[Threshold rat neonatal cardiomyocyte response to gradual cryptosporidial infection severity increase].

Infectious gastroenteritis is one of the common causes of tachyarrythmia, malabsorbtion and growth retardation in children. Our recent studies have indicated that neonatal.cryptosporidial gastroenteritis is associated with long-term cardiomyocyte abnormalities. The aim of the present study was to find out how neonatal cryptosporidiosis of various severities affects cardiac anatomy and cardiomyocyte polyploidization, remodeling and HIF-1α expression. Using real-time PCR, cytometry, immunohistochemistry, image analysis and interatrial septum visual examination, we revealed that gradual increase in cryptosporidial invasion was associated with threshold changes. At weak parasitic infection, interatrial septum was entire and there was no statistically significant change in cardiomyocytes. At moderate and severe infection, all changes in cardiac anatomy and cardiomyocytes were statistically significant and demonstrated approximately similar degree. Compared to control, heart were atrophied and elongated, interatrial septum contained a small window (patentforamrn ovale), and cardiomyocytes lost protein, became elongated, thin and accumulated additional genomes. Also we found HIF-1α mRNA hyperexpression. Notable, the threshold response to gradual stimulus is an important criterion of development programming since such a response is commonly a consequence of abnormal anatomic structure formation and cell differentiation failure. Our results can be interesting for physicians because they indicate that even moderate cryptosporidiosis can be dangerous for neonatal heart and can trigger neonatal programming of cardiovascular pathology. Also, our results for the first time demonstrate the association between gastroenteritis, patent foramen ovale and cardiomyocyte malfunction.

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

Disturbances at the childhood age increase risk of the appearance of cardiovascular diseases decades later. The nature of this interconnection called ontogenetic programming is not completely understood. Valuable sources of knowledge about mechanisms of ontogenetic programming are data of interspecies study of biology of the body life cycles and of heart physiological capabilities. 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 capability in adults. Basing on these data, we suggested that the neonatal gastroenteritis causing tachyarrhythmia, malabsorption, and the growth deceleration might lead to pathological changes in the heart. Our task was to evaluate the effect of cryptosporidial gastroenteritis of different degrees of severity on heart of the neonatal rats. By using methods of Real-Time PCR, immunocytochemistry, image analysis, and study of interatrial septum, we have established that a gradual increase of intensity of infestation with Cryptosporidium parvum oocytes causes sharp changes corresponding to the "all or nothing" response. At a weak infestation the interatrial septum was close (like in control), while significant changes in expression of isoforms of heavy chains of alpha- and beta-myosin were absent. At the intermediate and severe infestation, in the interatrial septum the foramen ovale was visualized and there were observed cardiac atrophy and a strong shift of ration of expression of myosin heavy chains toward the low-velocity beta chain. Thus, by disturbing the frequency-strength ratios and causing outflow of resources from the formed heart, the neonatal gastroenteritis produces pathological changes of the organ molecular and anatomical structures. Our results can be interest to evolutionary biologists and physicians, as they show importance of knowledge of evolutionary-comparative investigations for the search for novel risk factors of heart diseases and demonstrate interconnection between gastroenteritis, pathology of interatrial septum, and a change of composition of the main contractile proteins in cardiomyocytes.

[Rat cardiomyocyte remodeling after neonatal cryptosporidiosis. II. Elongation, excessive polyploidization and HIF-1alpha overexpression].

