**1. Introduction**

The cardiovascular system plays a vital for the whole organism. It performs many vital functions such as supplying organs and tissues with nutrients, hormones, carrying oxygen to the cells and maintaining physiological temperature.

According to the World Health Organization, diseases of the cardiovascular system are the main cause of lethality worldwide. Every year, more than 7.4 million people die from coronary heart disease worldwide, and these rates continue to grow. Congenital heart defects are also highly prevalent and may be a cause of serious complications in the future. Thus, the search for solutions to the cause and consequences of heart diseases is one of the most serious biomedical problems.

The possibility to regenerate the mammalian heart is still rarely studied; however, studying it would allow finding new ways to restore the heart after severe injuries.

A huge number of factors affect the functioning of the heart. One of them is cell aging, which manifests itself in the form of various disorders. Cellular aging is associated to a greater extent with the loss of telomere length and a decrease in telomerase activity.

Understanding the processes of regulation of telomere length and telomerase activity in the ontogenesis of cardiac tissue can help to understand the causes of heart disease in one or another period of development of the organism.

#### **2. Telomeres and Telomere Complex**

In 1961, Leonard Hayflick discovered the limit of somatic cell division in vitro. The limit is named after the scientist—the Hayflick limit. According to Hayflick's observations, human fibroblasts dividing in a cell culture died after approximately 50 divisions. The cells showed signs of aging, stopped dividing and underwent programmed cell death [1].

Ten years after the discovery of the Hayflick limit, Olovnikov hypothesized that DNA polymerase cannot copy a small region at the end of chromosome (telomere), which leads to terminal under-replication of DNA [2] (Figure 1).

**Figure 1.** Problems associated with chromosomes' terminal under-replication [2].

Scientists postulated that telomeres undergo shortening with each cell division and assumed that this phenomenon was associated with the cell division limit. This hypothesis was confirmed in 1985, when Greider and Blackburn (1985) identified an enzyme in the ciliate *Tetrahymena thermophila* that prevented the degradation of the ends of chromosomes. The enzyme was named telomerase [2].

Vertebrate telomeres consist of repeating TTAGGG sequences at the ends of chromosomes and maintain their integrity. Since DNA replication is asymmetric at both strands, the sequence at the 3 end would lose 30–200 nucleotides with each cycle of DNA replication and cell division. Telomeres have non-coding recurring sequences at the 3 end to prevent the loss of coding sequences during replication [3]. Moreover, telomeres are covered with Shelterin complex consisting of six proteins: TRF1 (telomere repeat binding factor 1), TRF2 (telomere repeat binding factor 2), TIN2 (TRF1-interacted nuclear protein 2), RAP1 (rif-associated protein), POT1 (protection of telomeres) and TPP1 (telomere protected protein 1). Telomeres end with a single-stranded 3-end, which has a compact T-loop structure that maintains their stability [4]. Telomeres were proposed as mitotic clocks that show how many times a cell has divided [5].

When telomeres shorten to a critical length, the cell goes into a state of senescence, which initiates a series of changes in gene expression patterns of cell cycle inhibitors, decreases cellular proliferative potential and activates apoptosis [6].

Telomerase is responsible for telomere elongation and consists of an RNA component (TERC) and telomerase reverse transcriptase (TERT), a catalytic component. TERT uses TERC as a template for synthesizing new repeats of telomeric DNA at the single-stranded ends of chromosomes [7]. Most somatic cells lack telomerase activity, but undifferentiated germ cells, stem cells, activated lymphocytes and most tumor cells have a high level of telomerase activity to overcome telomere contraction and maintain limitless cell division. However, differentiated resting cells usually have a low or undetectable level of telomerase activity [8].

#### **3. Embryonic Development of the Heart**

The heart begins to function in the early stages of development in both mammals and lower vertebrates such as *Danio rerio* (zebrafish) [9,10]. In mice, the level of proliferation of cardiomyocytes

(CM) is high in early embryogenesis, and then it gradually decreases until the 10th to 12th day of embryonic development (E10–12) when the heart is almost fully formed [9,11]. Similar dynamics are also shown for telomerase: its activity is detected in the heart tissue of the human fetus until the 12th week of embryonic development, which coincides with the histological differentiation of the myoblasts of the heart into cardiomyocytes [12]. This observation is consistent with the fact that, by the sixth month of prenatal development, the morphological appearance of the heart muscle is almost the same as that of an adult [12].

