Effects of mast cells on the behavior of isolated heart fibroblasts: modulation of collagen remodeling and gene expression

The extracellular matrix plays a critical role in the development and maintenance of the vertebrate heart. Changes in the accumulation, composition, or organization of the extracellular matrix are known to deleteriously affect heart function. Mast cells are thought to stimulate collagen expression and fibroblast proliferation accompanying fibrosis in some organs; however, the effects of mast cells on the heart interstitium are largely unexplored. The present studies were carried out to determine the effects of mast cells on isolated heart fibroblasts. Several in vitro assays were used including collagen gel contraction to examine the effects of mast cells on the function of isolated fibroblasts. Neonatal heart fibroblasts were cultured either with mast cells, mast cell-conditioned medium, or mast cell extracts, and their ability to contract collagen gels measured. Results from these experiments indicated that mast cells inhibit heart fibroblast migration and contraction of 3-dimensional collagen gels. Further experiments indicated that incubation of neonatal heart fibroblasts with extracts of mast cells altered the expression of collagen, matrix metalloproteases, and matrix receptors of the integrin family. These studies suggest that mast cells play an important role in the regulation of the cardiac interstitial matrix. Further studies are warranted to determine the mechanisms whereby mast cells modulate fibroblast activity.     

Isolation and low temperature preservation of adult rat heart cells

Adult rat heart cells were isolated by perfusion of the coronary system of the heart with a 0.05% collagenase solution.In one method (A), cells were finally isolated by shaking the heart fragments in a collagenase solution, after which the cells were washed and suspended in a Ca- and Mg-free buffered salt solution. The effect of different DMSO concentrations, 5, 10, 15, 20, 25, and 30% and the effect of the addition and dilution rate of DMSO on the number of trypan blue-excluding, intact, and contracting cells were studied. The highest DMSO concentration which was tolerated by the isolated adult heart cells was 15%. Variation of addition rate and the dilution rate of DMSO had no effect. After freezing at external cooling rates of 1, 5, 10, 30, and 50 °C/min to ?100 °C, and then rapidly to ?196 °C, in the presence of 5, 10, or 15% DMSO, reanimation of these cells was not achieved.In another method (B), heart fragments, after collagenase perfusion of the heart, were first treated with 5, 10, or 15% DMSO, after which the cells were isolated. If these cells were frozen at 1 °C/min with 10% DMSO, 15% of the cells, expressed as a percentage of the control, remained morphologically intact and 38% of the cells were contracting after thawing. Significantly higher survival percentages of 30 and 61%, respectively, were obtained if the heart fragments were left intact during freezing.     

The role of mononuclear phagocytes in cardiac allograft rejection in the rat: III. The effect of cells extracted from rat cardiac allografts upon beating heart cell cultures

Cells extracted from rat cardiac allografts were able to bring about cessation of beating of heart cell culture monolayers nonspecifically. Nonadherent populations, depleted of macrophages, were consistently less potent than unseparated cells in this assay. Cells extracted from isografts were totally ineffective. Allogeneically stimulated peritoneal cells were also nonspecifically active. Again, nonadherent cells were less efficient than unseparated cells at stopping heart cell monolayers from beating, while adherent cells, enriched for macrophages, were more efficient. Activated bone marrow culture macrophages syngeneic or allogeneic to the heart cultures were also highly potent in beating heart cell assays. Thus in all cases the predominant effector cell type was adherent and nonspecific in its action and therefore presumably a macrophage. Supernatants from wells in which no beating cells remained following incubation with each type of effector population tested were transferred undiluted to fresh wells. In all cases there was no effect at all upon the beating of heart cell monolayers. Antirat heart antiserum plus complement was able to bring about the cessation of beating of heart culture monolayers at a dilution of 1:64. Alloantibody plus complement did not bring about cessation of beating at any dilution, although nonmyocardial cells were killed. The possibility that macrophages are the chief effector cell type in a DTH-like mechanism for cardiac allograft rejection is discussed.     

