**7. Heart Aging**

Aging of the heart includes a number of physiological changes that increase the risk of developing diseases and conditions that are hazardous to health. Cardiac diseases are associated with age and have a detrimental impact on the whole organism. However, it remains unclear how cellular aging of heart tissue a ffects the appearance of heart diseases.

It is known that telomere length decreases with age. For human heart tissue, telomere loss is approximately 20 base pairs per year [39]. In addition to this, it was found that in old rats there was a tendency to reduce the length of terminal restriction fragments (TRF) related to the telomeric and subtelomeric region. Interestingly, only the heart showed a significant decrease in the average length of telomeres compared to the brain, kidney, lung and liver. [40]. A similar state of telomeres was found in coronary artery endothelial cells, where the T/C ratio (ratio of telomere length to centromeres) was reduced depending on the age of the donor [41]. At the same time, the reduction of the end sections of chromosomes is unlikely to be associated with cell division, as described above.

As for the age-dependent diseases, there is a correlation between the length of telomeres and the presence of one or another heart disease. For example, the risks of coronary heart disease are associated with telomeres shorter than 200 base pairs [42]. The mechanism of a possible causal link between short telomeres and ischemic disease has not been fully elucidated, but shorter telomeres are positively associated with the rapid formation of plaques observed in atherosclerosis and marked atherogenesis [42]. Indeed, direct measurement of telomere length in coronary endothelial cells supports the concept that telomere shortening in coronary endothelial cells with aging can contribute to the development of coronary endothelial dysfunction and the development of coronary heart disease in humans [41].

Dilated cardiomyopathy (DC) is characterized by an increase in cardiac ventricular volumes, thinning ventricular wall thickness, hypertrophy and impairment of cardiac function [43,44]. In an aging heart with DC, the forced entry of primitive cells, which express stem cell surface antigen c-kit, into irreversible quiescent state was identified by the expression of cell cycle inhibitor p16INK4a and by very short telomeres [45].

Telomeric shortening in CMs with age can be explained by the launch of miRNA-34a synthesis, the target of which is phosphatase 1 of the nuclear targeting subunit (PNUTS), which is involved in the maintenance of telomeres by interacting with TRF2. TRF2, together with PNUTS, is also involved in regulating the response to DNA damage and inhibiting the phosphorylation of Chk2 (checkpoint kinase), leading to apoptosis. Increased expression of PNUTS inhibits telomere depletion without telomerase involvement [46]. However, there are probably other molecular pathways that result in a reduction in telomere length. They can be associated, for example, with oxidative stress, leading to the accumulation of DNA damage in telomeric regions.

To determine the possible role of telomeres and telomerase in cardiac aging, mice with telomereinduced dysfunction were examined by knockout on telomerase subunit genes. Mice of the fifth generation (G5) with a *Terc* gene knockout (*Terc*-/-) suffered from severe left ventricular insufficiency and DC. Compared to WT mice, the masses of the heart and left ventricle were significantly reduced in G5 mice. Despite the decrease in heart weight in G5, hypertrophy was demonstrated, which was accompanied by a decrease in the number of CMs [47]. The phenotype was also characteristic for G4 *Tert*-/- mice: a decrease in body weight and endurance and an increase in free fatty acids and mitochondrial dysfunction. Mitochondrial dysfunction manifested as inhibition of PGC-1α and PGC-1β, key metabolic regulators. This led to a decrease of gluconeogenesis, a reduction in ATP synthesis, cardiomyopathy, and increased oxidative stress [48], which is a sign of tissue aging [49].

The tumor suppressor protein p53 is an important mediator of telomere dysfunction. An increase in this protein is observed in *Terc*-/- mice when telomeres reach a critically short length. These results are consistent with the notion that telomere loss in mice activates p53, which modulates both apoptosis and growth arrest [47]. In addition to these functions, the p53 protein is a link between telomere length and mitochondrial function: an increase in its synthesis leads to inhibition of the promoters *PGC-1*α and *PGC-1*β [48]. Thus, dysfunction of telomeres leads to premature aging of the heart, which manifests itself in the form of diseases dependent on age.

As described previously [50], premature aging of the cardiovascular system is induced by metabolic stress, obesity, hypertension, insulin resistance and type 2 diabetes [51–54]. Additionally, there is evidence that autophagy is important for longevity and health [55], and a change in autophagy contributes to heart aging [56–58]. Although it has been shown that autophagy inducers have a beneficial effect on life expectancy and slow down the aging of the cardiovascular system [56], there is still a contradiction between the protective and harmful effects of autophagy induction on aging [59]. There are many aging treatment options based on telomerase activation, NO modulation, antioxidants, PARP inhibition, senolytic therapy, plasma membrane redox system (PMRS) activators and stem cell therapy [55,60].

There are reports about the use of telomerase as a therapeutic tissue-specific target for diseases of the cardiovascular system [61,62]. Telomerase can be used to treat coronary heart disease due to protection against ROS [63,64]. With ischemia reperfusion injury, telomerase deficiency leads to heart failure [65]. Telomere depletion is a characteristic sign of cardiac hypertrophy. Shortening of telomeres in CMs is a marker of heart failure in humans, and shorter telomere length in CMs usually correlate with a reduced ejection fraction [66].

## **8. Heart Regeneration**

As we stated above, the regenerative potential of CMs, as well as telomerase activity, decreases in mammalian hearts shortly after birth [13].