However, a full picture of dynamics of telomerase activity during the cardiac embryonic development is still unclear. It is known that activity is registered during E11.5 [13] and E16.5 in mice [14], as well as on E10 and E20 in rats. Moreover, telomerase activities in developing rat hearts start to decline after E10 [15]. Dynamics of telomerase inactivation in developing hearts of rats and humans appear to have similar patterns since, in rats, the heart becomes a fully formed functional organ by E16 [16].

#### **4. Early Postnatal Heart Development**

Proliferation reaches the first minimum point in the heart of newborn mice (i.e., day 0 of postnatal development; P0) [17]. During this period, the system that is responsible for the cell cycle is transformed from embryonic to postnatal mode. Before birth, the number of CMs increases, and after birth, it remains almost unchanged. At the same period, tetraploid and binuclear CMs begin to appear [17]. At P3, the peak of mitotic activity appears again, which correlates with an increased number of binuclear CMs (up to 80%) and a decrease in the number of mononuclear CMs. At the same time, both in binuclear and mononuclear CM populations, there is a transition to the G1 phase and cessation of the cell cycle [17]. After P3 there is a sharp decrease in the number of CMs that have entered mitosis [17,18].

If we take a look at the activity of telomerase in the heart at this stage of development, we find a correlation both with a decrease of proliferation and with the advent of binuclear and polyploid CMs. Therefore, it can be speculated that negative telomerase regulation may be important for permanent stopping of the CM cell cycle [15]. Thus, in newborn mice, gradual suppression of telomerase activity occurs, and by P2 the activity decreases by more than 65% [14]. By the third month of postnatal development, only a very small number of *Tert*-expressing cells remain [19]. A number of studies have shown a sharp decline in telomerase activity in newborn mice relative to the hearts of E11.5, and after P10 it is almost undetectable [13]. *Tert* expression has a similar dynamic, which indicates a possible mechanism for the suppression of telomerase activity through the catalytic subunit of telomerase [13,20].

Similar to previous data, it was found that five days after birth, the activity of telomerase in the rat heart was only 20% of the activity at E10. Telomerase activity was absent in P20 heart and remained below the detection limit up to four months of age [15].

Regarding the distribution of telomerase activity in the heart, *TERT* expression is found in a population of cells, including CM, fibroblasts and endothelial cells [19].

The decrease in proliferation potential of CMs positively correlates with telomere depletion in newborn mice. Rapid reduction of telomere length occurs within the first two weeks after birth. Further, the length of the telomeres does not change, as the CMs leave the cell cycle, which contributes to the conservation of telomere reserves. In the first days after birth, the proliferating CMs have a longer telomere than non-proliferating CMs. However, after P15, these differences are already nullified [13].

There are several possible causes for the sharp drop in telomere length in newborn mice. Telomeres shorten during phase S due to the inability of the DNA replication mechanism to support the ends of linear DNA molecules [5]. Therefore, the absence of telomerase predetermines the loss of telomere reserves in CM during the period of their postnatal DNA replication.

More surprising is the high rate of loss of telomeres, starting with P1, which leads to a significant increase in DNA damage in telomeric sites in a one-week period [13]. In this regard, another cause of damage and further loss of telomeres may be the appearance of reactive oxygen species (ROS) in CM

after the metabolic transition from anaerobic glycolysis to mitochondrial oxidative phosphorylation during the first week after birth. So, it was found that the level of ROS increased in newborn mice, which probably leads to an increase in the number of DNA damage foci (replacement of guanine with 8-oxo-7,8-dihydroguanine) between P4 and P7 [21]. Such alterations have a particularly noticeable effect on the promoter regions of genes that have a high GC content [22]. Telomeres also have a high GGG content, which makes them an ideal target for ROS attacks [23]. Due to an increase in the level of oxidizers and disturbances in telomeres, the DNA damage response is activated, which leads to a halt in proliferation through activation of the cell cycle inhibitor p21 [13,21]. However, if the oxygen concentration or the ROS levels are reduced, then the proliferation window of the CMs in the early postembryonic period expands along with the increase in the numbers of mononuclear cells relative to two- and multi-nuclei CMs [21]. ROS can be associated with telomerase inhibition due to telomere DNA damage or deoxynucleotides oxidation that explains a sharp drop of telomerase activity after birth [24].

