Formation of tetraploid nuclei in regenerating rat liver]

The proliferation of binucleated cells in the liver of young Wistar rats after partial (2/3) hepatectomy was studied by means of autoradiography and cytophotometry. The analysis of the kinetics of 3H-thymidine labelled cells has shown that both the bi- and mononucleated cells proceed through the mitotic cycle and enter mitosis simultaneously. The nuclei of 2nX2 cells enter prophase simultaneously but fuse during metaphase, so that the subsequent division results in the formation of mononucleated tetraploid cells.     

Biochemical,morphological and flow cytometric evaluation of the effects of hexachlorobenzene on rat liver

This study investigated the ability of HCB (0.1% in the diet for 15 days) to cause early changes in the cellular ploidy of rat liver. Treatment caused marked hepatomegaly, increase of microsomal proteins and cytochrome P-450 content and reduction of hepatocyte microviscosity. Microscopic examination showed that the hepatocytes were enlarged, with hyaline cytoplasm and vacuoles. The size distribution of the isolated hepatocytes showed a larger percentage of bigger cells. Flow-cytometric DNA/protein analysis was performed on whole (fixed) cells and on nuclei. From the combined results of both analyses it was possible to exclude significant changes in the percentages of diploid, mononucleated tetraploid, binucleated tetraploid and octoploid hepatocytes. The DNA and protein content of each subpopulation remained unchanged. Our results suggest that HCB does not cause early diploidization of liver cells and that hepatomegaly and cytochrome P-450 induction seem not to be correlated with effects on total DNA and total protein contents.Abbreviations HCB hexachlorobenzene - PI propidium iodide - FITC fluorescein isothiocyanate - DN diploid nuclei - SN 2N-4N nuclei in S-phase - TN tetraploid nuclei - DC diploid cells - SDC 2N-4N diploid cells in S-phase - TC tetraploid cells - STC 4N-8N tetraploid cells in S-phase - OC octoploid cells - MDC mononucleated diploid cells - SMDC mononucleated diploid cells in S-phase - BOC binucleated octoploid cells - SBTC binucleated tetraploid cells in S-phase - BTC binucleated tetraploid cells - MTC mononucleated tetraploid cells.     

Effect of ionizing radiation on DNA synthesis in ataxia telangiectasia cells.

The effect of ionizing radiation on DNA synthesis in control and ataxia telangiectasia (AT) lymphoblastoid cell lines was determined. A dose dependent decrease in DNA synthesis was observed in control cells, and the rate and extent of thi decrease in synthesis increased with time after irradiation. No decrease in DNA synthesis was obtained in AT cells, immediately following irradiation, at doses up to 400 rads. At longer times postirradiation, inhibition of synthesis increased but the extent of inhibition was less in AT cell than controls at all doses used. An immediate depression of DNA synthesis was evident in control cells after a radiation dose of 200 rads reaching a maximum at 90 min postirradiation. Little or no decrease in DNA synthesis was evident in AT cells up to 60 min after the same radiation dose, but a decrease occurred between 60 and 90 min after irradiation. The rate of recovery of DNA synthesis to normal levels was more rapid in AT cells than in controls.     

Hepatocytes in heterokaryons can suppress entry into the S-period of fibroblast nuclei from murine NIH 3T3 cells

Mouse liver regeneration after partial hepatectomy can be considered as a spectacular example of controlled tissue increase. In this study serum-deprived (0.2%) resting and serum-stimulated (10%) proliferating NIH 3T3 mouse fibroblasts were fused with primary hepatocytes isolated from normal (intact) and regenerating adult mouse liver at different times after partial hepatectomy (1-15 days) to elucidate mechanisms of liver cell proliferation cessation at the regeneration end. DNA synthesis was investigated in the nuclei of heterokaryons and non-fused cells using radioautography. Hepatocytes isolated from regenerating liver within 1-12 days following operation did not retard the entry of stimulated fibroblast nuclei into the S-period. In contrast, hepatocytes isolated within 15 days after hepatectomy were found to have inhibitory effect on the entry of stimulated fibroblast nuclei into the S-period in heterokaryons. Preincubation of these hepatocytes with cyclocheximide for 2-4 h abolished their ability to suppress DNA synthesis in stimulated fibroblast nuclei in heterokaryons. Possible reasons of inhibitory effect of differentiated cells in heterokaryos are discussed. The data obtained enable us to conclude that the mechanism of proliferative process control in regenerating hepatocytes seems to be stopped being affected by the intracellular growth inhibitors, whose formation depends on protein synthesis.     

