Paradoxical relationship between protein content and nucleolar activity in mammalian cardiomyocytes.

It was recently demonstrated that polyploidization of the avian myocardium is associated with a reduction of cardiac aerobic capacity evaluated by the heart mass to body mass ratio (heart index). To investigate possible cellular correlates of polyploidization, the protein content and nucleolar activity per cell and per genome were examined by image cytometry in 21 mammalian species, differing in the degree of heart polyploidization and heart index. We found that average cardiomyocyte ploidy level correlates negatively with the animal heart index (r = -0.75, p < 10(-4)), i.e., the large heart of athletic mammals is polyploidized to a lesser degree than the relatively smaller heart of sedentary species, which confirms the picture observed in birds. The protein content per genome decreased with the elevation of cardiomyocyte ploidy level. This inverse correlation was especially pronounced with the removed effect of body mass (r = -0.79, p < 10(-4)). Surprisingly, these changes were accompanied by the increase of nucleolar activity per genome (r = 0.61, p < 10(-3)). In the two species, for which the microarray gene expression data were available (human and mouse), this increase was paralleled by the elevated expression of ribosomal protein genes (but there was no increase in the expression of tissue-specific genes). Thus, in the polyploid cardiomyocytes there is a misbalance between protein content per genome and ribosome biogenesis. The reduction of protein content (per genome) of polyploid cardio my ocytes should further curtail heart functionality (in addition to reduction of heart index), because it is known that cardio myocyte protein content consists of more than 90% contractile proteins. This finding makes doubtful a widespread notion that polyploidization is necessary for cell function. Because somatic polyploidization is associated with stressful conditions and impaired energetics, we suppose that additional genomes can serve for cell regeneration and as a defense against oxidative damage in the organs that work at the limit of their metabolic capacity.     

Ploidies of cardiomyocytes in human myocardial hypertrophy]

DNA cytophotometry has been performed in ventricular cardiomyocytes of hypertrophic human hearts. In the cases of hypertrophy in adults (generalized atherosclerosis, postinfarct scars), polyploidy expression did not exceed the limits of normal variability developed during childhood. In the cases of hypertrophy caused by congenital heart defects, high polyploidy has been revealed (the mean level 20c and more, where c is haploid DNA content), which considerably exceeded the upper limit of normal variability (approximately 10c). Our hypothesis has confirmed that heart hypertrophy in adults proceeds in conditions of stable genome rather than due to redundant polyploidization of the ventricular myocytes. The same idea assumes enhanced polyploidization of the myocytes in childhood in humans with congenital heart diseases.     

Biological significance of liver cell polyploidy: an hypothesis

The liver cell polyploidy phenomenon, a characteristic of many species of mammals, is reviewed. The liver parenchyma of adult animals represents a mixed population of mononuclear and binuclear cells with different number of chromosome sets and, therefore DNA content per nucleus. The polyploid hepatocytes are formed during postnatal liver growth as a result of a change from normal mitoses to polyploidizing ones. Hence, the polyploidization of hepatocytes is regarded as an equivalent of cell multiplication.An hypothesis of the biological significance of liver cell polyploidy is based on the fact of a high level of spontaneous chromosome aberrations in mitotic hepatocytes. Ploidy increase is known to give resistance against different kinds of genome alteration. Polyploidization of the liver cells ensures protection against deleterious consequences of the aberrant genome formation resulting from aberrant mitoses.Some implications of the hypothesis are discussed: the reasons for species-specific differences of liver cell polyploidy; the mechanisms of hepatocyte radioresistance; the relation of polyploidy to liver cell aging. The prerequisite factors for unbalanced cell genome formation are adduced: DNA and chromosome damage as the first step in the process, stimulation of mitosis as the second one. The aberrant polyploid genome of hepatocytes is assumed to be the cytogenetic basis for some chronic liver diseases in man.     

