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Comparative Study
. 2011 Jul;179(1):349-66.
doi: 10.1016/j.ajpath.2011.03.036. Epub 2011 May 19.

Effects of Age and Heart Failure on Human Cardiac Stem Cell Function

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Free PMC article
Comparative Study

Effects of Age and Heart Failure on Human Cardiac Stem Cell Function

Daniela Cesselli et al. Am J Pathol. .
Free PMC article

Abstract

Currently, it is unknown whether defects in stem cell growth and differentiation contribute to myocardial aging and chronic heart failure (CHF), and whether a compartment of functional human cardiac stem cells (hCSCs) persists in the decompensated heart. To determine whether aging and CHF are critical determinants of the loss in growth reserve of the heart, the properties of hCSCs were evaluated in 18 control and 23 explanted hearts. Age and CHF showed a progressive decrease in functionally competent hCSCs. Chronological age was a major predictor of five biomarkers of hCSC senescence: telomeric shortening, attenuated telomerase activity, telomere dysfunction-induced foci, and p21(Cip1) and p16(INK4a) expression. CHF had similar consequences for hCSCs, suggesting that defects in the balance between cardiomyocyte mass and the pool of nonsenescent hCSCs may condition the evolution of the decompensated myopathy. A correlation was found previously between telomere length in circulating bone marrow cells and cardiovascular diseases, but that analysis was restricted to average telomere length in a cell population, neglecting the fact that telomere attrition does not occur uniformly in all cells. The present study provides the first demonstration that dysfunctional telomeres in hCSCs are biomarkers of aging and heart failure. The biomarkers of cellular senescence identified here can be used to define the birth date of hCSCs and to sort young cells with potential therapeutic efficacy.