The mechanism that promotes cell cycle arrest in postnatal CMs can also be associated with the gap-fusion-bridge cycle, which leads to tetraploidization, appearance of binucleated cells and inhibition of proliferation [25]. In the gap-fusion-bridge cycle, chromosomes with nonfunctional telomeres merge with each other, forming bridges during mitosis. These chromosome bridges may eventually collapse under the action of forces emanating from the anaphase poles, and further proliferation is inhibited. In murine CMs, decrease in telomeres is associated with the appearance of chromosomal bridges between the daughter nuclei: eight days after birth, CMs display the presence of chromosomal bridges, which correlates with a decrease in telomere length. A potential genomic imbalance caused by the breakdown of chromosomal bridges in binucleated CMs can be a barrier to proliferation [13].

The knockout for the *Terc* gene confirms the role of telomeres in proliferation. Third-generation *Terc*-null mice have shorter telomeres, anaphase bridges and a lower proliferation level than wild-types (WTs) at P1 [13].

It should be noted that in lower vertebrates, such as *D. rerio*, telomerase activity in many tissues is sufficiently high both during embryonic and postnatal development [26]. This observation is used to study the proliferative potential of CMs in the regenerative process [27]. In addition, it was shown that CMs of *D. rerio* are single-nucleated and diploid [28,29], which may be associated with high telomerase activity capable of maintaining sufficient telomere length for normal proliferation [28,29].

## **5. Prepubertal Period**

CMs of mammals lose their ability to proliferate after birth due to telomere dysfunction and reduced telomerase activity [13]. However, there is evidence of a proliferation surge in the prepubertal period. Thus, from P14 to P15, activation of mitosis with subsequent cytokinesis is observed in both mononucleated or binucleated CMs. Proliferation is accompanied by the expression of cell cycle regulating genes [30]. From this, it can be speculated that there is a molecular mechanism for overcoming the proliferative barrier associated with short telomeres and low telomerase activity. The authors of the study sugges<sup>t</sup> that the wave of synthesis of the hormone triiodothyronine is an impulse for the induction of mitosis [30]. On the other hand, the cause of the activation of proliferation can be telomere elongation due to the start of telomerase expression. This hypothesis is derived from data on increasing the length of telomeres and subsequently the level of proliferation due to the introduction of Tert in the heart of an adult mouse in response to heart damage [20]. At the same time, there is another point of view on this observation: during growth, the size of the heart increases almost exclusively due to hypertrophy, but not hyperplasia. It was shown that the increase in the number of CMs or proliferation rate was not observed between P13 and P100, and that no active DNA synthesis occurred [31]. Similar results were demonstrated for P14–P21 [32].

However, the contribution of hyperplasia to an increase in the size of the heart should not be completely ruled out, although it is probably not as high as was presented in the study of Naqvi et al., 2014 [30]. Indeed, it has been found that proliferating mononuclear cells with increased telomerase

activity are present in the hearts of young cats, and they have the physiological properties of immature cells in the form of calcium current in T-type channels [33]. Mitotic activity in human mononuclear CMs is shown during the first 20 years of life (1.9% of the total number of CM), which decreases, but is registered up to the age of 40 [34], as confirmed by earlier works [35]. There is also evidence of an increase in ploidy of CMs and the absence of growth in the number of binuclear cells during the first 20 years of life [35].

#### **6. Heart of Adult Vertebrates**

As described above, telomerase activity in mammals drops to a minimum shortly after birth, which causes telomere shortening. However, in the heart of adult animals, as shown in mice, telomerase activity is registered, both in CMs, and in fibroblasts and endothelial cells [19].

CMs of adult mice vary in size: binucleated and multinucleated CMs undergo hypertrophy, and small mononucleated CMs show features of proliferating cells. The length of telomeres in these classes of CMs is inversely related to cell size. In addition, *p16CDKN2* expression is observed in large binucleated and multinucleated CMs with the shortest telomeres [36]. P16CDKN2 specifically binds and inhibits cyclin-dependent kinases CDK4 and CDK6, which act as regulators of the progression of the cell cycle in G1, contribute to the phosphorylation of the retinoblastoma protein (pRB) and induce cell cycle arrest [37].

With a TRF assay, it has been demonstrated that, unlike mammals, telomerase activity in the hearts of *D. rerio* is high throughout life, and telomere length remains almost unchanged [38]. Further studies were conducted on changes in telomere length in fish of di fferent ages using the Q-Fish method. It was shown that in *D. rerio* the activity of telomerase and the length of telomeres in CMs also varied with age, and the aging of fish leads to a decrease of telomere length and telomerase activity [26].