Cellular aspects of pathogenesis of hypertrophic cardiomyopathy: Role of cardiomyocyte polyploidy and activation of nuclear antigen of the proliferating cell in myocardium

For a comparative analysis of cytomorphological characteristics of hypertrophied interventricular septum (IVS), both patients different ages with severe courses of obstructive hypertrophic cardiomyopathy (OHCMP) were examined, including children, and patients with essential arterial hypertension (EAH). The course of OHCMP in children as compared with adults was found to be characterized by considerable IVS hypertrophy that was accompanied by an acceleration of cardiomyocyte polyploidization. The mean ploidy level of cardiomyocytes in children with OHCMP was higher than in adult patients. The mean ploidy level of nuclei, the number of prolipherative cell nuclear antigen (PCNA)-positive nuclei, and the number of polyploid cardiomyocyte nuclei in adult patients with OHCMP were significantly higher statistically than in patients with EAH. The PCNA-positive labels in stromal cells were revealed only in patients with OHCMP. The obtained data indicate an important role of cardiomyocyte polyploidy and of activation of the proliferating cell nuclear antigen in development of myocardial hypertrophy in patients with OHCMP.     

植物多倍体基因组的形成与进化

多倍化是植物进化变异的自然现象,也是促进植物发生进化改变的重要力量。在被子植物中,约 70％的种类在进化史中曾发生过一次或多次多倍化的过程。目前的研究结果表明,自然界绝大多数多倍体是通过未减数配子的融合而形成的,并且很多多倍体种是通过多次独立的多倍化过程而重复发生的。由多倍化所导致的重复基因在多倍体基因组中可能有三种不同的命运,即：保持原有的功能、基因沉默或分化并执行新的功能。多倍化以后,重复基因组的进化动态则主要表现在染色体重排和“染色体二倍化”、不同基因组之间的相互渗透、以及核-质之间的相互作用等方面。     

Methodological aspects of flow cytometric analysis of DNA polyploidy in human heart tissue

The purpose of the study was to investigate the possibilities of flow cytometry (FCM) for the analysis of DNA polyploidy in human heart tissue. Suspensions of single nuclei were prepared with the detergent-trypsin procedure and stained with propidium iodide. A mathematical correction procedure was developed to correct for background and clumping. For diploid model populations of chicken and trout red blood cells this correction reduced artifactual fractions in the FCM DNA profile to less than 0.5%, indicating that interference by background and clumping was almost completely overcome by this correction procedure. FCM DNA profiles were obtained from 12 hypertrophic and 7 normal human adult hearts. Clear differences were found between DNA profiles from the normal and the hypertrophic hearts, the latter showing a higher degree of polyploidization. From the corrected DNA profiles, six different polyploidization parameters were computed, all of which showed a significant correlation with at least three out of four different parameters for heart hypertrophy. FCM appears to be a reliable method for the measurement of polyploidization in heart tissue, provided background and clumping are corrected for.     

Polyploidization and endomitosis in giant cells of rabbit trophoblast

Morphological and cytophotometric investigations have been performed on giant cells of the rabbit trophoblast to reveal a mechanism of nuclei polyploidization and define the level of polyploidy. The character of endomitotic chromosomes is found to differ and depend largely on the degree of nuclei polyploidy. Small chromosomes were found in nuclei with low levels of polyploidy. For highly polyploid nuclei, two stages are distinguished. In the first case condensed chromosomes join into bundles resembling Riesenchromosomen in plants, whereas in the second, decondensed chromosomal threads separate and disperse in the karyoplasm. The splitting does not involve nuclei-forming chromosomes in the region of the nucleolar organiser. The degree of polyploidy was determined on the 15th day of development. It was found that giant cell nuclei contain DNA in amounts corresponding to 32-512 chromosomal sets. Most of the nuclei have levels of 128c and 256c. Highly-polyploid nuclei disintegrate into small nuclei with the degree of polyploidy varying from 1c to 32c. Di- tri- and tetraploid nuclei predominate.     

Methodological aspects of flow cytometric analysis of DNA polyploidy in human heart tissue

Summary The purpose of the study was to investigate the possibilities of flow cytometry (FCM) for the analysis of DNA polyploidy in human heart tissue. Suspensions of single nuclei were prepared with the detergenttrypsin procedure and stained with propidium iodide. A mathematical correction procedure was developed to correct for background and clumping. For diploid model populations of chicken and trout red blood cells this correction reduced artifactual fractions in the FCM DNA profile to less than 0.5%, indicating that interference by background and clumping was almost completely overcome by this correction procedure. FCM DNA profiles were obtained from 12 hypertrophic and 7 normal human adult hearts. Clear differences were found between the DNA profiles from the normal and the hypertrophic hearts, the latter showing a higher degree of polyploidization. From the corrected DNA profiles, six different polyploidization parameters were computed, all of which showed a significant correlation with at least three out of four different parameters for heart hypertrophy. FCM appears to be a reliable method for the measurement of polyploidization in heart tissue, provided background and clumping are corrected for.In honour of Prof. P. van Duijn     

