Sci. Aging Knowl. Environ., 14 May 2003
Vol. 2003, Issue 19, p. pe11
[DOI: 10.1126/sageke.2003.19.pe11]

PERSPECTIVES

Telomerase and the Aging Heart

E. Kevin Heist, Fawzia Huq, and Roger Hajjar

The authors are at the Cardiovascular Research Center, Massachusetts General Hospital, Boston, MA 02129, USA. E-mail: hajjar{at}cvrc.mgh.harvard.edu (R.H.)

http://sageke.sciencemag.org/cgi/content/full/sageke;2003/19/pe11

Key Words: cardiovascular disease • telomerase • telomere • Terc-/- mice • cardiomyocyte • replicative senescence

Introduction

As our population ages and the average life span increases, so does the burden of cardiovascular disease. Although other risk factors such as lifestyle patterns, genetic traits, blood lipid levels, and diabetes can contribute to the development of this condition, advancing age unequivocally remains the most powerful predictor of developing cardiac disease (1). Pathologic changes in cardiac phenotype and function, such as diastolic dysfunction, reduced contractility, left ventricular hypertrophy, and heart failure, all increase in incidence with age. Given the limited capacity for cardiac regeneration, reversing or slowing the progression of these abnormalities poses a major challenge. In this article, we review how one of the critical mechanisms underlying cellular senescence--telomere shortening--is implicated in cardiac dysfunction and how manipulations of telomere length affect cardiac structure and function.

Telomeres are highly conserved unique structures that cap the ends of linear chromosomes and function to protect chromosomes from recombination, end-to-end fusion, and recognition as damaged DNA (2). The telomere hypothesis of cell aging states that the life span of a cell is restricted by the integrity of its telomeres (3) (see "More Than a Sum of Our Cells"). Normal somatic cells have limited replicative potential, and the shortening of telomeres with successive cell divisions during DNA replication is thought to be the crucial mechanism through which this "mitotic clock" operates. After a limited number of replicative cycles, a critical telomere length is reached (less than 5 kb in humans), triggering irreversible arrest of cell growth (4). Certain tumors and immortalized cell lines are able to maintain telomere length in the absence of detectable telomerase activity (5). This process, termed "alternative lengthening of telomeres," has not been observed in normal tissue, and so its relevance outside of the field of oncology is unknown.

Telomere length varies between species and may even be different between members of the same species (6). In humans, telomere length is 10 to 15 kb in the germ line, in contrast to the situation in somatic tissues, where the telomere length is significantly shorter (7). Laboratory mice (Mus musculus) have relatively long telomeres (30 to 150 kb) in somatic (telomerase-negative) cells, whereas the related mouse species Mus spretus has shorter telomeres, closer in length to those in humans (6), when assessed by Southern blotting. However, recent studies analyzing telomere length using quantitative fluorescence in situ hybridization have shown significantly shorter telomere lengths in Mus musculus (10 to 60 kb) (8), suggesting that artifacts in length detection methodology might contribute to perceived differences in telomere length.

The primary enzyme responsible for maintaining intact telomeres over successive cell divisions is telomerase, an RNA-dependent DNA polymerase that is found predominantly in germ cells, tumor cells, and stem cells (which have limitless proliferative capacity) (9). Telomerase consists of two essential components: a functional RNA that serves as a template for the synthesis and restoration of the DNA tandem repeat (9) and a catalytic subunit with reverse transcriptase activity, hTERT (2). Expression of hTERT is closely associated with telomerase activity in cells; it is generally repressed in normal cells and up-regulated in immortal cells (2). In fact, most normal human cells, including cardiac myocytes, do not express hTERT and have undetectable telomerase activity (9). In contrast, a number of mouse tissues do display telomerase activity (the testis, liver, kidney, and spleen) (10). In the myocardium, telomerase activity is detected in embryonic and neonatal myocardium but is undetectable in adult (aged 8 weeks) cardiac tissue. Whether telomerase is expressed in the embryonic or neonatal human myocardium is unknown, but differences in telomerase expression between humans and mice may be a limitation in using mouse models to study the role of telomere shortening in age-associated conditions in humans.

