by Olga ANATSKAYA, Cand. Sc. (Biol.), Alexander VINOGRADOV, Cand. Sc. (Biol.), RAS Institute of Cytology (St. Petersburg)

A chicken will never outstrip an eagle and a pig will never run faster than a deer-their hearts will not tolerate the stress. On a more serious note-what determines the endurance (the "capacity") of that most vitally important organ? Why this capacity is reduced in animals and humans after certain ailments? Answers to these questions are provided by genetic research conducted at the Institute of Cytology of the Russian Academy of Sciences.

WHAT IS SOMATIC POLYPLOIDY?

To begin with, let us remember that body tissues of animals and plants consist of cells. Each cell usually has one nucleus with two genomes with recordings of hereditary information (obtained one from each parent). These are diploid cells that multiply by division, which is preceded by duplication of the parental genomes and division of the nucleus. But if this standard process is stopped due to some reasons at the latter stage, then instead of two cells only one is produced, with two nuclei. Also, a cell can be formed with a large undivided nucleus containing four genomes (tetraploid one). If the rounds of incomplete divisions repeat many times over, there appear cells with many diploid nuclei or a large polyploid one (containing dozens of genomes). The increase of genome number in cells of some tissues is called "somatic polyploidy".

This process was described back in the middle of the 19th century by the German biologist Theodor Swann.

He examined the epithelium of a frog under the microscope and noted that almost every second cell contained two nuclei. A little later, in 1865 his compatriot, the Foreign Member of St. Petersburg Academy, Rudolph Virkhoff demonstrated in his book "Cellular Pathology" the pictures of multi - and binuclear cells from normal and pathologically modified human tissues. Later on, many researchers noted prevalence of this phenomenon in animals and plants.

In 1985 these scattered data were summed in a monograph by two researchers of the RAS Institute of

Articles in this rubric reflect the authors' opinion. - Ed.

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Polyploidy in cardiomyocytes of mammals: A - cardiomyocyte of a pig with 24 genomes concentrated in 12 diploid nuclei; B - binuclear cardiomyocyte of a deer with diploid nuclei; C - diploid cardiomyocyte of a wolf; D - cardiomyocyte of a man with one giant nucleus containing 16 genomes.

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Biology of Development - Vsevolod Brodsky and Irina Uryvaeva. They demonstrated that multiple duplication of genetic information in cells of somatic tissues is a "common" biological phenomenon in moss, lichens, algae, flowering plants and also invertebrate and vertebrate animals. The authors were the first to emphasize the fact that some mammals have organs which almost completely consist of polyploid cells. This phenomenon was discovered in particular in the placenta and the liver of rodents and also... in the human heart. It was established that in the norm it contains up to 95 percent of polyploid cardiomyocytes (cardiac muscle cells) with 4, 8, 16 and 32 genomes.

In spite of being so common, somatic polyploidy did not attract special attention of researchers for a long time because results of some studies indicated that polyploid cells are an approximate equivalent of the corresponding number of diploid ones. And it followed that the increase of genome number does not cause basic changes in the properties of cells, which means that it does not influence the physiology of the organs they constitute. The appearance of additional "genomic copies" was perceived as a manifestation of the process of differentiation or as a way of saving time and resources (in cell divisions).

But in 1991 a team of researchers from our Institute, headed by Inessa Yerokhina, and scientists from the RAS Institute of Biology of Development headed by Vsevolod Brodsky, discovered that polyploid cardiomyocytes of man are quite different from the corresponding number of diploid ones. The former were found to contain much less protein per genome. With the increasing cell ploidy in a row of 2 - 4 - 8 - 16 - 32 - 64 genomes, the amount of protein increased only as 2 - 3 - 5.8 - 7.8 - 13 - 16.8. And since 95 percent of protein in cardiomyocites is represented by myofibrils (fibers responsible for the contraction of muscles), it became clear that polyploidy must influence the functional potential of the heart. To what degree - it was to be found out.