Fine structural examination of the single heart tube in the fifteen day ferret embryo

Summary The different segments of the embryonic heart tube of the ferret were examined with light and transmission electron microscopy. The cells of bulbus cordis, bulboventricular junction, primitive ventricle, atrioventricular junction, and primitive atria were in the process of differentiating into myocardial cells. The ventricular muscle cells were the most developed cells; the least mature muscle cells were those located at the arterial and venous ends of the heart tube. The cells between the ventricle and the two ends of the heart tube showed a spectrum of developmental stages, especially with respect to the morphological development of the myofibrils. Other organelles as well as surface specializations did not permit a distinction between cells of the different regions of the heart tube. There was no striking difference in the size and shape of the developing muscle cells of the atrioventricular and bulboventricular junctions compared to the developing ventricular muscle cells. Morphologically there was no evidence to suggest that the tissue of the atrioventricular and bulboventricular rings was specialized or different from any of the other segments of the single heart tube of the embryonic ferret.This work was supported by NIH Grant#HL26893-01 and by Biomedical Research Support Grant S07RR05417 from the Division of Research Resources, NIH     

Cryopreservation of human heart cells

Single cells were isolated from human heart specimens and preserved at -56 degrees C in a cryoprotecting solution containing dimethyl sulfoxide. The cells were reanimated by rapid thawing and the properties of the cells were evaluated in a physiological solution. The reanimated cells showed morphological and physiological properties similar to those seen in normal, freshly isolated cells. These results demonstrate the feasibility of storing human heart cells for long-term studies.     

Catecholamines are present in larval Xenopus laevis: a potential source for cardiac control

Changes in noradrenaline (NA), adrenaline (A), and dopamine (DA) levels in the heart, kidneys, and whole body (without heart and kidneys) during embryonic development were investigated in the frog, Xenopus laevis using high-performance liquid chromatography (HPLC). In addition, the presence of cells immunoreactive to tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and/or phenylethanolamine-N-methyltransferase (PNMT) in the heart of Xenopus larvae was investigated using immunohistochemical techniques. The presence of nerve fibers was visualized using antibodies against acetylated tubulin (AcT). NA and DA concentrations in the heart were low and steady in NF 40-56, showed an increased value at NF 57, and decreased again in froglets. A trend toward higher concentrations of A was observed at NF 43-49 and NF 57. Cells immunoreactive to TH, DBH, and PNMT were found in the heart from NF 40, and the TH immunoreactive cells became more abundant in the whole heart at later stages. The presence of catecholamines in the non-innervated larval heart together with the finding of TH/DBH/PNMT immunoreactive cells suggests that catecholamines are synthesized and stored in the heart and could therefore have a paracrine role in cardiac control in Xenopus larvae. Detectable concentrations of catecholamines were also found in kidneys and whole bodies (except heart and kidneys). Therefore, catecholamine-producing cells outside the heart can be an important source of circulating catecholamines involved in adrenergic cardiac control in Xenopus larvae.     

The distribution of centrosomes in endothelial cells of non-wounded and wounded aortic organ cultures

Summary The distribution of centrosomes in porcine vascular endothelial cells of the thoracic aorta maintained in organ culture was determined in en face preparations using immunofluorescence. Rectangular pieces of aorta that had the distal half (with respect to the heart) of their endothelial surface gently denuded with a scalpel blade and pieces with intact endothelium were cultured for up to 96 h. At time 0, centrosomes were found to be preferentially oriented toward the heart, both in the cells of intact monolayers and in cells at the wound edge. This distribution was maintained in the intact monolayers for at least 24 h, but by 72 h the number of centrosomes in the center of the cells exceeded the number oriented toward the heart as the cells changed from a fusiform to a polygonal shape. The centrosomes of most endothelial cells at the wound edge began to redistribute themselves within the first 24 h in culture, moving from a position toward the heart to a position either in the center of the cell or away from the heart. By 72 h, the majority of centrosomes in endothelial cells at the wound edge were oriented away from the heart toward the denuded region. It is concluded that the centrosomes in the endothelial cells maintained in organ culture respond to injury in a manner similar to those grown in monolayer cell culture except that the reorientation of centrosomes occurs more slowly.     

Selective killing of fibroblast-like cells in cultures of mouse heart cells by treatment with a Ca ionophore, A23187

Myocardial cells uncontaminated by fibroblast-like cells could be obtained by treating mouse heart cell cultures with a Ca ionophore, A23187. When trypsin-dissociated mouse heart cells were cultivated for 1 day or more on Petri dishes, both myocardial and fibroblast-like cells were detected on the dish under a phase contrast microscope. However, when the cultured heart cells were treated for a short period (e.g. 10 min) at 25 °C with A23187, only fibroblast-like cells exhibited morphological degeneration, such as nuclear pycnosis, cytoplasmic condensation and bleb- and balloon-formation. The myocardial cells maintained their normal morphology during the treatment; transient irregular beating was observed, but disappeared after removal of A23187. Thus treatment of heart cell cultures with A23187 can yield a large amount of myocardial cells without contamination by fibroblast-like cells. The possible mechanism for this selective effect is discussed.     