Retrospective epidemyological studies evidence that infant diseases leave survivors with an increased susceptibility to cardiovascular diseases in later life. At the same time, the mechanisms of this link remain poorly understood. Based on medical statistics reporting that infectious gastroenteritis is the most common cause of maladies in babies, infants and children, we analysed the effects of moderate cryptosporidial gastroenteritis on the heart and ventricular cardiomyocyte remodelling in rats of the first month of life. The disease was challenged by a worldwide human protozoic pathogen Cryptosporidium parvum (Apicomplexa, Sporozoa). The main symptoms manifested in the growth retardation moderate diarrhea. Using real-time PCR, cytophotometry, confocal microscopy and image analysis, we indicated that cryptosporidiosis was associated, with the atrophy heart and the elongation, narrowing, protein content decrease and hyperpolyploidization of cardiomyocytes and the moderate overexpression of hypoxia inducible factor 1alpha (HIF-1alpha) mRNA. Cardiomyocyte shape remodeling and heart atrophy presented in all age groups. The severity of these changes, hovewer, declined gradually from younger to older groups. In contrast, hyperpolyploidization and HIF-1alpha mRNA overexpression were registered mainly among animals aged between 6 and 13 days, and were barely detected and non-significant in older age groups. In the rat the time period covering 6-13 days after birth is known to coincide with the intensive cardiomyocyte polyploidization and the switch from proliferation to hypertrophy. Thus, our data indicate that neonatal cryptosporidiosis may be potential cardiovascular diseases risk factor and that one of the critical time windows for the growing heart covers the time period when cardiomyocyte undergo polyploidization.

[Cardiomyocyte myosin heavy chain composition change after cryptosporidial gastroenteritis].

Cardiovascular diseases are the most common case of human death in developed countries. Thus, the discovering of their new risk factors is of primary importance. Based on epidemiology studies, vertebrate life-history traits comparison and cross-species cardiomyocyte transcriptome analysis, we suggest that one of these factors could be infectious gastroenteritis. This disease outflows recourses from cardio-vascular system and triggers pathological stimuli, like tachyarrhythmia, inflammation, malapsorption and energy depletion thereby disturbing cardiomyocyte metabolism and function. To test this hypothesis, we challenged gastroenteritis in neonatal rats with widespread human parasite Cryptosporidium parvum (Apicomplexa, Sporozoa). The results obtained by the methods of immunocytochemistry, quantitative morphometry and real-time PCR, indicate that moderate cryptosporidiosis lasting four days induces dramatic shift in myosin isoform expression ration toward isoform beta (with low ATPase activity) both at mRNA (by 1.7-4.5 folds) and protein (by 2.5-6 folds) levels. Antithetical manner of this shift and coherence between changes in mRNA and protein suggest that cryptosporidiosis affects all main steps of a complex myosin heavy chain regulatory network. Since the overexpression of myosin heavy chain beta (showing several times lower ATPase activity than myosin heavy chain alfa) is a generally accepted marker of human cardiac failure, we can consider cryptosporidial gastroenteritis as a new risk factor of cardiac contractile ability impairment. Our data can be interesting for clinicians.

[Somatic polyploidy associated metabolic changes revealed by modular biology].

Excessive somatic polyploidy usually accompanies physiologic and pathologic overload and it is generally accepted as a symptom of pathology. At the same time, polyploidy cells exist in most fungal, plant, mollusk, fish, bird and mammalian tissues confirming their great evolutionary success. The secret of this success remains enigmatic. Since transcriptome rearrangements usually start with metabolic flux redistribution, we decided to investigate firstly the effects of polyploidy on cell metabolism. Using multitest approach of modular biology and databases Entrez Gene, RefSeq, GNF SymAtlas, Gene Ontology, KEGG, BioCarta; MsigDb, Reactome, GenMAPP, and HumanCyc, we performed detailed comparison of metabolic genes expression in human and mouse organs with reciprocal pattern of polyploidy (i. e. in the heart and in the liver). Pairwise criss-cross comparison of diploid vs. polyploid organs allowed removing species- and tissue-specific effects. From our results, polyploidy is associated with rearrangements of main metabolic pathways. We found deep depression of mitochondrial processes, features of autophagia, and increased carbohydrate degradation and lipid biosynthesis. Taken together, these changes pointed to the energy and oxygen deprivation. We also found clear indications of enhanced oxidative stress protection. The major of them are triggering of pentose-phosphate pathway, depression of mitochondria-cytoplasm electron shuttles, and impartment of electron flows across 1 (NADH dehydrogenase) and IV (cytochrome c-oxydase) breath complexes. We suggest that all these changes are necessary for the increase in metabolic plasticity and for the protection of replicating DNA from oxidative damage.