DNA synthesis in cultures of atrial and ventricular cardiomyocytes in the rat

By means of 3H-thymidine autoradiography DNA replicative activity has been studied in cultured atrial and ventricular myocytes, and non-muscle cells from hearts of 2-week-old rats (age when cell proliferation in the myocardium is already significantly depressed). PAS-reaction was used as a cytochemical marker of cardiomyocytes: atrial myocytes are richer in glycogen than ventricular cells. Labeling indices of atrial myocytes after a 24 hour exposure to 3H-thymidine were higher than ventricular ones: on day 6 of culturing--47 and 5%, and on day 11-34 and 8%, respectively. After 10 days of culturing the number of binucleated atrial myocytes, non-typical for atrial myocardium in vivo, increased by 25-40% as compared with 8-13% on days 2-3 in culture. In 10-day cultures, 3- and 4-nucleated atrial myocytes were observed. Both mononucleated and binucleated atrial and ventricular myocytes incorporated 3H-thymidine. To find out whether the deeper inhibition of replicative activity in ventricular myocytes influences fibroblasts and endothelial cells from ventricles, the proliferative activity of non-muscle cells was studied. Non-muscle cells, both in atrial and ventricular cultures, behaved as a totally proliferating population (labeling indices on the 6th day are about 75-90%) and their growth rate decreased during the formation of the contact-inhibited monolayer. These cells, contrary to myocytes, are predominantly mononucleated in all the periods studied. The deeper depression of replication in ventricular myocytes appears to be related with their higher level of differentiation as compared to myocytes of the atrial myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of DNA replication by ultraviolet light.

DNA replication in ultraviolet-irradiated HeLa cells was studied by two different techniques: measurements of the kinetics of semiconservative DNA synthesis, and DNA fiber autoradiography. In examining the kinetics of semiconservative DNA synthesis, density label was used to avoid measuring the incorporation due to repair replication. The extent of inhibition varied with time. After doses of less than 10J/m2 the rate was initially depressed but later showed some recovery. After higher doses, a constant, low rate of synthesis was seen for at least the initial 6 h. An analysis of these data indicated that the inhibition of DNA synthesis could be explained by replication forks halting at pyrimidine dimers. DNA fiber autoradiography was used to further characterize replication after ultraviolet irradiation. The average length of labeled segments in irradiated cells increased in the time immediately after irradiation, and then leveled off. This is the predicted pattern if DNA synthesis in each replicon continued at its previous rate until a lesion is reached, and then halted. The frequency of lesions that block synthesis is approximately the same as the frequency of pyrimidine dimers.     

Gene expression comparisons performed for biodosimetry purposes on in vitro peripheral blood cellular subsets and irradiated individuals

