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T F Robinson

Publications and source records attributed to T F Robinson.

36 records · Page 2Linked to original sources
PubMed1987

Enzyme-antibody histochemistry. A method for detection of collagens collectively.

Different types of distinct molecular forms of collagen are components of the extracellular matrix in most tissues. The common types can usually be detected by immunohistochemical methods but others may escape detection for lack of specific antisera. However, all these collagens are substrates for the collagenase of Clostridium histolyticum. In this report we describe a method that allows the visualization of collagens, collectively, in a tissue preparation. The method is based on the affinity between clostridial collagenase and collagen on one hand, and collagenase and its antibody on the other. Under the conditions of low temperature used in the procedure, collagenase binds to collagen, but digestion does not occur. Subsequent reaction of the bound collagenase with the specific collagenase antibody is followed by reaction with a tagged anti-IgG reagent. This allows the visualization of the enzyme-substrate complex. The procedure is illustrated in sections of the heart and the aorta, as well as in the isolated cardiomyocytes and the collagen distribution is verified using collagens type I and IV specific antibodies. In all instances the collagenase staining pattern includes all structural features seen individually with the type specific anticollagen antibodies.

Animals↗
PubMed1987

Profound structural alterations of the extracellular collagen matrix in postischemic dysfunctional ("stunned") but viable myocardium.

Ultrastructural studies of the extracellular collagen matrix were made on the "stunned" myocardium using scanning, conventional and high voltage transmission electron microscopy and light microscopy. Regional myocardial dysfunction was produced by 12 sequential 5 minute occlusions of the left anterior descending coronary artery, separated by 10 minute intervals of reperfusion. A final 90 minute reperfusion period documented persistent myocardial dysfunction. At the end of the final reperfusion period, the percent systolic shortening, measured by sonomicrometers, was depressed significantly to 35 +/- 9% of baseline. The heart was then perfusion fixed, and samples were taken from both control and stunned areas. No changes associated with irreversible cellular damage were noted in the stunned region. However, scanning electron microscopy of the stunned area showed that the extracellular collagen matrix underwent profound structural changes. Collagen cables were roughened, uncoiled and discontinuous. Linear grooves on the surface of the myocytes were frequently seen, indicating complete loss of collagen cables. The usual dense collagen weave surrounding myocytes became patchy or absent. Myocyte to myocyte struts were sparse and frequently absent, with remnant nodular or nublike structures indicative of breakage. High voltage electron microscopy of the stunned area showed that the collagen struts were discontinuous and vacuolated with rounded tips. Light microscopy of silver-stained sections of the stunned tissue demonstrated large patchy areas that were devoid of silver, indicating absence of the collagen matrix. There was a progressive increase in percent systolic bulging during each sequential coronary occlusion, suggesting increasing myocardial compliance. These results indicate that the myocardial collagen matrix is severely damaged from reversible ischemic cell injury. The greater myocardial compliance and less effective contractile effort in the stunned myocardium might be explained on a structural basis: disruption of the mechanical coupling function provided by the extracellular collagen matrix.

Animals↗
PubMed1987

Alterations of the myocardial skeletal framework in acute myocardial infarction with and without ventricular rupture. A preliminary report.

Thinning and dilatation (expansion) of the infarct region and complete rupture of the ventricular wall are significant complications of acute transmural myocardial infarction associated with increased morbidity and mortality. The pathogenesis of these related events is unknown. Recent studies of myocardial connective tissue have delineated an extensive array of intercellular and pericellular structures which serve as a skeletal framework and which may modulate contractile activity. We have employed a modified silver impregnation method to visualize the connective tissue components by light microscopy. To explore whether the skeletal framework is altered in acute myocardial infarction with and without ventricular rupture, we studied 9 human hearts at autopsy, and 4 canine infarcts of known duration. The human infarctions included 4 nonruptured cases with infarcts 1-5 days old, and 5 ruptured cases with infarcts 3-10 days old. Sections from normal, lateral, and central infarct or ventricular rupture sites were stained with silver. The normal tissue from each heart served as a control. Silver staining was moderately decreased in the lateral infarct zones, and markedly decreased in the central non-ruptured infarct zones. In the 5 ventricular rupture cases, the rupture site had no silver staining. A similar pattern was observed in the 4 canine infarcts. Thus, we conclude that the skeletal framework is markedly altered in the central zone of acute myocardial infarction. The acute changes of silver stained connective tissue may contribute significantly to the development of infarct expansion or ventricular wall rupture.