[Neonatal gastroenteritis triggers long-term cardiomyocyte atrophy, remodeling and irreversible hyperpolyploidization].

Growth retardation, inflammation and cardiac overload in early childhood are linked with hypertension and infarction in adults. This link was termed as developmental programming. Exact mechanisms and critical time frames for development of the heart are still unknown. To elucidate these questions, we developed a model of moderate cryptosporidial gastroenteritis triggering main programming factors. Sliding the time point of infection day by day (from day 4 to day 18), we tested complete rat neonatal period. Also, we repeated all experiments 30 days after infection. Using methods of cytometry, immunocytochemistry and confocal microscopy, we compared sensitivity of ventricular cardiomyocyte shape, protein content and ploidy. Our data indicated that gastroenteritis lasting four days triggered cardiomyocyte atrophy, almost doubling cell length to width ratio, and premature and excessive polyploidization. Surprisingly, nucleus and cytoplasm reacted to the disease differently. Cardiomyocytes accumulated genomes only when the disease covered the time period between 6 and 14 days after birth, when cells substitute proliferative growth with hypertrophy. Contractile proteins and cell shape on the contrary, showed high sensitivity in the course of complete neonatal period. After restoration, ploidy did not regress, whereas cell shape and protein content revealed moderate restoration. Taking into account that somatic polyploidy is irreversible and that it alters global gene expression pattern, we may suggest that genome duplication is one of the instruments of developmental programming and that gastroenteritis is one if the triggers of this programming.

[Intestinal cryptosporidiosis at an early age and its negative consequences].

This review calls the attention of physicians, primarily pediatricians, to cryptosporidiosis, a still little known intestinal infection caused by the protozoan pathogen Cryptosporidium parvum (Coccidia, Sporozoa). By using 10--14-day rats as a model, the authors have first provided evidence that even 4-day intestinal cryptosporidiosis may trigger obvious negative changes in the liver and heart, i.e. in the organs where the parasite does not develop. In the infected rats, growth retardation was registered, in addition to liver hypertrophy and partial heart atrophy, and growth retardation. Light and electron microscopies, absorption and fluorescence cytometry, quantitative morphometry, and image analysis were applied. In both hepatocytes and cardiomyocytes, the polyploid cell fraction was seen much increased, with the occurrence of 4c, 8c, and even 16c nuclei. Besides, in the hepatocytes, the amount of glycogen decreased whereas the level of protein increased, along with enhanced nucleolar activity in the nuclei. Unlike, the cardiomyocytes of the infected rats were characterized by protein decrease, in addition to almost two-fold cell body elongation. This is the first documented evidence for serious pathological changes in the extraintestinal organs, caused by the intestinal pathogen C. parvum. Within the first 4 days of infection, both the liver and heart of the host seem to work under stress. It is plausible that on modulating liver and heart ploidy, the intestinal parasitic infection (cryptosporidiosis) may bring about functional impairments of these organs, untypical of early age, leading eventually to long-term consequences in further life of formerly infected individuals.

[Polyploidy: significance for cardiomyocyte function and heart aerobic capacity]. / Poliploidiia: znachenie dlia funktsii kardiomiotsitov i potentsiala raboty serdtsa.

