Beating of multinucleated giant myocardial cells in culture

Cultured mouse myocardial cells grown as cell sheets in Petri dishes fused together and formed multinucleated giant cells on treatment with HVJ (Sendai virus). The giant cells had well organized myofibrils and beat spontaneously and rhythmically. The spontaneous beating activity of the giant cells changed in response to changes of the external potassium and calcium concentrations and on addition of ouabain in the same way as the beating of cultured myocardial cells not treated with HVJ. When a microelectrode was inserted into giant cells that exhibited spontaneous beating, action potentials were easily recorded.     

Maintenance and characterization of spontaneous contraction rhythm in cultured cardiac myocytes fused with cardiac fibroblasts

Cardiomyocytes (CMs) fuse with various cells including endothelial cells, cardiac fibroblasts (CFs). In addition, recent studies have shown that stem cells fuse spontaneously with cells remaining in the damaged tissues, and restore tissue functions after myocardial infarction. In this study, we investigated whether cultured cardiomyocytes fused with proliferative cardiac fibroblasts maintained the phenotype of functional myocytes by analyzing the spontaneous contraction rhythm after fusion with CFs lacking a beating capability. CMs and CFs cultured for 4 days in vitro were used in this study. The fusion of cultured CMs and CFs was achieved with polyethylene glycol (PEG) and hemagglutinating virus of Japan (HVJ). Analyses of CMs fused with CFs by using either PEG or HVJ to imitate spontaneous fusion in vivo demonstrated that CMs and CFs actually fused together and fused cells expressed lineage marker proteins of both CMs and CFs. In addition, fused cells reentered the G2-M phase of the cell cycle. Furthermore, fused cells retained the spontaneous contraction activity. The present study demonstrated that CMs fused with proliferative CFs showed the phenotype of both CMs and CFs and spontaneous rhythmic contraction.     

Dopa oxidase expression in fibroblast x melanoma fusions. Extinction in heterokaryons and maintenance in non-dividing cybrids

Pigmented B-16 mouse melanoma cells were fused with chick embryo fibroblasts or fibroblast cytoplasts and maintained as heterokaryons or non-dividing cybrids, respectively. These single cells were examined ultrastructurally for evidence or pigment gene expression using a cytochemical test for dopa oxidase, the initial enzyme in the conversion of dopa to melanin. Heterokaryons showed significantly less enzyme activity than control cells, whereas non-dividing cybrids showed no significant difference. Therefore, the presence of the intact nuclear membranes in the heterokaryons did not serve as a barrier to the interactions resulting in extinction of differentiated function(s). However, the presence of the fibroblast nucleus was necessary to elicit continued response.     

The isolation of heterokaryons and hybrids by a selective system using irreversible biochemical inhibitors

A general method for isolating heterokaryons between any type of cells was developed using irreversible biochemical inhibitors. Cells were treated with a lethal dose of a reagent, washed free of unreacted inhibitor, and then fused to cells treated in a similar fashion with an agent of different specificity. Unfused parental cells or homokaryons do not have their damaged molecules replaced and thus die. Only heterokaryons receive a full complement of those molecules necessary for cell survival. Heterokaryons formed by this technique are viable, divide, and give rise to hybrid populations. Although more efficient inhibitors may be found, iodoacetamide and diethylpyrocarbonate provide a workable combination that demonstrates the feasibility of the approach. Since cells with heritable selective markers are not required, this technique should greatly expand the range of cell types suitable for hybridization experiments. In addition, it permits studies on pure populations of heterokaryons before any cell division has occurred.     

