S-nitrosylation and apoptosis
S-nitrosylation is an important biological reaction of nitric oxide; it refers to the conversion of thiol groups, including cysteine residues in proteins, to form S-nitrosothiols. S-Nitrosylation is a mechanism for dynamic, post-translational regulation of most classes of protein. NO has been shown to regulate apoptosis through S-nitrosylation of protein.
Research hasrevealed that S-nitrosylation mainly antangonizes apoptosis by targeting many elements in both extrinsic and intrinsic pathways (Iyer et al., 2008). Fas, as a cell surface receptor, is a member of the tumor necrosis receptor superfamily that induces apoptosis when cross-linked by Fas ligand or by Fas agonist antibody (Park et al., 2006; Lavrik et al., 2005; Mannick & Schonhoff, 2004). Regulation of cell signaling by protein nitrosylation is well exemplified in the Fas signalling pathway (Mannick & Schonhoff, 2004). Consistent with receptor-mediated apoptosis, two main pathways of Fas-mediated apoptosis have been identified (Park et al., 2006). In type1 cells caspase-8 directly cleaves caspase-3, which starts the death cascade. In type2 cells the quantity of caspase-8 is insufficient to directly activate the executioner caspase-3. Instead, it involves (activates) tBid- mediated cytochrome c (Cyto-C) release from mitochondria followed by apoptosome formation (Park et al., 2006; Lavrik et al., 2005). In resting cells caspase-3 zymogens in mitochondria are kept inactive via S-nitrosylation of their catalytic site cysteine. Caspase-3 may be S-nitrosylated in mitochondria due to an association between S-nitrosylated caspase-3 and NOS. Moreover, S-nitrosylated but not denitrosylated caspase-3 associates with acid sphingomyelinase (ASM) in mitochondria. The association of S-nitrosylated caspase-3 with ASM provides another level of apoptosis regulation by inhibiting capase-3 cleavage and activation by initiator caspases. When cells are stimulated by Fas ligand, caspase-3 becomes denitrosylated. Denitrosylation stimulates caspase-3 activity by two mechanisms. First, denitrosylation allows the catalytic site of caspase-3 to function. In addition, denitrosylated caspase-3 presumably dissociates from ASM, allowing initiator caspases to cleave caspase-3 to its fully active form. Thus S-nitrosylation/denitrosylation serves as an off/on switch for caspase-3 function during apoptosis. Cyto-C activity is also regulated by nitrosylation during Fas-induced apoptosis. However, in contrast to caspase-3, Cyto-C is not nitrosylated in resting cells. Instead, when cells receive an apoptotic stimulus, Cyto-C is nitrosylated on its heme iron in mitochondria and then is rapidly released into the cytoplasm. In the cytoplasm, hemenitrosylated Cyto-C stimulates caspase-3 cleavage by the apoptosome. Thus, coordinated denitrosylation of caspase- 3 and hemenitrosylation of Cyto-C serves to enhance caspase activation and Fas-induced apoptosis. It remains to be determined if denitrosylation of caspase-3 is directly linked to nitrosylation of Cyto-C in mitochondria via a direct transfer of a NO+ group from the catalytic site cysteine of caspase-3 to the heme iron of Cyto-C (Mannick et al., 1997; Mannick & Schonhoff, 2004; Schonhoff et al., 2003; Stamler et al., 2001(Mannick et al., 1997; Mannick & Schonhoff, 2004; Schonhoff et al., 2003; Stamler et al., 2001). NO can also inhibit apoptosis by direct nitrosylation of caspase-9 ( Torok et al., 2002).
In intrinsic apoptosis pathways, Cyto-C is released from mitochondria into cytoplasm initiates the apoptotic signals (Brune, 2003; Schonhoff et al., 2003) and has been suggested as the commitment step for apoptosis (Gaston et al., 2003). Previous studies suggest that nitrosylation of Cyto-C is a novel mechanism of apoptosis regulation in cells and a very early event in apoptotic signalling (Schonhoff et al., 2003). However, the critical commitment step in the mitochondrial pathway of apoptosis has not been firmly established. Several recent findings suggest that caspase-9 activation is essential for, and likely represents, the commitment step for the mitochondrial pathway of apoptosis. Nitrosylation of caspase-9 by induced (i) NOS generated NO inhibits apoptosis downstream of Cyto-C release and would appear to be another mechanism negatively regulating this pathway of apoptosis (Torok et al., 2002). Besides nitrosylation of caspases, another mechanism underlying the anti-apoptotic effects of NO via S-nitrosylation includes stimulation of the anti-apoptotic activity (function) of thioredoxin (Trx), which depends on S-nitrosylation at Cys69 (Haendeler et al., 2002). S-nitrosylation and inhibition of Apoptosis signal regulating kinase (ASK1) (in L929 cells) at Cys869 also lead to anti-apoptosis (Park et al., 2004).
