Senescence and death of primitive cells and myocytes lead to premature cardiac aging and heart failure

Chronological myocardial aging is viewed as the inevitable effect of time on the functional reserve of the heart. Cardiac failure in elderly patients is commonly interpreted as an idiopathic or secondary myopathy superimposed on the old heart independently from the aging process. Thus, aged diseased hearts were studied to determine whether cell regeneration was disproportionate to the accumulation of old dying cells, leading to cardiac decompensation. Endomyocardial biopsies from 19 old patients with a dilated myopathy were compared with specimens from 7 individuals of similar age and normal ventricular function. Ten patients with idiopathic dilated cardiomyopathy were also analyzed to detect differences with aged diseased hearts. Senescent cells were identified by the expression of the cell cycle inhibitor p16INK4a and cell death by hairpin 1 and 2. Replication of primitive cells and myocytes was assessed by MCM5 labeling, myocyte mitotic index, and telomerase function. Aged diseased hearts had moderate hypertrophy and dilation, accumulation of p16INK4a positive primitive cells and myocytes, and no structural damage. Cell death markedly increased and occurred only in cells expressing p16INK4a that had significant telomeric shortening. Cell multiplication, mitotic index and telomerase increased but did not compensate for cell death or prevented telomeric shortening. Idiopathic dilated cardiomyopathy had severe hypertrophy and dilation, tissue injury, and minimal level of p16INK4a labeling. In conclusion, telomere erosion, cellular senescence, and death characterize aged diseased hearts and the development of cardiac failure in humans.     

Telomeres, stem cells, and hematology

Telomeres are highly dynamic structures that adjust the cellular response to stress and growth stimulation based on previous cell divisions. This critical function is accomplished by progressive telomere shortening and DNA damage responses activated by chromosome ends without sufficient telomere repeats. Repair of critically short telomeres by telomerase or recombination is limited in most somatic cells, and apoptosis or cellular senescence is triggered when too many uncapped telomeres accumulate. The chance of the latter increases as the average telomere length decreases. The average telomere length is set and maintained in cells of the germ line that typically express high levels of telomerase. In somatic cells, the telomere length typically declines with age, posing a barrier to tumor growth but also contributing to loss of cells with age. Loss of (stem) cells via telomere attrition provides strong selection for abnormal cells in which malignant progression is facilitated by genome instability resulting from uncapped telomeres. The critical role of telomeres in cell proliferation and aging is illustrated in patients with 50% of normal telomerase levels resulting from a mutation in one of the telomerase genes. Here, the role of telomeres and telomerase in human biology is reviewed from a personal historical perspective.     

Telomerase activity in rat cardiac myocytes is age and gender dependent

Telomerase replaces telomeric repeat DNA lost during the cell cycle, restoring telomere length. This enzyme is present only during cell replication and its activity reflects the extent of proliferation. Whether cardiac myocytes are terminally differentiated cells is still a highly controversial issue, and the possibility of myocyte division is frequently rejected. On this basis, telomerase was measured in pure preparations of myocytes, isolated from rats throughout their lifespan. Fetal and neonatal rat myocytes were used as positive control cells. Contrary to expectation, the authors report that telomerase activity was detectable in pure preparations of young adult, fully mature adult, and senescent ventricular myocytes, defeating the dogma that this cell population is permanent and irreplaceable. Aging decreased 31% telomerase activity in male myocytes. An opposite effect occurred in female myocytes in which this enzyme increased 72%. This differential adaptation between the two genders in the rat model may be relevant to observations in humans; myocyte loss occurs in men as a function of age, whereas myocyte number is preserved in women. The greater growth potential of female myocytes may be critical for the longer lifespan and decreased incidence of heart failure in women.     

Vascular smooth muscle cells undergo telomere-based senescence in human atherosclerosis: effects of telomerase and oxidative stress