Pattern of chick gene activation in chick erythrocyte heterokaryons

The reactivation of chicken erythrocyte nuclei in chick-mammalian heterokaryons resulted in the activation of chick globin gene expression. However, the level of chick globin synthesis was dependent on the mammalian parental cell type. The level of globin synthesis was high in chick erythrocyte-rat L6 myoblast heterokaryons but was 10-fold lower in chick erythrocyte-mouse A9 cell heterokaryons. Heterokaryons between chick erythrocytes and a hybrid cell line between L6 and A9 expressed chick globin at a level similar to that of A9 heterokaryons. Erythrocyte nuclei reactivated in murine NA neuroblastoma, 3T3, BHK and NRK cells, or in chicken fibroblasts expressed less than 5% chick globin compared with the chick erythrocyte-L6 myoblast heterokaryons. The amount of globin expressed in heterokaryons correlated with globin mRNA levels. Hemin increased beta globin synthesis two- to threefold in chick erythrocyte-NA neuroblastoma heterokaryons; however, total globin synthesis was still less than 10% that of L6 heterokaryons. Distinct from the variability in globin expression, chick erythrocyte heterokaryons synthesized chick constitutive polypeptides in similar amounts independent of the mammalian parental cell type. Approximately 40 constitutive chick polypeptides were detected in heterokaryons after immunopurification and two-dimensional gel electrophoresis. The pattern of synthesis of these polypeptides was similar in heterokaryons formed by fusing chicken erythrocytes with rat L6 myoblasts, hamster BHK cells, or mouse neuroblastoma cells. Three polypeptides synthesized by non-erythroid chicken cells but less so by embryonic erythrocytes were conspicuous in heterokaryons. Two abundant erythrocyte polypeptides were insignificant in non-erythroid chicken cells and in heterokaryons.     

Characterization of heterokaryons between skeletal myoblasts and somatic cells formed by fusion with HVJ (Sendai virus); effects on myogenic differentiation

In skeletal myogenic differentiation, myoblasts fuse with myogenic cells spontaneously, but do not fuse with non-myogenic cells either in vivo or in vitro, suggesting that the fusion of myoblasts with non-myogenic cells is unsuitable for differentiation. To understand the inevitability of the fusion among myoblasts, we prepared heterokaryons in crosses between quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) and rodent non-myogenic cells, such as tumor cells, fibroblasts, or neurogenic cells by HVJ (Sendai virus) and examined how myogenic differentiation was influenced in the prepared heterokaryons, focusing on myogenin expression and myofibril formation as markers of differentiation. When presumptive QM-RSV cells were fused with non-myogenic cells by HVJ and induced to differentiate, both myogenin expression and myofibril formation were suppressed. When myotubes of QM-RSV cells that had already expressed myogenin and formed myofibrils were fused with non-myogenic cells, both myogenin and myofibrils disappeared. Especially, fibrous structures of myofibrils were significantly lost and dots or aggregations of F-actin were formed within 24 hr after formation of heterokaryons. However, the fusion of presumptive or differentiated QM-RSV cells with rodent myoblasts did not disturb myogenin expression or myofibril formation. These results suggest that mutual fusion of myoblasts is indispensable for normal myogenic differentiation irrespective of the species, and that some factors inhibiting myogenic differentiation exist in the cytoplasm of non-myogenic cells, but not in myoblasts.     

The Mechanism of Heterokaryotic Growth in VERTICILLIUM DAHLIAE

Heterokaryons of Verticillium dahliae, forced between complementary auxotrophs, were stable at 21° and resembled the wild type morphologically. In such heterokaryons the hyphal cells were predominantly uninucleate, and no nuclear migration from cell to cell was observed. Heterokaryosis was apparently confined to binucleate, interhyphal, anastomosed cells that arose 1-2 mm behind the colony front. Such anastomosed cells thereby fed and maintained large homokaryotic areas including the colony edge. This stable mosaic colony is in sharp contrast to the heterokaryon of Neurospora.—Heterokaryons of V. dahliae cannot continue growth at 30° because the high temperature prevents hyphal anastomosis. Heterozygous diploids sector out from heterokaryons after 8-12 days at 30°. Interhyphal anastomosed cells are apparently the site of karyogamy.     

Heterokaryon analysis of muscle differentiation: regulation of the postmitotic state

MM14 mouse myoblasts withdraw irreversibly from the cell cycle and become postmitotic within a few hours of being deprived of fibroblast growth factor (Clegg, C. H., T. A. Linkhart, B. B. Olwin, and S. D. Hauschka, 1987, J. Cell Biol., 105:949-956). To examine the mechanisms that may regulate this developmental state of skeletal muscle, we tested the mitogen responsiveness of various cell types after their polyethylene glycol-mediated fusion with post-mitotic myocytes. Heterokaryons containing myocytes and quiescent nonmyogenic cells such as 3T3, L cell, and a differentiation-defective myoblast line (DD-1) responded to mitogen-rich medium by initiating DNA synthesis. Myonuclei replicated DNA and reexpressed thymidine kinase. In contrast, (myocyte x G1 myoblast) heterokaryons failed to replicate DNA in mitogen-rich medium and became postmitotic. This included cells with a nuclear ratio of three myoblasts to one myocyte. Proliferation dominance in (myocyte x 3T3 cell) and (myocyte x DD-1) heterokaryons was conditionally regulated by the timing of mitogen treatment; such cells became postmitotic when mitogen exposure was delayed for as little as 6 h after cell fusion. In addition, (myocyte x DD-1) heterokaryons expressed a muscle-specific trait and lost epidermal growth factor receptors when they became postmitotic. These results demonstrate that DNA synthesis is not irreversibly blocked in skeletal muscle; myonuclei readily express proliferation-related functions when provided with a mitogenic signal. Rather, myocyte-specific repression of DNA synthesis in heterokaryons argues that the postmitotic state of skeletal muscle is regulated by diffusible factors that inhibit processes of cellular mitogenesis.     