Although most of the reports have proven that S-nitrosylation mainly inhibits apoptosis, there are also data showing that S-nitrosylation could induce apoptosis as well. The mechanisms underlying the pro-apoptotic effects of NO via S-nitrosylation include inhibition of the anti-apoptotic transcription factor NF-kB through a variety of mechanisms, including S-nitrosylation of NF-kB (in A549 cells) or nitrosylation of the target cysteine in the IkB kinase complex (IKK) (in Jurkat cells) leading to decreased NF-KB-mediated transcription and decreased Bcl-2 expression (Marshall & Stamler, 2002; Schonhoff et al., 2006). p21ras, JNK kinase, and the p50 monomer (of p50-p65) have been identified as sites of S-nitrosylation that mediate the stimulation or inhibition of NF-kB by NO (Marshall and Stamler, 2002). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is S-nitrosylated by NO, which initiates an interaction with the E3 ligase Siah1, leading to nuclear translocation and ubiquitin-mediated degradation of nuclear target proteins (Benhar & Stamler, 2005). Hara et al. has demonstrated that deprenyl and TCH346 are neuroprotective by preventing the S-nitrosylation of GAPDH and inhibiting GAPDH/Siah cell death cascade (Hara et al., 2006). NO can also enhance apoptosis by NO-induced persistent inhibition and nitrosylation of mitochondrial Cyto-C oxidase in lung endothelial cells (Zhang et al., 2005). The work done by Gu et al. has illustrated that S-Nitrosylation activated Matrix metalloproteinase-9 in vitro was implicated in the pathogenesis of neurodegenerative diseases, stroke, and induced neuronal apoptosis (Gu et al., 2002).
S-Glutathionylation and apoptosis
S-thiolation refers to the incorporation of a low-molecular-mass (LMM) thiol to a protein via formation of a mixed disulfide bridge between a cysteine residue and the LMM thiol. In the intracellular environment in which GSH is the major thiol present, its incorporation results in a PTM named S-glutathionylation or S-glutathiolation, or more commonly S-thiolation (Martinez-Ruiz et al., 2011). Protein S-glutathiolation, the reversible covalent addition of glutathione to cysteine residues on target proteins, is emerging as a candidate mechanism by which both changes in the intracellular redox state and the generation of reactive oxygen and nitrogen species may be transduced into a functional response (Klatt & Lamas, 2000). S-glutathionylation is a redox signaling mechanism that can be produced without the concourse of NO. However, evidence for S-glutathionylation induced by NO and/or RNS has accumulated, linking this modification with NO signalling (Giustarini et al., 2004). At least two mechanisms explain the link between RNS production and S-glutathionylation. One is the observed glutathionylation induced by peroxynitrite. The other is a nitrosylated protein cysteine may react with GSH, or S-nitrosoglutathione can be formed and react with the cysteine thiol, both leading to S-glutathionylation (Martinez-Ruiz et al., 2011).
Vikas Anathy et al. demonstrated that stimulation with Fas ligand (FasL) induces S-glutathionylation of Fas at cysteine 294 independently of nicotinamide adenine dinucleotide phosphate reduced oxidase – induced ROS. Instead, Fas is S-glutathionylated after caspase-dependent degradation of Grx1, increasing subsequent caspase activation and apoptosis (Anathy et al., 2009). Suparna Qanungo et al. indicated S-glutathionylation of p65-NFKB as a major mechanism underlying the inhibition of the NF_B survival pathway and promotion of apoptosis after GSH supplementation in hypoxic pancreatic cancer cells (Qanungo et al., 2007). Therefore, RNS could regulate apoptosis through S-glutathionylation of protein
Tyrosine nitration and apoptosis
RNS-mediated nitration modifications include nitration of tyrosine, tryptophan, amine, carboxylic acid, and phenylalanine groups. However, nitration of tyrosine residues to produce nitrotyrosine has recently received much attention. Protein tyrosine nitration is a covalent protein modification resulting from the addition of a nitro (-NO2) group onto one of the two equivalent ortho carbons of the aromatic ring of tyrosine residues (Gow et al., 2004). Biological nitration of tyrosine depends largely on free radical chemistry. There are two main key nitration pathways that operate in vivo and involve peroxynitrite and hemoperoxidase-catalyzed nitration (Peluffo & Radi, 2007). Tyrosine nitration is a two-step process where the initial reaction is the oxidation of the aromatic ring of tyrosine to yield tyrosyl radical (Tyr^) (oxidation step), which in turn adds ^NO2 (addition step) to yield 3-NO2-Tyr (Peluffo & Radi, 2007).