Somatic polyploidy, defined as genome multiplication, was found in all differentiated mammalian tissues. The highest level of such a polyploidy was found in the myocardium. This phenomenon was shown to be associated with changes in the pattern of gene expression. Hence, polyploidization may create cells with new physiology. The effect of polyploidy on the heart function has never been studied. The aim of the present study was to investigate the effect of polyploidy on cardiomyocyte functioning and heart aerobic capacity. DNA and the total protein content, nucleolar activity reflecting the rate of rRNA synthesis and, consequently, ribosome biogenesis, were measured in ventricular myocytes isolated from the human and from 21 mammalian species by image cytometry and microscopic morphometry. The total protein content was estimated after staining slides with naphtol-yellow dye. For measurement of DNA and nucleolar area, staining with Hoechst and AgNO3 was applied. Cardiac aerobic capacity was evaluated by the heart mass to body mass ratio. A negative correlation between the heart index and the average cell ploidy was revealed (r = -0.79; P < 0.0001). The average genome number per myocyte was registered to be higher by approximately 35% in the sedentary mammals, with the heart index about 0.4% from body mass, than in the athletes with heart index about 0.6% of body mass. Polyploidization was shown to be associated with a sharp decrease in the protein/DNA ratio in cardiomyocytes. As a result, cardiomyocytes in the athletic mammals with poorly polyploid hearts have much higher protein content per genome than do cells in the sedentary species with highly polyploid hearts. Surprisingly, despite decreased protein/DNA ratio, the nucleolar area per genome significantly increased with polyploidization, indicating the imbalance between the cellular protein content and the rate of ribosome biogenesis. Such an imbalance should obviously impair cardiac function, because the additional genomes take some valuable space and biological resources from the cell, which could have been otherwise directed to the maintenance of cardiomyocyte contractile machinery. It is generally accepted that somatic polyploidy is associated with oxidative stress and energetic starvation. Thus, we suppose that additional genomes may serve for cardiomyocyte protection from oxidative damage in the hearts.

[Cell response of rat liver parenchyma to the infection by the intestinal protozoan pathogen Cryptosporidium parvum (Sporozoa, Coccidia)]. / Reaktsiia kletok parenkhimy pecheni krys na zarazheniie kishechnym protozoinym patogenom Cryptosporidium parvum (Sporozoa, Coccidia).

In the present work, the authors' previous studies of a "distant action", exerted by an intestinal pathogen (Cryptosporidium parvum) on the liver of experimentally infected baby rats, were extended to include shifts in the quantity of glycogen, protein and nuclear DNA in the host liver at different degrees of infection. One of the outcomes of this work is the discovery of a very quick response of hepatocytes and a high sensitivity of rat liver to parasitic invasion even at a weak intensity of infection. 85-90 h after oocyst feeding to rats, glycogen quantity in their livers was 2.5 times lower that in the control. This suggests that the infected host liver worked under energetic starvation conditions. The proposed coefficients of general infection (I) and infection with intracellular stages (F) made it possible to distinguish between the total abundance of parasites in the host intestine during the whole period of infection, and the number of feeding intracellular stages available by the moment of autopsy. The glycogen amount in rat hepatocytes does not depend on I, and negatively correlates with F. Unlike, the protein content in hepatocytes positively correlates with I, being independent of F. Despite the obvious deficiency of amino acids in the infected rats, as a consequence of cryptosporidiosis-induced malabsorption, the protein synthesis in their hepatocytes was not at all inhibited but, on the contrary, much activated. This is a most characteristic feature of the distant action of C. parvum on the liver of parasitized host. With C. parvum infection, the share of polyploid hepatocytes does not correlate with either I, or F. However, compared to the control, the mean values of relative numbers of polyploid cells in weakly, moderately, and heavily infected animals (according to I values) were higher by 20, 100 and 100%, respectively. In hepatocyte nuclei of C. parvum infected rats, the total area of nucleoli increases almost by 30%. The above changes are discussed in terms of both the liver compensatory response to the existing pathology (diarrhea), and the host-parasite relationships. Studies into the distant action of an intestinal pathogen (C. parvum) on non-intestinal organs (liver) of the infected host may be qualified as a new and original approach to pathogenesis of protozoan infections (coccidioses sensu lato), to which young host specimens are known to be most susceptible.

