Comparison of mitosis in binucleated and mononucleated newt cardiac myocytes.

BACKGROUND: The cultured adult newt ventricular myocyte has been shown to undergo mitosis and cytokinesis in a fully differentiated state. Insight into its proliferation and cellular changes during the repair process involves obtaining a better understanding of the nuclear pattern (mononucleated, binucleated, or multinucleated) resulting from mitotic events. Mitosis is easily observable in cultured newt cardiac myocytes using phase-contrast microscopy. METHODS: From days 8-19 in culture, the process of mitosis in mononucleated and binucleated newt ventricular myocytes was recorded and timed by using time-lapse video microscopy. Cultured cardiac myocytes were double-stained for myosin and F-actin by using fluorescein isothiocyanate (FITC)-labeled MF20 and rhodamine phalloidin. RESULTS: Mitotic, mononucleated myocytes produced mononucleated daughter cells in 80% of the cases, whereas 20% were single, binucleated myocytes, In binucleated myocytes, only 32% underwent complete cytokinesis to produce two binucleated daughter cells, whereas 68% resulted in variably nucleated myocytes. Mononucleated and binucleated myocytes undergoing mitosis had similar time intervals for the period from nuclear breakdown (prometaphase) to the start of anaphase (108.7 minutes and 94.5 minutes, respectively), but the period between anaphase and midbody formation was significantly shorter in binucleated than in mononucleated myocytes (43.5 minutes and 69.3 minutes, respectively). The myofibrillae were not as well organized in binucleated myocytes as those observed in mononucleated myocytes. CONCLUSIONS: Mitosis in vitro appears to proceed more rapidly in binucleated newt cardiac myocytes, which have more poorly organized myofibrillae than mononucleated myocytes. Mitosis of cultured binucleated myocytes commonly results in variably nucleated daughter cells, whereas mononucleated myocytes produce predominantly mononucleated daughter cells.

Effect of hypoxia on the proliferation of retinal microvessel endothelial cells in culture.

BACKGROUND: To determine if hypoxia stimulates the proliferation of retinal microvessel endothelial cells in culture. METHODS: Bovine retinal microvessel endothelial cells were cultured in normoxic (95% air, 5% CO2) and hypoxic (2% O2, 5% CO2, 93% N2) conditions. Endothelial cells were identified by acetylated LDL and Factor VIII-related antigen immunocytochemical staining. Cells from passages three to eight were used in these experiments. Proliferation assays included cell counts by hemocytometer and autoradiographic analysis of incorporated 3H-thymidine (3H-TdR). RESULTS: At day 4, cell counts of endothelial cells in hypoxia showed a 133% increase over those grown in normoxic conditions (N = 25, P < 0.01). Cell counts per day for 5 days were 121-181% greater in hypoxia. Autoradiography of endothelial cells exposed to 3H-TdR and counted every 12 hours for 60 hours exhibited labeling indices 112-118% higher in hypoxic conditions (P < 0.0001). Endothelial cells cultured under hypoxic conditions were smaller and spindle-shaped, whereas those grown under normoxic conditions were larger and more polygonal. CONCLUSIONS: Hypoxia increases DNA synthesis and stimulates proliferation of retinal microvessel endothelial cells in vitro and induces alterations in morphology. These results may be relevant to microvessel angiogenesis, which occurs in vivo under ischemic conditions.

Factors altering DNA synthesis in the cardiac myocyte of the adult newt, Notophthalmus viridescens.

To better understand the mechanisms governing the proliferation of cardiac myocytes it is important to identify the factors controlling this phenomenon, and to characterize their actions. DNA synthesis was quantified in vitro in ventricular myocytes from the adult red-spotted newt, Notophthalmus viridescens. Ventricles were enzymatically separated and plated onto laminin. Myocytes were fed modified L-15 medium with 10% fetal bovine serum, and were variously treated with transforming growth factor-beta, transforming growth factor-beta combined with platelet-derived growth factor, acidic fibroblast growth factor, basic fibroblast growth factor, 12-0-tetradecanoylphorbol-13-acetate, heparin, or conditioned medium from ventricular myocytes or non-myocytes (primarily endothelial cells). With their final feeding the cells were given 1 mu Ci/ml of tritiated thymidine, and 24 hours later were fixed and stained. Dishes were coated with photographic emulsion, exposed, and developed. The percent of cells with labeled nuclei was determined. Experimental media that significantly increased DNA synthesis included those containing acidic fibroblast growth factor (121% of control), basic fibroblast growth factor (119% of control), 12-0-tetradecanoylphorbol-13-acetate (233% of control) and conditioned medium from ventricular myocytes (230% of control) or non-myocytes (128% of control). Media significantly inhibiting DNA synthesis were those containing heparin (31% of control), transforming growth factor-beta (38% of control), non-myocyte conditioned medium and heparin (75% of control), or transforming growth factor-beta and platelet-derived growth factor (63% of control).