RNS-mediated Tyrosine nitration induction of Apoptotic Signal Transduction
Work by Hortelano et al. indicates that nitric oxide-dependent apoptosis in macrophages occurs in the presence of a sustained increase of the mitochondrial transmembrane potential, and that the chemical modification and release of cytochrome c from the mitochondria precedes the changes of the mitochondrial transmembrane potential. NO-dependent apoptosis in macrophages involves a chemical modification of cytochrome c that alters its structure and facilitates release from the mitochondria, regardless of the changes of the mitochondrial transmembrane potential (Hortelano et al., 1999). Cassina et al. has shown that Tyr-67 is a preferential site of nitration among the four conserved tyrosine residues in cytochrome c. Cytochrome c3+ was more extensively nitrated than cytochrome c2+ by mitochondrial but also cytosolic or extracellular derived ONOO- diffusing to the intermembrane space (Cassina et al., 2000). Tao et al. proved that nitrative inactivation of Trx plays a proapoptotic role in postischemic myocardium (Tao et al., 2006). Studies conducted by Li et al. demonstrated that there exists a TNF-a-initiated, cardiomyocyte iNOS/NADPH oxidase-dependent, peroxynitrite-mediated signaling pathway that contributes to posttraumatic myocardial apoptosis. In this paper nitrotyrosine content acted as a footprint of in vivo peroxynitrite formation (Li et al., 2007).
RNS-Mediated tyrosine nitration inhibition of apoptotic signal transduction
Although many studies have shown that RNS-mediated tyrosine nitration mainly induce apoptosis. Some research indicated that it also can inhibit apoptosis. Work by Sonsoles Reinehr et al. indicates that CD95 nitration as a novel mechanism for apoptosis inhibition by NO, which competes with pro-apoptotic CD95-tyrosine phosphorylation(Reinehr et al,2004). The study of Nakagawa et al determined that cytochrome c nitrated by continuous treatment with peroxynitrite lost its ability to cause caspase cascade activation in vitro, whereas cytochrome c nitrated by a bolus peroxynitrite treatment had preserved activity (Nakagawa et al, 2007).
Previous data have shown that Reactive Nitrogen Species can either stimulate (pro-apoptosis) or prevent apoptosis (anti-apoptosis) (Boyd & Cadenas, 2002; Brune, 2003; Choi et al., 2002; Patel et al., 1999). The concentrations and local environments including cellular redox state and the presence of free radicals of NO and RNS play a key role in determining whether they stimulate or inhibit apoptosis (Brune, 2003). Peroxynitrite(ONOO-), an important RNS, is formed by the reaction between high concentrations of NO and superoxide . High concentrations of NO or ONOO-can induce apoptosis (Choi et al., 2002). Liang et al. demonstrated for the first time that L-arginine administered at different time points during I/R exerted different effects on post-ischemic myocardial injury and suggests that stimulation of eNOS reduces nitrative stress and decreases apoptosis whereas stimulation of iNOS increases nitrative stress and enhances myocardial reperfusion injury (Liang et al., 2004). But, Rus et al.’s results demonstrated that inhibition of iNOS raises the peroxidative and apoptotic level in the hypoxic heart indicating that this isoform may have a protective effect on this organ against hypoxia/ reoxygenation injuries, and this challenges the conventional wisdom that iNOS is deleterious under these conditions (Rus et al., 2010). So, the effect on apoptosis of RNS and its regulation need further clarification.
RNS signaling and aging myocardial ischemic injury
Accumulated data have shown that nitric oxide derived reactive nitrogen intermediates are critical contributors in controlling apoptosis which determine the susceptibility of aging hearts to myocardial ischemic Injury.
