Telomere shortening is an in vivo marker of myocyte replication and aging - PubMed (original) (raw)

Telomere shortening is an in vivo marker of myocyte replication and aging

J Kajstura et al. Am J Pathol. 2000 Mar.

Abstract

To determine whether adult cardiac myocytes are capable of multiple divisions and whether this form of growth is restricted to a subpopulation of cells that retain this capacity with age, telomere lengths were measured in myocyte nuclei isolated from the left ventricle of fetal and neonatal Fischer 344 rats and rats at 4, 12, and 27 months after birth. Two independent methodologies were used for this analysis: laser scanning cytometer and confocal microscopy. In each case, fluorescence intensity of a peptide nucleic acid probe specific for telomeric sequence was evaluated. The two techniques yielded comparable results. Telomeric shortening increased with age in a subgroup of myocytes that constituted 16% of the entire cell population. In the remaining nondividing cells, progressive accumulation of a senescent associated nuclear protein, p16(INK4), was evidenced. In conclusion, a significant fraction of myocytes divides repeatedly from birth to senescence, counteracting the continuous death of cells in the aging mammalian rat heart.

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Figures

Figure 1.

Percoll-treated myocytes (A). Nuclei are stained by PI (yellow fluorescence) and the cytoplasm by α-sarcomeric actin (red fluorescence). Isolated myocyte nuclei are depicted by red fluorescence of PI (B). Nuclei (blue fluorescence) after in situ hybridization with a PNA probe specific for telomeric sequence (C). Red fluorescent dots correspond to individual telomeres. Confocal microscopy; original magnifications, ×150 (A), ×300 (B), ×1500 (C).

Figure 2.

Bivariate distribution of DNA content and telomere length in myocyte nuclei from the left ventricle of fetal (F) and 1-day-old neonatal (N) rats, and rats at 4 (4M), 12 (12M), and 27 (27M) months of age.

Figure 3.

Measurements of the percentage of myocyte nuclei with shorter telomeres by laser scanning cytometer and confocal microscopy (see Figure 2 ▶ for symbols). Asterisks indicate statistical differences (P < 0.05) from F (*), N (**), 4M (***), and 12M (****) values; n = 5 in each group.

Figure 4.

Detection of p16INK4 in myocyte nuclear proteins by immunoprecipitation and Western blot. See Figure 2 ▶ for symbols.

Figure 5.

Localization of p16INK4 in myocyte nuclei by confocal microscopy in a rat heart at 27 months. A illustrates by red fluorescence nuclei stained by propidium iodide and B shows by green fluorescence p16INK4 labeling of the majority of nuclei. In C, red fluorescence corresponds to α-sarcomeric actin antibody staining of the myocyte cytoplasm and yellow fluorescence reflects the combination of PI and p16INK4 labeling of nuclei. Arrows, p16-positive myocyte nuclei; arrowheads, p16-positive nonmyocyte nuclei. Original magnifications, ×1200.

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Telomere shortening is an in vivo marker of myocyte replication and aging - PubMed (original) (raw)