RESUMEN
OBJECTIVES: The depressed myocardial function observed in brain dead organ donors has been attributed to massive sympathetic discharge and catecholamine cardiotoxicity. Because elevated catecholamines are associated with altered myocardial gene expression, we investigated whether acute brain death from increased intracranial pressure alters the expression of myocardial gene products important in contractility. METHODS: A balloon expansion model was used to increase intracranial pressure in rabbits (n = 22). At timed intervals after brain death, mean arterial pressure, heart rate, electrocardiograms, histologic myocardial injury, and systemic catecholamines were assessed. Messenger RNA levels encoding myofilaments, adrenergic receptors, sarcoplasmic reticulum proteins, transcription factors, and stress-induced programs were measured with blot hybridization of total left ventricular RNA. RESULTS: Increased intracranial pressure induced an immediate pressor response that temporally coincided with diffuse electrocardiographic ST segment changes. Systemic epinephrine and norepinephrine levels concurrently increased (5- to 8-fold within 1 minute), then fell below baseline within 2 hours, and remained depressed at 4 hours. By 1 hour, histologic injury was evident. Four hours after the induction of increased intracranial pressure, levels of messenger RNA-encoding skeletal and cardiac alpha-actins, egr-1, and heat shock protein 70 were significantly increased. Sham-operated animals did not exhibit these changes. CONCLUSIONS: Select changes in myocardial gene expression occur in response to increased intracranial pressure and implicate ventricular remodeling in the myocardial dysfunction associated with acute brain death.
Asunto(s)
Muerte Encefálica/metabolismo , Regulación de la Expresión Génica/fisiología , Miocardio/metabolismo , Citoesqueleto de Actina/metabolismo , Análisis de Varianza , Animales , Muerte Encefálica/fisiopatología , Catecolaminas/sangre , Regulación de la Expresión Génica/genética , Genes Inmediatos-Precoces/fisiología , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Presión Intracraneal , Miocardio/patología , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Conejos , Distribución Aleatoria , Factores de TiempoRESUMEN
We investigated the protective effect of heat stress and metabolic preconditioning in cultured adult rat cardiac myocytes and correlated this effect with induction of heat shock proteins (HSP). Myocytes were preconditioned with sublethal heat shock or metabolic preconditioning for 30 min. Twenty hours later, preconditioned myocytes were subjected to lethal heat shock (46 degrees C for 2 h) or ischemia by incubation in ischemic buffer for 2 h. Cellular injury index was reduced from 69 +/- 4.0% in lethally heat-shocked cells to 27.0 +/- 1.6% with heat shock preconditioning (mean +/- SE; P < 0.01) and 19.0 +/- 3.0% with metabolic preconditioning (P < 0.01). Cellular injury index was 81.0 +/- 1.0% in ischemic myocytes and was reduced to 25.9 +/- 2.7 and 21.4 +/- 2.6% in heat shock- and metabolic-preconditioned myocytes, respectively (P < 0.01). A significant cross-tolerance of myocytes against lethal injury was observed with the two preconditioning methods. Western blot analysis revealed 3.3- and 2.5-fold increases in HSP 90 and 500- and 15-fold increases in HSP 70 with heat shock and metabolic preconditioning, respectively. HSP 27 expression remained unaltered relative to control cells. We conclude that heat shock and metabolic preconditioning induce delayed tolerance against lethal injuries in adult cardiac myocytes with elevated levels of HSP 70 and HSP 90.
Asunto(s)
Proteínas HSP70 de Choque Térmico/fisiología , Proteínas HSP90 de Choque Térmico/fisiología , Precondicionamiento Isquémico Miocárdico , Miocardio/citología , Animales , Western Blotting , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Calor , Masculino , Microscopía Electrónica de Rastreo , Miocardio/patología , Ratas , Estrés Fisiológico/patología , Estrés Fisiológico/fisiopatología , Factores de TiempoRESUMEN
BACKGROUND: Sonic muscle fibers intrinsic to the swim bladder of the oyster toadfish Opsanus tau proliferate throughout adult life and have an unusual radial morphology: alternating ribbons of sarcoplasmic reticulum (SR) and myofibrils surround a central core of sarcoplasm. Large fibers in adults form multiple cores, fragment, and appear to divide into smaller, more energy efficient units. METHODS: We examined embryonic to adult development of sonic muscle using electron and light microscopy and focused on the incidence of satellite cells (SC). RESULTS: Muscle fibers form late in the larval period from myoblasts, which do not appear to fuse into myotubes, but enlarge and differentiate myofibrils in a single patch. The SR differentiates from the outside inward, separating the myofibrils into bundles of varying thickness, which often exceed the thickness seen in adults. SCs in juveniles and adults have a sparse cytoplasm and a heterochromatic nucleus. The % SC nuclei (SC nuclei/total nuclei) decreases from a high of 88% in larvae to a low of 1% in adults although the adult average is 10%. No embryonic type fibers in the process of differentiating myofibrils were seen in adults. Small immature fibers, which had not yet formed the central core, have a complete radially organized contractile cylinder. CONCLUSIONS: Immature muscle fibers formed embryonically in the larval period have a different morphology from immature fibers in adults, suggesting that splitting rather than SCs is a major source of new fibers in adults.