Gao et al’s results showed that the protective effects of adenosine on myocardial I/R injury are markedly diminished in aged animals and that the loss in NO release in response to adenosine may be at least partially responsible for this age-related alteration (Gao et al., 2000). Studies also show that increased susceptibility of the type 2 diabetic GK rat heart to ischemic injury is not associated with impaired energy metabolism. Reduced coronary flow, upregulation of eNOS expression, and increased total NOx levels confirm NO pathway modifications in this model, presumably related to increased oxidative stress. Modifications in the NO pathway may play a major role in I/R injury of the type 2 diabetic GK rat heart (Desrois et al., 2010). Our results show that aging induces phenotypic upregulation of iNOS in the heart, in which P-AR stimulation interacts with ischemia and triggers a markedly increased NO production, which creates a nitrative stress, generates toxic peroxynitrite, activates apoptosis, and eventually causes cardiac dysfunction and myocardial injury. An iNOS inhibitor-1400W can markedly attenuate these adverse effects in the aging heart (Li, 2006).
Thioredoxin and aging-related myocardial apoptosis
Thioredoxin (Trx) is a 12-kDa protein ubiquitously expressed in all living cells that fulfils 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 (Lincoln et al, 2003). Recent in vitro studies demonstrate that Trx interacts directly with, and inhibits, the activity of apoptosis-regulating kinase-1 (ASK1), a mitogen-activated protein (MAP) kinase that activates two proapoptotic kinases, p38 MAP kinase (MAPK) and c-Jun N-terminal kinase (JNK) (Liu & Min, 2002). 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, and Trx activity/ expression might be reduced in the aging heart, thus tilting the death/survival balance toward cell death and promoting ischemia/reperfusion injury.
Under physiologic conditions, ASK1 activity is inhibited by several cellular factors, including Trx, glutaredoxin, and phosphoserine-binding protein 14-3-3 (Bishopric & Webster, 2002). Previous studies have demonstrated that many cellular stresses and apoptotic stimuli activate mitochondrial-dependent apoptotic pathways by facilitating dissociation of ASK1 with its inhibitory protein. Trx is physically associated with ASK1 in cardiac tissues from young animals. However, Trx-ASK1 binding was reduced in cardiac tissue from aging animals. Therefore, it is likely that increased posttranslational Trx modification in aging hearts results in disassociation of Trx from ASK1, thus increasing postischemic myocardial apoptosis by increasing p38 MAPK activity.
Lots of studies have demonstrated that, in addition to upregulation or downregulation of Trx expression at the gene level, Trx activity is regulated by posttranslational modification. Three forms of posttranslational modification of Trx have been previously identified. These include oxidation, glutathionylation, and S-nitrosylation. Interestingly, all three forms of modification occur at cysteine residues but affect Trx function differently. Oxidation of the thiol groups of Cys-32 and-35 forms a disulfide bond which results in Trx inactivation. However, previous studies have demonstrated that administration of oxidized Trx-1 exerts significant antioxidant and cytoprotective effects unless intracellular Trx reductase is inhibited, indicating that oxidative Trx inhibition is reversible and this form of posttranslational modification may not be the major mechanism responsible for Trx inactivation in vivo (Andoh et al, 2003). Glutathionylation occurs at Cys-73, and this posttranslational modification significantly inhibits Trx activity (Casagrande et al, 2002). However, whether Trx glutathionylation may occur in vivo in diseased tissues remains completely unknown and the role of this form of posttranslational modification in regulating Trx function in vivo remains to be determined. S-nitrosylation has been reported to occur at either Cys-69 or Cys-73. In contrast to oxidation and glutathionylation, S-nitrosylation increases Trx activity and further enhances its antiapoptotic effect (Haendeler et al., 2004; Mitchell & Marietta, 2005).
In a recent study, it has been demonstrated that, in addition to three previously reported posttranslational Trx modifications which all occur at the cysteine residue, Trx can also be modified at the tyrosine residue (protein nitration) in a peroxynitrite-dependent fashion (Tao et al., 2006). More interestingly, in contrast to the reversible (by Trx reductase) oxidative Trx inactivation, nitrative modification of Trx results in an irreversible inactivation. Therefore, nitric oxide and its secondary reaction products, particularly peroxynitrite, exert opposite effects on Trx activity. Specifically, nitric oxide itself induces Trx S-nitrosylation and enhances its activity. In contrast, peroxynitrite results in Trx nitration and causes an irreversible inactivation. In Zhang et al.’s study, Trx activity was determined by using the insulin disulfide reduction assay. Compared with young animals, cardiac Trx activity is decreased in the aging heart before myocardial ischemia and reperfusion, and this difference can be further amplified after myocardial ischemia and reperfusion. However, Trx expression is slightly increased, rather than decreased, in aging hearts. These results indicate that it is posttranslational Trx modification rather than reduced protein expression that reduces Trx activity in the aging heart (Zhang et al., 2007).