We examined the benefit of gene expression analysis on peripheral blood cellular subsets of different radiosensitivity to elucidate their utility as biodosimeters for estimation of dose in irradiated individuals. Peripheral mononucleated cells were isolated from 18 healthy volunteers employing density separation in a CPT-NH tube. Peripheral mononucleated cells were cultured in RPMI 1640 medium containing 10% autologous serum and were irradiated with 0.1-1 Gy (240 kV, 13 mA, X rays at 1 Gy/min). A low-dose study was performed with isolated peripheral mononucleated cells from one healthy donor in three independent experiments. Peripheral mononucleated cells were irradiated at 0 (sham), 1, 2.5 and 5 cGy (70 kV, 13 mA X rays at 1 cGy/min) and gene expression was measured 24 and 48 h after irradiation. After irradiation, CD4(+) or CD8(+) cells were isolated by magnetic beads in independent experiments. RNA from lymphocyte subsets and peripheral mononucleated cells was isolated after 24 and 48 h and converted into cDNA. Gene expression of GADD45, CDKN1A, DDB2, PCNA, BAX and ATF3 were determined using RTQ-PCR. Data were analyzed employing linear and logistic regression analysis. The same examinations were performed in 5 individuals either diagnosed using CT scans (up to 4.3 cGy) or by administering (F-18)-fluoro-2-deoxy-d-glucose (F-18 FDG, 0.6 cGy). Methodological, intra- and inter-individual variability in 90-95% of measurements did not exceed the introduced twofold change over sham-irradiated control values in peripheral mononucleated cells and CD4(+) cells, and therefore no false positive results were observed. Dose reconstruction in peripheral mononucleated cells in opposite to CD4(+) lymphocytes required fewer genes and appeared more efficient (R-square = 84.8% compared to 51.8%). In vitro samples exposed to 10 cGy could be completely discriminated from sham-irradiated samples without individual pre-exposure controls, which coincided with our preliminary in vivo results. However, in vitro differential gene expression was measured relative to control values and did not differ significantly at 24 and 48 h after irradiation in contrast to our preliminary in vivo data. In addition, below 5 cGy in vitro data did not show reproducible significant changes in gene expression, which was opposite to our preliminary in vivo data. Therefore a twofold change in gene expression over control sufficiently controls for different sources of variance, and measuring gene expression in peripheral mononucleated cell for biological dosimetry purposes appears superior over measurements in lymphocyte subsets. The increased gene expression measured after low absorbed doses in vivo and in vitro might indicate a particular applicability of this method for a low-level radiation scenario in the absence of individual pre-exposure controls. However, the constant gene expression values measured up to 48 h in our in vitro model at doses >10 cGy, and the absence of reproducible and statistically significant gene expression changes below 5 cGy contrast to the preliminary in vivo results performed at similar doses. Therefore, measurements with our in vitro models should be interpreted cautiously.     

An analysis of the impulse character of cellular proliferation in the livers of sexually immature mice]

The quantitative ratio between labelled pre-and postmitotic nuclei of hepatocytes was studied 8 hr after H-3-thymidine administration in the 12-15 g mice. The karyoautoradiographic analysis showed that DNA-synthetizing cells are concentrated at a certain stage of S-period. This indicates the complete synchronization of DNA synthesis in the liver parenchyma. The times during which all the hepatocytes enter the S-period did not exceed 4 hr.     

DNA damage and repair induced in vitro by nitrilotriacetic acid (NTA) in human lymphocytes

In cultured human lymphocytes we determined the ability of nitrilotriacetic acid (NTA) to inhibit DNA replication and to stimulate DNA repair synthesis (UDS), as well as to influence the UDS induced by UV irradiation. In phytohemagglutinin-stimulated lymphocytes a strong inhibition of DNA replication was induced by NTA concentrations above 10(-3) M, which was accompanied by a marked cell lethality, whereas at lower doses the incorporation of tritiated thymidine (3H-TdR) into DNA or treated cells was slightly increased in comparison to untreated cells. When, after NTA pretreatment, UDS was determined by scintillation spectrometry or autoradiography in unstimulated G0 lymphocytes, UV-irradiated or unirradiated, an increased incorporation of 3H-TdR was observed, positively correlated with the NTA doses. This effect was only partially due to the expansion of the intracellular TdR pool as a consequence of the stimulation of 3H-TdR uptake by NTA. Even after normalization of the scintillometric data by the radioactivities of the soluble nucleotide fraction, significant increase of DNA repair synthesis was detected after treatment with 7.5 x 10(-3)-10(-2) M NTA.     

The effect of cycloheximide on the entry into the S period of the nuclei in heterodikaryons]

Serum-deprived (0.2%) resting NIH 3T3 mouse fibroblasts were fused with serum-stimulated (10%) proliferating cells to elucidate mechanisms of entering into S-period operating in the nuclei of the heterokaryons under the effect of cycloheximide--an inhibitor of protein synthesis. Using radioautography DNA synthesis was investigated in mono-, homo- and heterodikaryons. After short (0.5-3.0 h) depressing of protein synthesis, the nuclei of stimulated cells in heterokaryons were found to enter into S-period. Under these conditions no induction of DNA synthesis was found in the nuclei of resting cells in heterodikaryons. In other experiments, resting cells were under the effect of cycloheximide during 2-4 h before the fusion, that led to a great induction of DNA synthesis in the nuclei of these cells in heterodikaryons. The data obtained are consistent with the idea of fibroblast transition to the rest under the action of labile proteins-repressors.     