FACTORS OF ENDURANCE

In 1995 British physiologists Charles M. Bishop and Patrick J. Butler proved that the weight index of the heart (the ratio of its weight to that of the body) is directly proportional to its functional possibilities. In agile animals this index is higher than in slow-moving ones. For example, the heart of a sloth is about five-fold smaller than the heart of a dog of a similar weight, and a similar proportion is observed in comparison of a chicken with an eagle. The scientific world, however, did not at once accept the conclusion that such a simple parameter can have such fundamental significance. But the vast factual material, which included above hundred species of fishes, amphibians, reptiles, mammals and birds, confirmed the interconnection between the heart index and its functional potential. Today this is a commonly accepted parameter.

To understand what consequences follow from the polyploidy, we carried out a detailed inter-species study of vertebrate animals. We assessed the average number of genomes in cardiomyocytes of 40 species of birds and 30 species of mammals possessing different heart index, which reflect the ability of them to run, fly and swim without oxygen debt (i.e. without breath-choking).

It was found that in birds and mammals polyploidy is associated with a weak and relatively small heart (and an inactive way of life). Such animals cannot move actively for a long time, unlike the "athletic" species, and they are exposed to the risk of cardiovascular disorders. These facts were met with the extreme surprise by many biologists who regarded polyploidy as a mechanism of boosting of organ functions.

For example, the heart of birds, which do not fly actively and die from heart failures more often than others, weighs only 0.4 - 0.6 percent of body mass and have up to 24 genomes in each cell. The cells are often concentrated in small diploid nuclei surrounded by a small amount of cytoplasm. On the other hand, cardiomyocytes of "athletic" birds, which can sustain long workload with increased oxygen consumption (such as long high-speed flights), practically do not accumulate additional genomes. The hearts of "record-holders" like martin and falcon, which can fly non-stop for hours in search for food (sometimes at speed of up to 200 km/h), weigh respectively 1.6 and 1.1 percent of the body mass and consist mostly of diploid and (maximum) tetraploid cells, which have very powerful cytoplasm with a plenty of contractile elements.

Among mammals, the most polyploidized cardiomyocytes have been discovered in pigs-it has the lowest heart index (0.25 percent of body weight). And pigs, more than other animals are exposed to hypertension and sudden infarcts. About 70 percent of its cardiomyocytes contain 8 - 32 genomes located in a large number of nuclei. And mammals of similar weight, but with a more active way of life, like deers and wolves, which have heart indexes of 0.8 and 0.9 percent, have mostly cells with 2 - 4 genomes.

From this point of view, the man occupies a special place among mammals. He differs from other species by a great variability of ploidy and morphology of its cardiomyocytes. In some people, heart cells are similar to those of wolves-have powerful contractile fibrils, one or two diploid nuclei and a rectangular form. In others, the cardiomyocytes are like those of a deer, they have nuclei of average size (with 4 - 8 genomes) and a well-developed contractile system. In still others, they are of irregular shape, contain large nuclei (8 - 32 genomes) and relatively weak contractile apparatus. Representatives of all these categories are practically healthy - without clearly expressed pathologies. But heart disorders, especially those possessed by people since childhood, are associated with accumulation of additional genomes. In chronic patients, cardiologists often register in atria and ventricles the prevalence of cells with 64 and even 128 genomes. It is a paradox, but the contractile apparatus of cardiomyocytes with giant

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nuclei does not look powerful. On the contrary, their contractile fibrils are often rather weak.

PROTECTION FROM FUNCTIONAL STRESS

Why is polyploidization associated with a reduction of functional possibilities of the heart? To answer this question, we carried out an inter-species comparison of the characteristics of contractile ability of cardiomyocytes, which was estimated by the activity of mitochondria ("power stations" of the cell) and the level of protein per genome. And we established that these characteristics considerably decline with the accumulation of additional genomes. At the interspecific level this effect is expressed almost fivefold stronger than at the intra-organismal level. In mammals of about the same weight, with duplication of genome number in a cell, the amount of protein per genome is reduced by 250 percent and the fraction of the volume occupied by mitochondria - by 320 percent. As a result, the cardiomyocyte of a deer with 4 genomes contains about as much protein and mitochondria as that of a pig with 24 genomes (nuclei in such cells are distributed practically along the whole cytoplasm). And cardiomyocyte of man with 16 genomes - contain as much protein as diploid cardiomyocyte of a wolf.