A Mitochondrial pro-apoptotic protein, HtrA2/Omi, is another reason of enhanced MI/R injury in the aging heart
It is well known that apoptotic cell death is orchestrated by a family of caspases. X-chromosome linked inhibitor of apoptosis protein (XIAP), as a member of IAPs, was the most potent endogenous inhibitor of caspases in human beings. XIAP has three baculovirus IAP repeat (BIR1, BIR2, BIR3) domains and a really interesting new gene (RING) domain. Biochemical studies suggested BIR2 inhibits caspase-3 and caspase-7, whereas BIR3 inhibits caspase-9 (Deveraux et al., 1999). The RING domain is an E3 ligase that presumably directs targets to the ubiquitin-proteasome degradation system, such as caspase-3 (Salvesen & Duckett, 2002; Martin, 2002). The anti-apoptotic activity of XIAP is regulated by a group of proteins that bind to the BIR domains via N-terminal conserved 4-residue IAP-binding motif (Shi, 2002). Recently it has been shown that overexpression of XIAP via in vivo delivery in an adenovirus could reduce both myocardial apoptosis and infarction following I/R (Kim et al., 2011). Wang et al. the protein and mRNA content of XIAP in the heart after MI/R was decreased, while the protein content of XIAP showed positive correlation with cardiac function in 42 rats after MI/R(Wang et al., 2010). These findings suggested a link between myocardial apoptosis, and anti-apoptotic therapy was effective in reducing I/R injury. Meanwhile we found the degradation of XIAP in aging myocardium after MI/R was more than that in young myocardium after MI/R, which are consistent with previous results in which myocardial apoptosis was exaggerated with aging after MI/R. Additionally, the expression of XIAP was also significantly decreased than that in the young adult heart without the intervention of MI/R, which suggested that the decline of XIAP expression may be a major factor responsible for the increased susceptibility of the aging heart.
XIAP is regulated by two cellular proteins, Smac/DIABLO and HtrA2/Omi, which are nuclear-encoded mitochondrial proteins. The cleavage of their mitochondrial-targeting sequences inside mitochondria generates processed active Smac /DIABLO and HtrA2/Omi with new apoptotic N termini, named the IAP-binding motif (IBM) (Srinivasula et al., 2003). Stimulated by apoptotic triggers, Smac/DIABLO and HtrA2/Omi release into the cytosol and competitively bind to the BIR domains of IAPs via IBM, so that the BIR-bound caspases are released and reactivated, resulting in cell apoptosis (Wu et al., 2000, Suzuki et al., 2001). Unlike Smac/DIABLO, the pro-apoptotic activity of HtrA2/Omi involves not only IAP binding but also serine protease activity. Although Omi/HtrA2 and Smac/DIABLO both seem to target XIAP once released into the cytosol, increasing evidence suggests that Omi/HtrA2 may play a unique role in apoptosis. Several different Smac/DIABLO-deficient cells respond normally to various apoptotic stimuli, suggesting the existence of a redundant molecule or molecules compensating for a loss of Smac/DIABLO function (Okada et al., 2002). In contrast, Omi/HtrA2-knockdown cells have shown to be more resistant to apoptotic stimuli (Martins et al., 2002).