Characterization of myogenic cell membrane: spontaneous formation of heterokaryotic myotubes between two different kinds of myoblasts

In a previous study, it has been shown that presumptive mouse C2 myoblast cells are strongly resistant to HVJ (hemaglutinating virus of Japan, Sendai virus)-mediated cell fusion, but do become capable of fusion upon differentiation. Quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) also become more sensitive to HVJ-mediated cell fusion during differentiation. Investigations were undertaken to see whether heterokaryotic myotubes were formed spontaneously by co-culture of two different kinds of myogenic cells, QM-RSV cells and C2 cells. When both cells were committed to myotube formation, they spontaneously fused without HVJ on co-culture. On the other hand, when both or one of the cells were in the presumptive state, heterokaryons were not formed by co-culturing. Furthermore, committed QM-RSV cells did not fuse with non-myogenic cells. These results indicate that the membranes of myogenic cells change to become capable of fusion for myotube formation during differentiation.     

Further studies on preservation of the beating rhythm of myocardial cells in culture

1. 1. Single myocardial cells from fetal mouse heart beat spontaneously in monolayer culture. In standard medium they maintained a constant beating rate for at least 5 h. After the beating rate of individual cells had been accelerated for a short time by electrical stimulation, the original beating rate could be immediately restored by interrupting the stimulation. Quiescent myocardial cells from neonatal mouse atrium could be induced to beat by electrical stimulation and most of them ceased to beat again immediately by interrupting the stimulation.

2. 2. After the spontaneous beating of individual myocardial cells had been stopped or slowed down for a short time by incubation in medium of low temperature or high potassium or low calcium concentration, the original beating rate could be restored by replacing the cells in the original, normal medium.

3. 3. After the spontaneous beating of individual myocardial cells had been stopped by adding a metabolic inhibitor, such as 2,4-dinitrophenol or 2-deoxyglucose, the original beating rate could be restored by replacing the cells in the original, normal medium.

4. 4. Both single myocardial cells and cell clusters showed arrhythmia, including flutter and fibrillation, in medium of low potassium or high calcium concentration. After a short period of arrhythmia, the original beating rate could be restored by replacing the cells in the original, normal medium. The arrhythmia of cell clusters produced in either low potassium or high calcium medium was also corrected immediately by addition of quinidine sulfate.

     

粟长蠕孢菌的异核现象及异核体核型比影响因素的研究

本试验通过23株带有遗传标记的粟长蠕孢菌突变菌株,获得生理性状及生长势不同于亲本的异核体。利用营养缺陷型标记菌株研究的结果表明,粟长蠕孢菌异核体的形成及核型成分的变化受选择压力的影响。原生质体检测结果表明,在异核菌丝体中,异核细胞占46.7%,同核细胞占53.3%。分生孢子检测结果表明,只有0.06%的分生孢子保持异核状态。     

Extinction of muscle-specific properties in somatic cell heterokaryons

In studies of gene regulation using somatic cell fusion techniques, the analysis of heterokaryons circumvents several problematic aspects of the more traditional approach utilizing proliferating hybrid cells. We have analyzed the expression of muscle specific properties in heterokaryons between muscle and nonmuscle cells in order to investigate whether differentiating cells contain regulatory factors that repress the expression of alternative developmental pathways. Heterokaryons and cybrids were derived from polyethylene glycol-mediated fusion of differentiated mononucleate chicken myocytes with mouse melanoma cells, mouse melanoma cytoplasts, chicken fibroblasts, or other chicken myocytes. Our results demonstrate that fusion of a myocyte with a nonmyogenic cell generally results in extinction of muscle-specific properties in the immediate fusion product. Myocyte X melanoma heterokaryons ceased to express the skeletal muscle forms of myosin, desmin and creatine kinase, reinitiated DNA synthesis, and showed a loss of spontaneous fusion competence within 96 hr after their formation. Although chicken myocyte X mouse melanoma heterokaryons showed extinction of muscle specific properties, they continued to synthesize protein and to incorporate [3H]hypoxanthine, presumably due to the continued production of constitutive chicken HPRT. That presence of the melanoma nucleus was required for extinction to be observed was demonstrated by the continued expression of muscle proteins in cybrids between chicken myocytes and melanoma cytoplasts. Significantly, heterokaryons between chicken myocytes and chicken fibroblasts also exhibited extinction of muscle proteins, demonstrating for the first time that extinction is not restricted to fusions in which at least one parental cell type was derived from an established cell line. Our results strongly support the notion that extinction reflects cell-type specific gene regulatory mechanisms operative during development.     

