Thioredoxin reductase was nitrated in the aging heart after myocardial ischemia/reperfusion

Abstract

The age-related loss of anti-oxidant defense reduces recovery from myocardial ischemia/reperfusion injury (MI/R) in aged people. Our previous data showed that inactivation of thioredoxin (Trx) was involved in enhanced aging MI/R injury. Thioredoxin reductase (TrxR), the enzyme known to regulate Trx, is less efficient with age. The aim of the current study was to determine why TrxR activity was reduced and whether reduced TrxR activity contributed to enhanced aging MI/R injury. Both Trx and TrxR activity were decreased in the aging heart, and this difference was further amplified after MI/R. However, MI/R injury did not change TrxR expression between young and aging rats. Increased nitrogen oxide (NOx) but decreased nitric oxide (NO) bioavailability (decreased phosphorylated vasodilator-stimulated phosphoprotein) was observed in aging hearts. Peroxynitrite (ONOO⁻) was increased in aging hearts and was further amplified after MI/R. TrxR nitration in young and aging hearts was detected by immunoprecipitation (anti-nitrotyrosine) followed by immunoblotting (anti-TrxR). Compared with young hearts, TrxR nitration was increased in the aging hearts, and this was further intensified after MI/R. The ONOO⁻ decomposition catalyst (FeTMPyp) reduced TrxR nitration and increased TrxR and Trx activity. More importantly, FeTMPyp attenuated the MI/R injury in aging hearts as evidenced by decreased caspase-3 and malondialdehyde (MDA) concentration and increased cardiac function. Increased ONOO⁻ nitrated TrxR in the aging heart as a post-translational modification, which may be related to the enhanced MI/R injury of aging rats. Interventions that inhibit nitration and restore TrxR activity might be a therapy for attenuating enhanced MI/R injury in aging heart.

FIG. 1.

Aging rat hearts were more susceptible to myocardial ischemia/reperfusion injury. (A) Comparison of left ventricular systolic pressure (LVSP) in both the young rats and aging rats after myocardial ischemia/reperfusion (MI/R). (B) Left ventricular end diastolic pressure (LVDEP). (C and D) Maximal rate of rise/decrease of left ventricular pressure (±dp/dtmax). (E) MI/R-induced cardiomyocytes apoptosis with aging. Activity of caspase-3 was determined at the end of re-perfusion. (F) Malondialdehyde (MDA) concentration. MI/R indicates myocardial ischemia for 30 min and reperfusion for 3 hr. The Sham group underwent the same surgical procedures except that the left coronary artery was not occluded. Data are expressed as mean±standard error of the mean (SEM) (n=6–8 each). (*) p<0.05, (**) p<0.01 vs. Sham; (#) p<0.05 vs. Young.

FIG. 2.

Age reduced efficiency of thioredoxin (Trx) system in the myocardium. (A and B) Both Trx activity and thioredoxin reductase (TrxR) activity were significantly reduced in the aging rat heart either before or after myocardial ischemia/reperfusion (MI/R). (C) Age-dependent differences in TrxR1 protein expression. (D) Age-dependent differences in TrxR2 protein expression. Densitometry analysis for expression of TrxR1 and TrxR2 protein by western blot (n=8 each). Bar heights represent mean values and brackets indicate standard error of the mean (SEM). (*) p<0.05, (**) p<0.01 vs. Sham; (#) p<0.05 vs. Young. SY, Sham young; SA, Sham aged; RY, reperfused young; RA, reperfused aged.

FIG. 3.

Aging enhances nitrative stress in the heart subjected to myocardial ischemia/reperfusion (MI/R). (A) Total NOx production was increased in the aging heart. (B) Phosphorylated vasodilator-stimulated phosphoprotein (p-VASP) was reduced in heart tissues isolated from aged rat. (C) Nitrotyrosine content, as a marker for ONOO⁻, was increased in aging rat heart either before or after MI/R. (*) p<0.05, (**) p<0.01 vs. Sham; (#) p<0.05, (##) p<0.01 vs. Young.

FIG. 4.

The inactivation of thioredoxin reductase (TrxR) was related to its nitration. TrxR nitration in young and aging hearts was detected by immunoprecipitation (anti-nitrotyrosine) followed by immunoblotting (anti-TrxR). Nitration of TrxR1 (A) and TrxR2 (B) in the aging heart subjected to myocardial ischemia/reperfusion (MI/R). The peroxynitrite decomposition catalyst FeTMPyp decreased the nitration of TrxR1 (C) and attenuated its activity (D). (E) Treatment with FeTMPyp attenuated Trx activity in the aging heart subjected to MI/R. Ten minutes before reperfusion, aging rats were randomized to receive vehicle (saline) or FeTMPyp via intra-peritoneal injection (IP). (*) p<0.05 vs. Sham; (#) p<0.05 vs. Young; (Δ) p<0.05, (ΔΔ) p<0.01 vs. Vehicle. IB, immunoblot; SY, Sham young; SA, Sham aging; RY, reperfused young; RO, reperfused old; V, vehicle; Fe, FeTMPyp.

FIG. 5.

FeTMPyp attenuated the myocardial ischemia/reperfusion (MI/R) injury in the aging rats. Effect of FeTMPyp on caspase-3 activity (A) and malondialdehyde (MDA) concentration (B) in the aging heart subjected to MI/R. (C) Effects of FeTMPyp on left ventricular systolic pressure (LVSP) in the aging heart subjected to MI/R. (D) Left ventricular end diastolic pressure (LVDEP). (E and F) Maximal rate of rise/decrease of left ventricular pressure (±dp/dtmax). Ten minutes before reperfusion, rats were randomized to receive vehicle (saline) or FeTMPyp via intraperitoneal injection (IP). Data expressed as mean±standard error of the mean (SEM) (n=8 each). (Δ) p<0.05, (ΔΔ) p<0.01 vs. Vehicle.