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Sacos Aéreos/embriología , Sacos Aéreos/crecimiento & desarrollo , Peces/embriología , Peces/crecimiento & desarrollo , Desarrollo de Músculos , Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/embriología , Músculo Esquelético/crecimiento & desarrollo , Sacos Aéreos/citología , Animales , División Celular/fisiología , Núcleo Celular/ultraestructura , Citoplasma/ultraestructura , Embrión no Mamífero/embriología , Femenino , Masculino , Microscopía Electrónica , Músculo Esquelético/citología , Retículo Sarcoplasmático/ultraestructuraRESUMEN
Sounds of the channel catfish Ictalurus punctatus were found to consist of a rapid series of pulses produced by rubbing a ridged process on the first pectoral spine against the rough surface of a groove in the pectoral girdle during fin abduction. Although sounds can be made with either fin, approximately half of the individuals exhibited a fin preference, and 90% of these preferred the right fin. Unlike examples of handedness in other invertebrates and fishes, this preference is not simply a matter of anatomical asymmetry, but as in humans, reflects a preference between two equally developed limbs.
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Extremidades/fisiología , Lateralidad Funcional/fisiología , Sonido , Animales , IctaluridaeRESUMEN
Ischemia/reperfusion (I/R) and preconditioning of the heart by coronary artery occlusions increase expression of heat shock protein 70 (HSP 70). Because free radicals are generated during I/R, we hypothesized that the oxidant stress might contribute to an increased expression of HSP 70. Isolated rat hearts were perfused with free radical-generating systems such as xanthine/xanthine oxidase (X/XO), irradiated rose bengal (RB) generating singlet oxygen, and H2O2 for 15 min followed by 30 min of recovery period. Significant decrease in developed pressure and coronary flow occurred after perfusion with X/XO, H2O2, and RB. During I/R, the developed pressure and coronary flow were 60 +/- 8 and 80 +/- 5%, respectively, of control, which improved significantly with superoxide dismutase. The expression of HSP 70 mRNA increased over 13-fold in hearts perfused with X/XO, 6- to 7-fold with RB, and over 5-fold with H2O2. With I/R, an over 10-fold increase in HSP 70 mRNA was observed, which decreased significantly in the presence of superoxide dismutase. These results demonstrate that oxidant stress directly increases HSP 70 mRNA in the rat heart. It is concluded that one of the potential mechanisms of expression of HSP 70 by I/R may be oxygen radicals.
Asunto(s)
Proteínas HSP70 de Choque Térmico/genética , Miocardio/metabolismo , Estrés Oxidativo , ARN Mensajero/metabolismo , Animales , Circulación Coronaria , Radicales Libres , Peróxido de Hidrógeno/metabolismo , Técnicas In Vitro , Masculino , Contracción Miocárdica , Isquemia Miocárdica/metabolismo , Reperfusión Miocárdica , Oxígeno/metabolismo , Perfusión , Ratas , Ratas Sprague-Dawley , Rosa Bengala , Oxígeno Singlete , Xantina , Xantina Oxidasa/metabolismo , Xantinas/metabolismoRESUMEN
Activated neutrophils have been implicated in reperfusion injury and the no-reflow phenomenon of intramyocardial arterioles. This study tested the hypothesis that ischemia and activated neutrophils impair coronary endothelial and smooth muscle cell function of epicardial and intramyocardial coronary arteries. Alteration of smooth muscle and endothelial cell function in epicardial coronary arteries (3 mm diameter) and intramyocardial coronary arteries (0.3 mm diameter) was compared by means of a myograph after exposure to ischemia (epicardial, 160 minutes, intramyocardial, 30 minutes), activated neutrophils, and combined ischemia and activated neutrophils. Morphologic studies at the ultrastructural level were done by means of scanning electron microscopy. Epicardial coronary artery function was normal after ischemia, storage with activated neutrophils, and ischemia followed by storage