Stimulation of DNA synthesis by PDGF in the newt cardiac myocyte.

The adult newt cardiac ventricular myocyte has been successfully placed in cell culture and has been shown to undergo in vitro DNA synthesis. Although several growth factors have been reported to increase DNA synthesis in cardiac myocytes in vitro, PDGF has not been reported to do so, but has been shown to be active in other systems. Ventricles were removed from the adult red-spotted newt and were enzymatically and mechanically dissociated in a solution containing trypsin and collagenase. Cells were preplated on to plastic to remove non-myocytes. Myocytes were then plated onto laminin. Groups of myocytes were fed control medium and medium containing porcine PDGF. Myocytes were given 1 microCi/ml of tritiated thymidine 6 or 24 h before fixation. Control myocytes showed a peak DNA synthesis at 12-14 days in culture. One ng/ml of PDGF increased DNA synthesis significantly to 22% above control. Myocytes responded to PDGF with significantly increased DNA synthesis in about 12 h. PDGF did not induce earlier DNA synthesis, but increased synthesis at all days of culture tested. These results indicate that PDGF acts upon cardiac myocytes, increasing their DNA synthesis.

Analysis of DNA synthesis in cell cultures of the adult newt cardiac myocyte.

Cell division in the adult cardiac myocyte has been examined in a number of different species in vivo and in vitro. The newt cardiac myocyte responds to trauma in vivo with proliferation. It has recently been successfully placed into cell culture. The purpose of the present study was to analyze the process of DNA synthesis in these cultures. The myocytes were cultured in modified Leibovitz L-15 medium on a bovine corneal endothelial cell membrane carpet and were incubated with tritiated thymidine (1 microCi/ml) for 24 hr prior to fixation at 10, 15, 20 and 30 days. Labeling indices were determined to be 10.5 +/- 2.5, 16.5 +/- 2.8, 10.5 +/- 2.2, and 2.9 +/- 0.6, respectively. When myocytes were exposed to 1 microCi/ml tritiated thymidine continuously from the fifth to the thirtieth day in culture, the labeling index was 34.5 +/- 6.8. Comparison of DNA synthesis in the in vivo and in vitro systems indicated comparable patterns, although there was an earlier onset of activity in culture. Between 8 and 15 days in culture, myocyte mitoses were regularly observed. Myocytes in metaphase contained well-organized myofibrillae, suggesting that mitosis may occur with highly differentiated morphology in vitro. It appears that this system will be useful in the definition of mechanisms involved in both initiating and stopping proliferative events in the cardiac myocyte.

Nuclear characteristics of cardiac myocytes following the proliferative response to mincing of the myocardium in the adult newt, Notophthalmus viridescens.

Amphibian cardiac myocytes are predominantly mononucleated and have been demonstrated to respond to injury with DNA synthesis and mitosis. The nature of this response with regard to nuclear number and ploidy is unclear. In this study, the apex of the newt ventricle was minced and replaced, increasing the reactive area of the wound. At 45 days after mincing following multiple injections of tritiated thymidine (2.5 microCi/animal, 20 Ci/mM) 15 to 20 days after mincing, three ventricular zones were isolated and fixed: Zone 1, the minced area; Zone 2, extending approximately 500 micron proximally from the amputation plane; and Zone 3, the portion proximal to Zone 2. Myocytes separated in 50% KOH were examined for DNA synthesis by autoradiography and for nuclear number and DNA content using a scanning microdensitometer on Feulgen-Naphthol yellow S-stained cells. No labeled myocyte nuclei were found in control hearts and 98.3% of the myocytes were 2C. At 45 days, 46.78% of myocyte nuclei within Zone 1 were labeled, while 13% were non-diploid. In Zone 2, 9.25% were labeled with 4.8% non-diploid. In Zone 3, 1.1% were labeled, with 2.8% non-diploid. The newt ventricle's response to injury apparently may involve complete mitosis and cytokinesis, resulting in mononucleated diploid cells.

Changes in binucleation and cellular dimensions of rat left atrial myocytes after induced left ventricular infarction.

