Figure 1.
Impaired ALDH2 and SIRT1 activity in aged hearts.
(A) Representative gel blots depicting (B) p16 and (C) ALDH2 protein expression in young and aged hearts. Quantification showing (D) decreased ALDH2 activity; (E) increased 4-HNE protein adducts formed; (F) increased MDA levels; (G) increased protein carbonyl level; (H) decreased cardiac SIRT1 activity ; (I) decreased cardiac SIRT1 protein expression levels in aged heart. (n=8 per group. *P<0.05 vs. young).
Figure 2.
Alda-1 treatment prevented the harmful effect of 4-HNE on cardiomyocytes.
Isolated cardiomyocytes with or without Alda-1 treatment (20 µmol/L) were incubated with 4-HNE (10 µmol/L) or vehicle for 1 hr. (A) ALDH2 activity; (B) 4-HNE adduct protein; (C) ROS generation; (D) protein carbonyl formation and (F) cardiac SIRT1 activity were assessed by quantificational detection (n=8 per group. *P<0.05 vs. vehicle control, #P <0.05 vs. 4-HNE alone). (E) Representative immunoprecipitation (IP) picture was used to confirm carbonylation of SIRT1. (G) Cardiomyocytes were pretreated for 1 h with vehicle, SRT1720 (1 µmol/L), 4-HNE (10 µmol/L) or 4-HNE plus SRT1720 or Alda-1(20 µmol/L), and then with or without 1 hr of hypoxia and 1 hr reoxygenation (H/R). Quantification showing cardiomyocytes caspase-3 activity. (n=8 per group. *P<0.05 vs vehicle control, #P <0.05 vs. vehicle H/R, † P <0.05 vs SRT1720 H/R, ‡ P <0.05 vs. HNE H/R).
Figure 3.
Alda-1 treatment protected cardiomyocytes against H/R injury.
Cardiomyocytes were pretreated for 1 h with vehicle, Alda-1(20 µmol/L) or Alda-1 plus Ex-527 (10 µmol/L), and then with or without 1 hr of hypoxia and 1 hr reoxygenation (H/R). Quantification showing (A) endogenous 4-HNE adduct protein, (B) cardiomyocytes caspase-3 activity, (C) cardiomyocyte death. (n=8 per group. *P<0.05 vs. vehicle control; #P <0.05 vs. vehicle H/R, † P <0.05 vs. Alda-1 H/R).
Figure 4.
Alda-1 activated SIRT1 in aged hearts during I/R.
Young and aged C57BL/6 mice were subjected to 30-minute coronary artery ligation followed by 1-hour reperfusion in vivo, Alda-1 (16 µg/g) or vehicle was administered via tail vein 2 hr before ischemia. (A) ALDH2 activity and (B) HNE protein adducts formation were assessed. (C) Nuclear extracts from young and aged hearts were subjected to immunoprecipitation (IP) with SIRT1 antibody. The IP products were further analyzed by immunoblotting; anti-DNPH and anti-SIRT1 antibodies were reciprocally used to confirm carbonylation of cardiac SIRT1. Anti-TBP was used as a input ; bar graphs show relative levels of carbonylation of cardiac SIRT1 in young and aged hearts. (D) Nuclear and (E) cytoplasmic extracts from young and aged hearts were analyzed by immunoblotting. TBP and GAPDH were detected as nuclear and cytoplasmic loading control, respectively. Bar graphs show relative levels of nuclear SIRT1 and cytoplasmic SIRT1 from young and aged hearts. (F) Nuclear fractions of young and aged LVs were subjected to the SIRT1 activity assay. (n=6-8 per group. *P<0.05 vs. young sham; #P <0.05 vs. young I/R vehicle; † P <0.05 vs. Aging I/R vehicle).
Figure 5.
Alda-1 treatment improved the tolerance of aged hearts during I/R.
Young and aged C57BL/6 mice were subjected to 30-minute coronary artery ligation followed by 4-hour reperfusion, Alda-1 (16 µg/g) or vehicle was administered via tail vein 2 hr before ischemia. (A) Creatine kinase (CK) activity measurement were collected 4 h after reperfusion from young and aged I/R mice. (B) Young and aged C57BL/6 mice, Sirt1+/− heterozygous knockout mice, and Sirt1+/+ wild-type littermate mice (C57BL/6 background) were subjected to 30-minute coronary artery ligation followed by 4-hour reperfusion, Alda-1 (16 µg/g) or vehicle was administered via tail vein 2 hr before ischemia. Representative pictures and quantification of ratio of infarct size to total myocardium. (n=6-8 per group. *P<0.05 vs. young vehicle; #P <0.05 vs. Sirt1+/+ wild-type vehicle; † P <0.05 vs. Aging vehicle). (C) Isolated hearts from young and aged C57BL/6 mice or (D) Sirt1+/− heterozygous knockout mice and Sirt1+/+ wild-type littermate mice (C57BL/6 background) were subjected to ex vivo heart perfusion (Langendorff). Heart rate × left ventricular developed pressure was determined during baseline perfusion, global ischemia, and postischemic reperfusion with vehicle or Alda-1(20 µmol/L) administration. (n=6-8 per group. *P<0.05 vs. young or Sirt1+/+ wild-type vehicle; #P <0.05 vs. aged vehicle).