Restoration of metabolic cooperation in heterokaryons between HGPRT-deficient mouse A9 fibroblasts and chick embryo erythrocytes

Genetic determinants of metabolic cooperation were studied by fusing chick erythrocytes to HGPRT- mammalian cells. Heterokaryons were then tested for their ability to incorporate [3H]hypoxanthine and to transfer radioactive material to HGPRT- recipient cells. Chick erythrocytes (CE) have nuclei which are inactive but contain the HGPRT gene and some cytoplasmic HGPRT enzyme activity. They are unable, however, to cooperate with HGPRT- cells. Of the two mammalian cell lines used, the human GM29 line is HGPRT- and capable of functioning as a receptor cell in cooperation experiments with HGPRT+ cells. The HGPRT- mouse A9 line on the other hand is unable to cooperate. Immediately after fusion, both types of heterokaryons incorporated [3H]hypoxanthine, indicating the presence of some chick HGPRT enzyme contributed by the erythrocyte partner at the time of fusion. While the CE-GM29 heterokaryons participated in metabolic cooperation shortly after fusion, the CE-A9 heterokaryons did not. However, four days after fusion, i.e., at a time when the erythrocyte nucleus had been reactivated, the CE-A9 heterokaryons did cooperate. This suggests that in CE-A9 heterokaryons the genes required for metabolic cooperation are expressed by the previously dormant chick erythrocyte nucleus.     

The suppression of myogenic functions in heterokaryons formed by fusing chick myocytes to diploid rat fibroblasts

Heterokaryons were formed by fusing differentiated chick skeletal myocytes to fibroblasts derived from skin, lung or heart cultures. The heterokaryons were analyzed for the synthesis of skeletal myosin light chains, acetylcholine receptor, total CPK activity and the ability to spontaneously fuse to form myotubes. Whereas all of the above myogenic functions were expressed in control heterokaryons formed between myocytes and myoblasts, all were extinguished in the crosses between myocytes and fibroblasts. These results confirm that the suppression of myogenic functions previously observed in cell hybrids involving fibroblastoid tumor cells also occurs in heterokaryons isolated using biochemical inhibitors between diploid fibroblasts and chick skeletal myocytes.     

Mouse C2 myoblast cells resist HVJ (Sendai virus)-mediated cell fusion in the proliferating stage but become capable of fusion after differentiation

To investigate the mechanism of myoblast fusion, we attempted to prepare artificial myotubes of mouse C2 myoblast cells using the hemagglutinating virus of Japan (HVJ, Sendai virus). Proliferating C2 cells showed strong resistance to HVJ-mediated cell fusion and remained morphologically unchanged even though massive numbers of virions adsorbed onto their surface. They showed no membrane disruption, which occurs in the early stage of cell fusion induced by HVJ. These observations suggest that proliferating C2 cells are resistant to HVJ-mediated cell fusion. However, upon induction of differentiation, C2 cells gradually became capable of fusion induced by HVJ and then even generated heterokaryons with Ehrlich ascites tumor cells. When differentiated C2 cells that had become fusion-sensitive were treated with HVJ in the presence of EDTA, they did not fuse but degenerated, suggesting that their cell membranes were transiently disrupted by interaction with HVJ. These results suggest that the cell membranes of myoblasts change to a fusion-capable state during the process of differentiation.     

Reactivation of chick erythrocyte nuclei in heterokaryons with temperature-sensitive Chinese hamster cells.