The recovery of mammalian cells treated with methyl methanesulfonate, nitrogen mustard or UV light. I. The effect of alkylation products on DNA replication.

CHO cells were synchronized in G1 phase and treated with MMS or HN2. The subsequent rate of DNA replication was found to be reduced in a dose-dependent manner. In addition, 2 X 10(-3 M and 3 X 10(-3) M MMS resulted in a 3--4 h delay prior to the initiation of S phase. If the cells were held for 8 h in hydroxyurea after MMS treatment, no subsequent lag in DNA synthesis was seen after removal of the hydroxyurea. The entry of confluent cells into S phase was found to be delayed 7 h upon trypsinizing and replating. Treatment of these cells with MMS resulted in a reduced rate of DNA replication, but no further delay in its initiation. Repair replication was found to continue at a constant rate for at least 12 h following MMS treatment of cells under all of these conditions. At the concentrations used in these experiments MMS severely inhibited the rate of protein synthesis, but HN2 had little effect. By comparing both the kinetics of repair replication and recovery of protein synthesis with the rate of DNA replication, it was concluded that the initial, severe reduction in rate following MMS treatment was probably due to an inhibition of protein synthesis.     

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.     

DNA and histone synthesis rate change during the S-period in Ehrlich ascites tumor cells

Data from flow-cytometric analysis of DNA of Ehrlich ascites tumor cells were fitted using non-linear least squares curve fitting routines. Analysis of rates of synthesis from the derived S-period profiles revealed a pattern of changing rates of DNA synthesis during the S-period. Three main peaks are seen whose trough to through periods range from 0 to 16%, 16 to 65%, 65 to 100% of the DNA synthesized during S. The differences between the peak rates and rates in the intervening troughs are small, about 10% of the maximum, but these occur reproducibly. Some differences in the DNA distribution profiles, hence rate profiles, can be seen among samples taken at different times during the day. These are thought to reflect the effects of circadian rhythms, but they are not large enough to obscure the general pattern of rate shifts that occur during the S-period. Analyses of radioactivity of ³H-thymidine pulse labelled cells, sorted across the S-period, were in accord with the results obtained from the DNA distributions. A parallel analysis of DNA and histones showed a correspondence in the timing and direction of shifts in rate for both during the middle part of the S-period.     

A comparison of N-methyl-N-nitrosourea-induced chromatid aberrations and micronuclei in barley meristems using FISH techniques

Individuals can be exposed to high doses (more than 5Gy) during radiation accidents. It is, of course, helpful to the physician to have biological indicators also for such high doses. The problem with most cytogenetic indicators is, that the response levels off at doses starting around 5-7Gy of low LET radiation and that the dose-response curve even declines after doses exceeding about 10Gy. Thus, it may be difficult to decide, whether the dose was, for example, 8 or 14Gy. We studied how the micronucleus assay can be used to give information also in the high dose range. It turned out that micronucleus frequency itself cannot be used for the estimation of doses exceeding about 5-7Gy. There are, however, at least three other endpoints that can be determined in the cytochalasin B assay that can assist the decision in the high dose range: (1) the number of mononucleated cells; (2) the ratio of tri- to tetranucleated cells; (3) the average micronucleus frequency in micronucleus positive binucleated cells.     

Single-cell analysis of ploidy and centrosomes underscores the peculiarity of normal hepatocytes

Polyploidization is the most well recognized feature of the liver. Yet, a quantitative and behavioral analysis of centrosomes and DNA content in normal hepatocytes has been limited by the technical challenges of methods available. By using a novel approach employing FISH for chromosomes 18, X and Y we provide, for the first time, a detailed analysis of DNA copies during physiological development in the liver at single cell level. We demonstrate that aneuploidy and unbalanced DNA content in binucleated hepatocytes are common features in normal adult liver. Despite the common belief that hepatocytes contain 1, 2 or no more than 4 centrosomes, our double staining for centrosome associated proteins reveals extranumerary centrosomes in a high percentage of cells as early as 15 days of age. We show that in murine liver the period between 15 days and 1.5 months marks the transition from a prevalence of mononucleated cells to up to 75% of binucleated cells. Our data demonstrate that this timing correlates with a switch in centrosomes number. At 15 days the expected 1 or 2 centrosomes converge with several hepatocytes that contain 3 centrosomes; at 1.5 months the percentage of cells with 3 centrosomes decreases concomitantly with the increase of cells with more than 4 centrosomes. Our analysis shows that the extranumerary centrosomes emerge in concomitance with the process of binucleation and polyploidization and maintain α-tubulin nucleation activity. Finally, by integrating interphase FISH and immunofluorescent approaches, we detected an imbalance between centrosome number and DNA content in liver cells that deviates from the equilibrium expected in normal cells. We speculate that these unique features are relevant to the peculiar biological function of liver cells which are continuously challenged by stress, a condition that could predispose to genomic instability.     

