Structural and elemental characterization of heart cells grown in a collagen matrix

1989 ◽  
Vol 47 ◽  
pp. 992-993
Author(s):  
A. LeFurgey ◽  
L.A. Hawkey ◽  
M.C. Carney ◽  
P. Ingram ◽  
M. Lieberman
Keyword(s):  
Liquid Nitrogen ◽  
Collagen Matrix ◽  
Cardiac Cells ◽  
Heart Cells ◽  
Embryonic Chick ◽  
Culture Dish ◽  
Diffusion Limitations ◽  
Freeze Dried ◽  
Quick Freezing

Cultured embryonic chick heart cells have been utilized as a model system for characterization of various membrane transport mechanisms. One advantage of this system is that the cells may be grown with differing geometries to minimize diffusion limitations and to optimize the growth configuration for particular techniques, such as ion-selective microelectrode measurements, fluorescent dye indicators, patch clamp, etc. A spontaneously contracting strand of cells embedded in a collagen matrix has recently been developed for measurements of cytoplasmic free ions by nuclear magnetic resonance (NMR) spectroscopy. These same strands, which provide the large numbers of cells needed for NMR, can be subdivided into small fragments ideal for cryopreservation prior to electron probe X-ray microanalysis (EPXMA). The aims in this study were to characterize the ultrastructure of cardiac cells within the strand, to demonstrate the quality of preservation obtainable by quick freezing methods, and to quantitatively map with EPXMA the distribution of physiologically relevant elements in thin, freeze-dried cryosections of the cells.Cells were isolated by serial trypsinization of 11-day old embryonic chick hearts. Strands of cells approximately 100 cm in length and 0.2 mm in diameter were obtained by extrusion of a cell-collagen mixture through polyethylene tubing into media within a culture dish. Three to five millimeter segments of 1-day old strands which contracted spontaneously were preserved by rapid immersion in liquid nitrogen-cooled liquid propane at 〜-185°C and stored in liquid nitrogen prior to being (a) cryosectioned for subsequent EPXMA or (b) freeze-substituted for conventional transmission electron microscopy (CTEM). Segments of strands were also chemically preserved in 2.5% glutaraldehyde in 0.1 M sodium cacodylate and processed as above for comparative CTEM. Cryosections of the frozen strands were cut at <-140°C with a dry glass knife and placed directly onto pre-cooled, carbon-coated, 200 mesh, fine bar nickel grids with a precooled implement. The grids were transferred to a liquid nitrogen cooled copper well for freeze drying at 10‒3 Torr over 24 to 48 hours. Prior to EPXMA, the grids were coated with 〜1OOÅ carbon.

Characterization of a novel subset of cardiac cells and their progenitors in the Drosophila embryo

Development ◽  
2000 ◽  
Vol 127 (22) ◽  
pp. 4959-4969 ◽  
Author(s):  
E.J. Ward ◽  
J.B. Skeath
Keyword(s):  
Heart Development ◽  
Cell Types ◽  
Cardiac Cells ◽  
Lineage Tracing ◽  
Drosophila Embryo ◽  
Heart Cells ◽  
Pericardial Cells ◽  
Asymmetric Divisions ◽  
Genetic Mechanisms

The Drosophila heart is a simple organ composed of two major cell types: cardioblasts, which form the simple contractile tube of the heart, and pericardial cells, which flank the cardioblasts. A complete understanding of Drosophila heart development requires the identification of all cell types that comprise the heart and the elucidation of the cellular and genetic mechanisms that regulate the development of these cells. Here, we report the identification of a new population of heart cells: the Odd skipped-positive pericardial cells (Odd-pericardial cells). We have used descriptive, lineage tracing and genetic assays to clarify the cellular and genetic mechanisms that control the development of Odd-pericardial cells. Odd skipped marks a population of four pericardial cells per hemisegment that are distinct from previously identified heart cells. We demonstrate that within a hemisegment, Odd-pericardial cells develop from three heart progenitors and that these heart progenitors arise in multiple anteroposterior locations within the dorsal mesoderm. Two of these progenitors divide asymmetrically such that each produces a two-cell mixed-lineage clone of one Odd-pericardial cell and one cardioblast. The third progenitor divides symmetrically to produce two Odd-pericardial cells. All remaining cardioblasts in a hemisegment arise from two cardioblast progenitors each of which produces two cardioblasts. Furthermore, we demonstrate that numb and sanpodo mediate the asymmetric divisions of the two mixed-lineage heart progenitors noted above.

scholarly journals Chronic murine myocarditis due to Trypanosoma cruzi: an ultrastructural study and immunochemical characterization of cardiac interstitial matrix

1986 ◽  
Vol 81 (1) ◽  
pp. 29-41 ◽  
Author(s):  
Sonia G. Andrade ◽  
Jean Alexis Grimaud
Keyword(s):  
Mouse Model ◽  
Trypanosoma Cruzi ◽  
Ultrastructural Study ◽  
Inflammatory Process ◽  
Collagen Matrix ◽  
Cardiac Cells ◽  
Blood Capillary ◽  
Chagas Cardiomyopathy ◽  
Chagasic Cardiomyopathy

