| BackgroundWith the increasing of life span, the growing number of older adults increases demands on the public health system and on medical and social services, Cardiovascular Diseases (CVD) are the most common cause of death worldwide in older age. It is well recognized that age is a major independent risk factor for morbidity and mortality in ischemic heart disease. However, the mechanisms responsible for this age-related pathologic change remain elusive. Accumulating evidence indicates that myocardial apoptosis, a gene-regulated programmed cell death, markedly increases with aging. The molecular mechanisms and signaling transduction pathways which are responsible for increased susceptibility of cardiomyocytes to apoptosis with aging remain largely unidentified.Thioredoxin (Trx) is a 12-kDa protein ubiquitously expressed in all living cells that fulfills a variety of biological functions related to cell proliferation and apoptosis. It is involved not only in cytoprotective functions against oxidative stress but also in the regulation of cellular proliferation and the aging process. Clinical and experimental results have demonstrated that inhibition of Trx promotes apoptosis. Recent in vitro studies demonstrate that Trx interacts directly with, and inhibits, the activity of apoptosis-regulating kinase-1 (ASK1), a mitogenactivated protein kinase (MAPK) that activates two proapoptotic kinases, p38 MAPK and c-Jun N-terminal kinase (JNK). In aged mouse livers, the ratio of ASK1/Trx–ASK1 (free ASK1/Trx-binding ASK1) increases and this correlates with the increased basal activity of the p38 MAPK pathway. These results suggest that Trx may play critical roles in cell proliferation and cell death in aging. However, whether Trx activity is reduced in the aged heart and whether this alteration may contribute to increased cardiomyocyte apoptosis in the aged heart has never been previously investigated.Reactive oxygen species (ROS) have long been recognized to cause oxidative protein modification and to act as major mediators of the aging process. However, the increased susceptibility of the aging heart to reperfusion mediated myocardial injury (RMMI) and increased postischemic cardiomyocyte apoptosis in aging animals cannot be exclusively explained by ROS production, and many fundamental questions remain unresolved. Recent data have identified nitric oxide (NO)-derived reactive nitrogen species (RNS), such as peroxynitrite (ONOO-), as critical contributors of protein modification and cell injury, providing potential targets for therapeutic interventions. However, whether increased RNS production may increase cardiomyocyte apoptosis via nitrative modification of a specific protein remains unknown.Aims1. To determine whether Trx activity is reduced in the aging heart?2. To identify the mechanism responsible for aging-induced Trx alteration.3. To determine the signaling mechanism by which reduced Trx activity leads to apoptotic cardiomyocyte death in the aging heart.4. To investigate whether treatment with FP-15 (a peroxynitrite decomposition catalyst) attenuates apoptotic cardiomyocyte death in the aging heart. Methods1. Experimental protocols: Male adult (3-months-old) and aging (20-months-old) C57/B16 mice were anesthetized with 2% isoflurane. Mice were subjected to 30 mins of myocardial ischemia (MI) fellow 3 or 24 hrs reperfusion. At 10 min before reperfusion, mice were randomized to receive either vehicle PBS, pH 7.5), or FP-15 (a peroxynitrite decomposition catalyst, 5 mg/kg) by i.p. injection. At the end of the reperfusion period, the heart was quickly excised, and the ischemic/reperfused cardiac tissue was isolated and processed according to the procedures described below.2. Total NO assay: Cardiac tissue was homogenized with lysis buffer, and the supernatant was incubated with nitrate reductase for 2 h to reduce NO3 to NO2. Samples containing NO2 were then injected into the reducing solution (120 mM potassium iodide in glacial acetic acid), and NO was detected by chemiluminescence with a nitric oxide analyzer (NOA 280I; Sievers, Boulder, CO). Nitrotyrosine content was evaluated with ELISA.3. Detection of Trx nitration and Trx–ASK1 interaction: Endogenous Trx-1 was immunoprecipitated with a monoclonal anti-murine Trx-1 antibody (Redox Bioscience). After sample separation, Trx-1 nitration was detected with a monoclonal antibody against nitrotyrosine (Upstate), and the Trx-1–ASK1 interaction was determined by using a monoclonal antibody against ASK1 (Upstate). The blot was developed with Supersignal-Western reagent (Pierce).4. p38 MAPK activity assay: Heart tissue was homogenized in 0.5 ml ice-cold cell lysis