In addition, Liu et al. first provided direct evidence that a normal level of endogenously expressed HtrA2/ Omi contributes to apoptosis after MI/R in vivo (Liu et al., 2005). Althaus et al. have also suggested that HtrA2/Omi plays a decisive role in apoptosis after MI/R in young rats (Althaus et al., 2007). Then Wang et al showed that the release of HtrA2/Omi from mitochondria to cytosol was significantly increased in the old MI/R rat heart compared with that in the young MI/R rats (Wang et al, 2006). Meanwhile, cytosol was markedly increased in the old sham group compared with that in the young sham group. Taken together, these results reveal that HtrA2/Omi plays a causative role in increased post-ischemic cardiomyocyte apoptosis in the aging heart (Okada et al., 2002). In order to investigate whether increased HtrA2/Omi plays an important role in aged myocardial apoptosis resulting in myocardial dysfunction and increased susceptibility to MI/R injury, Wang et al observed the effect of ucf-101, a highly selective Omi/HtrA2 inhibitor, on MI/R injury. They have provided direct evidence in the current study that treatment with ucf-101 in aging MI/R animals reduced the caspase-3 activity and improved the cardiac functions. Their results demonstrated that translocation of Omi/HtrA2 from the mitochondria to the cytosol enhanced MI/R injury in aging heart via promoting myocardial apoptosis. These studies may provide some therapies to prevent the over-release of HtrA2/Omi from mitochondria with aging and reduce the risk for MI/R in the elderly. This could help to explain the loss of ventricular function with age and may lead to discoveries of specific therapeutic interventions that can attenuate this type of cell loss (Wang et al., 2010).
Prospect
Aging has become a major health issue and socioeconomic burden worldwide. Coronary heart disease is the leading cause of death worldwide, for patients presented with an acute myocardial infarction, early and successful myocardial reperfusion is the most effective interventional strategy for reducing infarct size and improving clinical outcomes. The process of myocardial reperfusion itself, however, can induce injury to the myocardium, thereby reducing the beneficial effects of myocardial reperfusion. Aging renders the heart more susceptible to cell death from ischemia/reperfusion. In order to develop strategies aimed to limit reversible and irreversible myocardial damage in older patients, there is a need to better understand how aging increases myocardial apoptosis in myocardial ischaemia/reperfusion.
This topic introduced that RNS signaling induced apoptosis contributes to increased susceptibility of aging hearts to myocardial ischemic injury, and the age-associated alterations in translocation of HtrA2/Omi from mitochondria to cytosol are implicated in the markedly increased risk for MI/R injury in old persons.
As mentioned above, three RNS signaling pathways have been recognized .On the one hand, many studies show that RNS can be pro-apoptotic; on the other hand, many studies show that RNS exert anti-apoptotic effects through the same signaling pathway. Further studies should continue to elucidate the many factors that determine how RNS promotes or inhibits apoptosis.
Wang et al. results provide strong evidence that HtrA2/Omi plays a causative role in increased post-ischemic cardiomyocyte apoptosis in the aging heart, but the mechanisms of age-associated alterations in translocation of HtrA2/Omi from mitochondria to cytosol need elucidation.
There is an urgent need for more research of myocardial ischemia/reperfusion conducted on senescent animals. Some researches about RNS signaling induced apoptosis contributes to increased susceptibility of aging hearts to myocardial ischemic injury have been performed in rats or mice. However, biological signaling pathways, proteolytic portfolios, and the overall response to myocardial injury can be quite different in these small rodents when compared to larger mammals. While these murine studies have provided invaluable insight and provoked new hypotheses, they must be carried forward using large animals that more closely recapitulate the clinically-relevant context and for carefully designed clinical trials involving aged human subjects. There also needs to be better coordinated efforts between basic science investigators, clinical trial managers and physicians. (Spinale, 2010, as cited in Bujak et al., 2008; Singh et al., 2010; Lindsey, 2005; Juhaszova et al. ,2005 as cited in Bolli et al., 2004).
Otherwise, most studies have been conducted on healthy aging animals. Ischemic heart disease develops as a consequence of a number of etiological risk factors and always coexists with other disease states. These include systemic arterial hypertension and related left ventricular hypertrophy, hyperlipidemia, and atherosclerosis, diabetes and insulin resistance, as well as heart failure. These systemic diseases with aging as a modifying condition exert multiple biochemical effects on the heart that can potentially affect the development of I/R injury and interfere with responses to cardioprotective interventions. Therefore, the development of rational therapeutic approaches to protect the ischemic heart requires preclinical studies that examine cardioprotection specifically in relation to complicating disease states and risk factors. Surprisingly, relatively little effort has been made to uncover the cellular mechanisms by which risk factors and systemic diseases such as hypertension, hyperlipidemia and atherosclerosis, diabetes, insulin resistance, and heart failure interfere with cardioprotective mechanisms of aging (Ferdinandy et al., 2007).
Although, as mentioned above, RNS and HtrA2/Omi may be critical contributors in controlling apoptosis which determine the susceptibility of aging hearts to myocardial ischemic injury.