Chinese hamster cell line K12 is temperature-sensitive for the initiation of DNA synthesis. K12 cells synchronized by serum deprivation were collected in early G1(G0). Heterokaryons were formed by fusing chick erythrocytes with serum-starved K12 cells through the use of UV-irradiated Sendai virus. At the permissive temperature (36.5 degrees C), erythrocyte nuclei in heterokaryons enlarged, the chromatin dispersed, and erythrocyte nuclei synthesized DNA at about the same time as the K12 nuclei. At the restrictive temperature (41 degrees C), erythrocyte nuclei enlarged, but neither erythrocyte nor K12 nuclei initiated DNA synthesis. When erythrocyte nuclei were fused with Wg-1A cells, the wild-type parent for ts K12 cells, both kinds of nuclei synthesized DNA at 36.5 degrees C and 41 degrees C. Activation of erythrocyte nuclei was inefficient in heterokaryons incubated in low-serum medium. The results indicate that serum factors and a cellular function defined by the K12 mutation are required for activation of chick erythrocyte nuclear DNA synthesis.     

Formation and characterization of spontaneously formed heterokaryons between quail myoblasts and 3T3-L1 preadipocytes: correlation between differential plasticity and degree of differentiation

Skeletal muscle cells and adipose cells have a close relationship in developmental lineage. Our previous study has shown that the heterokaryons between quail myoblasts and undifferentiated 3T3-L1 cells (preadipocytes) normally differentiated into myotubes, whereas the heterokaryons between myoblasts and differentiated 3T3-L1 cells (adipocytes) failed myogenic differentiation. These results suggest differences between preadipocytes and adipocytes. The purpose of this study was to clarify whether preadipocytes have flexibility in differentiation before terminal adipose differentiation. Presumptive quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) and mouse 3T3-L1 cells (either preadipocytes or adipocytes) were co-cultured for 48 h under conditions allowing myogenic differentiation. On co-culture between myoblasts and undifferentiated 3T3-L1 cells, heterokaryotic myotubes formed spontaneously, but not on co-culture with differentiated 3T3-L1 cells. In addition, the heterokaryotic myotubes expressed mouse myogenin derived from the 3T3-L1 cell gene. Our previous study indicated that the fusion sensitivity of differentiating myoblasts change with decreasing cholesterol of the cell membrane during myoblast fusion. Thus we compared the level of membrane cholesterol between undifferentiated and differentiated 3T3-L1 cells. The result showed that the level of membrane cholesterol in 3T3-L1 cells increases during adipose differentiation. Corresponding to the increase in membrane cholesterol content, differentiated 3T3-L1 cells had lower sensitivity to HVJ (Sendai virus)-mediated cell fusion than undifferentiated 3T3-L1 cells. This study demonstrated that 3T3-L1 cells at an undifferentiated state have a capacity for spontaneous fusion with differentiating myoblasts following myogenic differentiation, and that the capacity is lost after terminal adipose differentiation.     

Stability of inactive X-chromosome in mouse embryoid body-mule cell and transformed mouse cell-mule cell heterokaryons

Cloned fibroblast cells of female mule derivation, expressing only the horse G6PD phenotype, were fused with established mouse cells, with spontaneously and virally transformed mouse cells, and with embryoid bodies from a transplantable mouse teratoma. Heterokaryons were harvested at various intervals after fusion, and tested for their G6PD activity patterns by electrophoresis on Cellogel sheets. No expression of the donkey G6PD phenotype in these heterokaryons could be detected, although hybrid G6PD bands formed between mouse and horse subunits were clearly visible. These observations imply that neither the cytoplasm of the embryoid bodies, nor of the DNA and RNA tumour virus-transformed mouse cells can induce depression of the G6PD locus on an inactive X-chromosome and provide further evidence for the genetic stability of the inactive X-chromosome in mammalian somatic cells.     

Globin synthesis in fibroblasts fused with erythroblasts. I. Avian fibroblasts.

Heterokaryons of chick embryo erythroblasts fused with other avian fibroblasts were studied with regard to globin production. After the incorporation of radioactive amino acids, soluble proteins were separated on SDS-urea polyacrylamide gels. There was a striking increase in radioactivity above background in the globin region from lysates of fusion cultures when compared with fibroblast cultures. This was maximal at 24 hours after fusion, and then declined. Electrophoresis on acid-or alkaline-urea gels further identified the material as globin chains. Tryptic digestion and fingerprinting revealed methionine-labeled peptides characteristic of chick embryo erythroblast globin. An apparent stimulation of globin chain synthesis by heterokaryons compared to erythroblasts was found to be due to a difference in the specific activity of the precursor amino acid pools in the different cell types.