buffer. Immunoprecipitation was performed by adding monoclonal antibody against phospho-p38 MAP kinase (Thr180/Tyr182) to lysate containing proteins. The kinase reactions were carried out in the presence of 200μM ATP and 2μg ATF-2 fusion protein at 30°C for 30 min. After incubation, the samples were separated by SDS–PAGE, and ATF-2 phosphorylation was measured by Western immunoblotting using a monoclonal antibody against phosphorylated ATF-2 followed by an enhanced chemiluminescent detection.5. Determination of ischemic/reperfusion-induced cardiomyocyte apoptosis: Myocardial apoptosis was analyzed by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining and caspase-3 activity assay.6. Measurement of infarct size: At 24 h after reperfusion, mice were anesthetized, the ligature around the coronary artery was retied, and 2% Evans blue dye was injected into the left ventricular cavity. and the hearts were excised. Myocardial infarct size was determined by using Evans blue-2,3,5-triphenyltetrazolium chloride staining.Results1. Aging-enhanced myocardial RMMI: RMMI was enhanced in aging mice as evidenced by increased apoptosis (TUNEL, 5.0±1.8% vs. 8.8±0.9%; caspase 3 activity, 18.0±0.9 nmol/h/mg protein vs. 27.4±1.9 nmol/h/mg protein, P<0.01, n=8) and enlarged infarct size (38.5±2.7% vs. 52.0±1.8%, P<0.01, n=8).2. Trx activity is reduced but its expression is increased in the aging heart: Compared with young animals, cardiac Trx activity was decreased in the aging heart before MI/R (2.57±0.07μmol/min/mg vs. 1.85±0.05μmol/min/mg,n=8,P<0.01), and this difference was further amplified after MI/R (1.14±0.05μmol/min/mg vs. 0.50±0.05μmol/min/mg,n=8,P<0.01). Surprisingly, Trx expression was slightly increased(4.04±0.15 AU vs. 4.79±0.16 AU,n=6-8,P<0.05), rather than decreased in aging hearts. These results indicate that reduced Trx activity in the aging heart is caused by posttranslational Trx modification rather than by reduced protein expression.3. Production of reactive nitrogen species and Trx nitration are increased, Trx–ASK1 interaction is inhibited, and p38 MAPK activity is increased in the aging heart: Total NO production and cardiac nitrotyrosine were determined, a greater than 1.5-fold increase in both NOx and nitrotyrosine contents was observed in cardiac tissues obtained from aging animals even before the hearts were subjected to ischemia/reperfusion. Moreover, ischemia/reperfusion-induced overproduction of peroxynitrite was further amplified in the aging heart (0.26±0.01 pmol/mg protein vs. 0.20±0.01 pmol/ mg protein, P<0.05, n=8). Trx nitration was not detected in cardiac tissue obtained from young animals. In contrast, clear Trx nitration was detected in cardiac tissues from the aging heart before ischemia/reperfusion(0.69±0.28 AU, n=6), and this aging-induced Trx nitration was further intensified after ischemia/reperfusion(0.69±0.28 AU vs. 7.44±0.31 AU, P<0.01,n=6). Trx is physically associated with ASK1 (anti-Trx-1 immunoprecipitation and anti-ASK1 immunoblotting) in cardiac tissues isolated from young animals, and this protein–protein interaction was significantly decreased in aging animals(28.35±2.56 AU vs. 21.35±2.75 AU, P<0.01,n=6). Consequently, activity of p38 MAPK, a proapoptotic downstream molecule for ASK1, was significantly enhanced in the aging heart compared with the young heart(4.24±0.47 AU vs. 7.44±1.56 AU, P<0.01,n=8).4. Treatment with a peroxynitrite decomposition catalyst blocked Trx nitration and protected the aging heart from RMMI: Treatment with FP-15 shortly before reperfusion reduced Trx nitration, preserved Trx activity, restored Trx–ASK1 interaction, reduced P38 MAPK activity, attenuated caspase 3 activation, and reduced infarct size in aging animals (from 52±2.2% to 27.9±1.9%, a 46% reduction, n=8, P<0.01).Conclusion1. Our present study provided the first evidence that the activity of Trx, a critical antioxidant and antiapoptotic molecule, is significantly reduced in aging hearts even before they are subjected to myocardial ischemia/reperfusion;2. Moreover, we have demonstrated that it is the posttranslational nitrative modification of Trx, not its expression, which is responsible for this reduced Trx activity in the aging heart.3. In addition, we have demonstrated that increased nitrative modification of Trx results in a decreased Trx–ASK1 binding in the aging heart before myocardial ischemia/reperfusion. Treatment with a novel peroxynitrite decomposition catalyst shortly before reperfusion blocked nitrative Trx inactivation, attenuated ASK1 activation, and reduced postischemic myocardial apoptosis in the aging heart.
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