| BackgroundGrowing evidence from both animal experiments and clinical observations indicates that myocardial infarction after myocardial ischemia/reperfusion (MI/R), the single-most important cause of death in the world, is caused not only by necrosis but also by apoptosis. However, the signaling mechanisms leading to post-ischemic apoptosis are incompletely understood. The role of nitric oxide (NO) in apoptosis signaling has been one of the most intensely studied topics in biomedical research in the past few years. However, published results are rather confusing and often controversial.Thioredoxin is a ubiquitous thiol oxidoreductase that regulates cellular reduction/oxidation (redox) status as well as cell proliferation/cell survival. It participates in redox reactions by reversible oxidation of its active center dithiol to disulfide and catalyzes dithio-disulfide exchange reactions involving many thiol-dependent processes. Thus, the Trx system is considered to constitute an endogenous antioxidant system in addition to theglutathione and superoxide dismutase systems. In addition to its activity as an oxidoreductase, recent in vitro studies demonstrate that reduced Trx (but not oxidized) binds to and inhibits activity of apoptosis-regulating kinase-1 (ASK-1), a MAP kinase kinase kinase (MAPKKK) that activates two pro-apoptotic kinases, p38 MAPK and JNK.Numerous experimental results have demonstrated that low concentrations of NO produced from endothelial NO synthase (eNOS) or pharmacological concentrations of exogenous NO produced by NO donors reduce apoptosis. Recent in vitro studies in cultured cells suggested that S-nitrosylation, a posttranslational protein modification that involves the covalent attachment of an NO group to a cysteine thiol, may be involved in the antiapoptotic effect of NO. Although it is known that human Trx contains multiple cysteines, some or all of which can be S-nitrosylated, the functional significance of this posttranslational modification has not been evaluated in a pathologically relevant model in vivo.Exposing cultured adult cardiomyocytes to high concentrations of nitric oxide causes significant apoptosis. However, this pro-apoptotic effect of NO was completely abolished when a cell permeable SOD mimic was added together with NO. This result strongly suggests that the reaction product between NO and "02", peroxynitrite (ONOO"), but not NO itself, is responsible for nitric oxide's pro-apoptotic effect. However, the mechanisms by which ONOO" at pathologically relevant concentrations stimulate cardiomyocyte apoptosis remain largely unknown. The nitration of protein tyrosine residues (nitrotyrosine formation) has been used extensively as a footprint for in vivo production of ONOO". However, emerging evidence indicates that nitrative modification of signaling proteins results inalterations of their functions, thus contributing to the development and advancement of cell death and tissue injury.Aim1. To determine whether systemic administration of Trx reduces myocardial reperfusion injury by reducing the degree of postischemic myocardial apoptosis.2. To elucidate whether the antiapoptotic effects of Trx can be modified by S-nitrosylation, investigate the mechanism(s) by which S-nitrosylation may modulate the function of Trx.3. To determine whether nitration of Trx plays a causative role in myocardial apoptosis signaling, and if inhibiting Trx nitration may be a novel therapeutic strategy to reduce myocardial reperfusion injury.Methods1. Adult mice were subjected to 30 min of MI and treated with either vehicle or human Trx (hTrx, 2mg_kg, i.p.) 10 min before reperfusion. Myocardial infarction(24 h after reperfusion) and myocardial apoptosis (both DNA laddering and TUNEL analysis) were determined 3 h after reperfusion. The location of exogenous hTrx was detected with immunohistochemical staining.2. The animal model was the same as that in the first part. Mice received one of the following treatments: experiment 1, intraperitoneal injection of PBS, hTrx, hTrx—SNO, GSNO; or experiment 2, PBS, eTrx, eTrx+GSNO. MI/R injury as determined by TUNEL staining, DNA fragmentation, caspase-3 activity, and infarct size. S-NO content in Trx, S-nitrosated Trx, p38 MAPK activity were detected.3. The animal model was the same as that of the first part. Adult mice were subjected to 30 min of MI and treated with either PBS, hTrx, SIN-1-hTrx, SIN-1, SIN-1-Hb-hTrx or SIN-1-SOD-hTRX 10 min before reperfusion. Myocardial infarction was detected with Evans blue-TTC staining and myocardial apoptosis were determined by both DNA laddering and TUNEL analysis. Nitrated Trx and nitrotyrosine content were evaluated with ELISA or Western blot.Results1. Systemic administration of Trx in vivo leads to uptake of this protein by cardiomyocytes where it exerts significant cardioprotective effects when given before reperfusion, as evidenced by reduced myocardial apoptosis and decreased myocardial infarct size.2. Direct evidence in vivo provided that S-nitrosation, likely occurring at Cys-69, significantly enhances the cardioprotective efficacy of human Trx.3. Trx exerts its protective effect, at least in part, by inhibiting p38-MAPK activation, an effect that is enhanced after S-nitrosation of human Trx.4. Exogenously administered S-nitrosated hTrx can function as a nitrosothiol source to replenish depleted endogenous protein S-nitrosation in the heart.Cone I us i on1. Systemic administration of Trx during early stages of ischemic events, regardless of whether it is S-nitrosated or not, can protect myocardial tissue from reperfusion-related damage, offering a potentially attractive... |
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