Deficiency of telomere-associated repressor activator protein 1 precipitates cardiac aging in mice via p53/PPARα signaling

Abstract

Background: Telomere shortening and dysfunction may cause metabolic disorders, tissue damage and age-dependent pathologies. However, little is known about the association of telomere-associated protein Rap1 with mitochondrial energy metabolism and cardiac aging. Methods: Echocardiography was performed to detect cardiac structure and function in Rap1^+/+ and Rap1^-/- mice at different ages (3 months, 12 months and 20 months). Telomere length, DNA damage, cardiac senescence and cardiomyocyte size were analyzed using the real-time PCR, Western blotting, senescence associated β-galactosidase assay and wheat germ agglutinin staining, respectively. Western blotting was also used to determine the level of cardiac fatty acid metabolism related key enzymes in mouse and human myocardium. Chromatin immunoprecipitation assay was used to verify the direct link between p53 and PPARα. The p53 inhibitor, Pifithrin-α and PPARα activator WY14643 were utilized to identify the effects of Rap1/p53/PPARα signaling pathway. Results: Telomere was shortened concomitant with extensive DNA damage in aged Rap1^-/- mouse hearts, evidenced by reduced T/S ratios and increased nuclear γH2AX. Meanwhile, the aging-associated phenotypes were pronounced as reflected by altered mitochondrial ultrastructure, enhanced senescence, cardiac hypertrophy and dysfunction. Mechanistically, acetylated p53 and nuclear p53 was enhanced in the Rap1^-/- mouse hearts, concomitant with reduced PPARα. Importantly, p53 directly binds to the promoter of PPARα in mouse hearts and suppresses the transcription of PPARα. In addition, aged Rap1^-/- mice exhibited reduced cardiac fatty acid metabolism. Pifithrin-α alleviated cardiac aging and enhanced fatty acid metabolism in the aged Rap1^-/- mice. Activating PPARα with WY14643 in primarily cultured Rap1^-/- cardiomyocytes restored maximal oxygen consumption rates. Reduced Rap1 expression and impaired p53/PPARα signaling also presented in aged human myocardium. Conclusion: In summary, Rap1 may link telomere biology to fatty acid metabolism and aging-related cardiac pathologies via modulating the p53/PPARα signaling pathway, which could represent a therapeutic target in preventing/attenuating cardiac aging.

Keywords: PPARα; Rap1; cardiac aging; fatty acid metabolism; p53.

Figure 1

Protein expression of Rap1 was reduced with concomitantly enhanced p53 in the aged heart of mouse and human. (A) Representative Western blots of Rap1 and p53 expression in myocardium of mice at different ages (3-month, 12-month, 24-month). n = 6; (B) Representative Western blots of Rap1 and p53 in human myocardium with different age groups (young group, < 40 years, n = 7; middle age/aged group, > 40 years, n = 5). Protein presence of Rap1 and p53 was normalized to GAPDH. Data are shown as means ± S.E.M; ^*P < 0.05 12-month or 24-month vs. 3-month; or middle age/aged group vs. young group; ^#P < 0.05 24-month vs. 12-month.

Figure 2

Loss of Rap1 exacerbated cardiac hypertrophy, senescence and global cardiac performance. Transthoracic echocardiography was performed to evaluate left ventricular mass (A) in Rap1^+/+ and Rap1^-/- mice at different ages (3-month, n = 9; 12-month, n = 14; 20-month, n = 9). (B) Representative pictures from whole heart of Rap1^+/+ and Rap1^-/- mice at 12-month. (C) Heart weight/body weight ratio and (D) heart weight/tibia length ratio of Rap1^+/+ and Rap1^-/- mice at 12-month, n = 7; (E) Left ventricular end-diastolic diameter (LVIDd) in Rap1^+/+ and Rap1^-/- mice at different ages (3-month, n = 9; 12-month, n = 14; 20-month, n = 9). (F) Representative pictures of wheat germ agglutinin staining and quantification of cardiomyocyte area (n = 7) in the heart of Rap1^+/+ and Rap1^-/- mice at 20-month (n = 5). Myocardial performance index (MPI, G), isovolumic relaxation time (IVRT, H) and ratio of the early to late ventricular filling velocities (E/A ratio, I) in Rap1^+/+ and Rap1^-/- mice at different ages (3-month, n = 9; 12-month, n = 14; 20-month, n = 9). (J) Representative pictures of SA-β-Gal-stained heart sections and quantification of SA-β-Gal-staining positive area in the heart of Rap1^+/+ and Rap1^-/- mice at 20-month (n = 5). (K) Ejection fraction (EF) in Rap1^+/+ and Rap1^-/- mice at different ages (3-month, n = 9; 12-month, n = 14; 20-month, n = 9). Magnification 400×: Scale: 100 μm. Data are shown as means ± S.E.M; ^*P < 0.05 12-month or 20-month vs. 3-month; ^#P < 0.05 Rap1^-/- vs. Rap1^+/+; ^†P < 0.05 20-month vs. 12-month.