Although human atherosclerosis is associated with aging, direct evidence of cellular senescence and the mechanism of senescence in vascular smooth muscle cells (VSMCs) in atherosclerotic plaques is lacking. We examined normal vessels and plaques by histochemistry, Southern blotting, and fluorescence in situ hybridization for telomere signals. VSMCs in fibrous caps expressed markers of senescence (senescence-associated beta-galactosidase [SAbetaG] and the cyclin-dependent kinase inhibitors [cdkis] p16 and p21) not seen in normal vessels. In matched samples from the same individual, plaques demonstrated markedly shorter telomeres than normal vessels. Fibrous cap VSMCs exhibited markedly shorter telomeres compared with normal medial VSMCs. Telomere shortening was closely associated with increasing severity of atherosclerosis. In vitro, plaque VSMCs demonstrated morphological features of senescence, increased SAbetaG expression, reduced proliferation, and premature senescence. VSMC senescence was mediated by changes in cyclins D/E, p16, p21, and pRB, and plaque VSMCs could reenter the cell cycle by hyperphosphorylating pRB. Both plaque and normal VSMCs expressed low levels of telomerase. However, telomerase expression alone rescued plaque VSMC senescence despite short telomeres, normalizing the cdki/pRB changes. In vivo, plaque VSMCs exhibited oxidative DNA damage, suggesting that telomere damage may be induced by oxidant stress. Furthermore, oxidants induced premature senescence in vitro, with accelerated telomere shortening and reduced telomerase activity. We conclude that human atherosclerosis is characterized by senescence of VSMCs, accelerated by oxidative stress-induced DNA damage, inhibition of telomerase and marked telomere shortening. Prevention of cellular senescence may be a novel therapeutic target in atherosclerosis.     

Developmental and tissue-specific regulation of mouse telomerase and telomere length.

Telomere shortening and telomerase activation in human somatic cells have been implicated in cell immortalization and cellular senescence. To further study the role of telomerase in immortalization, we assayed telomere length and telomerase activity in primary mouse fibroblasts, in spontaneously immortalized cell clones, and in mouse tissues. In the primary cell cultures, telomere length decreased with increased cell doublings and telomerase activity was not detected. In contrast, in spontaneously immortalized clones, telomeres were maintained at a stable length and telomerase activity was present. To determine if telomere shortening occurs in vivo, we assayed for telomerase and telomere length in tissues from mice of different ages. Telomere length was similar among different tissues within a newborn mouse, whereas telomere length differed between tissues in an adult mouse. These findings suggest that there is tissue-specific regulation of mouse telomerase during development and aging in vivo. In contrast to human tissues, most mouse tissues had active telomerase. The presence of telomerase in these tissues may reflect the ease of immortalization of primary mouse cells relative to human cells in culture.     

Overexpression of insulin-like growth factor-1 in the heart is coupled with myocyte proliferation in transgenic mice.

Transgenic mice were generated in which the cDNA for the human insulin-like growth factor 1B (IGF-1B) was placed under the control of a rat alpha-myosin heavy chain promoter. In mice heterozygous for the transgene, IGF-1B mRNA was not detectable in the fetal heart at the end of gestation, was present in modest levels at 1 day after birth, and increased progressively with postnatal maturation, reaching a peak at 75 days. Myocytes isolated from transgenic mice secreted 1.15 +/- 0.25 ng of IGF-1 per 10(6) cells per 24 hr versus 0.27 +/- 0.10 ng in myocytes from homozygous wild-type littermates. The plasma level of IGF-1 increased 84% in transgenic mice. Heart weight was comparable in wild-type littermates and transgenic mice up to 45 days of age, but a 42%, 45%, 62%, and 51% increase was found at 75, 135, 210, and 300 days, respectively, after birth. At 45, 75, and 210 days, the number of myocytes in the heart was 21%, 31%, and 55% higher, respectively, in transgenic animals. In contrast, myocyte cell volume was comparable in transgenic and control mice at all ages. In conclusion, overexpression of IGF-1 in myocytes leads to cardiomegaly mediated by an increased number of cells in the heart.     

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) induces cancer cell senescence by interacting with telomerase RNA component

Oxidative stress regulates telomere homeostasis and cellular aging by unclear mechanisms. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a key mediator of many oxidative stress responses, involving GAPDH nuclear translocation and induction of cell death. We report here that GAPDH interacts with the telomerase RNA component (TERC), inhibits telomerase activity, and induces telomere shortening and breast cancer cell senescence. The Rossmann fold containing NAD(+) binding region on GAPDH is responsible for the interaction with TERC, whereas a lysine residue in the GAPDH catalytic domain is required for inhibiting telomerase activity and disrupting telomere maintenance. Furthermore, the GAPDH substrate glyceraldehyde-3-phosphate (G3P) and the nitric oxide donor S-nitrosoglutathione (GSNO) both negatively regulate GAPDH inhibition of telomerase activity. Thus, we demonstrate that GAPDH is regulated to target the telomerase complex, resulting in an arrest of telomere maintenance and cancer cell proliferation.     