Animals↗
PubMed1987

Intrinsic connective tissue abnormalities in the heart muscle of cardiomyopathic Syrian hamsters.

Significant connective tissue abnormalities occurring in hearts of cardiomyopathic Syrian hamsters are reported. These abnormalities include a pronounced loss of the intrinsic connective tissue skeletal framework around foci of myocytolytic necrosis within the non-necrotic myocardium. These changes were demonstrated by a silver impregnation technique, and they were confirmed by scanning electron microscopy. Quantitation demonstrated more than a twofold increase in the area of ventricular wall affected by pathologic changes, when the connective tissue alterations were included with the myocardial necrosis. In addition, the authors also observed focal, thick "tethering" connective tissue fibers at the termini of necrotic lesions, seemingly connecting them to normal muscle. These connective tissue abnormalities may contribute to the progressive loss of ventricular function that occurs in this model of cardiomyopathy. They may permit greater wall thinning than would occur with focal necrosis alone, and they may increase focal mural stiffness in the tethered regions. Further investigation of the pathogenesis of these changes and their mechanical significance is indicated.

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PubMed1985

Extracellular structures in heart muscle.

The extracellular matrix of heart muscle contains a considerable variety of structures. We have systematically studied the morphology of these structures using several methods of fixation and microscopy. Endomysial connections between cells are comprised of struts of collagen [1] as well as combinations of elastin fibers, collagen fibers, and microfibrils. The rest of the extracellular matrix is filled with a polyanionic lattice of unit collagen fibrils, microthreads, and granules. In the course of these investigations, we have observed regions of structural continuity across the sarcolemma, from endomysial collagen struts to Z-bands. We have also correlated the mechanical resistance to stretch with orientation of epimysial collagen fibers and sarcomere lengths in living as well as fixed rat papillary muscles. Our observations suggest that the extracellular skeletal framework plays an important role in normal cardiac function.

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PubMed1985

Mechanical properties of developing swine myocardium.

Mechanical responses of myocardium from 16 piglets were studied from 18 hr to 12 days after birth. Tension, time and velocity parameters of contraction and relaxation were determined for every contraction cycle. Increasing the frequency of stimulation in step-changes induced negative inotropy in some muscles regardless of age. Doubling extracellular calcium ion concentration induced a positive force-frequency response in all muscles. Epinephrine increased tension and velocities without affecting contraction time. The ultrastructure was immature even on the 12th postnatal day. We concluded that in newborn piglet hearts, the mechanisms for calcium delivery are not fully developed. Thus, the heart undergoes a transient phase during which at least a principal portion of calcium for the myofibers is supplied by the extracellular fluid. While receptors for catecholamines are present, the time course for their response is immature.

Animals↗
PubMed1985

Size and shape of enzymatically isolated ventricular myocytes from rats and cardiomyopathic hamsters.

Rod-shaped and branched ventricular myocytes from rats and cardiomyopathic hamsters (strain 53.58) were isolated enzymatically, and their widths and lengths were measured in physiological salt solutions containing normal levels of calcium (2.5 mmol). In rats of approximately 200 g body weight, the average myocyte width and length are 25 micron and 115 micron. The isolated cells are also classified according to shape with nearly 50% branched or otherwise irregular. Myocytes of the hearts of the 53.58 strain of cardiomyopathic hamsters at 7 months of age are significantly larger than control hamsters of the same age, indicating that cellular hypertrophy has occurred. Estimates of the number of cells in the ventricles indicate that there is a cell loss of nearly 13% in the myopathic heart. A consideration of the significance of wider and longer myocytes with undiminished myofibrillar mass lead to the conclusion that the decreased contractility displayed by the cardiomyopathic hamster heart must be due, at least in part, to functional defects in the myofibrillar apparatus, in the system of activation, or in cellular integration.

Age Factors↗
PubMed1984

Myofilament diameters: an ultrastructural re-evaluation.