[Morphofunctional changes in hepatocytes during the early postnatal development of rats experimentally infected with the intestinal protozoan pathogen, Cryptosporidium parvum (Coccidia, Sporozoa)]. / Morfofunktsional'nye izmeneniia pecheni v rannem postnatal'nom razvitii u krys, éksperimental'no zarazhennykh kishechnym protozoinym patogenom Cryptosporidium parvum (Coccidia, Sporozoa).

Morphofunctional changes in hepatocytes of 10-14-day old rats were followed in norm and after experimental infection with different doses of oocysts of Cryptosporidium parvum. The liver index (ratio between the liver and body masses) varied with the intensity of invasion on the background of slowing down up to the total cessation of animal growth rates, and all this obviously pointed to severe pathology. In the infected rats, some cytological indices were shifted compared to the norm: protein amount and the average number of genomes per hepatocyte were seen to increase, the normal ratio between cells with different ploidy levels being violated. The particular correlation analysis was employed to distinguish between the ontogenetic (animal growth related) and pathologic (related to the infection intensity) polyploidization and hypertrophy in hepatocytes. In 10-14-day old rats, the former is affected primarily by the increase in the share of multinuclear hepatocytes, whereas the latter is accomplished by the increase in the number of cells with polyploid nuclei (4c and 4c x 2 cells). In the heavily infected rats, the ontogenetic polyploidy was almost totally suppressed due, presumably, to their growth rate inhibition, the rise in hepatocyte ploidy resulting form the obvious pathological changes in the liver. In the infected rats, the ontogenetic hypertrophy of hepatic parenchymatous cells was not manifested, and the observed protein accumulation in hepatocytes also resulted from the pathological changes in the liver. It is obvious that changes in cell hypertrophy (protein content) may serve as a more susceptible tool that readily perceives the host's stress experienced due to the parasitic infection (cryptosporidiosis), than cell ploidy: the levels of the respective responses of these two parameters differing by 4 times. However, due to the known reversible nature of hypertrophy, it cannot be used for the aims of a long-term prediction about the future mode of liver functioning in the animal that survived cryptosporidiosis. Unlike, such a parameter as frequencies of hepatocytes with different ploidy levels is much more useful in this respect.

[Interconnection of parameters of the mitochondrial and myofibrillar apparatus of cardiomyocytes and ploidy and hypertrophy in certain mammalian species, differing in body mass]. / Vzaimosviaz' parametrov mitokhondrial'nogo i miofibrilliarnogo apparatov k-ardiomiotsitov s urovnem ikh ploidnosti i gipertrofii u nekotorykh vidov mlekopitaiushchikh, razlichaiushchikhsia po masse tela.