Morphofunctional parameters of the nucleoli during development of polyploid nutrient cells of gonads from the snail Succinea lauta

Ag-protein contents, integral area and number of nucleoli in polyploidizing nuclei of gonadal nutrient cells of the snail Succinea lauta were estimated on the squashed preparations by means of morphometry and cytophotometry. 8 NORs of different size were found in haploid chromosome set of prophase spermatocytes (n = 22), but usually 1-2 nucleoli per 2c DNA are present in the nutrient cell nuclei. During genome multiplication from 2c to 32c-64c the Ag-proteins content of nucleoli increased proportionally to gene dosage, but irregularly: before 8c-level the coefficient of increasing in each endocycle was more than 2; from 8c to 16c it was 2; after 16c-level it usually decreased to 1.6-1.3. This dynamics reflects the effects of several factors on nucleolar activity: endomitotic polyploidy (gene dosage effect), differentiation and rhythmic functioning of tissue. Increasing indexes of integral area and the number of nucleoli during polyploidization were significantly less, than increasing index of Ag-proteins. The lag of nucleolar area for 4 cycles (2c-32c) was 32%, and number of nucleoli per diploid set decreased from 2 to 1. It may be due to NOR aggregation corresponding chromosomes. The photometric index of Ag-protein content more adequately reflects in the nucleolar activity during development and functioning of tissues.     

Dexamethasone Treatment of Newborn Rats Decreases Cardiomyocyte Endowment in the Developing Heart through Epigenetic Modifications

The potential adverse effect of synthetic glucocorticoid, dexamethasone therapy on the developing heart remains unknown. The present study investigated the effects of dexamethasone on cardiomyocyte proliferation and binucleation in the developing heart of newborn rats and evaluated DNA methylation as a potential mechanism. Dexamethasone was administered intraperitoneally in a three day tapered dose on postnatal day 1 (P1), 2 and 3 to rat pups in the absence or presence of a glucocorticoid receptor antagonist Ru486, given 30 minutes prior to dexamethasone. Cardiomyocytes from P4, P7 or P14 animals were analyzed for proliferation, binucleation and cell number. Dexamethasone treatment significantly increased the percentage of binucleated cardiomyocytes in the hearts of P4 pups, decreased myocyte proliferation in P4 and P7 pups, reduced cardiomyocyte number and increased the heart to body weight ratio in P14 pups. Ru486 abrogated the effects of dexamethasone. In addition, 5-aza-2''-deoxycytidine (5-AZA) blocked the effects of dexamethasone on binucleation in P4 animals and proliferation at P7, leading to recovered cardiomyocyte number in P14 hearts. 5-AZA alone promoted cardiomyocyte proliferation at P7 and resulted in a higher number of cardiomyocytes in P14 hearts. Dexamethasone significantly decreased cyclin D2, but not p27 expression in P4 hearts. 5-AZA inhibited global DNA methylation and blocked dexamethasone-mediated down-regulation of cyclin D2 in the heart of P4 pups. The findings suggest that dexamethasone acting on glucocorticoid receptors inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via increased DNA methylation in a gene specific manner.     

多倍体植物中基因表达模式的变化

植物杂交和多倍化能导致基因组结构发生变化,并显著影响了基因表达,因此认为杂交和多倍化是促进植物进化的一个重要力量。近些年大量的研究表明植物多倍化后基因表达模式发生了复杂的改变,包括基因沉默、基因表达的基因组偏向性及组织特异性、基因激活等现象,本文对这些现象及其特点和机制进行了综述。     

Depression of proteasome activities during the progression of cardiac dysfunction in pressure-overloaded heart of mice

The ubiquitin-proteasome system contributes to regulation of apoptosis degrading apoptosis-regulatory proteins. Marked accumulation of ubiquitinated proteins in cardiomyocytes of human failing hearts suggested impaired ubiquitin-proteasome system in heart failure. Since cardiomyocyte apoptosis contributes to the progression of cardiac dysfunction in pressure-overloaded hearts, we investigated the role of ubiquitin-proteasome system in such conditions. We found that proteasome activities already depressed before the onset of cardiac dysfunction in pressure-overloaded hearts of mice. Cardiomyocyte apoptosis was observed along with depression of proteasome activities and elevation of proapoptotic/antiapoptotic protein ratio in failing hearts. In cultured cardiomyocytes, pharmacological inhibition of proteasome accumulated proapoptotic proteins such as p53 and Bax. Gene silencing of these proapoptotic proteins by RNA interference prevented the accumulation of respective proteins and attenuated cardiomyocyte apoptosis induced by proteasome inhibition. We conclude that depression of proteasome activities contributes to cardiac dysfunction resulting from cardiomyocyte apoptosis through accumulation of proapoptotic proteins by impaired degradation.     