Our colleague biophysicists, observing this unusual phenomenon on the photographs, refused to believe their eyes and said that it was impossible because it contradicted the main principle of cell energetics - it is senseless for the cell to feed these extra nuclei. Nevertheless, polyploid cells with such seemingly strange physiological properties are very common in nature, which indicates their adaptive significance.

A comparative analysis of the levels of gene expression* in polyploid and diploid cardiomyocites enabled us to identify the possible causes of duplication of genetic information. Using bioinformatics methods, we compared the activity of more than 10,000 genes. It turned out that polyploidization substantially changed the expression level of several hundreds of them. Why? With the increasing number of genomes, the hereditary information does not change, and the qualitative composition of genes remains the same, like in diploid cells.

Our data indicate: genes regulated by ploidy change their activity to help the cell to protect itself from the apoptosis (programmed cell death), the effect of toxic substances, infections and oxidative stress. It is quite probable that it is these features that make it possible for the heart to function under extreme conditions, and the reduction of its potential is the "payment" for this possibility. That is why the highest ploidy of cardiomyocytes was found in birds-remote migrants like geese, ducks and swans. And although they are incapable of aerobic (oxygen-consuming) activities over long time, their organism is a miracle of adaptivity to high workload under conditions of reduced oxygen level. It is known that during migration, the geese, ducks and swans fly for many hours under the extreme conditions-at altitudes of 8 - 10 km above sea level, in the ratified atmosphere (with oxygen concentration below 30 percent of usual level) and at temperature of -40C, where additional requirements are applied to protective mechanisms of the cell.

REGULATORS OF PLOIDY

The connection of polyploidy with the change of cardiomyocyte properties, which leads to a reduction of functional potential of the heart, sets special importance to the question: what factors determine the level of ploidy? It was established: in mammals the additional genomes appear during feeding with milk (in rats from day 7 till day 14 after birth, in pigs - from the age of one week to one month), in birds they are accumulated in the period from birth to maturity. In man polyploidization appears from birth to the age of 11 years. Consequently, factors regulating this process in adult animals should be traced to the early postnatal age.

We compared in details the features of the growth and "the way of life" of "athletic" and low-moving mammals and birds. It turned out that they strongly differ by the degree of independence and mobility just afterbirth. The first (with hearts of low ploidy) are growing fast, they are "altricial", i.e. born blind and immature, with non-stabilized maintenance of body temperature, and remain practically immobile for a long time. It is the "parents" who take care of their food and health. The most typical representatives of this group are predators. On the contrary, the low-moving species (with highly polyploidized hearts) demonstrate slow growth and "precocial" development. From the first minutes of life they look at the world with wide-open eyes, actively move around and take care of their feeding. The parents take practically no care of them, showing interest in their progeny only during first few days. As a result, the heart of such babies is developing under conditions of a much higher workload than in "athletic" species.

The obtained data led us to conclusion that there is an inversion of workload on the heart during formation of the individual (i.e. in ontogenesis). The heart with a high functional potential in the adult state is formed under relatively low workload and good supply of the organism with resources. And the picture is different if the heart works at the end of its possibilities from the animal's birth and under conditions of a shortage of resources. The role of workload on the baby's heart in programming of its adult ploidy and aerobic capability can be seen in the mammals and birds with a high ploidy of cardiomyocytes. For example, the hearts of pigs and chicken are overloaded during their early development because of an artificially accelerated gain of weight. In ducks and geese this happens because they perform their first migration (which means rising to the height of

* Level of gene expression - the amount of matrix RNA (m-RNA) of a given gene in the cell, reflecting the intensity of gene working. - Auth.

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10 km) still being nestlings. An additional workload on the heart in the ontogenesis of man is associated with the upright gait (walking).

