Aged-senescent cells contribute to impaired heart regeneration

Abstract

Aging leads to increased cellular senescence and is associated with decreased potency of tissue-specific stem/progenitor cells. Here, we have done an extensive analysis of cardiac progenitor cells (CPCs) isolated from human subjects with cardiovascular disease, aged 32-86 years. In aged subjects (>70 years old), over half of CPCs are senescent (p16^INK4A , SA-β-gal, DNA damage γH2AX, telomere length, senescence-associated secretory phenotype [SASP]), unable to replicate, differentiate, regenerate or restore cardiac function following transplantation into the infarcted heart. SASP factors secreted by senescent CPCs renders otherwise healthy CPCs to senescence. Elimination of senescent CPCs using senolytics abrogates the SASP and its debilitative effect in vitro. Global elimination of senescent cells in aged mice (INK-ATTAC or wild-type mice treated with D + Q senolytics) in vivo activates resident CPCs and increased the number of small Ki67-, EdU-positive cardiomyocytes. Therapeutic approaches that eliminate senescent cells may alleviate cardiac deterioration with aging and restore the regenerative capacity of the heart.

Keywords: aging; cardiac regeneration; cardiac repair; myocardial infarction; p16INK4a; progenitor cells; senescence; senescence-associated secretory phenotype; senolytics.

Figure 1

Over half of cardiac progenitor cells (CPCs) in the aged human heart are senescent (a) Representative immunofluorescence images and quantification of c‐kit^pos p16^INK4Apos CPCs, (n = 35 donors), (b) c‐kit^pos SA‐β‐gal^pos CPCs (*p = 0.0014; n = 2–4), (c) c‐kit^pos γ‐H2AX^pos p16^INK4A‐expressing CPCs (*p = 0.0264; n = 5 donors). (d) Q‐FISH telomere length of single c‐kit^pos CPCs (n = 100 cells per group, 20 cells per donor). Representative immunofluorescence images of telomere staining, L5178Y‐S (10 kb) and L5178Y‐R (79 kb) are mouse cell lines with known telomere length. Frequency distribution histogram of CPC telomere length (n = 5 donors/group). Nuclei stained in blue by DAPI. All data are mean ± SEM

Figure 2

Cardiac progenitor cells (CPCs) isolated from aged hearts exhibit diminished proliferation, clonogenicity and cardiomyocyte differentiation potential (a) Quantification of CPC proliferation (*p = 0.0266; n = 3–4 donors), (b) single CPC‐derived clonal efficiency (*p = 0.0008, n = 4–5 donors), (c) CPC spherogenesis number (*p = 0.0051) and (d) size (*p = 0.01), (n = 2–3 donors). (e) Representative immunofluorescence images of undifferentiated and differentiated CPCs from old and middle‐aged donors. Nuclei stained in blue by DAPI. (f) Quantification of Nkx2.5^pos (*p = 0.0078; n = 3 donors) and (g) α‐sarcomeric actin expression (*p = 0.0140; n = 3 donors). All data are mean ± SEM

Figure 3

Aged‐senescent CPCs show decreased reparative potential (a) In vivo MI experimental design. (b) Representative confocal images of PKH26^pos CPCs engrafted in the myocardium 4 days post‐MI. (c) Quantification and representative confocal images of engraftment of PKH26^pos cells 28 days post‐MI (*p = 0.015 vs. c‐kit^neg cells, n = 4–5 mice). (d) Echocardiography measurements of LV ejection fraction (EF), fractional shortening (FS), left ventricular end‐diastolic diameter (LVEDD) and left ventricular end‐systolic diameter (LVESD) at baseline (BL) before MI, 7 and 28 days after MI and cell injection (*p < 0.05 vs. Sham, †p < 0.05 vs. Cycling‐CPCs, Δp < 0.05 vs. dox‐induced Sen‐CPCs, n = 5–7 mice). (e) Representative micrographs and quantification of average LV fibrosis (*p < 0.05 vs. Sham; †p = 0.0112 vs. Cycling‐CPCs; n = 5–6 mice). (f) Representative confocal images of PKH26 co‐expression with α‐sarcomeric actinin and vWF (arrowheads) 28 days post‐MI. (g) Quantification and representative confocal images of BrdU^pos/α‐sarcomeric actinin^pos cardiomyocytes and BrdU^pos/vWF^pos capillaries (*p < 0.05 vs. all; n = 5–7 mice). Nuclei stained in blue by DAPI. All data are mean ± SEM