Cardiomyocyte ploidy levels in birds with different growth rates

Cytofluorimetric study of ploidy levels in ventricular cardiomyocytes was carried out on 36 adult bird species belonging to 10 orders as well as on the quail Coturnix coturnix, of different ages. It was shown that polyploidization of quail cardiomyocytes occurs during the first 40 days after hatching and ends by the time growth is completed. In adult birds, the cardiomyocyte ploidy hardly changed at all. Interspecies comparison revealed that in the adult bird myocardium 2cx2 myocytes are predominant, accounting for at least 50% of the cell population. Multinuclear cells with three to eight diploid nuclei were widespread. The percentage of such cells was five to six times higher in precocial species than in altricial birds of the same weight. Myocytes with polyploid nuclei were rare. A significant interspecies variability of cardiomyocyte ploidy levels was observed. The most prominent differences were found between the precocial and the altricial birds. The mean number of genomes in cells correlated both with the body mass and with the growth rate of the birds. The differences between the precocial and altricial birds disappeared when a statistical method was used to eliminate the effect of the growth rate, but did not when the effect of body mass was eliminated. Among the altricial birds, which are generally immobile during growth, the cardiomyocyte ploidy levels also correlated more closely with growth rate than with body mass. The opposite was observed in the precocial birds, which are highly mobile from the first minutes of life. We conclude that the interspecies variability of bird cardiomyocyte ploidy levels is a result of changes in the balance between the cardiac functional load and the growth rate; this is manifested at the cellular level as a competition between the proliferation and differentiation of cardiomyocytes. J. Exp. Zool. 289:48-58, 2001.     

Both Nucleolar Organizers Are Replicated in Dipteran Polyploid Tissues: A Study at the Level of Individual Nuclei

Working with the Dipteran Calliphora erythrocephala, we have tested the hypothesis that only one nucleolar organizer region (NO) is replicated during polyploidization. NO replication was examined in two very different highly polyploid nuclear types: salivary gland nuclei and nurse cell nuclei. Two strains of the organism containing NO regions with highly diagnostic nontranscribed spacer (NTS) polymorphisms were prepared and reciprocal single pair-matings between members of the strains were performed. The representation of the two distinguishable NOs in diploid and polyploid DNAs of individual F1 progeny from each cross was then examined. DNA from a total polyploid nuclear DNA preparation and from individual polyploid nuclei of both tissue types was analyzed. Our results show conclusively that both genomic NOs are replicated in individual polyploid nuclei of both types. Further, evidence for variation in the relative replication of cistrons from the two NOs by individual nuclei was obtained. The cistron types present in the NOs of both strains showed differential replication upon polyploidization. In general, the patterns of differential cistron replication seen in salivary gland and nurse cell nuclei were similar.     

Upregulation of Nox4 in the aging vasculature and its association with smooth muscle cell polyploidy

Our recent reports indicated that polyploidization of aortic vascular smooth muscle cells (VSMC) serves as a biomarker for aging, and that the polyploid state is linked to a higher incidence of senescence in vivo. Here, we found that NADPH oxidase 4 (Nox4) expression is augmented in VSMC from aortas of old rats and that Nox4 levels are increased in polyploid VSMC in comparison to diploid cells in vivo. Seeking to determine if Nox4 upregulation plays a causal role in the accumulation of polyploid cells, we performed ploidy analysis on primary VSMC transduced with Nox4 adenovirus. We observed a consistent accumulation of polyploid cells and a concomitant decrease in the percentage of diploid cells in Nox4 overexpressing cells in comparison to controls or to cells overexpressing dominant negative Nox4. Further exploration of this phenomenon in VSMC cultures identified a Nox4-induced decrease in the chromosome passenger protein, survivin, whose absence and mislocalization during polyploidization was previously shown to induce VSMC polyploidy. Taken together, our study is the first to show increased Nox4 levels in VSMC during aging, and to demonstrate its role in induction of polyploidy in this lineage.     