Physiologists use the quantitative criterion of workload on the formatting organs - the rate of postnatal growth. The main natural retardant of the growth is mobility: the more independent is an animal and the more it moves around, the slower its growth. The helpless and low-moving nestlings of birds and mammals grow 3 - or 4-fold faster than the "independent" and active nestlings of a similar weight. But why the mobility is the main "regulator" of this process? Because muscle system is the biggest organ of vertebrates - amounting to about 40 percent of the body weight and consuming the greatest part of resources. During active moving it spends up to 90 percent of all body energy, and nearly 50 percent during moderate moving. Thus, the rate of postnatal growth is a good indicator of workload on the growing heart: the faster the animal is growing (under natural conditions), the lesser it is moving around and the lower workload it experiences.

We investigated the influence of growth rate on the polyploidization of cardiomyocytes and heart index of adult mammals. It turns out that with acceleration of growth the first factor decreases and the second increases. But only the period of accumulation of genomes is important for both these parameters. Later on, the growth rate has practically no effect on heart formation.

Summing up, the morphological and functional features of the heart at the cellular and organ levels are programmed by the functional load during post-natal development, which is determined by the growth rate. The programming takes place during car-diomyocyte polyploidization. Therefore, it is this time frame that can be regarded as a critical period for formation of this organ.

MEDICAL ASPECTS

In order to understand to what extent the regularities discovered by us are applicable to the medicine, we studied how the increased heart workload caused by ailments of digestive system in the critical period of growth are later reflected in the heart of adult animals (these experiments were performed together with researchers of our Institute - Tamara Beyer and Nina Sidorenko). The situation was provoked by experimental diarrhea infections, which is associated with acceleration of heartbeats. Engaged in the experiment were 10-day old rats, which were ill for 4 days. The age of their contamination corresponded to the period of most active polyploidization of cardiomyocytes. The kind of disorder was chosen because it is associated with a high workload on the heart and because it is one of the most frequent causes of retardation of growth in children.

Diarrhea was caused by the common protozoan pathogen Cryptosporidium parvum. It is most dangerous for children of up to 10 years of age whose heart is still forming and whose immune system is immature. In Europe and the United States doctors dealing with intestinal disorders often register this ailment. In Asian countries - India, Thailand, Sri Lanka - this infection has already reached an epidemic scale.

Our studies indicate that cryptosporidiosis retards the growth of young rats and causes a considerable atrophy of the heart. Heart index in animals after the disease was 30 - 40 percent lower than in control animals. We observed the excessive number of cardiomyocytes with 4, 8 and even 16 genomes. The level of protein in cells was considerably reduced (by 40 - 60 percent), cardiomyocytes had strongly elongated shape, which should reduce the strength of heartbeats. We checked whether these changes were retained 20 days after the recovery (practically till sexual maturation). We found that the differences in cell ploidity and heart index remained stable during this time. We discovered that the effect of diarrhea manifests itself by the principle "all or nothing", i.e. in the cases of strong, medium and medium-weak contamination the effect was similar, while in the weaker cases it was practically absent. It is possible that defense systems of the organism fail when a certain level of infection is exceeded. All this confirmed the results of our inter-species studies and proved that even a small additional workload on the heart during the critical period of its formation leads to irreversible reduction of its future functional potential.

We think we begin to understand the great variability of cardiomyocyte ploidy of the normal human hearts. An important role in this variability belongs to ailments suffered by a child during the critical period of heart development (from birth to 11 years, and the most sensitive period is after 7 years). Practically all infections can be dangerous in this time, as well as disorders of the digestive, nervous, respiratory, muscular, excretory or endocrinal systems. Even a usual acute respiratory - viral disorder with a slight rise of temperature can accelerate the heartbeats. The understanding of the early changes, which precede the clinical manifestations of cardiovascular ailments, is necessary for development of preventive measures. Our comparative studies demonstrated the need for maximal parental care in the critical period of heart formation in children. At that time the heart should be protected during any ailment.

This work was supported by the Russian Foundation for Basic Research (RFBR) and by the Programme of the Presidium of the Russian Academy of Sciences "Molecular and Cellular Biology" (MCB RAS).

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