In newborn mice (P1), in response to injury, accelerated differentiation of CM occurs [67,68]. Following the P1 period, the regenerative potential is quickly lost, and a similar injury on P7 leads to fibrosis instead of regeneration [67]. Tetraploidy, binucleation, diminished telomerase activity and telomere shortening during this period can be causative of a loss of proliferative potential of CMs in response to injury [13]. A recent study showed that co-cultivation of mononucleated and bi/multinucleated CMs from adult and newborn animals, respectively, led to the de-differentiation and proliferation of not only mononucleated, but also bi/multinucleated CMs, although to a lesser extent [69].

Telomerase expression in the mammalian heart was investigated using transgenic mice expressing green fluorescence protein (GFP) driven by the promoter for murine telomerase reverse transcriptase (mTert), which is a necessary and rate-limiting component of telomerase [19]. Local proliferation of *mTert-GFP*-expressing cells in the adult heart suggests the existence of a subpopulation of mTERT-positive cells that display a phenotype similar to stem cells. This observation is supported by the expression of the heart-specific transcription factors NKX2.5 and GATA4 in these cells, which are necessary for the differentiation into CM lineage. These factors are described as distinctive features of the native stem cells of the heart. A marked local increase in their number in response to trauma in the adult heart indicates their role in regeneration [19]. A similar increase in the number of stem-like cells with the surface antigen Sca-1 and c- kit and their proliferation was observed during a heart attack, which was accompanied by an increase in telomerase activity [70]. Telomerase delays growth arrest, aging and prevents cell death. It may also be involved in the fight against mechanical and oxidative stress [71], which increases with a concomitant increase in ROS during necrosis and inflammation [72].

Conditions of hypoxia can reduce the oxidative stress after induced myocardial infarction. This leads to an increase in proliferation, which further helps to reduce the area of fibrosis and improves systolic function [73]. In addition to these data, the possibility was found of increasing the regenerative potential of hearts at one week of postnatal development of mice after administration of the antioxidant *<sup>N</sup>*-acetyl-*L*-cysteine [21].

Cardiac muscle regeneration after an injury is complicated by an "irreversible" exit of CMs from the cell cycle. A forced expression of *Tert* in the cardiac muscle in mice is sufficient to restore telomerase activity and telomere length. This, in turn, can delay the exit from the cell cycle in the cardiac muscle, cause hypertrophy in postmitotic cells and contribute to CM survival [14].

To elucidate a role of telomerase in cardiac regeneration, *Tert* was overexpressed in mouse hearts by adeno-associated viral delivery [14,20]. Mice were subjected to experimental myocardial infarction (MI). Upon MI, *Tert*-expressing hearts showed attenuated cardiac dilation, improved ventricular function and smaller infarct scars concomitant with increased survival by 17% compared with controls. Cardiac transcriptome analysis revealed an increase of epidermal growth factor receptor (EGF) in *Tert*-expressing hearts. Signaling through EGF is cardio-protective, emphasizing defensive function of Tert. Tert therapy also leads to activation of pathways associated with extracellular matrix remodeling (an increase in serum MMP-9 and TGF-b). TGF-b has a pleiotropic effect on almost all cell types involved in the repair and heart remodeling after injury. Long-term activation of these genes may also be a consequence of enhanced heart regeneration, which requires matrix remodeling to integrate new CMs. Indeed, the CM can re-enter the cell cycle after injury. Expression of Ki-67 and the presence of phosphorylated forms of histone H3 (proteins, the maximum expression of which coincides with mitosis) was found to increase the number of proliferating CMs near the infarct zone in the Tert-treated group. According to these results, it is plausible that activation of Tert may assist cardiac regeneration [13].

After injury, sufficient telomere length is required for proliferation. In P1 G3 *Terc*-null newborn mice, the proliferative capacity is lower than that of WT controls. Instead of increasing proliferation, CMs of G3 *Terc*-/- mice grow hypertrophic. The aggravation of telomere shortening after cryogenic damage at P1 in the heart of G3 mice causes an increase of p21 levels compared to WTs, which indicates the activation of the DNA damage response. This leads to the cessation of the cell cycle. A reliable proliferative response was observed in CMs of p21-/- mice that were seven days old. This is the age at which the CMs of WT mice lose the ability to divide after injury. This observation further emphasizes the participation of telomerase inhibition through the expression of p21 in stopping cell cycle and inhibition of the regeneration reaction after injury in the early postnatal period [13].

The proliferative ability is obviously associated with increased telomerase activity relative to mammals; therefore, the *D. rerio* model is interesting from the point of view of the possibility of regeneration of CMs.

In a recent study, it was shown that cryogenic damage to the heart leads to an increase in telomerase activity in WT zebrafish, which was associated with an increase in *tert* gene expression. To determine the role of telomeres in regeneration, damage to the heart was performed with *tert*-/- and WT *D. rerio*. It was shown that the cardiac output was not restored, and the area of damage did not decrease in *tert*-/- fish, which indicates the need for telomerase during regeneration. However, the level of the inflammatory response, an important process for regeneration, was the same for WT and *tert*-/- fish. Similarly, dedi fferentiation of CMs in response to an injury occurs normally in the absence of telomerase. The length of telomeres increases only in WT CMs during regeneration, and this is characteristic of both actively proliferating cells and nondividing cells. Therefore, we can conclude that telomere elongation is important for CM regeneration [27].

Induction of polyploidy in the heart of zebrafish leads to a loss of regenerative potential after injury [29]. Consequently, for recovery of the myocardium, diploid CMs are required, the number of which decreases in the mammalian heart with age as a result of telomere depletion [13].