Figure 3

Loss of Rap1 led to shorter telomere and triggers DNA damage in the aging heart. In the heart of Rap1^+/+ and Rap1^-/- mice at 12-month of age, (A) the ratio of telomeric repeats with single-copy gene (T/S ratio) n = 7; (B) Representative Western blots of nuclear γH2AX expression, n = 6; (C) Representative pictures of dihydroethidium (DHE) staining, n = 7; (D) mRNA expression of thioredoxin-1 (Trx1), thioredoxin reductase (TrxR) and glutathione reductase (GR), n = 6; (E) mRNA expression of p53, n = 6; Representative Western blots of p21, p53 (F), acetyl-p53 (G) and nuclear p53 (H) expression, n = 6. (I) Representative field of view and quantification of immunofluorescent staining of p53 (red), α-actinin (green) and nucleus (DAPI, blue) in adjacent heart sections. Magnification 630×, scale bars: 20 µm; n = 8. Protein presence of nuclear γH2AX and nuclear p53 was normalized to Histone H3. Protein presence of p21, p53 and acetyl-p53 was normalized to GAPDH. mRNA levels are expressed as fold changes against those mRNA expressions in the myocardium of Rap1^+/+ mice. Data are shown as means ± S.E.M; ^#P < 0.05 Rap1^-/- vs. Rap1^+/+.

Figure 4

Loss of Rap1 reduced fatty acid metabolism with aging via PPARα. (A) Representative Western blots of CD36, ACC, CPT1α and ACADL expression (n = 6) and (B) ATP production (n = 7) in the heart of Rap1^+/+ and Rap1^-/- mice at 12-month of age; (C) Representative electron micrographs of mitochondria in the hearts of Rap1^+/+ and Rap1^-/- mice at 12-month of age with low (2200×) and high (5200×) magnifications. White arrow indicates more cristae fragmentation and vacuolization in Rap1^-/- mice. (D) Representative Western blots of CD36, ACC, CPT1α and ACADL expression (n = 6) in the primary cardiomyocytes from the hearts of Rap1^+/+ and Rap1^-/- mice at 12-month of age. Representative Western blots of PGC1α, PGC1β and PPARα expression (E) and nuclear PGC1α and PPARα expression (F) in the hearts of Rap1^+/+ and Rap1^-/- mice at 12-month of age, n = 6. Protein presence of CD36, ACC, CPT1α, ACADL, PGC1α, PGC1β and PPARα was normalized to GAPDH. Protein presence of nuclear PGC1α and PPARα was normalized to Histone H3. (G) mRNA expression of PPARα, PGC1α, CD36, ACOX1, CPT1α, Cyp4a10, FADS1 and FADS2 in the hearts of Rap1^+/+ and Rap1^-/- mice at 12-month of age, n = 5. (H) mRNA expression of PPARα in the hearts of Rap1^+/+ and Rap1^-/- mice at 3-month of age, n = 5. mRNA levels are expressed as fold changes against those mRNA expressions in the myocardium of Rap1^+/+ mice. (I) Plasma triglyceride level in the Rap1^+/+ and Rap1^-/- mice at 12-month of age, n = 7. Data are shown as means ± S.E.M; ^#P < 0.05 Rap1^-/- vs. Rap1^+/+.

Figure 5

p53 mediates the effect of Rap1 loss on fatty acid metabolism and cardiac aging. (A) Representative Western blots of p53 expression in primary cardiomyocytes from the hearts of Rap1^+/+ and Rap1^-/- mice at 12-month of age, n = 6. (B) Representative Western blots of p53, CD36, ACC, CPT1α and ACADL expression in the hearts of Rap1^+/+ and Rap1^-/- mice at 12-month of age, with or without treatment of PFTα (1.1 mg/kg/day, 6 weeks), n = 5. Protein presence of p53, CD36, ACC, CPT1α and ACADL was normalized to GAPDH. Transthoracic echocardiography was performed to evaluate left ventricular mass (C), left ventricular end-diastolic diameter (LVIDd, D), ejection fraction (EF, E) and myocardial performance index (MPI, F) in Rap1^+/+ and Rap1^-/- mice at 12-month of age, with or without treatment of PFTα, n = 7. Data are shown as means ± S.E.M; ^*P < 0.05 PFTα vs. Vehicle; ^#P < 0.05 Rap1^-/- vs. Rap1^+/+.

Figure 6

p53 directly suppresses transcription of PPARα. (A) Chromatin immunoprecipitation (ChIP) showing p53 binding on the promoters of PPARα at indicated sites, n = 5; (B) Representative Western blots of p53 and PPARα in primary cardiomyocytes from the heart of Rap1^+/+ mice at 12-month of age, with or without treatment of Nutlin 3a (10 µg/mL, 6 h). Protein presence of p53 and PPARα was normalized to GAPDH. n = 6; (C) mRNA expression of PPARα in primary cardiomyocytes from the heart of Rap1^+/+ mice at 12-month of age, with or without treatment of Nutlin 3a. mRNA levels were expressed as fold changes against that mRNA expression in the primary cardiomyocytes without treatment of Nutlin 3a, n = 5; (D) Oxygen consumption rates (OCR) was measured under basal, palmitate-BSA (0.5 mM), oligomycin (1 µM), FCCP (1 µM) and rotenone/antimycin A (1 µM)-stimulated conditions at indicated time points in the primary cardiomyocytes from the hearts of Rap1^+/+ and Rap1^-/- mice at 12-month of age, with or without treatment of WY14643 (10 µM, 3 h). Data are shown as means ± S.E.M; ^#P < 0.05 Rap1^-/- vs. Rap1^+/+; ^*P < 0.05 Nutlin 3a vs. Vehicle or Rap1^-/-+WY14643 vs. Rap1^-/-.

Figure 7

Impaired PPARα signaling in human myocardium with aging. (A) Representative Western blots and quantification of PPARα (B), CD36 (C), ACADL (D) and ACC (E) in human myocardium with different age groups (young group, n = 7; middle age/aged group, n = 5). Protein presence of PPARα, CD36, ACADL and ACC was normalized to GAPDH. Data are shown as means ± S.E.M; ^*P < 0.05 middle age/aged group vs. young group.