with activated neutrophils. Intramyocardial artery function, however, was altered. Contraction to a 45 mmol/L concentration of potassium chloride after ischemia and storage with activated neutrophils was increased (p = 0.06). Smooth muscle relaxation was significantly decreased after ischemia, but storage with activated neutrophils did not further decrease smooth muscle relaxation. Endothelium-dependent relaxation to bradykinin was significantly decreased after combined ischemia and incubation with activated neutrophils (p < 0.05). Sensitivity to bradykinin was decreased after both ischemia alone (p < 0.05) and activated neutrophils alone (p < 0.05). Similar morphologic alterations were found in epicardial and intramyocardial arteries after ischemia. Activated neutrophils alone minimally damaged endothelial cells of nonischemic intramyocardial and epicardial arteries. Endothelial cells of both arteries exposed to ischemia alone showed evidence of ischemic damage, including endothelial cell blebbing, nuclear bulging, and appearance of large holes in the cell surface. Severe endothelial cell damage was found after combined ischemia and storage with neutrophils: total destruction of cells and exposure of the basal lamina. Endothelial damage, therefore, correlated with artery function in intramyocardial but not in epicardial arteries. These results indicate that ischemia is a prerequisite for severe neutrophil injury of intramyocardial artery endothelium-mediated relaxation. This may explain no-reflow phenomenon in arterioles concurrent with myocardial reperfusion injury.
Asunto(s)
Vasos Coronarios/fisiopatología , Endotelio Vascular/fisiopatología , Isquemia/fisiopatología , Activación Neutrófila , Animales , Vasos Coronarios/ultraestructura , Endotelio Vascular/ultraestructura , Isquemia/patología , Microcirculación , Microscopía Electrónica de Rastreo , Contracción Miocárdica , Óxido Nítrico/fisiología , PorcinosRESUMEN
BACKGROUND: Differences in the cytoskeletal protein actin in cells from the zona glomerulosa and zona fasciculata would be of considerable interest because there is persuasive evidence that rat corticosteroids are secreted by mechanisms that are somewhat zone-specific. We have previously shown evidence that actin may be involved in steroid secretion, possibly in connection with changes in adrenocortical microvilli. However, the cells upon which the data were based were not separated according to zone of origin. METHODS: Immunogold electron microscopy and morphometric procedures were used to determine whether ACTH-induced changes in the peripheral cytoplasm of isolated adrenocortical cells occur in both zona fasciculata and zona glomerulosa cells. RESULTS: Actin immunoreactivity was more concentrated in the cytoplasm adjacent to the plasma membrane (including the cytoplasm within the microvilli) than it was in the internal cytoplasm in cells from both zones (4-6 times more concentrated in zona glomerulosa cells and 3-6 times more concentrated in zona fasciculata cells). However, the mean aggregate microvillar surface length (microvillar index) of untreated zona fasciculata cells (previously reported (Loesser and Malamed, 1987)) was 23% greater than that of untreated zona glomerulosa cells. Although ACTH (at a maximal steroidogenic concentration) had no effect on the peripheral cytoplasmic actin concentration of zona glomerulosa cells, there was a 24% increase in the aggregate microvillar length. In contrast, in zona fasciculata cells, ACTH treatment was accompanied by an increase in peripheral cytoplasmic actin concentration of 58-64% and an increase in aggregate microvillar surface length of 40% (previously reported (Loesser and Malamed, 1987)), almost twice that for zona glomerulosa cells. CONCLUSION: The results suggest that ACTH-induced hormone release from zona fasciculata cells is mediated by increases in peripheral cytoplasmic actin and aggregate microvillar length; in zona glomerulosa cells such changes are small or absent.