Morphological and cytofluorometric study of the giant cells of the trophoblast of the common vole]

The primary and secondary giant cells of trophoblast in placenta Microtus arvalis were studied. The giant polyploid nuclei are formed in result of series of successively proceeding endomitotic polyploidization of chromosomes. Two stages of endomitosis are described: endointerphase with the uniform net of thin chromatin threads and the stage when small round or rod-shaped paired chromosomes gather mostly under the nuclear membrane. Great number of round, oval, and complex-shaped nucleoli may be seen in nuclei during both stages of endomitosis, the number growing during polyploidization. The morphology of the chromosome-nucleolar apparatus involves peculiarities of the polyploidization mechanism in placenta Microtus arvalis trophoblast. Endomitosis occurs both in low and high-polyploid nuclei. Cytofluorometric determination of the DNA amount in nuclei polyploid nature. The degree of polyploidy of the trophoblast giant cells nuclei during terminal differentiation of placenta corresponds to 128c-512c, and some nuclei contain the DNA amount corresponding to 1024 and 2048 chromosomal sets. The cause of origin of the polyploid cells in trophoblast of rodents placenta is discussed.     

Heart-type fatty acid binding proteins are upregulated during terminal differentiation of mouse cardiomyocytes,as revealed by proteomic analysis

At birth, the cardiomyocytes in the mouse neonatal heart still retain their ability to proliferate. However, this lasts only a few days and then the cardiomyocytes irreversibly lose their potential to divide. It is still not fully understood what factors are involved in the cessation of cardiomyocyte proliferation. Using proliferating cell nuclear antigen (PCNA) antibodies, we established that cardiomyocytes could divide extensively in 2-day-old mouse neonatal hearts and to a lesser extent in 6-day-old hearts. By 13 days, the cardiomyocytes have mostly stopped dividing. Comparative two-dimensional gel electrophoresis (2-DE) was performed on total proteins extracted from the 2-day- and 13-day-old hearts, in order to identify peptides that might be involved in the inhibition of cardiomyocyte proliferation. Using matrix-assisted laser desorption ionization mass spectroscopy (MALDI-TOF), we identified two protein spots that have the same molecular weight (approximately 14 kDa) but different pIs (5.9 and 6.1). Mass spectra analysis determined the proteins to be isoforms of the heart-type fatty acid binding protein (H-FABP). The pI 6.1 H-FABP is also known as mammary-derived growth inhibitor (MDGI; Specht et al. 1996). MGDI is a breast tumour growth suppressor gene capable of inhibiting tumour cell proliferation (Huynh et al. 1995). Both H-FABP isoforms were expressed in 2-day-old hearts but became strongly upregulated in 13-day-old hearts. We examined whether H-FABPs and PCNA were coexpressed in 2-, 6- and 13-day-old heart histological sections, using MDGI antibodies. The antibody could detect both forms of H-FABPs. It was established that there was a correlation between an increase in H-FABP expression and a decrease in PCNA expression. Hence, we tentatively propose that H-FABP isoforms are involved in regulating cardiomyocyte growth and differentiation in mouse neonatal hearts.This project was supported by a grant from the National Natural Science Foundation of China (Project No. 30340038).     

Polyploidizing mitoses and the biological meaning of polyploidy in liver cells]

The ontogenetic polyploidization of hepatocytes is regarded, within which normal mitoses are changed to polyploidizing mitoses, and diploid hepatocytes transform into polyploid mono- and binuclear cells. A new hypothesis is put forward of the biological significance of the liver cell polyploidy. The hypothesis takes into account a high level of spontaneous chromosomal aberrations in mitotic hepatocytes. The chromosome structural changes interfere with mitosis resulting in the chromosomal imbalance. Polyploidy bestows for hepatocytes a tolerance towards a chromosomal imbalance. Some implications of the hypothesis are discussed: unbalanced genome of hepatocytes after the treatment with mutagens and mitotic stimulators; the reasons of liver cell polyploidy differences in mammalian species; mechanisms of radioresistance of hepatocytes. Chromosomal imbalance of polyploid hepatocytes is assumed to be the basis for wome chronic liver diseases in man.