In situ, ultrastructural measurements of diameters of contractile filaments in skeletal and heart muscle differ considerably from those previously reported. Past measurements have been made in thin, transverse epoxy sections that were non-specifically stained with heavy metal salts to overcome background scattering of epoxy polymer. In our images from transverse de-embedded sections, the hexagonal lattice has some considerable differences from that seen in epoxy sections. Muscle samples from rat atrium and frog sartorius were fixed, dehydrated, embedded in polyethylene glycol, and sectioned. Sections were de-embedded in graded polyethylene glycol/ethanol, mounted on coated grids, critical point dried, and viewed in the electron microscope without staining. The backbone diameters of thick filaments were measured in the M band region and have an average value, after correction for shrinkage, of 25 nm. Thin filament diameters range from 6.5-9.5 nm. In regions of overlap of thin and thick filaments, the thick filament profiles varied from circular to asymmetric; diameters range up to 36 nm and yield eccentricity ratios varying from 1.5 to 1.0 (circular profiles). Portions of thick filaments touch or partially envelope neighboring thin filaments. The relative contributions of cytoskeletal components to these images of overlap regions remains to be determined, but the backbone diameters in glycerinated frog sartorius are not significantly different from control samples. The present results are consistent with those reported for rotary shadowed thick filaments; from recent experiments in muscles whose myofilament lattice is osmotically compressed; and with estimates of A band mass. This lattice geometry yields relatively low surface-to-surface distances between filaments. Steric considerations and their implications for cross bridge theory are discussed.

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PubMed1983

Skeletal framework of mammalian heart muscle. Arrangement of inter- and pericellular connective tissue structures.

We have studied the connective tissue of mammalian heart muscle in order to obtain an integrated description of extracellular structures and their dispositions relative to cardiac myocytes. Light microscopy and several types of electron microscopy have been employed in these investigations. The epimysium, the sheath of connective tissue that surrounds the muscle, contains relatively large fibers of collagen and elastin. In papillary muscles of rat, the large collagen fibers of the epimysium form a weave pattern at slack length (sarcomere lengths 1.8 to 2.0 micron) but are well aligned in states of stretch along the long axis of the muscle (sarcomere length 2.3 to 2.5 micron). We propose that the epimysial collagen network protects the sarcomeres from being stretched beyond lengths favorable to maximal force production. The endomysium is defined as the connective tissue that surrounds and interconnects myocytes; it consists of intercellular struts (bundles of collagen fibrils, often attached near Z-band level), a weave of bundles of collagen fibrils that envelopes myocytes, and a collagen fibril-microthread-granule lattice that bridges cells and fills the extracellular matrix. In contracted muscles festoons of sarcolemma are attached to Z-bands, thus forming regions for transmission of force across the sarcolemma. Perimysial bundles of collagen connect epimysium to endomysium and surround groups of myocytes. Collagen fibers often have a twisted configuration, probably for enhanced tensile strength. Superimposed on the large extracellular structures is the polyanion-rich lattice comprised of unit collagen fibrils, microthreads, and granules. Amorphous ground substance forms a matrix in which the fibrils of collagen fibers are embedded; it appears continuous with the cell coat in regions of fiber attachment. Elastic fibers interconnect cells and helically wind around myocytes. Circumferential forces from elastin stretched about shortened, thickened myocytes in systole should promote elongation in tandem with intramyocyte forces of elongation.

Animals↗
PubMed1981

Oxygen requirements, morphology, cell coat and membrane permeability of calcium-tolerant myocytes from hearts of adult rats.

The morphological, functional, and biochemical properties of freshly isolated heart muscle cells were examined. A reproducible method for the separation and purification of such cells isolated from adult rat heart was developed. It yields an average of 5 X 10(6) striated rectangular cells which retain normal morphology (range 2.5 to 11 X 10(6) and 4 X 10(6) calcium-tolerant cells (range 2.5 to 5.5 X 10(6) per heart. After purification, 85 to 95% of the cells retain normal morphology in solutions of calcium ion activity equal to 10 microM, and 65 to 79% of the cells are rectangular in solutions of calcium ion activity equal to 1 mM. Under the light microscope we were able to identify functionally intact individual cells that are calcium-tolerant and contract only in response to electrical stimulations, as well as dying myocytes that beat spontaneously. The examination of such cells under the electron microscope permitted us to address the question: What is the sequence of structural changes in a dying cell? The sarcomere lengths measured both in the living state and after preparation for electron microscopy are in the physiological range. In steady states of oxygen tension, respiration of the intact cells is undiminished from 50 torr to 2 torr. The oxygen tension for half maximal respiration is 0.15 torr. Therefore, the limitation of oxygen diffusion to the mitochondria of isolated heart muscle cells must be remarkably small.

Animals↗
PubMed1981

Isolated heart myocytes: ultrastructural case study technique.