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Corteza Suprarrenal/citología , Hormona Adrenocorticotrópica/farmacología , Actinas/análisis , Corteza Suprarrenal/efectos de los fármacos , Corteza Suprarrenal/ultraestructura , Animales , Citoplasma/química , Citoplasma/efectos de los fármacos , Citoplasma/ultraestructura , Inmunohistoquímica , Masculino , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Microvellosidades/química , Microvellosidades/efectos de los fármacos , Microvellosidades/ultraestructura , Ratas , Ratas Sprague-Dawley , Zona Fascicular/citología , Zona Fascicular/efectos de los fármacos , Zona Fascicular/ultraestructura , Zona Glomerular/citología , Zona Glomerular/efectos de los fármacos , Zona Glomerular/ultraestructuraRESUMEN
We investigated the efficacy of histidine in reducing ischemia-reperfusion (I/R)-induced myocardial injury in isolated perfused rat hearts. In I/R hearts, the contractile function and coronary flow were 59 +/- 10 and 78 +/- 6% of control. Perfusion with histidine resulted in significant increase in contractility (94 +/- 4%) and coronary flow (92 +/- 4%). The incidence of arrhythmias during reperfusion was 100% (10 out of 10) in the I/R hearts with an average duration of 12.22 +/- 1.55 (SE) min. The duration of arrhythmias was shortened to 8.24 +/- 1.46, 2.15 +/- 0.9, and 2.49 +/- 1.50 min with 10, 25, and 50 mM histidine, respectively. The duration of sinus rhythm increased from 6.26 +/- 1.56 min in I/R hearts to 10.66 +/- 1.55, 14.99 +/- 1.61, and 17.18 +/- 0.95, and 11.73 +/- 0.93 min after perfusion with 10, 25, and 50 mM histidine, and superoxide dismutase (SOD)-catalase-mannitol, respectively. Electron microscopy revealed significant ultrastructural damage of myocytes in I/R hearts, which included swelling of the mitochondria and disruption of both the sarcolemma and the myofibrils. Histidine reduced the ultrastructural damage in a dose-dependent fashion. In general, the protective effect of histidine was superior than SOD-catalase-mannitol. We conclude that histidine protects myocardium against I/R damage most likely by a singlet oxygen scavenging mechanism.
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Corazón/efectos de los fármacos , Histidina/farmacología , Isquemia Miocárdica/fisiopatología , Daño por Reperfusión Miocárdica/prevención & control , Reperfusión Miocárdica , Animales , Arritmias Cardíacas/etiología , Técnicas In Vitro , Masculino , Microscopía Electrónica , Isquemia Miocárdica/complicaciones , Isquemia Miocárdica/patología , Miocardio/ultraestructura , Perfusión , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Factores de TiempoRESUMEN
The structure and disposition of the feet occupying the junctions between sarcoplasmic reticulum (SR) and surface membrane/transverse tubules were studied in muscles from a variety of invertebrates. Feet were imaged by rotary shadowing of isolated junctional SR vesicles and by filtering of micrographs from grazing views of the junction in thin sections. The overall size and shape of invertebrate feet is the same as that of feet in skeletal and cardiac muscle of vertebrates. However, the arrangement of feet in invertebrate muscles differs from that in vertebrates. These findings are discussed in terms of known variations in properties of excitation-contraction coupling of the two phyla.
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Artrópodos/ultraestructura , Calcio/metabolismo , Moluscos/ultraestructura , Sarcolema/ultraestructura , Retículo Sarcoplasmático/ultraestructura , Animales , Astacoidea/ultraestructura , Canales de Calcio/ultraestructura , Saltamontes/ultraestructura , Insectos/ultraestructura , Microscopía Electrónica , Receptores Colinérgicos/ultraestructura , Receptores Purinérgicos/ultraestructura , Canal Liberador de Calcio Receptor de Rianodina , Escorpiones/ultraestructura , Especificidad de la EspecieRESUMEN
Certain pathophysiologic conditions are able to shift normal oxygen metabolism towards univalent reduction, resulting in the production of reduced oxygen intermediates, including free radicals and their metabolites. Oxygen free radicals have been implicated in a number of pathologic conditions, including myocardial injury. The possible sources of oxygen free radicals, the detection of the radicals during ischemia/reperfusion and possible mechanisms of free radical damage are discussed. The clinical implications of free radical damage and the experimental drug therapies at present under investigation are also reviewed.
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Daño por Reperfusión Miocárdica/etiología , Miocardio/metabolismo , Oxígeno/efectos adversos , Animales , Arritmias Cardíacas/tratamiento farmacológico , Soluciones Cardiopléjicas , Radicales Libres , Humanos , Contracción Miocárdica/fisiología , Infarto del Miocardio/tratamiento farmacológico , Daño por Reperfusión Miocárdica/prevención & controlRESUMEN
To assess myofilament overlap during locomotion, we estimated the length of myosin and actin filaments in axial red and white muscle of carp. Myosin filament lengths were 1.52 +/- 0.009 and 1.50 +/- 0.037 micron (means +/- SD) in the red and white muscle, respectively, as measured from thin sections. After correction for shrinkage (using the troponin-based 385-A axial periodicity), thin filaments were 0.96 +/- 0.009 and 0.97 +/- 0.023 micron in the red and white muscles, respectively. Filaments were also isolated from the white muscle and negatively stained. Myosin filaments were 1.56 +/- 0.025 microns, and actin filaments were 0.99 +/- 0.024 micron in length. The data from thin sections and isolated filaments agreed within 2% for actin and 4% for myosin filaments. The number of actin filament periods (24 for the red and white muscle) and the length of the filaments are the same as in frog. This suggests that the classic sarcomere length-tension curve of frog muscle may be used to estimate the functional properties of carp red and white muscle.