The cell composition in the lymphocyte population and the radiation adaptive response

Using lymphocytes of 5 healthy individuals the ability to adaptive response (AR), cell composition of population after PHA stimulation, changes in cell composition population after irradiation in the dose of 1.0 Gy and after irradiation in adaptive (0.05 Gy) and challenge (1.0 Gy) doses have been studied. AR observed in 2 of the 5 individuals only. After PHA stimulation the persons with AR have the total amount of cells after mitosis or during mitosis (the number of binucleated cells + the number of multinucleated cells + the whole cells with micronuclei + the number of mitotic cells) on average is higher than in persons without AR. In individuals with AR the linear correlation between the number of binucleated cells with micronuclei (on the 1000 scored binucleated cells) and the part of binucleated cells in the population is observed with coefficients of correlation -0.89 and -0.91. In the humans without AR this correlation is absent. The correlation observed permits to suppose that AR may occur at the expense of not only the decrease in number of damaged lymphocytes, but also the increase in the share of not damaged binucleated cell with the stable number of damaged cells.     

Arginase as one of the inhibitory principles in the density-dependent as well as plasma membrane-mediated inhibition of liver cell growth in vitro

Liver cells isolated from the adult rat livers under mild conditions were preincubated for 1 day with Williams medium E (WE) containing serum, dexamethasone and insulin, and then the cells (monolayered) were incubated for 2-3 days with WE (1 ml) containing only insulin to measure DNA synthesis and/or mitosis. DNA synthesis of cultured liver cells was dependent on cell densities within a region from 0.1 X 10(6) to 1.0 X 10(6) nuclei/dish (Falcon, diameter 35 mm). The addition of EGF from the beginning of preincubation stimulated DNA synthesis (or replication) as well as cell proliferation in vitro, but the density-dependent inhibition of DNA synthesis was observed similarly in the presence of EGF. In contrast to the low and high density cultures, DNA synthesis in the intermediary density cultures was enhanced by enlarging the medium volume or by adding ornithine (arginase inhibitor). DNA synthesis in low density cultures was inhibited by liver plasma membranes in a concentration-dependent fashion. The inhibition of DNA synthesis by liver plasma membranes in low concentrations (less than 30 micrograms protein/ml) was reduced by adding either extra arginine or ornithine. DNA synthesis of cultured liver cells (low density) was inhibited by replacing arginine in WE with equimolar ornithine and urea or by adding a commercial arginase (bovine liver). These, together with earlier findings indicating the presence of arginase in liver plasma membranes (outer leaflet), seem to support the idea that arginase may be involved in density-dependent as well as plasma membrane-mediated inhibition of DNA synthesis of cultured liver cells. However, this does not exclude possible involvement of other inhibitory principle(s), such as direct cell-to-cell or cell-to-plasma membrane interactions, especially in higher cell densities or larger plasma membrane concentrations.     

Escape from X-ray-induced arrest for lens cells stimulated from quiescence: time relationship to RNA, protein, and DNA synthesis

Quiescent cells of the central zone region of the rat lens epithelium were stimulated to enter the proliferation cycle by wounding. RNA synthesis and a corresponding increase in poly(A)+/total RNA reached a peak by Hour 4. Cells progressed into the G1B compartment by Hour 10. A rise in protein synthesis began at Hour 8, and onset of DNA synthesis occurred by Hour 14. The timing of cell cycle progression that allowed escape from a dose of X irradiation that completely inhibited DNA synthesis was investigated. A growth-arrest point was identified at Hour 9 where 10 GY of X irradiation given before, but not after, completely inhibited earliest responding cells from entering DNA synthesis on schedule. Increased quantities of cells entered DNA synthesis on schedule as timing of the X irradiation was moved closer to the end of G1. Based on time relationships, the rise in protein synthesis is correlated with the "sufficient" event for the escape.     

