mechanisms and to directly relate changes in intercellular coupling at gap junctions with electrophysiological alterations, we studied the effects of cAMP, a mediator of cardiac hypertrophy, on action potential conduction velocity and connexin expression in neonatal rat ventricular myocyte cultures. Conduction velocity was measured with an optical activation mapping technique in cells loaded with the voltage-sensitive dye RH-237. Action potentials were conducted 24% to 29% more rapidly (P < .005) after incubating cultures for 24 hours with the cAMP analogue dibutyryl cAMP (db-cAMP, 1 mmol/L). However, db-cAMP caused no change in the maximum rate of rise of the action potential upstroke, Vmax. Electron and immunofluorescence microscopy revealed a significant increase in the number and size of gap junctions in db-cAMP-treated cells. Immunoblotting showed that the total amounts of the ventricular gap junction proteins connexin43 and connexin45 (Cx43 and Cx45, respectively) increased 2- to 4-fold. Immuno-precipitation of metabolically labeled connexin proteins revealed a dose-dependent increase in the rate of Cx45 protein synthesis in myocytes exposed to db-cAMP ( > 2-fold after a 4-hour exposure) but no change in the Cx43 synthesis rate. Northern blot analysis demonstrated a time-dependent increase in the amount of Cx43 mRNA, with a maximum 3.3-fold increase after 4 hours of exposure to 1 mmol/L db-cAMP; cycloheximide did not block this effect. In contrast, Cx45 mRNA levels were not altered significantly after db-cAMP treatment. Thus, cAMP causes a significant increase in conduction velocity that appears to be attributable largely to enhanced expression of proteins responsible for intercellular communication. Cx43 and Cx45 levels appear to be upregulated by cAMP by disparate molecular mechanisms.
Asunto(s)
Bucladesina/farmacología , Comunicación Celular/fisiología , Conexinas/metabolismo , Miocardio/citología , Conducción Nerviosa , Potenciales de Acción/efectos de los fármacos , Animales , Células Cultivadas/efectos de los fármacos , Conexina 43/biosíntesis , Conexina 43/genética , Conexinas/biosíntesis , Conexinas/genética , Miocardio/ultraestructura , ARN Mensajero/metabolismo , Ratas , Factores de TiempoRESUMEN
The role of tissue discontinuities in anisotropic impulse propagation was assessed in two-dimensional anisotropic monolayers of neonatal rat myocytes cultured on a growth-directing substrate of collagen. Activation spread and distribution of maximal upstroke rate of rise (Vmax) of the action potential were measured with an optical system using a voltage-sensitive fluorescent dye (RH-327) and a 10x10 photodiode array with a spatial resolution ranging from 7 to 15 microns. Activation maps were compared with the cellular architecture and the distribution of gap junctions obtained from immunostaining the gap junction protein connexin43 (Cx43). Four types of structures were studied: (1) dense cell cultures, (2) cultures with anisotropic intercellular clefts of variable size, (3) discontinuities created by inclusion of nonmyocyte cells, and (4) discontinuities resulting from nonuniform expression of gap junctions. In dense monolayers, activation spread was continuous with microinhomogeneities in both longitudinal and transverse directions. The average cell dimensions in such monolayers were smaller than in adult canine myocardium. However, the degree of cellular anisotropy (length-to-width ratio of 5.3 +/- 1.4) and connectivity were similar. The presence of small intercellular clefts (less than one cell in length) did not disturb the general pattern of transverse or longitudinal activation spread, but it was associated with wave front microcollisions during transverse propagation and a concomitant increase of Vmax beyond the cleft. Long intercellular clefts caused discontinuous transverse propagation. Conduction velocity and Vmax decreased significantly at narrow isthmuses formed by closely apposed clefts, rendering such sites susceptible for conduction block. In contrast Vmax increased when the wave front faced the borders of the clefts. Nonmyocyte cells were electrically connected to myocytes and served as sinks for electrotonic currents, thereby producing localized conduction slowing and a decrease in Vmax. Localized inhomogeneity in Cx43 distribution correlated accurately with circumscribed conduction block and changes in Vmax. Our results provide direct experimental evidence that the cellular structure and gap junction distribution correlate with action potential propagation and distribution of Vmax. We suggest that in tissue with a nonuniform anisotropy, connective tissue separating fiber bundles or sites of inhomogeneous connexin distribution may represent predilective sites for block in transverse direction.