Immunocytochemical detection of enhanced expression of c-myc protein in the heart of cardiomyopathic hamster

An immunocytochemical study was performed to examine the expression of cellular c-myc protein in the heart of 30-, 120- and 180-day-old cardiomyopathic Syrian UM-X7.1 hamsters. The heart of age- and sex-matched BIO-RB hamster was used as normal control. In paraffin sections, an immunostaining for c-myc was markedly increased in cytoplasm of cells from the UM-X7.1 heart as compared with that of the BIO-RB heart which showed a weak staining. However, c-myc was localized in nuclei of cells in frozen sections of the heart. Specific cell types of the heart were differentiated with anti-vimentin, and we found that the increased expression of c-myc was present in nuclei of muscle cells of the UM-X7.1 myocardium. A quantitative study of c-myc-positive nuclei of muscle and nonmuscle cells was carried out by a video micrometer. The mean number of c-myc-positive nuclei of muscle cells was significantly higher in the cardiomyopathic heart than in the control heart from hamsters of all ages studied. These results suggest that the increase of c-myc protein may relate to the pathological state or pathogenesis of the hereditary cardiomyopathy.     

Atrial natriuretic peptide in the granular cells of the snail heart

Cardiomyocytes of vertebrates combine contractile and endocrine functions. They synthesize and secrete atrial natriuretic peptide (ANP), which is localized in their specific granules. The presence of ANP has been shown in some tissues of invertebrates, including the heart of molluscs. We have studied localization of ANP in cells of the snail heart. METHOD: The atrial and ventricular tissues of the snail Helix pomatia were studied by electron microscope immunocytochemistry, using anti-ANP antibodies. ANP-immunoreactivity has been detected in granules of granular cells located on the luminal surface of the snail myocardium. These cells are abundant in the atrium being very rare in the ventricle. Granular cells at different stages of maturation were revealed. Immature granular cells have light granules of moderate size with homogeneous tight content, while mature granular cells are huge in size and all their granules are fused together. The material of these granules loosens up and almost completely fills up the cytoplasm. No ANP-immunoreactivity was observed in muscle cells or nerve fibers. A possible origin of granular cells from the cardiac endothelial cells is discussed. The molluscan heart, similar to that of vertebrates, is a bifunctional organ. However, contrary to the heart of vertebrates, in the molluscan heart contractile and endocrine functions are separated between different types of cells.     

Heterogeneous cellular expression of creatine kinase isoenzyme during normal rat heart development

The degree to which developmentally related alterations in cardiac creatine kinase (CK) activity reflect modification of CK isoenzyme gene expression remains uncertain. The present studies addressed this question by assessing multiple aspects of CK in rat heart during the perinatal to adult transition. In addition to whole tissue, isolated and purified muscle and nonmuscle cells were studied, as well as myofibrillar, mitochondrial, and cytosolic subcellular fractions. Whole homogenate CK enzyme specific activity nearly doubled during the weanling to adult developmental period. Muscle cell CK activity increased by a similar magnitude. Nonmuscle cell activity decreased. In the adult heart, both myofibrillar and mitochondrial CK activities were augmented versus the weanling heart. The cytoplasmic fraction activity held constant during development. Electrophoretic isoenzyme analyses of both weanling and adult cardiac muscle cells indicated the presence of mitochondrial CK and MM-CK isoforms. Weanling heart nonmuscle cells contained mitochondrial, MM, MB, and BB isoforms; however, BB isoform was not detected in the adult heart nonmuscle cells. Arrhenius plots provided information regarding heart muscle and nonmuscle cell alterations during development. CK activation energies were also determined for whole tissue, muscle/nonmuscle cells, myofibrils, mitochondria, and cytosol. Results demonstrate that heterogeneous muscle/nonmuscle cellular composition and differential myofibrillar/mitochondrial subcellular composition account for normal, developmentally related changes in heart CK enzyme activity. CK isoenzyme gene expression changes were not detected in cardiac muscle cells, and transition of CK-B to CK-M gene expression is limited to nonmuscle cells during normal, weanling to adult development in the rat heart.     

Phagocytic capacity of invasive malignant cells in three-dimensional culture

To study the phagocytic capacity of invasive malignant cells, fragments of the hypoblast from chick blastoderms were confronted in three-dimensional culture with spheroidal aggregates of 1) malignant virally transformed C3H mouse cells (MO4), 2) HeLa cells and 3) embryonic chick heart cells. The hypoblast was used because it contains yolk, a marker that is absent in the confronting cells and that can be identified histologically and ultrastructurally. The confronting tissues were incubated on semi-solid agar-agar medium or in fluid medium on a gyrotory shaker. Cultures were followed for 1 to 7 days by stereomicroscopy, cinemicrophotography, light and transmission electron microscopy. Confrontation with MO4 cells of HeLa cells, known to be invasive in vitro, led to complete disappearance of the hypoblast. The fragments of hypoblast were well conserved when cultured alone or confronted with aggregates of chick heart cells. Degeneration of the hypoblast is shown at the area of contact with MO4-cell or HeLa-cell aggregates, in contrast to heart cells. Filopodia-like extensions from the MO4 or HeLa cells penetrate intercellularly, transcellularly and intracellularly into the hypoblast. Phagosomes, containing yolk and unidentified debris are observed in MO4 cells and in HeLa cells, but not in heart cells. These observations demonstrate the phagocytic capacity of invasive malignant cells.     