Effects of carcinogen treatment on rat liver DNA synthesis in vivo and on nascent DNA synthesis and elongation in cultured hepatocytes

One objective of this study was to determine the effects of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) treatment on DNA synthesis in regenerating rat liver. Rats were subjected to a two-thirds hepatectomy followed 20 h later by i.p. injection of N-OH-AAF. 4 h after carcinogen injection, it was found that N-OH-AAF caused a dose-dependent inhibition of [3H]thymidine incorporation into liver DNA. This inhibition was followed by a gradual, but incomplete recovery beginning 28 h after carcinogen treatment. Radioimmunoassay of deoxyguanine-C8 adducts remaining in liver DNA indicated that the recovery began prior to detection of adduct removal. The second objective of the study was to determine the effects of DNA damage on the size distribution and elongation of nascent hepatocyte DNA. Hepatocytes, which have been shown to demonstrate a pattern of inhibition and subsequent recovery of DNA synthesis following UV irradiation similar to that seen in vivo upon treatment with N-OH-AAF (Zurlo and Yager, 1984), were cultured under conditions that promote replicative DNA synthesis. The size distribution of nascent DNA after UV irradiation was determined by pH step gradient alkaline elution analysis. [3H]Thymidine pulse times and subsequent chase times were adjusted to equalize amounts of DNA synthesis in control and UV-irradiated cells. The results show that UV irradiation caused a dose-dependent decrease in the size distribution of nascent DNA suggesting an inhibition of elongation. Pulse-chase studies revealed that subsequent joining of nascent chains in UV-irradiated hepatocytes occurred at a rate comparable to or faster than controls and that this could be inhibited by caffeine. The results obtained from both the in vivo and in vitro studies show that resumption of DNA synthesis and nascent strand elongation occur on damaged templates. These observations along with our previous studies demonstrating the ability of UV-irradiated hepatocytes to carry out enhanced reactivation of UV-irradiated herpes virus lend support to the idea that DNA damage leading to inhibition of DNA synthesis may induce SOS-type processes which if mutagenic may play a role in the initiation of carcinogenesis.     

Mononucleated and binucleated cardiomyocytes in left atrium and pulmonary vein have different electrical activity and calcium dynamics

Cardiomyocytes consist of single- and bi-nucleus myocytes. However, the electrophysiological characteristics of mononucleated and binucleated myocytes have never been elucidated. Left atrium (LA) and pulmonary veins (PVs) are important substrate and initiators of atrial fibrillation. The purposes of this study were to evaluate the electrical properties and calcium homeostasis in mononucleated and binucleated cardiomyocytes in the LA and PVs. A whole-cell clamp, fluo-4 fluorescence, and immunocytostaining were used to investigated mononucleated and binucleated cardiomyocytes in the LA and PVs. Both mononucleated PV and LA cardiomyocytes had more positive resting membrane potential than respective PV and LA binucleated cardiomyocytes. Additionally, mononucleated PV cardiomyocytes (n = 36) had faster beating rates (2.1 ± 0.2 Hz versus 1.0 ± 0.2 Hz, P < 0.05) than binucleated (n = 10) PV cardiomyocytes. The PV (n = 18) and LA (n = 15) mononucleated cardiomyocytes had larger [Ca2?](i) transients (F/F? 1.64 ± 0.09 versus 1.20 ± 0.03 IU, P < 0.05; 1.52 ± 0.06 versus 1.19 ± 0.05 IU, P < 0.05) than the binucleated PV (n = 10) and LA (n = 10) cardiomyocytes. The immunostaining showed that mononucleated cardiomyocytes had lower Kir 2.3 and higher ryanodine receptor densities than did binucleated cardiomyocytes both in the PV and LA. In conclusions, mononucleated PV and LA cardiomyocytes contain distinctive electrophysiological characteristics with a higher arrhythmogenic activity, which indicates that cell nucleus number may potentially determine the electrical activity and calcium handling in cardiomyocytes.