We have developed a method for performing case studies of heart muscle cells enzymatically isolated from the ventricular walls of rats that is a simple and inexpensive adaptation of procedures developed for the examination of monolayers of attached, cultured cells. The technique represents a marked departure from published accounts of electron microscopic studies of pellets or monolayers from a population of potentially heterogeneous isolated myocytes. Here we report the method, which we have used under controlled conditions with 0 mmol and 1 mmol added CaCl2, to correlate sarcomere length and electrical stimulatibility in the living state with ultrastructural features that include the relative disposition of myofilaments and the integrity of the cell coat. The degree of shrinkage during the preparative steps in less than 5%, as directly determined from photographs of striations in the living, fixed, and embedded states.

Animals↗
PubMed1980

Lateral connections between heart muscle cells as revealed by conventional and high voltage transmission electron microscopy.

The lateral surfaces of heart muscle cells are interconnected by a varied and extensive network of structures that exist in addition to intercalated discs. Ultrastructural images of this network are vastly improved over those from epoxy-embedded material, particularly for low density components, through the application of a method for removing the embedding matrix from thin or thick sections that are then stereoscopically analyzed with standard or high voltage transmission electron microscopy. The connections include cables, 3-20 nm in diameter, multi-strand cables, 10-40 nm-granules, meshlike mats, and sheets, all extensively interwoven. It is suggested that intercellular connections of varying strength and distribution aid in the integration of mechanical performance of the large population of myocytes during the contractile cycle of the heart.

Animals↗
PubMed1979

The measurement and dynamic implications of thin filament lengths in heart muscle.

1. The lengths of the thin filaments in amphibian and mammalian cardiac muscle have been determined from electron micrographs of serial transverse sections. Thin filament lengths in frog atrial trabeculae range from 0.8 to greater than 1.3 micrometers, with a maximum possible error of 0.14--0.15 micrometer. In rat atrial tissue the span is from 0.6 to more than 1.1 micrometer, whereas in rat papillary muscle the breadth of the distribution is much narrower, from 0.9 to greater than 1.1 micrometer. Double overlap of thin filaments should, therefore, exist over a wide range of sarcomere lenghts. Thin filaments from opposite halves of a sarcomere accommodate each other by flexing up to an angle of about 2 degrees and moving from the trigonal position among the thick filaments to the centre of the region between two thick filaments. Such rearrangement probably contributes to the internal resistance to shortening in the muscle. 2. Except for the variation in thin filament lengths, the over-all morphology of the cardiac sarcomere is generally similar to that found in skeletal muscle. Thick filaments in heart muscle are uniform in length, and their profiles change along their lengths. They are generally round in the M band, triangular adjacent to the M band, round again in the overlap region, and either round or triangular near the tapered tips. The M bridges in rat cardiac tissue link neighbouring thick filaments to form a symmetric hexagonal array, whereas in the frog atrium, the M bridge connexions are incomplete and often form isolated triangular clusters. 3. Computed sarcomere length-developed tension curves were calculated using the thin filament length distributions and the assumptions basic to the sliding filament theory of muscle contraction. The curves for atrial tissue have plateau regions approximately as wide as the one-half micron variation in thin filament length. 4. Work done against the internal loads during systole may be stored as potential energy and released during diastole to produce sarcomeric re-extension.

Animals↗
PubMed1978

Force generation among cells in the relaxing heart.

When an isolated bundle of mammalian heart cells has been soaked for several hours in a solution containing 3 mM EGTA, a calcium chelator, 2 important changes in the tissue occur: (1) the surface membrane becomes very permeable to small ions and molecules so that the contractile proteins can be directly activated by Ca ions added to the bathing solution; and (2) all intercalated discs open. In this preparation direct Ca activation can produce the transmission of even greater forces among the cells than can be achieved by electrical stimulation of the intact bundle in the presence of catecholamines, high Ca and low Na. Some structure other than the intercalated discs must be transmitting the force. A careful examination of the ultrastructure of the mammalian myocardium has revealed a highly organized network of microfilaments which run between the basement membranes of adjacent cells and from the basement membranes to the collagen fibers. This hitherto undescribed network has all the structural appearances of a major force-bearing structure, and it is probably the primary pathway for force transmission during systole as well as the restoration to the diastolic configuration during relaxation.

Animals↗
PubMed1975

Coagulation and fibrinolysis in the dog.

A range of tests of coagulation and fibrinolysis was measured in "normal" dogs and compared with values obtained in "normal" humans by the same methods. The hematocrit platelet count, fibrinogen and plasminogen were similar in dogs and in humans. The prothrombin and partial thromboplastin times were considerably shorter in the dog than in man but the thrombin clotting time was comparable. Fibrinolysis was more active in dogs but the levels of fibrin degradation products were low, suggesting that there was no significant fibrin deposition and lysis occurring in vivo.

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