Sensitivity of rat heart endothelial and myocardial cells to alloimmune lymphocytes and to alloantibody-dependent cellular cytotoxicity.

We have attempted to define the structural target components for the two cytotoxic mechanisms in rejecting rat heart allograft: antibody-independent (direct) and antibody-dependent (ADCC) cellular cytotoxicity. Immune spleen cells and alloantibody were obtained at 1 and 2 weeks after heart allotransplantation, respectively. The cytotoxicity of immune spleen cells, with and without alloantibody, was tested against endothelial- and myocardial-enriched heart cell populations. We found endothelial cells to be sensitive to direct cellular cytotoxicity, most likely mediated by T lymphocytes, while myocardial cells were sensitive to ADCC. Both reactions were shown to be immunologically specific.     

The use of fluorescent antimyosin and DNA labeling for the estimation of the myoblast and myocyte population of primary rat heart cell cultures

Cell division in heart muscle cells progressively ceases during the development of the rat heart, leading to an adult stage with muscle cells incapable of cell division. We have quantitatively determined the number of dividing and nondividing heart muscle cells in cultures derived from different stages of the developing rat heart with the use of ³HTdR continuous labeling and fluorescent antimyosin staining. The cultures were derived from 14 and 17 day postcoital (dPC) rat embryos and from 1 and 4 day postnatal (dPN) rats. The percent nondividing cells increased with development and the age of the postnatal rat. The percent nondividing cells in 14 dPC equalled 21%, 17 dPC equalled 25%, 1 dPN equalled 44%, and 4 dPN equalled 60%. This method for the quantitative determination of dividing and nondividing cells in the developing rat heart provides a model that is useful for the study of the mechanism of the loss of cell division capacity.     

Monolayer co-culture of rat heart cells and bovine adrenal chromaffin paraneurons

Summary This paper describes a method for the preparation of co-cultures of rat heart cells and bovine adrenal chromaffin paraneurons. The most suitable condition for heart cell isolation was when a combination of paraneurons. The most suitable condition for heart cell isolation was when a combination of trypsin-DNAse I in Locke's solution was used for digestion. The best co-culture conditions were obtained when 10⁶ heart cells were plated on 7- to 8-d-old adrenal chromaffin paraneuron cultures containing 0.5×10⁶ cells per 35-mm diameter culture dishes. Measurements of DNA (heart cells and chromaffin paraneurons), monitoring of beating frequency (heart cells), and catecholamine (chromaffin paraneurons) levels and release indicated that both cell types remain viable and functional, for several weeks. Heart cells started their characteristic contractile activity 24 h earlier when plated either on viable or lysed chromaffin paraneurons, an effect apparently due to faster surface adhesion of heart cells. The beating frequency of heart cells increased after treatment of co-cultures with either noradrenaline or nicotine, with the latter agent acting indirectly through, the release of chromaffin paraneuron catecholamines. Propranolol produced a dose-related inhibition of the responses to either noradrenaline or nicotine, thus suggesting that the increase in myocyte's beating activity was mediated through β-receptors. Anti-myosin and anti-dopamine-β-hydroxylase immunostaining was used for cell type identification and for the demonstration of body-to-body and process-to-process contacts between adrenal chromaffin paraneurons and heart cells. This co-culture system will serve as a starting point of further studies directed to understand a) the influence of a cell type on the development and on the phenotypic characteristics of a second cell type and b) the interaction of cells derived from different organs and species. This study was supported by grant PG-20 from Medical Research Council of Canada and a grant from the Ontario Heart and Stroke Foundation. M. L. N. is a postdoctoral fellow of the MRC.     