The left atrium of young rats has previously been demonstrated to respond with DNA synthesis and binucleation 11 days after left ventricular infarction. This investigation was designed to examine the hypertrophic response of the left atrial myocyte of the rat at 20 and 60 days after ventricular infarction. Male Sprague-Dawley rats were subjected to left coronary artery ligation (CAL) or sham operation. Following enzymatic separation, left atrial myocytes were examined at 20 and 60 days postoperation for number of nuclei and cellular dimensions (cell length, width and area, and nuclear area). Results demonstrated that the level of binucleation at 20 days (77.3%) and 60 days (71.3%) was nearly twice that observed in sham-operated animals, which were 33.1% binucleated at 20 days and 43.5% binucleated at 60 days. In both mononucleated and binucleated myocytes, the mean lengths, widths, and cell areas from CAL hearts were significantly greater than those of corresponding sham-operated animals. In all cases, these values were larger in binucleated myocytes than in mononucleated cells. The mean area of CAL cells was approximately twice that of sham-operated myocytes. With regard to mean lengths and widths, although both were greater in the CAL animals, there was a decrease in length and increase in width between 20 and 60 days in the CAL group. Mean nuclear areas were significantly greater in CAL myocytes than in those from the sham-operated group. These increases in nuclear number and cellular dimensions of the atrial myocyte are prominent features of the response to the stress imposed by left ventricular infarction.

Isolation of cardiac myocytes from the adult newt, Notophthalmus viridescens. an electron microscopic and quantitative light microscopic analysis.

This report describes a technique which permits a high yield of viable adult cardiac myocytes from the adult newt using enzymatic separation techniques at low temperature and high enzyme concentrations. Observations by light microscopy showed the isolated myocytes to have a distinctively slender morphology which consisted of a variable number of arm-like appendages radiating from the center of cells which were predominantly mononucleated. Atrial myocytes were typically observed to have two to three arm-like appendages while ventricular myocytes typically had three to six appendages. The majority of myocytes displayed normal fine structure when examined by transmission electron microscopy. Computerized image analysis revealed that atrial cells were significantly greater in cell length (192.9 +/- 53.4 microns) and in nuclear length (25 +/- 5.3 microns) and perimeter (59.2 +/- 10.7 microns) than were ventricular cells (162.8 +/- 39 microns, 23.6 +/- 5.1 microns and 57.4 +/- 11.1 microns, respectively), while cell widths and areas were greater in ventricular cells (16.5 +/- 4.7 microns and 1839.8 +/- 585.0 microns, respectively) than in atrial cells (13.2 +/- 3.1 microns and 1520.3 +/- 527.6 microns, respectively). Comparison of these data with previous descriptions of isolated amphibian and mammalian cardiac myocytes emphasizes species-related differences.

Ultrastructural studies on migrating epidermal cells during the wound healing stage of regeneration in the adult newt, Notophthalmus viridescens.

The ultrastructure of the epidermal cells which migrate over the wound surface of the amputated limb of the adult newt, Notophthalmus viridescens, was observed with transmission (TEM) and scanning (SEM) electron microscopy. In order to aid in the visualization of polyanionic surface materials on the wound epithelium and wound surface with TEM, the basic dye, ruthenium red, was introduced into the fixatives and buffer. Control limbs were processed without ruthenium red. Shortly after amputation, basal cells at the wound margin possessed elongated, flattened profiles with long pseudopodial projections (lamellipodia and filopodia) that appeared to make contact with the fibrin exudate covering the stump tissues. Epidermal cells proximal to the site of amputation were also in a state of mobilization. Large intercellular spaces and a reduction in the number of desmosomes were observed in the migrating cells. Epidermal cell nuclei became characteristically euchromatic with well-developed nucleoli. Microfilaments were seen within the cytoplasm, extending toward the plasma membrane of cellular processes. Phagocytosed material was also present in the migrating cells. By approximately 9 hours post-amputation, wound closure was complete, and the wound epithelium consisted of three to four cell layers of a non-cornified epidermis. Generally, the amount of extracellular material present on the surface and in the enlarged intercellular spaces of migrating epidermal cells remained the same throughout the period of wound closure. A layer of polyanionic material was observed consistently over the fibrin meshwork covering the wound surface with TEM.

Scanning electron microscopy of epidermal cell migration in wound healing during limb regeneration in the adult newt, Notophthalmus viridescens.

The epidermal cells which migrate over the wound surface of the amputated limb of the adult newt were examined using the scanning electron microscope. Specimens were prepared routinely for scanning electron microscopy or were embedded in Epon 812 for light microscopic observations. A cuff of epidermal cells was seen at the edge of the wound, from which cells appeared to migrate over the wound surface. As early as five hours after transection of the limb, the basal layers of this cuff appeared to send out pseudopodial projections. These seemed to establish a physical contact with a fibrin-like substratum, which apparently served as a means of support for the migrating cells. Subsequently, the epidermal cells became elongate and had the appearance of streaming toward the center of the wound. Between 10 and 13 hours post-amputation, the cells in the central region of the stump were rounded up and some possessed microappendages resembling microplicae and microvilli. Throughout the entire period of wound coverage, the cells seemed to maintain contact with the fibrin network, which appeared to be the first structural element of wound architecture. As a result of these observations, the mechanism by which the epidermal cells migrate has been clarified.