Olga V. Anatskaya

Genome size and metabolic intensity in tetrapods : a tale of two lines

We show the negative link between genome size and metabolic intensity in tetrapods, using the heart index (relative heart mass) as a unified indicator of metabolic intensity in poikilothermal and homeothermal animals. We found two separate regression lines of heart index on genome size for reptiles–birds and amphibians–mammals (the slope of regression is steeper in reptiles–birds). We also show a negative correlation between GC content and nucleosome formation potential in vertebrate DNA, and, consistent with this relationship, a positive correlation between genome GC content and nuclear size (independent of genome size). It is known that there are two separate regression lines of genome GC content on genome size for reptiles–birds and amphibians–mammals: reptiles–birds have the relatively higher GC content (for their genome sizes) compared to amphibians–mammals. Our results suggest uniting all these data into one concept. The slope of negative regression between GC content and nucleosome formation potential is steeper in exons than in non-coding DNA (where nucleosome formation potential is generally higher), which indicates a special role of non-coding DNA for orderly chromatin organization. The chromatin condensation and nuclear size are supposed to be key parameters that accommodate the effects of both genome size and GC content and connect them with metabolic intensity. Our data suggest that the reptilian–birds clade evolved special relationships among these parameters, whereas mammals preserved the amphibian-like relationships. Surprisingly, mammals, although acquiring a more complex general organization, seem to retain certain genome-related properties that are similar to amphibians. At the same time, the slope of regression between nucleosome formation potential and GC content is steeper in poikilothermal than in homeothermal genomes, which suggests that mammals and birds acquired certain common features of genomic organization.

Organismal complexity, cell differentiation and gene expression: human over mouse

We present a molecular and cellular phenomenon underlying the intriguing increase in phenotypic organizational complexity. For the same set of human–mouse orthologous genes (11 534 gene pairs) and homologous tissues (32 tissue pairs), human shows a greater fraction of tissue-specific genes and a greater ratio of the total expression of tissue-specific genes to housekeeping genes in each studied tissue, which suggests a generally higher level of evolutionary cell differentiation (specialization). This phenomenon is spectacularly more pronounced in those human tissues that are more directly involved in the increase of complexity, longevity and body size (i.e. it is reflected on the organismal level as well). Genes with a change in expression breadth show a greater human–mouse divergence of promoter regions and encoded proteins (i.e. the functional genomics data are supported by the structural analysis). Human also shows the higher expression of translation machinery. The upstream untranslated regions (5′UTRs) of human mRNAs are longer than mouse 5′UTRs (even after correction for the difference in genome sizes) and contain more uAUG codons, which suggest a more complex regulation at the translational level in human cells (and agrees well with the augmented cell specialization).

Somatic polyploidy promotes cell function under stress and energy depletion: evidence from tissue-specific mammal transcriptome

Polyploid cells show great among-species and among-tissues diversity and relation to developmental mode, suggesting their importance in adaptive evolution and developmental programming. At the same time, excessive polyploidization is a hallmark of functional impairment, aging, growth disorders, and numerous pathologies including cancer and cardiac diseases. To shed light on this paradox and to find out how polyploidy contributes to organ functions, we review here the ploidy-associated shifts in activity of narrowly expressed (tissue specific) genes in human and mouse heart and liver, which have the reciprocal pattern of polyploidization. For this purpose, we use the modular biology approach and genome-scale cross-species comparison. It is evident from this review that heart and liver show similar traits in response to polyploidization. In both organs, polyploidy protects vitality (mainly due to the activation of sirtuin-mediated pathways), triggers the reserve adenosine-5′-triphosphate (ATP) production, and sustains tissue-specific functions by switching them to energy saving mode. In heart, the strongest effects consisted in the concerted up-regulation of contractile proteins and substitution of energy intensive proteins with energy economic ones. As a striking example, the energy intensive alpha myosin heavy chain (providing fast contraction) decreased its expression by a factor of 10, allowing a 270-fold increase of expression of beta myosin heavy chain (providing slow contraction), which has approximately threefold lower ATP-hydrolyzing activity. The liver showed the enhancement of immunity, reactive oxygen species and xenobiotic detoxication, and numerous metabolic adaptations to long-term energy depletion. Thus, somatic polyploidy may be an ingenious evolutionary instrument for fast adaptation to stress and new environments allowing trade-offs between high functional demand, stress, and energy depletion.

Neonatal cardiomyocyte ploidy reveals critical windows of heart development.