In an attempt to define the mouse-model for chronic Chagas' disease, a serological, histopathological and ultrastructural study as well as immunotyping of myocardium collagenic matrix were performed on Swiss mice, chronically infected with Trypanosoma cruzi strains: 21 SF and mambaí (Type II); PMN and Bolivia (Type III), spontaneously surviving after 154 to 468 days of infection. Haemagglutination and indirect immunofluorescence tests showed high titres of specific antibodies. The ultrastructural study disclosed the cellular constitution of the inflammatory infiltrate showing the predominance of monocytes, macrophages with intense phagocytic activity, fibroblasts, myofibroblasts and abundant collagen matrix suggesting the association of the inflammatory process with fibrogenesis in chronic chagasic cardiomyopathy. Artertolar and blood capillary alterations together with dissociation of cardiac cells from the capillary wall by edema and inflammation were related to ultrastructural lesions of myocardial cells. Rupture of parasitized cardiac myocells contribute to intensify the inflammatory process in focal areas. Collagen immunotyping showed the predominance of Types III and IV collagen. Collagen degradation and phagocytosis were present suggesting a reversibility of the fibrous process. The mouse model seems to be valuable in the study of the pathogenetic mechanisms in Chagas cardiomyopathy, providing that T. cruzi strains of low virulence and high pathogenecity are used.

The Effects of Drying Techniques on Clay-Rich Soil Texture

1974 ◽  
Vol 32 ◽  
pp. 466-467
Author(s):  
T. G. Naymik
Keyword(s):  
Critical Point ◽  
Liquid Nitrogen ◽  
Liquid Nitrogen Temperature ◽  
Drying Methods ◽  
Air Drying ◽  
Freeze Dried ◽  
Critical Point Drying ◽  
Set Up ◽  
The Relationship ◽  
Quartz Grains

Three techniques were incorporated for drying clay-rich specimens: air-drying, freeze-drying and critical point drying. In air-drying, the specimens were set out for several days to dry or were placed in an oven (80°F) for several hours. The freeze-dried specimens were frozen by immersion in liquid nitrogen or in isopentane at near liquid nitrogen temperature and then were immediately placed in the freeze-dry vacuum chamber. The critical point specimens were molded in agar immediately after sampling. When the agar had set up the dehydration series, water-alcohol-amyl acetate-CO2 was carried out. The objectives were to compare the fabric plasmas (clays and precipitates), fabricskeletons (quartz grains) and the relationship between them for each drying technique. The three drying methods are not only applicable to the study of treated soils, but can be incorporated into all SEM clay soil studies.

How to sample the entire length of a single muscle fiber quick-frozen after electrical point stimulation for high resolution EM

1992 ◽  
Vol 50 (1) ◽  
pp. 776-777
Author(s):  
Joachim R. Sommer ◽  
Teresa High ◽  
Betty Scherer ◽  
Isaiah Taylor ◽  
Rashid Nassar
Keyword(s):  
Skeletal Muscle ◽  
High Resolution ◽  
Action Potential ◽  
Muscle Fiber ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Electrical Field Stimulation ◽  
Skeletal Muscle Fiber ◽  
Freeze Dried ◽  
Quick Freezing

We have developed a model that allows the quick-freezing at known time intervals following electrical field stimulation of a single, intact frog skeletal muscle fiber isolated by sharp dissection. The preparation is used for studying high resolution morphology by freeze-substitution and freeze-fracture and for electron probe x-ray microanlysis of sudden calcium displacement from intracellular stores in freeze-dried cryosections, all in the same fiber. We now show the feasibility and instrumentation of new methodology for stimulating a single, intact skeletal muscle fiber at a point resulting in the propagation of an action potential, followed by quick-freezing with sub-millisecond temporal resolution after electrical stimulation, followed by multiple sampling of the frozen muscle fiber for freeze-substitution, freeze-fracture (not shown) and cryosectionmg. This model, at once serving as its own control and obviating consideration of variances between different fibers, frogs etc., is useful to investigate structural and topochemical alterations occurring in the wake of an action potential.

Confocal imaging of calcium in intact ventricular muscle provides a new view of excitation-contraction coupling

1996 ◽  
Vol 54 ◽  
pp. 738-739
Author(s):  
W.G. Wier
Keyword(s):  
Local Control ◽  
Cardiac Tissue ◽  
Cell Function ◽  
Isolated Heart ◽  
Cardiac Cells ◽  
Confocal Imaging ◽  
Ion Concentration ◽  
Heart Cell ◽  
Free Calcium ◽  
Heart Cells

A fundamentally new understanding of cardiac excitation-contraction (E-C) coupling is being developed from recent experimental work using confocal microscopy of single isolated heart cells. In particular, the transient change in intracellular free calcium ion concentration ([Ca2+]i transient) that activates muscle contraction is now viewed as resulting from the spatial and temporal summation of small (∼ 8 μm3), subcellular, stereotyped ‘local [Ca2+]i-transients' or, as they have been called, ‘calcium sparks'. This new understanding may be called ‘local control of E-C coupling'. The relevance to normal heart cell function of ‘local control, theory and the recent confocal data on spontaneous Ca2+ ‘sparks', and on electrically evoked local [Ca2+]i-transients has been unknown however, because the previous studies were all conducted on slack, internally perfused, single, enzymatically dissociated cardiac cells, at room temperature, usually with Cs+ replacing K+, and often in the presence of Ca2-channel blockers. The present work was undertaken to establish whether or not the concepts derived from these studies are in fact relevant to normal cardiac tissue under physiological conditions, by attempting to record local [Ca2+]i-transients, sparks (and Ca2+ waves) in intact, multi-cellular cardiac tissue.

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