Telomerase and Senescence

The relationship between replicative senescence (a condition in which cells no longer divide) and telomerase is an intriguing one. Shortening of telomeres in cultured cells lacking telomerase has been shown to result in both senescence-associated gene expression and inhibition of cell replication (11-14). Although the precise mechanisms underlying how shortened telomeres trigger the onset of cellular senescence are unknown (see "Senescence Tip-Off"), a number of pathways have been implicated in this process. These include the p53 and Rb tumor suppressor pathways, activation of which induces cell cycle arrest or apoptosis in response to cell injury (15, 16) (see Campisi Perspective). If successive shortening of telomeres leads to cellular senescence, then the corollary of this hypothesis of cell aging would suggest that maintaining telomere length through successive generations should immortalize the cell. There is considerable evidence to support this hypothesis. Expression of hTERT via the use of a viral vector in human somatic cells (retinal pigment epithelium and foreskin fibroblasts), which results in the maintenance of telomere length in these cells through successive generations, has shown that the replicative limit of cells can be considerably extended in situ (by up to 20 doublings) (7). Not only is the number of cell divisions increased but the cells are "younger" as assessed by their patterns of gene expression, morphology, and physiological markers.

Terc-/- Mice and Cardiac Function

So what are the implications of telomere shortening in cardiac myocytes? In order to define further the role of telomerase in physiologic and pathologic processes, a mouse strain was engineered in which the gene encoding the telomerase RNA component was deleted (Terc-/- mice) (17). The first generation of this murine line is relatively normal with regard to both telomere length and overall phenotype, despite the lack of telomerase activity. Successive generations, however, display progressively shorter telomeres (such that 3 to 5 kb of telomeric DNA are lost per generation) and suffer from more severe phenotypic abnormalities, with complete loss of viability after three to six generations, depending on the genetic background (18, 19). Intermediate generations demonstrate marked abnormalities in rapidly dividing tissues, which are presumably most dependent on telomere function, such as the gonads and the bone marrow. Signs of premature aging, such as the early graying of hair, are also evident (18, 20).

A recent paper by Leri and colleagues (21) focuses attention on the cardiac phenotype associated with telomerase ablation (see "Heartsick Chromosomes"). Although the ability of mature cardiomyocytes to replicate or be repopulated via migration of stem cells is controversial (22-24) (see Edelberg Perspective), the heart, like all organs, is critically dependent on cell replication during organogenesis. As with other tissues, the heart in Terc-/- mice demonstrates an increasingly abnormal phenotype (as described below), progressive telomere shortening, and a reduction in total cardiomyocyte numbers with successive generations. Although enlargement of cardiomyocytes (cellular hypertrophy) does occur in the affected mice, this change only partially compensates for the substantial loss of cells as the total heart mass is reduced in later-generation Terc-/- mice as compared to controls. These mice demonstrate a classic dilated cardiomyopathy (severe heart failure) that is functionally similar to that generated by a number of established methods of inducing heart failure. Abnormalities in both heart contraction and in relaxation are present in these animals. Analysis of isolated cardiomyocytes from these mice demonstrates decreasing telomere length with successive generations and up-regulation of p53 expression, specifically in the individual cells with the shortest telomeres (Fig. 1). Because p53 can induce apoptosis and growth arrest, this observation suggests an intriguing molecular explanation for the reduced cell number seen in the hearts of late-generation Terc-/- mice. Nuclear labeling studies in cells from these animals reveal increased cellular apoptosis but comparatively little cellular necrosis, substantiating the idea that increased p53 expression is responsible for the reduction in the number of cardiomyocytes (21). An important caveat to the findings relative to the cardiac system in late-generation Terc-/- mice is that cardiac development has taken place in animals that contain abnormalities within virtually all tissues as a result of the Terc gene knockout. Thus, the absence of telomerase activity in the heart itself and the general tissue abnormalities in the whole organism might both contribute to the cardiac phenotype. The relative importance of each of these factors remains to be determined, and so it cannot yet be said with certainty that the lack of cardiac telomerase specifically leads to the heart failure phenotype observed in these animals.