BACKGROUND The aim of our study was to find out, whether cardiomyocyte genome duplication participates in developmental programming of adult hypertension and impaired heart aerobic capacity, and if it does, whether ploidy-related programming is reversible and what are the timeframes of the most critical window. For this propose we studied the effect of the well-known factors of programming, including growth retardation, infection, and cardiac overload on the level of neonatal cardiomyocyte ploidy, protein content and shape. METHODS Using the model of rat cryptosporidial gastroenteritis, we shifted the time point of infection day by day through the neonatal period and traced the immediate and postponed effects of disease on isolated cardiomyocyte ploidy, phenotype, and protein content. RESULTS We found that gastroenteritis caused cardiac atrophy and a burst-like premature genome accumulation, elongation, narrowing and protein loss in the cardiomyocytes. These changes resulted in sharp increase of DNA content at the expense of contractile proteins. We also revealed clear indications of critical window of heart development during the peak of cardiomyocyte transition from proliferation to hypertrophy. After the rehabilitation, the atrophy of heart and cardiomyocyte remodelling showed a conspicuous restoration, whereas the hyperpolyploidization did not regress. An irreversible manner of excessive genome duplication and its well-known ability to alter gene expression confirm our suggestion that it is implicated in the ontogenetic programming of heart development. CONCLUSION We provided the first evidence that developmental programming can operate through cardiomyocyte genome duplication and that the critical window coincides with cell transition from proliferation to hypertrophy. Our data help determine the timing of critical window for human heart and would allow successful prevention of human cardiac abnormalities even before they become tangible.

Impact of neonatal cryptosporidial gastroenteritis on epigenetic programming of rat hepatocytes

Inflammation, malnutrition and growth retardation during critical time‐windows of development play a powerful role in ontogenetic programming of the life‐long risk to many adult diseases (including metabolic syndrome, obesity and diabetes). Cellular mechanisms and the accurate timing and duration of critical periods for the liver remain obscure. To resolve this problem, we developed a postnatal suckling‐weanling rat model of mild, moderate, and acute gastroenteritis challenged by a protozoan parasitic spread throughout the whole world, namely Cryptosporidium parvum. The physiological state of the liver was evaluated by hepatocyte ploidy and protein content that were measured by cytophotometry and image analysis on isolated cells. Hepatocyte ploidy is known to irreversibly increase after stress and is associated with the decrease in liver physiological capacity. Hepatocyte hypertrophy reflects cell functional loading. From our results, cryptosporidiosis is able to provoke a burst in premature hepatocyte polyploidization and hypertrophy (in proportion to parasitic load), and thus plays an important role in epigenetic programming of hepatocyte structure and function. We revealed two sensitive periods in liver growth. The first period (the less sensitive) covers the time before the establishment of homoiothermy, i.e. 6–9 days after birth. The second period (the more sensitive) covers the time of weaning when the change of type of nutrition and the peak of hepatocyte polyploidization and differentiation occurs. Thus, our data provide direct evidence that phenomenon of ontogenetic programming is reflected at the cellular level.

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.

Somatic polyploidy is associated with the upregulation of c-MYC interacting genes and EMT-like signature

The dependence of cancer on overexpressed c-MYC and its predisposition for polyploidy represents a double puzzle. We address this conundrum by cross-species transcription analysis of c-MYC interacting genes in polyploid vs. diploid tissues and cells, including human vs. mouse heart, mouse vs. human liver and purified 4n vs. 2n mouse decidua cells. Gene-by-gene transcriptome comparison and principal component analysis indicated that c-MYC interactants are significantly overrepresented among ploidy-associated genes. Protein interaction networks and gene module analysis revealed that the most upregulated genes relate to growth, stress response, proliferation, stemness and unicellularity, as well as to the pathways of cancer supported by MAPK and RAS coordinated pathways. A surprising feature was the up-regulation of epithelial-mesenchymal transition (EMT) modules embodied by the N-cadherin pathway and EMT regulators from SNAIL and TWIST families. Metabolic pathway analysis also revealed the EMT-linked features, such as global proteome remodeling, oxidative stress, DNA repair and Warburg-like energy metabolism. Genes associated with apoptosis, immunity, energy demand and tumour suppression were mostly down-regulated. Noteworthy, despite the association between polyploidy and ample features of cancer, polyploidy does not trigger it. Possibly it occurs because normal polyploidy does not go that far in embryonalisation and linked genome destabilisation. In general, the analysis of polyploid transcriptome explained the evolutionary relation of c-MYC and polyploidy to cancer.