Figures

Figure 1
Figure 1
Human cardiac stem cell (hCSC) niches. A: Atrial myocardium containing a cluster of six c-Kit-positive cells (green). Five are negative for α-sarcomeric actin (α-SA; red), and one has a minimal amount of this contractile protein (red spot indicated by arrow), a myocyte precursor. B–D: Four c-Kit-positive cells are present in this section of atrial myocardium. One cell is positive for OCT3/4 (yellow, indicated by arrow) (B); all four c-Kit-positive cells express NANOG (magenta). Myocytes are labeled by α-SA (white). E: Number of lineage-negative (Linneg), c-Kit-positive (c-Kitpos) hCSCs in the atria of donor (Don) and explanted (Exp) hearts. Data are reported as means ± SD. *P < 0.05 versus Don. F–I: Validation of immunolabeling by spectral analysis of c-Kit-positive cells (F), OCT3/4-positive nuclei (G), NANOG-positive nuclei (H), and α-SA-positive myocytes (I); corresponding images show examples of cells used in this assay.
Figure 2
Figure 2
Properties of hCSCs. A: Surface phenotype of small cardiac cells isolated from donor and explanted hearts. B–D: Growth properties of hCSCs. Data are reported as means ± SD. *P < 0.05 versus Don. E: Representative amplification curves of c-Kit and HPRT (housekeeping gene) expression in cell line A172 (red), which was used as positive control and in hCSC samples (black). In the histogram, solid bars indicate the relative expression of c-Kit in A172 cells (red) and in hCSCs (black), and open bars indicate the expression of c-Kit in hCSCs normalized versus A172 cells. Transcripts for OCT3/4, NANOG, SOX2, and KLF4 are present in hCSCs obtained from donor (Don-hCSCs) and explanted (Exp-hCSCs) hearts. The neuronally committed human teratocarcinoma cell line NT2 was used as positive control. PCR products were run on 1.8% agarose gel.
Figure 3
Figure 3
Clonal growth of hCSCs. A: Single cell-derived clone composed of c-Kit-positive hCSCs (green) expressing NANOG in their nuclei (magenta). B: Cloning efficiency of hCSCs from donor (Don) and explanted (Exp) hearts, reported as means ± SD. *P < 0.05 versus Don.
Figure 4
Figure 4
hCSC differentiation. A–H: Differentiating hCSCs acquire the myocyte lineage and express Nkx2.5 (white) (A), α-SA (red) (A, C, and D), α-cardiac actinin (red) (B), and connexin 43 (green, marked by arrow) (E). They also express the EC markers Ets1 (magenta), and von Willebrand factor (vWf, yellow) (F) and the SMC proteins GATA6 (yellow) (G) and α-smooth muscle actin (α-SMA) (magenta) (G and H). I: Differentiation of hCSCs from donor (Don) and explanted (Exp) hearts, reported as means ± SD. Scale bars: 10 μm (A, B, F, G); 20 μm (C, D, E, H).
Figure 5
Figure 5
Properties of differentiating myocytes . A: Ca2+ oscillations in hCSCs at baseline (undifferentiated) and after exposure to dexamethasone (differentiating) in the absence (Tyrode) and presence of caffeine (Caffeine). B: Fraction of cells displaying Ca2+ oscillation (Active Cells) and frequency of oscillatory events over a period of 33 minutes, with amplitude and duration of the oscillatory episodes. Data are reported as means ± SD. *P < 0.05 versus Tyrode. C: Ca2+ events in differentiating myocytes from donor and explanted hearts. *P < 0.05 versus Tyrode. D: Fraction of migrated hCSCs from donor (Don) and explanted (Exp) hearts. Data are reported as means ± SD. *P < 0.05 versus Don.
Figure 6
Figure 6
Telomere-telomerase axis in hCSCs from donor (Don) and explanted (Exp) hearts. A: In hCSC classes, products of telomerase activity display a 6-bp periodicity. Heat-inactivated lysates were used as negative control (lane 2 for each sample). TSR8 indicates telomerase control template; the 1301 cell line was used as a positive control (POS), and the primer-dimer lane as negative control (NEG). B: Dot plots of hCSCs hybridized without (mock-hybridization) and with the peptide nucleic acid (PNA) telomere probe. Gates were set around cells in the G0/G1 phase for both hCSCs (red) and control cells, tetraploid 1301 cell line (green). PI, propidium iodide. Relative telomeric length was computed as the ratio of the telomere signal of hCSCs and control cells. C–F: Four examples of telomere dysfunction-induced foci (TIFs) in which 53PB1 (red) colocalizes with telomere hybridization spots (green). Detail of interest (arrowhead, inset) is shown at higher magnification in the corresponding inset. G: γ-H2AX (green) and 53BP1 (red) colocalize at sites of DNA damage. The area included in the rectangles is shown at higher magnification on the right (yellow merged signals). In histograms, data are reported as means ± SD. H: Fraction of hCSCs with 1 to 5 TIFs. *P < 0.05 versus Don. Scale bars: 10 μm.
Figure 7
Figure 7
Senescence-associated proteins in hCSCs from donor (Don) and explanted (Exp) hearts. A and B: Phosphorylation of p53 at serine 15 by immunolabeling (red) (A) and Western blotting (B); Ran indicates loading conditions. C and D: Expression of p21Cip1 (yellow) (C) and p16INK4a (green) (D) in nuclei of hCSCs. Arrows represent positive nuclei. In histograms, data are reported as means ± SD. *P < 0.05 versus Don. Scale bars: 10 μm.
Figure 8
Figure 8
Biomarkers of senescence: telomere length in hCSCs from explanted and donor hearts. Linear regression of various parameters of hCSC function impairment.
Figure 9
Figure 9
Biomarkers of senescence: telomerase activity in hCSCs from explanted and donor hearts. Linear regression of various parameters of hCSC function impairment.
Figure 10
Figure 10
Biomarkers of hCSC senescence: telomere dysfunction-induced foci (TIFs) in hCSCs from explanted and donor hearts. Linear regression of various parameters of hCSC function impairment.
Figure 11
Figure 11
Biomarkers of hCSC senescence as a function of age in hCSCs from explanted and donor hearts. Linear regression of various parameters of hCSC function impairment.
Figure 12
Figure 12
Senescence of hCSCs from donor (Don) and explanted (Exp) hearts. A: Biomarkers of cellular senescence in hCSCs obtained from age-matched donor and explanted hearts are shown quantitatively. Data are reported as means ± SD. *P < 0.05 versus Don. B: Fraction of clonogenic hCSCs with TIFs, or expressing p16INK4a and p21Cip1.

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