Aging and atherosclerosis: mechanisms, functional consequences, and potential therapeutics for cellular senescence

Atherosclerosis is classed as a disease of aging, such that increasing age is an independent risk factor for the development of atherosclerosis. Atherosclerosis is also associated with premature biological aging, as atherosclerotic plaques show evidence of cellular senescence characterized by reduced cell proliferation, irreversible growth arrest and apoptosis, elevated DNA damage, epigenetic modifications, and telomere shortening and dysfunction. Not only is cellular senescence associated with atherosclerosis, there is growing evidence that cellular senescence promotes atherosclerosis. This review examines the pathology of normal vascular aging, the evidence for cellular senescence in atherosclerosis, the mechanisms underlying cellular senescence including reactive oxygen species, replication exhaustion and DNA damage, the functional consequences of vascular cell senescence, and the possibility that preventing accelerated cellular senescence is a therapeutic target in atherosclerosis.     

The young mouse heart is composed of myocytes heterogeneous in age and function

The recognition that the adult heart continuously renews its myocyte compartment raises the possibility that the age and lifespan of myocytes does not coincide with the age and lifespan of the organ and organism. If this were the case, myocyte turnover would result at any age in a myocardium composed by a heterogeneous population of parenchymal cells which are structurally integrated but may contribute differently to myocardial performance. To test this hypothesis, left ventricular myocytes were isolated from mice at 3 months of age and the contractile, electrical, and calcium cycling characteristics of these cells were determined together with the expression of the senescence-associated protein p16(INK4a) and telomere length. The heart was characterized by the coexistence of young, aged, and senescent myocytes. Old nonreplicating, p16(INK4a)-positive, hypertrophied myocytes with severe telomeric shortening were present together with young, dividing, p16(INK4a)-negative, small myocytes with long telomeres. A class of myocytes with intermediate properties was also found. Physiologically, evidence was obtained in favor of the critical role that action potential (AP) duration and I(CaL) play in potentiating Ca(2+) cycling and the mechanical behavior of young myocytes or in decreasing Ca(2+) transients and the performance of senescent hypertrophied cells. The characteristics of the AP appeared to be modulated by the transient outward K(+) current I(to) which was influenced by the different expression of the K(+) channels subunits. Collectively, these observations at the physiological and structural cellular level document that by necessity the heart has to constantly repopulate its myocyte compartment to replace senescent poorly contracting myocytes with younger more efficient cells. Thus, cardiac homeostasis and myocyte turnover regulate cardiac function.     

Morphological and functional alterations in ventricular myocytes from male transgenic mice with hypertrophic cardiomyopathy

Familial hypertrophic cardiomyopathy (FHC) is a human genetic disorder caused by mutations in sarcomeric proteins. It is generally characterized by cardiac hypertrophy, fibrosis, and myocyte disarray. A transgenic mouse model of FHC with mutations in the actin-binding domain of the alpha-myosin heavy chain (MyHC) gene displays many phenotypes similar to human FHC. At 4 months, male transgenic (TG) mice present with concentric cardiac hypertrophy that progresses to dilation with age. Accompanying this latter morphological change is systolic and diastolic dysfunction. Left ventricular (LV) myocytes from male TG and wild-type (WT) littermates at 5 and 12 months of age were isolated and used for morphological and functional studies. Myocytes from 5- and 12-month-old TG animals had shorter sarcomere lengths compared with WT. This sarcomere length difference was abolished in the presence of 2,3-butanedione monoxime, suggesting that the basal level of contractile element activation was increased in TG myocytes. Myocytes from 12-month-old TG mice were significantly longer than those from age-matched WT controls, and TG myocytes exhibited Z-band disorganization. When cells were paced at 0.5 Hz, TG myocyte relengthening and the fall in intracellular [Ca2+] were slowed when compared with cells from age-matched WT controls. Moreover, an increased amount of beta-myosin heavy chain protein was found in hearts from TG compared with WT. Thus, myocytes from the alpha-MyHC TG mouse model display many morphological and functional abnormalities that may help explain the LV dysfunction seen in this TG mouse model of FHC.     