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Fig. 1. Telomere length and p53 expression in myocyte nuclei. Myocyte nuclei are stained by a PNA (peptide nucleic acid) probe to detect telomere length (red fluorescence) and antibodies that recognize p53 (green fluorescence). Blue fluorescence corresponds to propidium iodide (a nonspecific nuclear stain). (A and B) Myocyte nuclei from the left ventricle of wild-type mice. (C and D) Myocyte nuclei from the left ventricle of fifth-generation (G5) Terc–/– mice. Arrowheads point to the nuclei that have short telomeres and are positive for p53. Scale bar, 10 µm. (E) The frequency distribution of telomere length in wild-type, G2 Terc–/–, and G5 Terc–/– mice (open bars). The corresponding values for each subclass of nuclei expressing p53 are shown by solid bars. [Figure is reproduced, and legend is reproduced with slight modifications, with permission from (21)]

 
Telomerase-Directed Therapeutic Intervention

Clearly, complete loss of the Terc gene has profoundly negative implications for successive generations of mice. More interesting from a therapeutic perspective, however, is whether there is a potential benefit to be gained from telomerase replacement or other telomerase-targeted therapies in aging-associated diseases such as heart failure. Previous studies have shown that overexpression of the telomerase catalytic subunit in the mouse germ line causes delayed exit from the cell cycle and hence greater proliferation of cardiac myocytes during cardiac development (25). This results in a normal-sized heart at young ages that consists of a larger number of smaller cardiomyocytes as compared to those in control animals. At later ages, these animals develop hypertrophy at the level of both the individual cardiomyocyte and the overall heart. Because telomerase-directed therapy would probably be administered relatively late in the course of a person's life, and certainly well past the stage of cardiogenesis, it is important to understand the effects of inducing telomerase expression in nondividing cells, which represent a large fraction of the cardiomyocyte cell population at this stage of life.

Expression of the catalytic subunit of telomerase in rat cardiomyocytes that no longer divide in response to serum stimulation has been achieved by adenoviral gene transfer (25). Although this manipulation did not cause cell division to resume in these postmitotic cells, it did stimulate hypertrophic growth of the infected cells, which could prove to be beneficial or detrimental depending on the clinical situation to which it might be applied. Interestingly, telomerase expression reduced apoptosis in these cultured myocytes, an effect that could potentially slow the progression of human heart disease. The effects of telomerase expression were also examined in transgenic mice. In these mice, overexpression of telomerase was found to provide partial protection from experimentally induced ischemia-reperfusion injury (achieved by coronary artery ligation) and limited the size of the "heart attack" produced, potentially through its antiapoptotic effect (25).

Conclusions

Further studies will be necessary to determine whether telomerase-directed therapy will prove to be an effective treatment for human heart failure and aging-associated cardiac dysfunction. In vivo gene transfer allows expression of exogenous genes in cardiomyocytes (Fig. 2) and so this technique could potentially allow for telomerase expression to treat a variety of human cardiac diseases in the future. Current evidence suggests that telomerase expression is unlikely to create new cardiomyocytes through the initiation of cell division in quiescent heart cells, but it might serve to induce compensatory cellular hypertrophy and slow the progression of heart failure through protection from apoptosis (25). Other potential benefits that have been observed in experimental systems after the induction of telomerase expression include improved endothelial function and increased angiogenesis (the development of new blood vessels), which might enhance the heart's blood supply (26-29). Certainly, targeting telomerase is an intriguing potential treatment for cellular senescence in many aging-associated diseases. Despite the promise, however, there are concerns about the consequences of telomerase-directed therapies for the treatment of cardiovascular disease. The long-term effects of inducing cardiomyocyte hypertrophy in this manner are not yet clear. Furthermore, telomerase expression in cardiac fibroblasts could potentially exacerbate cardiac fibrosis by promoting fibroblast cell division and thereby worsen cardiac dysfunction related to age or heart failure. Telomerase expression can induce perpetual cell division in cultured cells (7, 30), so the possibility that this therapy might cause cancer must be considered in the risk-benefit analysis of any potential therapy. Although cell immortalization generated by telomerase expression produces fewer karyotypic and phenotypic abnormalities than immortalization generated by expression of the SV40 oncogene, cells immortalized by telomerase are nonetheless karyotypically abnormal (31). Given the differences in telomere length and structure between mice and humans, the applicability of studies done in mice to human disease have yet to be determined. Only time and further study will determine how telomerase-targeted approaches will stack up against the multitude of therapies currently being developed to fight the ravages of aging in the heart and throughout the body.



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Fig. 2. Cardiomyocytes isolated from an aging rat infected with adenovirus expressing green fluorescent protein (GFP). A phase-contrast micrograph is shown on the left, and a fluorescence micrograph to detect GFP is shown on the right. Scale bar, 100 µm.

 


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Citation: E. K. Heist, F. Huq, R. Hajjar, Telomerase and the Aging Heart. Sci. SAGE KE 2003, pe11 (14 May 2003)
http://sageke.sciencemag.org/cgi/content/full/sageke;2003/19/pe11








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