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It is well recognized that apoptosis, a gene-regulated cell suicide process, contributes significantly to cardiomyocyte loss and the development of cardiac dysfunction in a variety of pathologic conditions, including myocardial ischemia/reperfusion (MI/R) and heart failure (Cohn et al., 1997; Gill et al., 2002). The search for treatments that reduce apoptotic myocyte death may offer novel therapeutic options to reduce myocardial reperfusion injury and improve cardiac function (Ma et al., 1999; Webster and Bishopric, 2003). In a recent study (Tao et al., 2004), we have demonstrated that exogenously administered recombinant human thioredoxin (rhTrx), a 12 kDa antioxidant protein (Holmgren, 1985; Yamawaki et al., 2003), preferentially entered ischemic/reperfused cardiomyocytes and markedly reduced post-ischemic myocardial apoptosis. However, the mechanisms responsible for Trx's cardioprotective effects remain unclear.
Reactive oxygen species have long been recognized to cause oxidative protein modification and to act as the major mediator of ischemia/reperfusion injury (Dart and Sanders, 1988). However, myocardial reperfusion injury cannot be explained exclusively by oxidative stress because multiple enzymatic and non-enzymatic pathways exist that can effectively reduce oxidized molecules (Willerson, 1997). Most recent data have suggested that nitric oxide (NO)-derived reactive nitrogen intermediates (reactive nitrogen species, RNS) may contribute to pathologic tissue injury by nitrative protein modification (nitrative stress), providing potential targets for therapeutic interventions (McCann, 1997; Bauer, 2000; Brookes and Darley-Usmar, 2002; Ischiropoulos and Beckman, 2003). One of the most toxic RNS, peroxynitrite (ONOO−), is formed by NO and superoxide (O•−2) at a near diffusion-limited rate. Considerable evidence now exists that peroxynitrite plays a causative role in post-ischemic myocardial apoptosis and necrosis (Virag et al., 2003; Liang et al., 2004). Although in vitro experiments have demonstrated that Trx is a powerful antioxidative molecule that participates in the detoxification of H2O2 to H2O, whether Trx may have anti-nitrative effects, thus reducing post-ischemic myocardial apoptosis, has not been previously determined.
Therefore, the aims of the present study were (1) to determine the effect of Trx on protein nitration (nitrative stress) in ischemic/reperfused hearts, (2) to identify the mechanisms by which Trx may reduce nitrative stress (i.e., inhibiting NO or superoxide production) and (3) to establish a causative link between Trx's anti-nitrative and anti-apoptotic effects.1
Figure 1. Experimental protocol. Male adult mice were anesthetized and surgical procedure (Surgical Pro.) was performed to induce MI. After 30 min of MI, the myocardium was reperfused for 3 h (for apoptosis, Western blot and biochemical analysis) or 24 h (for infarct size). Ten minutes before reperfusion, mice were randomized to receive either vehicle (PBS, pH 7.5) or recombinant human Trx (rhTrx, 0.7–6 mg kg−1) by i.p. injection.
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- Materials and methods
- Conflict of interest
We have recently reported that administration of rhTrx before reperfusion exerts significant cardioprotective effects after myocardial ischemia and reperfusion (Tao et al., 2004). However, the mechanism by which Trx may exert its anti-apoptotic effect was not directly determined. The present study provides the first direct evidence that treatment with rhTrx significantly reduces nitrotyrosine content in ischemic/reperfused cardiac tissue, suggesting that Trx may exert its anti-apoptotic effect via a novel anti-nitrative property. In this connection, we have also provided direct evidence that in vitro treatment with rhTrx blocks SIN-1 (a peroxynitrite donor)-induced cardiomyocyte apoptosis. Moreover, we have demonstrated for the first time in an in vivo animal model that treatment with thioredoxin reduces post-ischemic nitrative stress via upregulation of mSOD and inhibition of superoxide content, rather than by inhibition of NO production.
The Trx system, including Trx, Trx reductase and Trx peroxidase, has been shown to play a critical role in maintaining physiologic cardiovascular function as well as protecting cells from oxidative injury under a variety of pathologic conditions (Yamawaki et al., 2003). Our present experimental results provide direct evidence that exogenous rhTrx, when administered before reperfusion, significantly reduces myocardial apoptosis and infarct size. This result suggests that Trx may have great clinical applications in the treatment of cardiovascular diseases where apoptosis plays a pathogenic role.