bcl-2 overexpression promotes myocyte proliferation

To determine the influence of Bcl-2 on the developmental biology of myocytes, we analyzed the population dynamics of this cell type in the heart of transgenic (TG) mice overexpressing Bcl-2 under the control of the alpha-myosin heavy chain promoter. TG mice and non-TG (wild type, WT) mice were studied at 24 days, 2 months, and 4 months after birth. Bcl-2 overexpression produced a significant increase in the percentage of cycling myocytes and their mitotic index. These effects were strictly connected to the expression of the transgene, as demonstrated in isolated myocytes. The formation of mitotic spindle and contractile ring was identified in replicating cells. These typical aspects of mitosis were complemented with the demonstration of karyokinesis and cytokinesis to provide structural evidence of cell division. Apoptosis was low at all ages and was not affected by Bcl-2. The higher cell replication rate in TG was conditioned by a decrease in the expression of the cell-cycle inhibitors, p21(WAF1) and p16(INK4a), and by an increase in Mdm2-p53 complexes. In comparison with WT, TG had 0.4 x 10(6), 0.74 x 10(6), and 1.2 x 10(6) more myocytes in the left ventricle at 24 days, 2 months, and 4 months, respectively. Binucleated myocytes were 12% and 25% larger in WT than in TG mice at 2 and 4 months of age. Taken together, these observations reveal a previously uncharacterized replication-enhancing function of Bcl-2 in myocytes in vivo in the absence of stressful conditions.     

Adolescent feline heart contains a population of small, proliferative ventricular myocytes with immature physiological properties

Recent studies suggest that rather than being terminally differentiated, the adult heart is a self-renewing organ with the capacity to generate new myocytes from cardiac stem/progenitor cells (CS/PCs). This study examined the hypotheses that new myocytes are generated during adolescent growth, to increase myocyte number, and these newly formed myocytes are initially small, mononucleated, proliferation competent, and have immature properties. Ventricular myocytes (VMs) and cKit(+) (stem cell receptor) CS/PCs were isolated from 11- and 22-week feline hearts. Bromodeoxyuridine incorporation (in vivo) and p16(INK4a) immunostaining were measured to assess myocyte cell cycle activity and senescence, respectively. Telomerase activity, contractions, Ca(2+) transients, and electrophysiology were compared in small mononucleated (SMMs) and large binucleated (LBMs) myocytes. Heart mass increased by 101% during adolescent growth, but left ventricular myocyte volume only increased by 77%. Most VMs were binucleated (87% versus 12% mononucleated) and larger than mononucleated myocytes. A greater percentage of SMMs was bromodeoxyuridine positive (SMMs versus LBMs: 3.1% versus 0.8%; P<0.05), and p16(INK4a) negative and small myocytes had greater telomerase activity than large myocytes. Contractions and Ca(2+) transients were prolonged in SMMs versus LBMs and Ca(2+) release was disorganized in SMMs with reduced transient outward current and T-tubule density. The T-type Ca(2+) current, usually seen in fetal/neonatal VMs, was found exclusively in SMMs and in myocytes derived from CS/PC. Myocyte number increases during adolescent cardiac growth. These new myocytes are initially small and functionally immature, with patterns of ion channel expression normally found in the fetal/neonatal period.     

Diabetes promotes cardiac stem cell aging and heart failure, which are prevented by deletion of the p66shc gene

Diabetes leads to a decompensated myopathy, but the etiology of the cardiac disease is poorly understood. Oxidative stress is enhanced with diabetes and oxygen toxicity may alter cardiac progenitor cell (CPC) function resulting in defects in CPC growth and myocyte formation, which may favor premature myocardial aging and heart failure. We report that in a model of insulin-dependent diabetes mellitus, the generation of reactive oxygen species (ROS) leads to telomeric shortening, expression of the senescent associated proteins p53 and p16INK4a, and apoptosis of CPCs, impairing the growth reserve of the heart. However, ablation of the p66shc gene prevents these negative adaptations of the CPC compartment, interfering with the acquisition of the heart senescent phenotype and the development of heart failure with diabetes. ROS elicit 3 cellular reactions: low levels activate cell growth, intermediate quantities trigger cell apoptosis, and high amounts initiate cell necrosis. CPC replication predominates in diabetic p66shc-/-, whereas CPC apoptosis and myocyte apoptosis and necrosis prevail in diabetic wild type. Expansion of CPCs and developing myocytes preserves cardiac function in diabetic p66shc-/-, suggesting that intact CPCs can effectively counteract the impact of uncontrolled diabetes on the heart. The recognition that p66shc conditions the destiny of CPCs raises the possibility that diabetic cardiomyopathy is a stem cell disease in which abnormalities in CPCs define the life and death of the heart. Together, these data point to a genetic link between diabetes and ROS, on the one hand, and CPC survival and growth, on the other.     