Figure 4

Aged‐senescent CPCs have a SASP (a) Transcript SASP factor expression of dox‐induced Sen‐CPCs relative to Cycling‐CPCs (control). (b) SASP factor protein levels quantified by Luminex of unconditioned media (UM), Cycling‐CPC (CM) and dox‐induced Sen‐CPC (Sen CM) conditioned media (*p < 0.05 vs. UM and CM). Conditioned media applied to cycling‐competent CPCs and the following analyses performed; (c) Quantification and representative staining of CPC proliferation (*p < 0.001; n = 5 replicates), (d) p16^INK4A‐pos CPCs (*p < 0.0001; n = 5 replicates), (e) SA‐β‐gal^pos CPCs (*p < 0.0001; n = 5 replicates), (f) γH2AX^pos CPCs (*p < 0.001; n = 5 replicates). All data are mean ± SEM

Figure 5

Senolytic clearance abrogates the SASP (a) Viability (Crystal violet) and SA‐β‐gal quantification of dox‐induced Sen‐CPCs and Cycling‐CPCs exposed to various concentrations of D + Q for 3 days. (b–f) Quantification of 7 days of co‐culture of Cycling‐CPCs with dox‐induced Sen‐CPCs for (b) viability (*p = 0.0068); (c) proliferation (*p = 0.0056); (d) p16^INK4A (*p = 0.0025); (e) SA‐β‐gal (*p = 0.0025); (f) γH2AX (*p = 0.0002). CTRL is Cycling‐ CPCs alone. (n = 5 replicates). (g) Representative SA‐β‐gal staining after clearance of dox‐induced Sen‐CPCs from co‐culture by D + Q treatment. (h–k) Quantification of 17 days of co‐culture of Cycling‐CPCs with dox‐induced Sen‐CPCs or co‐culture with D + Q treatment for (h) viability (*p < 0.0001 vs. CTRL 17 days; †p = 0.0001 vs. co‐culture 17 days); (i) proliferation (*p < 0.0001 vs. CTRL 17 days; †p < 0.0001 vs. co‐culture 17 days); (j) p16^INK4A (*p < 0.0001 vs. CTRL 17 days; †p < 0.0001 vs. co‐culture 17 days) and (k) SA‐β‐gal (*p < 0.0001 vs. CTRL 17 days; †p < 0.0001 vs. co‐culture 17 days). CTRL is Cycling‐CPCs alone. (n = 5 replicates). (l) SASP factor protein levels quantified by Luminex from each treatment condition (*p < 0.0001 vs. CTRL; †p < 0.01 vs. co‐culture 7 days and 17 days). (n = 2 replicates). All data are mean ± SEM

Figure 6

Clearance of senescent cells stimulates new cardiomyocyte formation in the aged heart (a) In vivo senescent cell clearance experimental design. (b) Total cardiac p16^Ink4a gene expression following clearance (*p < 0.01 vs. Vehicle; n = 5 mice). (c) Quantification of CPCs following clearance, (*p < 0.0001 vs. Vehicle; †p = 0.0453 vs. AP; n = 10–11 mice). (d) Frequency distribution histogram of cardiomyocyte diameter, (n = 6 mice). (e) A Ki67^pos/α‐sarcomeric actin^pos cardiomyocyte (arrowhead) in the LV of a 32 month D + Q‐treated mouse. (f) Quantification of Ki67^pos cardiomyocytes, (*p < 0.0001 vs. Vehicle; n = 10 mice). (g) An EdU^pos/α‐sarcomeric actin^pos cardiomyocyte in the LV of a 22 month INK‐ATTAC AP‐treated mouse. Nuclei are stained by DAPI in blue. (h) Quantification of EdU^pos cardiomyocytes (*p < 0.0001 vs. Old + Vehicle; †p < 0.0001 vs. Young + AP; n = 5 mice). (i) Quantification of LV fibrosis (*p < 0.05 vs. Vehicle; n = 3 mice). All data are mean ± SD