NF-κB-Specific Suppression in Cardiomyocytes Unveils Aging-Associated Responses in Cardiac Tissue

Figures

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  Figure 1. Comparison of WT and 3M groups. (a) Survival analysis of WT and 3M animal groups using a Kaplan–Meier curve (p = 0.0167; n = 28 for each group); (b) monitoring of body weight (g) over the 18 months of WT and 3M animals (n = 12 for each group); values are expressed as mean ± SD.

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  Figure 2. Analysis of cardiac trophism in WT and 3M mice. (a) Representative image of hearts collected during euthanasia of young and old WT mice (left) and young and old 3M mice (right); a grid sheet, scaled to 1mm per side of each square, is included in the background image to facilitate visual comparison of heart sizes. (b) Heart weight-to-tibia length ratio (HW/TL) and (c) heart weight-to-body weight ratio (HW/BW), n = 10 per group. (d) Representative images of the histological analysis of cardiac hypertrophy and (e) quantification of the cross-sectional area of cardiomyocytes in the experimental groups (n = 4–5 in each group). Uncropped images provided in Figure S1. Crops of the entire original histological images were made to highlight and better represent the corresponding quantitative findings. Scale bar = 50 µm. Values are expressed as mean ± SD; * p < 0.05 vs. young WT, & p < 0.05 vs. young 3M, # p < 0.05 vs. old WT.

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  Figure 3. Histological analysis of cardiac fibrosis. (a) Representative images and (b) quantification of the collagen area fraction in picrosirius-stained slides from young and old WT and 3M groups. Values are expressed as mean ± SD. * p < 0.01 vs. young WT; n = 4 in each group. Scale bar = 100 µm.

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  Figure 4. Evaluation of apoptosis through the TUNEL assay. (a) Representative images; (b) quantification of the percentage of TUNEL-positive nuclei by the total number of nuclei in young and old WT and 3M groups. Values are expressed as mean ± SD. * p < 0.01 vs. young WT; # p < 0.05 vs. old WT; n = 4 in each group. Scale bar = 50 µm.

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  Figure 5. Analysis of cellular senescence markers. Quantitative analysis of mRNA levels by real-time PCR and protein expression by Western blot of p21 (a,c) and p53 (b,d) in young and old WT and 3M mice. (e) Representative image of the obtained blots. Uncropped images provided in Figure S2. * p < 0.05 vs. young WT; # p < 0.05 vs. old WT (n = 5–8 for real-time PCR experiments and n = 9–10 for Western blot experiments).

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  Figure 6. Lipofuscin pigments and senescence-associated β-galactosidase (SA-β-gal) activity. (a) Representative images of histological slides stained with Sudan Black B (SBB), with arrows indicating lipofuscin pigments. (b) Quantification of the percentage of lipofuscin (n = 4 in each group); * p < 0.01 vs. young WT; & p < 0.01 vs. young 3M. (c) Macroscopic visualization of SA-β-gal activity in transverse sections of hearts from young and aged WT and 3M mice.

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  Figure 7. Analysis of relative telomere length and DNA damage. (a) Quantification of relative telomere length, assessed by real-time PCR, in cardiac tissue samples from young and old WT and 3M mice (n = 8 in each group). (b) Representative image of the obtained blots and quantification of phosphorylated histone H2A.X (Ser 139) protein expression, considered a marker of DNA damage (n = 7 in each group). Uncropped images provided in Figure S2. * p < 0.05 vs. young WT.