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Corazón/fisiología , Óptica y Fotónica , Animales , Anisotropía , Células Cultivadas , Conexina 43/metabolismo , Perros , Fibroblastos/fisiología , Uniones Comunicantes/fisiología , Sistema de Conducción Cardíaco/fisiología , Técnicas Inmunológicas , Miocardio/citología , Miocardio/metabolismo , Conducción Nerviosa , Ratas , Ratas Wistar , Coloración y EtiquetadoRESUMEN
Transforming growth factor beta 1 (TGF beta 1)-null mice die fro complications due to an early-onset multifocal inflammatory disorder. We show here that cardiac cells are hyperproliferative and that intercellular adhesion molecule 1 (ICAM-1) is elevated. To determine which phenotypes are primarily caused by a deficiency in TGF beta 1 from those that are secondary to inflammation, we applied immunosuppressive therapy and genetic combination with the severe combined immunodeficiency (SCID) mutation to inhibit the inflammatory response. Treatment with antibodies to the leukocyte function-associated antigen 1 doubled longevity, reduced inflammation, and delayed heart cell proliferation. TGF beta 1-null SCID mice displayed no inflammation or cardiac cell proliferation, survived to adulthood, and exhibited normal major histocompatibility complex II (MHC II) and ICAM-1 levels. TGF beta 1-null pups born to a TGF beta 1-null SCID mother presented no gross congenital heart defects, indicating that TGF beta 1 alone does not play an essential role in heart development. These results indicate that lymphocytes are essential for the inflammatory response, cardiac cell proliferation, and elevated MHC II and ICAM-1 expression, revealing a vital role for TGF beta 1 in regulating lymphocyte proliferation and activation, which contribute to the maintenance of self tolerance.
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Antígenos CD11/inmunología , Inflamación/inmunología , Linfocitos/inmunología , Miocardio/inmunología , Inmunodeficiencia Combinada Grave/inmunología , Factor de Crecimiento Transformador beta/deficiencia , Factor de Crecimiento Transformador beta/genética , Animales , Animales Recién Nacidos , Anticuerpos/uso terapéutico , Secuencia de Bases , Cartilla de ADN , Genotipo , Heterocigoto , Homocigoto , Inmunoterapia , Inflamación/genética , Recuento de Leucocitos , Activación de Linfocitos , Ratones , Ratones SCID , Datos de Secuencia Molecular , Miocardio/patología , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/terapiaRESUMEN
Electrical activation of the heart requires transfer of current from one discrete cardiac myocyte to another, a process that occurs at gap junctions. Recent advances in knowledge have established that, like most differentiated cells, individual cardiac myocytes express multiple gap junction channel proteins that are members of a multigene family of channel proteins called connexins. These proteins form channels with unique biophysical properties. Furthermore, functionally distinct cardiac tissues such as the nodes and bundles of the conduction system and atrial and ventricular muscle express different combinations of connexins. Myocytes in these tissues are interconnected by gap junctions that differ in tissue-specific manner in terms of their number, size, and three-dimensional distribution. These observations suggest that both molecular and structural aspects of gap junctions are critical determinants of the anisotropic conduction properties of different cardiac tissues. Expression of multiple connexins also creates the possibility that "hybrid" channels composed of more than one connexin protein type can form, thus greatly increasing the potential for fine control of intercellular ion flow and communication within the heart.
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Uniones Comunicantes/fisiología , Sistema de Conducción Cardíaco/fisiología , Anisotropía , Comunicación Celular , Conexinas/genética , Conexinas/fisiología , Uniones Comunicantes/ultraestructura , Cardiopatías/patología , Cardiopatías/fisiopatología , Humanos , Miocardio/citología , Miocardio/metabolismo , Miocardio/ultraestructura , FenotipoRESUMEN
Three gap junction proteins have been identified in mammalian cardiac myocytes: connexin43 (Cx43), connexin45 (Cx45), and connexin40 (Cx40). These proteins form channels with different electrophysiological properties and have different distributions in cardiac tissues with disparate conduction properties. We characterized the expression, phosphorylation, turnover, and subcellular distribution of these connexins in primary cultures of neonatal rat ventricular myocytes. Cx43, Cx45, and Cx40 mRNA were specifically detected in RNA blots. Immunofluorescent staining with antibodies specific for Cx43 and Cx45 revealed punctate labeling at appositional membranes, but no immunoreactive Cx40 was detected. Double-label immunofluorescence confocal microscopy of cultured myocytes revealed colocalization of Cx43 and Cx45. Cx43 and Cx45 were both identified by immunoprecipitation from [35S]methionine-labeled cultures, but anti-Cx40 antibodies did not precipitate any radiolabeled protein. Phosphorylated forms of both Cx45 and Cx43 were immunoprecipitated from cultures metabolically labeled with [32P]orthophosphate. Phosphoamino acid analysis demonstrated that Cx45 was modified on serine residues, and Cx43 was phosphorylated on serine and threonine residues. Pulse-chase labeling experiments demonstrated that the half-lives of Cx43 and Cx45 were 1.9 and 2.9 hours, respectively. Thus, both Cx43 and Cx45 turn over relatively rapidly, suggesting that myocardial gap junctions have the potential for dynamic remodeling. The results implicate multiple mechanisms of gap junction regulation that may differ for different connexins.