Using cytophotometry and interferometry, ploidy levels and dry weights were determined in cardiac atrium and ventricle myocytes in various mammalian species. Besides, in the same species, using electron microscopy and image analysis, myofibril volume density (MFVD) and mitochondrial volume density (MTVD), as well as the total length of internal mitochondrial membranes (IMM) per cell area unit were measured. The total of 14 mammalian species were studied, with approximately 100,000-fold interspecies differences in the body weight. The dry weights of the left ventricle myocytes in different mammalian species have been shown to vary from 3660 +/- 127 to 8890 +/- 160 pg. Somewhat smaller were the right ventricle myocytes; their dry weight varied from 3598 +/- 134 to 8189 +/- 160 pg. The atrium myocytes were significantly smaller than the ventricle myocytes in all the mammalian species studied. The lowest dry weight of the left atrium myocytes was revealed in the mouse (2415 +/- 96 pg), while the largest weights of the left atrium myocytes were found in the pig (5530 +/- 138 pg). Myocytes of the right atrium, with their mean dry weights in different species varying from 2379 +/- 93 (in the mouse) to 5123 +/- 124 pg (in the pig), were the smallest among all cardiac chamber myocytes. The data obtained indicate that differences in size between the ventricles and atria in mammals are owing predominantly to differences in the number of cardiomyocytes in different parts of the heart rather than to the size of these cells. The dry weight ratio between the right and left ventricle myocytes in various mammalian species was, on average, 0.937 +/- 0.02, and between the right and left atrium myocytes 0.938 +/- 0.04. This ratio between the left atrium and left ventricle myocytes was 0.717 +/- 0.03 and that between the right atrium and right ventricle was 0.722 +/- 0.02. The data obtained indicate that the ratios of cardiomyocyte sizes in different heart parts are rather stable parameters in the mammalian evolution. The mean ploidy levels in myocytes in different parts of the heart corresponded to the mean sizes of the cells. In all the species studied in this work, the ploidy of myocytes of the right ventricle was lower, on average, by 7% compared to myocytes of the left ventricle. The atrial cardiomyocytes had a markedly lower ploidy than the ventricular cardiomyocytes, the myocyte ploidy levels in the left atrium being in all the species higher than in the right atrium. In spite of a higher ploidy level in the ventricular than in the atrial myocytes, this difference in ploidy was less pronounced than the corresponding difference in the myocyte sizes in the same heart parts. In the majority of mammalian species studied, myocyte polyploidization in different parts of the heart combined two ways promoting the increase in the number of genomes in the cells: (1) acytokinetic mitoses in binuclear and multinuclear cells, i. e. mitotic divisions of diploid nuclei without cytoplasm division; in this case no increase in the tissue cell number occurred, (2) alternation of acytokinetic mitoses in mononuclear cells and of bimitoses in binuclear cells; in this case tissue cells increased in number. The ratio of these two ways of polyploid cell formation differed in various species. The former way appeared to be the only in the nutria and horse, whereas the latter way was found in the shrew and arctic fox. In pigs, whose polyploid cardiomyocytes are also formed mainly through the increase in number of diploid cell nuclei, the multinuclear cardiomyocytes contained, apart from diploid nuclei, a small amount of polyploid 4c and 8c nuclei. The formation of such cells is due presumably to the two mechanisms: the asynchronous DNA synthesis in one of the nuclei in a multinuclear cell, and the subsequent incomplete polyploidizing mitosis. (ABSTRACT TRUNCATED)

[The polyploidization characteristics of the hepatocytes of the mouse-like hamster Calomyscus mystax]. / Osobennosti poliploidizatsii gepatotsitov myshevidnogo khomiachka Calomyscus mystax.

A cytophotometric measurement of DNA content in hepatocytes of maturing mouse-like hamsters was made. Cells belonging to ordinary mammalian ploidy classes 2c, 2c x 2, 4c, and 4c x 2 made about 90% of the hepatocyte population. The share of binucleated cells wa high (about 80%), the majority of these cells being 2c X 2 hepatocytes. Binucleated cells with tetraploid and diploid nuclei occur in almost every animal. An average hepatocyte ploidy level in mouse-like hamster is 4.6c. The main peculiarity of parenchymal liver cell populations is that up 5% of hepatocytes contain 3--11 nuclei of different ploidy classes. Multinucleated cells increase in number from 1.5% to 4% within the period from one year (the age of maturation) to two years. Later on their percentage does not change. It is found that in binucleated and multinucleated hepatocytes DNA synthesis can proceed asynchronously. Asynchrony in DNA synthesis elevates as the number of nuclei increases. Among the 2c x 2 and 2c x 3 cells an uneven distribution of 3H-thymidine label can occur, respectively, in 5 and in 50% cases, whereas all the cells with more than 3 nuclei display an uneven an uneven 3H-thymidin label distribution. The formation of multinucleated cells is supposed to be associated with asynchrony in DNA-synthesis in binucleated cells and with the restitution of mitosis.