Senescence research from historical theory to future clinical application

Historically, the findings from cellular lifespan studies have greatly affected aging research. The discovery of replicative senescence by Hayflick developed into research on telomeres and telomerase, while stress-induced senescence became known as a telomere-independent event. Senescence-inducing signals comprise several tumor suppressors or cell cycle inhibitors, e.g., p53, cyclin-dependent kinase inhibitor p16 Ink4a and others. Stress-induced senescence serves as a physiological barrier to oncogenesis in vivo, while it activates senescence-associated secretary phenotype, inducing chronic inflammation. Thus, beside telomere length, p16, p53 and inflammatory cytokines have been utilized as biomarkers for cellular senescence. Telomere lengths in human leukocytes correlate well with events of aging-related lifestyle diseases, indicating the importance of cellular senescence in organismal aging. As such, the development of senescence research will have significant future clinical applications, e.g., senolysis. Geriatr Gerontol Int 2021; 21: 125–130 .     

Telomerase expression and activity are coupled with myocyte proliferation and preservation of telomeric length in the failing heart

The role and even the existence of myocyte proliferation in the adult heart remain controversial. Documentation of cell cycle regulators, DNA synthesis, and mitotic images has not modified the view that myocardial growth can only occur from hypertrophy of an irreplaceable population of differentiated myocytes. To improve understanding the biology of the heart and obtain supportive evidence of myocyte replication, three indices of cell proliferation were analyzed in dogs affected by a progressive deterioration of cardiac performance and dilated cardiomyopathy. The magnitude of cycling myocytes was evaluated by the expression of Ki67 in nuclei. Ki67 labeling of left ventricular myocytes increased 5-fold, 12-fold, and 17-fold with the onset of moderate and severe ventricular dysfunction and overt failure, respectively. Telomerase activity in vivo is present only in multiplying cells; this enzyme increased 2.4-fold and 3.1-fold in the decompensated heart, preserving telomeric length in myocytes. The contribution of cycling myocytes to telomerase activity was determined by the colocalization of Ki67 and telomerase in myocyte nuclei. More than 50% of Ki67-positive cells expressed telomerase in the overloaded myocardium, suggesting that these myocytes were the morphological counterpart of the biochemical assay of enzyme activity. Moreover, we report that 20--30% of canine myocytes were telomerase competent, and this value was not changed by cardiac failure. In conclusion, the enhanced expression of Ki67 and telomerase activity, in combination with Ki67-telomerase labeling of myocyte nuclei, support the notion that myocyte proliferation contributes to cardiac hypertrophy of the diseased heart.     

Telomerase expression in chickens: Constitutive activity in somatic tissues and down-regulation in culture

Although human and rodent telomeres have been studied extensively, very little is known about telomere dynamics in other vertebrates. Moreover, our current dependence on mice as a model for human tumorigenesis and aging poses a problem because human and mouse telomere biology is very different. To explore whether chickens might provide a more useful model, we have examined telomerase activity and telomere length in chicken tissues as well as in primary cell cultures. Although chicken telomeres resemble human telomeres in that they are 8–20 kb in length, the distribution of telomerase activity in chickens resembles what is found in mice. Active enzyme is present in germline tissue as well as in a wide range of somatic tissues. Because chicken cells exhibit extremely low rates of spontaneous immortalization, this finding indicates that constitutive telomerase expression does not necessarily lead to an increased immortalization frequency. Finally, we found that telomerase activity is greatly down-regulated when primary cultures are established from chicken embryos. Although this down-regulation explains the telomere loss and replicative senescence that we observed in fibroblast cultures, it raises questions concerning how relevant studies of senescence in primary cell cultures are to aging in whole animals.     

Myocyte death and renewal: modern concepts of cardiac cellular homeostasis

The adult mammalian myocardium has a robust intrinsic regenerative capacity because of the presence of cardiac stem cells (CSCs). Despite being mainly composed of terminally differentiated myocytes that cannot re-enter the cell cycle, the heart is not a postmitotic organ and maintains some capacity to form new parenchymal cells during the lifespan of the organism. Myocyte death and formation of new myocytes by the CSCs are the two processes that enable this organ to maintain a proper and uninterrupted cardiac output from birth to adulthood and into old age. CSCs are activated in response to pathological or physiological stimuli, whereby they enter the cell cycle and differentiate into new myocytes (and vessels) that significantly contribute to changes in myocardial mass. The future of regenerative cardiovascular medicine is arguably dependent on our success in dissecting the biology and mechanisms regulating the number, growth, differentiation, and aging of CSCs. This information will generate the means to manipulate CSC growth, survival, and differentiation and, therefore, will provide the tools for the design of more physiologically relevant clinical regeneration protocols. In this article, we review the developments in cardiac cell biology that might, in our opinion, have a broad impact on cardiovascular medicine.