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Citoesqueleto de Actina/ultraestructura , Locomoción , Músculos/ultraestructura , Citoesqueleto de Actina/fisiología , Actinas/análisis , Animales , Carpas , Microscopía Electrónica , Músculos/fisiología , Miosinas/análisis , NataciónRESUMEN
We present a simple approach for effective freeze-drying and rotary shadowing of large molecules, molecular assemblies, and cell organelles. Simply, a suspension of specimen is adsorped to a glass coverslip, stabilized, and rinsed with 30% methanol. A second coverslip is "sandwiched" on top, and excess methanol is withdrawn from the edges then frozen by plunging into liquid nitrogen and split. Following either rotary or unidirectional shadowing and replication, the coverslip is dissolved in hydrofluoric acid. In addition to avoiding the problems encountered with air-drying specimens for rotary shadowing, the technique also reproducibly provides the thin layer of solution necessary for proper freeze-drying, regardless of how hydrophobic the sample is. The "glass sandwich" technique allows modification of the glass substrate (making it hydrophobic with carbon or hydrophilic by soaking it in alcian blue) which clearly alters the shape of macromolecular assemblies such as myosin filaments and decorated thin filaments.
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Citoesqueleto/ultraestructura , Liofilización , Sustancias Macromoleculares , Músculos/ultraestructura , Proteínas/análisis , Actinas/análisis , Animales , Astacoidea , ATPasas Transportadoras de Calcio/análisis , Membrana Celular/ultraestructura , Miosinas/análisisRESUMEN
The localization of actin and the effect of ACTH on its concentration was examined in freshly isolated rat adrenocortical cells. Lowicryl K4M-embedded cells were used for the immunoelectron localization of actin; gold was used as a label for immunoreactive sites. Actin was at least 4 times as concentrated at the cortical cytoplasm as in the lipid droplets and at least 5 times as concentrated in the microvilli as in the lipid droplets. ACTH stimulation approximately doubled the concentration of actin in the cortical cytoplasm and increased by 50% the concentration of actin in the microvilli. The microvillar contribution to the cell surface area was 40% higher in ACTH-stimulated cells than it was in unstimulated cells. These results provide quantitative evidence suggesting that actin and the microvilli participate in steroid secretion by the adrenocortical cell.
Asunto(s)
Actinas/metabolismo , Corteza Suprarrenal/metabolismo , Hormona Adrenocorticotrópica/farmacología , Corteza Suprarrenal/ultraestructura , Animales , Inmunohistoquímica , Masculino , Microscopía Electrónica , Concentración Osmolar , Ratas , Ratas EndogámicasRESUMEN
We have modified the Lowicryl K4M low-temperature dehydration and embedding procedure for immunoelectron microscopy to provide improved ultrastructural detail and facilitate the localization of actin and tubulin in isolated rat adrenocortical cells, chick spinal cord with attached dorsal root ganglia (SC-DRG), and cultured dorsal root ganglia (DRG). Cells and tissues were fixed for immunocytochemistry either in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde (0.1 M PIPES buffer, pH 7.3) or in a mixture of 0.3% glutaraldehyde and 1.0% ethyldimethylaminopropylcarbodiimide (0.1 M phosphate buffered saline, pH 7.3). Dehydration was in ethanol at progressively lower temperatures to -35 degrees C. Infiltration at -35 degrees C was followed by ultraviolet polymerization at -20 degrees C. Comparable samples were fixed in glutaraldehyde and osmium tetroxide and embedded in Epon 812 or Epon-Araldite. Post-embedding immunostaining of thin sections utilized commercially available monoclonal antibodies to tubulin and actin followed by the protein A-gold technique (Roth et al., Endocrinology 108:247, 1981). Actin immunoreactivity was observed at the periphery of mitochondria and between mitochondria and lipid droplets in rat adrenocortical cells and at the periphery of neuronal cell processes of SC-DRG. Tubulin immunoreactivity was associated with microtubules throughout neurites of cultured DRG. Our modified technique allows preservation of ultrastructural details as well as localization of antigens by immunoelectron microscopy.