Loss of protein interactions and regulatory divergence in yeast whole-genome duplicates

Whole-genome duplications are important for the growth of genome complexity. We investigated various factors involved in the evolution of yeast whole-genome duplicates (ohnologs) making emphasis on the analysis of protein interactions. We found that ohnologs have a lower number of protein interactions compared with small-scale duplicates and singletons (by about -40%). The loss of interactions was proportional to their initial number and independent of ohnolog position in the protein interaction network. A faster evolving member of an ohnolog pair has a lower number of interactions compared to its counterpart. The Gene Ontology mapping of non-overlapping and overlapping interactants of paired ohnologs reveals a sharp asymmetry in GO terms related to regulation. The fraction of these terms is much higher in non-overlapping interactants (compared to overlapping interactants and total dataset). Network clustering coefficient is lower in ohnologs, yet they show an increased density of protein interactions restricted within the whole ohnologs set. These facts suggest that subfunctionalization (or subneofunctionalization) reflected in the loss of protein interactions was a prevailing process in the divergence of ohnologs, which distinguishes them from small-scale duplicates. The loss of protein interactions was associated with the regulatory divergence between the members of an ohnolog pair. A small-scale modularity (reflected in clustering coefficient) probably was not important for ohnologs retention, yet a larger-scale modularity could be involved in their evolution.

Changes in the heart of neonatal rats after cryptosporidial gastroenteritis of different degrees of severity

Disturbances at the childhood age increase risk of appearance of cardiovascular disease decades later. The nature of this interconnection called ontogenetic programming is not completely understood. Valuable source of knowledge about mechanisms of ontogenetic programming are data of interspecies study of biology of the body life cycles understanding on the triggers and mechanisms are the cross-species comparative data on life-cycle and heart aerobic capacity. Taken into account the interspecies differences, these data allow finding the correct direction of experimental investigations. Results of studies of almost 100 homoiothermal species have shown the slow growth and a high loading on the heart at postnatal development to decrease its aerobic capacity in adults. Basing on these data, we suggested that the neonatal infectious gastroenteritis causing tachyarrhythmia, malabsorption, and the growth deceleration might lead to pathologic changes in the heart. Our task was to evaluate the effect of cryptosporidial gastroenteritis of different degrees of severity on heart of neonatal rats. By using methods of Real-Time PCR, immunocytochemistry, image analysis, and study of atrial septum (ostuim primum), we have established that a gradual increase of intensity of infestation with Cryptosoridium parvum oocysts causes sharp changes corresponding to “all-or-nothing” response. At a weak infestation the atrial septum was close (like in control), while significant changes in expression of isoforms of heavy chains of α- and β- myosin were absent. At the intermediate and severe infestation, in the atrial septum the foramen ovale was visualized and there were observed the cardiac atrophy and a strong shift of ration of expression of myosin heavy chains toward the low-velocity of β-chain. Thus, by disturbing the frequency-strength ratios and causing the outflow of resources from the formed heart, the neonatal cryptosporidiosis produces pathological changes of the organ molecular and anatomical structure. Our results can be interest to evolutionary biologists and physicians, as they show the importance of knowledge of evolutionary-comparative investigations for search for novel risk factors of heart diseases and demonstrate interconnection between gastroenteritis, pathology of atrial septum, and a change of composition of the main contractile proteins in cardiomyocytes.

Remodeling of rat cardiomyocytes after neonatal cryptosporidiosis. I. Change of ratio of isoforms of myosin heavy chains

Diseases of the human cardiovascular system are the main cause of death in developed countries. Therefore, searching for new risk factors thereof is of particular interest. Upon comparing epidemiological data with data of transcriptome of cardiomyocytes and comparative physiology of vertebrate ontogenesis, we have come to the conclusion that one such factor may be gastroenteritis. This disease includes at once several stimuli able to cause functional and metabolic alterations in the heart: tachycardia, hormonal and ionic misbalance, and outflow of resources from the cardiovascular system. Using the model of rat neonatal gastroenteritis caused by the widespread human and animal enteropathogen Cryptosporidium parvum (Apicomplexa, Sporozoa), we studied the change of expression of α- and β-myosin heavy chains after the developed cryptosporidiosis. Online data obtained by methods of immunocytochemistry, quantitative morphometry, and polymerase chain reaction not only have confirmed our suggestion, but also have shown that moderate 4-day-long cryptosporidiosis is sufficient for producing a significant (1.7- to 4.5-fold) shift in the ratio of myosin isoforms toward the β-isoform beta at the level of mRNA and at the level of protein (2.5–6 times). The reciprocity of the changes, as well as their clear similarity at the level of mRNA and of protein, indicates that the cryptosporidial gastroenteritis involves all the main chains of a complex network of regulation of expression of the myosin heavy chains. A shift of the ratio of myosin isoforms toward the β-isoform that has an ATPase activity several times lower than the α-isoform is the commonly accepted indicator of human heart failure; therefore, the cryptosporidial gastroenteritis can be considered a novel risk factor for decrease of the heart’s contractile ability. Our data may be of interest for clinical and preventive medicine.