Mesenchymal stem cell administration at coronary artery reperfusion in the rat by two delivery routes: a quantitative assessment

AIMS: Ideally, mesenchymal stem cells (MSC) home to and/or remain at the site of damaged myocardium when administered after myocardial infarction. However, MSC may not remain in the heart, but instead relocate to other areas. We investigated quantitatively the distribution of labeled rat MSC, given by two routes after coronary artery occlusion/reperfusion in rats. MAIN METHODS: Rats were subjected to 45 min of coronary artery occlusion and 7 days of reperfusion. Before reperfusion rats received 2x10(6) MSC, labeled with europium, injected directly into the ischemic region of the heart (n=9) or intravenously (n=8). After 1 week tissues were analyzed for label content together with a standard curve of known quantities of labeled MSC. KEY FINDINGS: In rats receiving cells injected directly into the myocardium, 15% of labeled cells were retained in the heart. When the cells were administered intravenously, no MSC were detected in the heart. The route of administration did not affect distribution to other organs, as the number of MSC in liver, spleen and lung was similar with both routes of delivery. SIGNIFICANCE: Even with direct intramyocardial injection, only a small proportion of the cells are retained in the heart, instead traveling to other organs. With intravenous injection there was no evidence that cells "homed" to the damaged heart. Although cell delivery to the heart was significantly affected by the route of administration, the distribution of cells to other organs was similar with both routes of administration.     

Nestin (+) stem cells independently contribute to neural remodelling of the ischemic heart

Recent studies have revealed the existence of multipotent nestin-immunoreactive cells in the adult mammalian heart. These cells were recruited to infarct site following ischemic injury and differentiated to a vascular lineage leading to de novo blood vessel formation. Here, we show that a sub-population of cardiac resident nestin((+)) cells can further differentiate to a neuronal-like fate in vivo following myocardial infarction. In the ischemically damaged rat heart, neurofilament-M((+)) fibres were detected innervating the peri-infarct/infarct region and the preponderance of these fibres were physically associated with processes emanating from nestin((+)) cells. One week after isogenic heterotopic cardiac transplantation, the beating transplanted rat heart was devoid of neurofilament-M((+)) fibre staining. The superimposition of an ischemic insult to the transplanted heart led to the de novo synthesis of neurofilament-M((+)) fibres by cardiac resident nestin((+)) cells. Nerve growth factor infusion and the exposure of normal rats to intermittent hypoxia significantly increased the density of neurofilament-M((+)) fibres in the heart. However, these newly formed neurofilament-M((+)) fibres were not physically associated with nestin((+)) processes. These data highlight a novel paradigm of reparative fibrosis as a subpopulation of cardiac resident nestin((+)) cells directly contributed to neural remodelling of the peri-infarct/infarct region of the ischemically damaged rat heart via the de novo synthesis of neurofilament-M fibres.     

Culture of insect cells contracting spontaneously; research moving toward an environmentally robust hybrid robotic system

Here we propose an environmentally robust hybrid (biotic-abiotic) robotic system that uses insect heart cells. Our group has already presented a hybrid actuator using rat heart muscle cells, but it is difficult to keep rat heart muscle cells contracting spontaneously without maintaining the culture conditions carefully. Insect cells, by contrast, are robust over a range of culture conditions (temperature, osmotic pressure and pH) compared to mammalian cells. Therefore, a hybrid robotic system using not mammalian cells but insect cells can be driven without precise environmental control. As a first step toward the realization of this robotic system, the larvae of two lepidopteran species, Bombyx mori (BM) and Thysanoplusia intermixta (TI) were excised and the culture conditions of their dorsal vessel (insect heart) cells were examined. As a result, spontaneously contracting TI cells derived from the dorsal vessel were obtained. The contraction of TI cells started on the 7th day and continued for more than 18 days. Spontaneously contracting BM cells were not obtained in this study. These experimental results suggest the possibility of constructing an environmentally robust hybrid robotic system with living cells in the near future.     

Immunostimulation effects of yellowtail heart extracts in vitro and in vivo

The immunostimulation effects of yellowtail heart extracts were examined. Screening various parts of the yellowtail viscera, we found that extracts from the yellowtail heart enhanced IgM production by human hybridoma HB4C5 cells. Yellowtail heart extracts heated at 121°C for 20 min and dialyzed showed the highest IgM production-stimulating activity toward HB4C5 cells. Also, immunoglobulin production by mouse spleen lymphocytes was stimulated by yellowtail heart extracts in vitro, and lymphocytes derived from mice administered the extract for 20 d were activated in vivo. Yellowtail heart extracts were partially purified by anion-exchange chromatography, and fractions containing a 33 kDa-protein exhibited immunostimulating activity. LC-MS/MS analysis revealed that the 33 kDa-protein was most similar to tropomyosin-4 from various fishes. Purified tropomyosin from porcine muscle enhanced IgM production by HB4C5 cells. This means that tropomyosin-4 is one of the immunostimulating substances in the yellowtail heart.