It has been recognized for many years that superoxide production is markedly increased in ischemic/reperfused cardiac tissue and that subsequent formation of H2O2 and HO. has been thought to be the primary mechanism responsible for reperfusion injury (Dart and Sanders, 1988). However, antioxidant treatment has not been proven beneficial in several clinical trails (Willerson, 1997). Accumulating evidence from recent biochemical and biological studies indicates that besides oxidative stress, NO-initiated nitrative stress also plays a critical role in cell death and tissue injury associated with a variety of cardiovascular diseases (Zweier et al., 2001; Ferdinandy and Schulz, 2003). High concentrations of NO have been reported to induce apoptotic cell death in cultured cells (Chung et al., 2001). The reported mechanisms involve DNA strand breaks (Richard et al., 1995; Pieper et al., 1999), p53 accumulation (Kim et al., 1999; Chung et al., 2001), cdc42 activation (Thomas et al., 2000), p38 mitogen activated protein kinase activation (Ghatan et al., 2000) and mitochondrial permeability transition formation (Hortelano et al., 1997). However, in a recent study, we have demonstrated that in cultured cardiomyocytes, neither superoxide nor NO alone results in significant apoptosis. Specifically, exposing cardiomyocytes to high concentrations of pyrogallol (>100 μM), a superoxide donor, or SNAP (>300 μM), an NO donor, induces significant apoptosis. However, pyrogallol-induced apoptosis is blocked not only by Tiron, a cell-permeable superoxide scavenger, but also by L-NMMA, a non-selective NOS inhibitor (administered 10 min before pyrogallol treatment). On the other hand, SNAP-induced apoptosis at high concentrations is blocked not only by hemoglobin, an NO scavenger, but also by Tiron. Moreover, exposing cultured cardiomyocytes to SIN-1, an ONOO− donor, at concentrations that caused an increase in nitrotyrosine content comparable to that seen in the ischemic/reperfused heart, resulted in significant cardiomyocyte apoptosis (Ma et al., 2004). This result strongly suggests that superoxide and NO induce cardiomyocyte apoptosis by a peroxynitrite-dependent mechanism. Therefore, it is conceivable that therapeutic interventions that block excessive production of NO or superoxide, or directly inhibit ONOO−-induced nitrative stress, may exert a significant cardioprotective effect.
Peroxynitrite is the bi-radical reaction product of NO and O2•− at a diffusion-limited rate (Ferdinandy, 2006). Peroxynitrite has been reported to increase apoptotic cell death in a variety of cell types. The proapoptotic mechanisms of ONOO− include protein and DNA oxidation (Chiarugi and Moskowitz, 2002), lipid peroxidation (Ushmorov et al., 1999), protein nitration (Francescutti et al., 1996; Gow et al., 1996; MacMillan-Crow et al., 1998), apoptosis-inducing factor release (Zhang et al., 2002) and endoplasmic reticulum stress with the subsequent release of caspase 12 (Oyadomari et al., 2001). Our present experiment demonstrated for the first time that administration of rhTrx markedly reduced cardiac nitrotyrosine content and reduced apoptosis. However, treatment with rhTrx failed to inhibit iNOS expression and NO production in ischemic/reperfused cardiac tissue, indicating that Trx blocks nitrative stress and subsequent cell death by mechanisms other than direct inhibition of NO production. In this connection, we have provided direct evidence that treatment with rhTrx caused a significant upregulation of mSOD and markedly reduced superoxide content in the ischemic/reperfused heart. As the production of potent nitrative products, such as ONOO−, requires simultaneous production of NO and superoxide, the upregulation of mSOD and inhibition of superoxide content by Trx provides a likely explanation for Trx's anti-nitrative and anti-apoptotic effects.
Another interesting finding of the present study is that the addition of rhTrx in cultured cardiomyocytes blocked SIN-1-induced cardiomyocyte apoptosis. This result provides the strongest evidence that Trx may exert its anti-apoptotic effect via a novel anti-nitrative mechanism. There are two possible explanations for this novel phenomenon. First, by its antioxidant property, Trx may scavenge SIN-1-released superoxide, thus preventing peroxynitrite formation. However, as NO reacts with superoxide at a diffusion-limited rate, it is unlikely that Trx may effectively compete with NO to react with superoxide. Another possible explanation is that exogenously administered rhTrx may compete with other endogenous anti-apoptotic molecules (such as SOD and endogenous Trx) to react with peroxynitrite, thus preventing their nitrative inactivation as previously reported (Guo et al., 2003). More experiments are needed to provide direct evidence to support this hypothesis.
In summary, we have demonstrated in the present study for the first time that Trx reduces nitrative stress by multiple mechanisms that involve blocking superoxide production (thus preventing production of potent nitrative species) and possibly by directly inhibiting peroxynitrite-induced nitrative inactivation of endogenous anti-apoptotic molecules. As recent studies have clearly demonstrated that nitrative stress plays a critical pathogenic role in many cardiovascular diseases, the novel anti-nitrative property of Trx provides a likely explanation for its cardiovascular protective effect as previously reported. Therefore, Trx may prove to have therapeutic value in cardiovascular diseases where ONOO− generation is increased and apoptotic cell death occurs.