Ischemia‐reperfusion and cardioprotection: A delicate balance between reactive oxygen species generation and redox homeostasis

Abstract

Ischemia‐reperfusion injury of the myocardium has long been a subject of intense research. Cardiac preconditioning, an associated phenomenon, has also been critically investigated over the past two decades. Although the biochemistry of ischemia‐reperfusion and its association with oxidative metabolism has long been established, recent studies have further revealed a more intricate role of a number of reactive oxygen‐nitrogen species in those processes. Emerging evidence suggests that an elaborate network of enzymes (and other biomolecules) dedicated to the generation, utilization, and diminution of reactive oxygen‐nitrogen species maintains the redox homeostasis in the myocardium, and any perturbation of its status has distinctive effects. It thus appears that while excessive generation of reactive species leads to cellular injury, their regulated generation may cause transient and reversible modifications of cellular proteins leading the transmission of intracellular signals with specific effects. Taken together, generation of reactive oxygen‐nitrogen species in the myocardium plays a nodal role in mediating both ischemic injury and cardioprotection.

• Cardiac ischemia
• cardiac preconditioning
• reactive oxygen species
• reactive nitrogen species
• redox signaling
• reperfusion injury

Abbreviations
GSH	=	glutathione
GSSG	=	glutathione disulfide
Akt	=	protein kinase B
PI	=	phosphatidylinositol
MAPK	=	mitogen‐activated protein kinase
iNOS	=	inducible nitric oxide synthase
GPCR	=	G protein couple receptor
Ang	=	angiotensin
IGF	=	insulin‐like growth factor
EGF	=	epidermal growth factor
HO	=	heme oxygenase
Nrf	=	NF‐E2‐related factor‐2
ECH	=	epithelioid cell histiocytoma

Cardiac ischemia—the leading cause of heart failure, acute myocardial infarction, and arrhythmias—has long been a major contributor towards infirmity in the industrialized societies and thus is a subject of intense research 1. Reperfusion injury, an essential consequence of the restoration of blood supply to the ischemic myocardium, has also been a subject of intense interest 2. Advantageously, following the seminal observation in the mid‐80s that several cycles of brief ischemia followed by reperfusion renders the heart more resistant towards reperfusion injury had led to the concept of ‘ischemic preconditioning’ and the feasibility of ‘cardioprotection’ 3–6. However, in spite of the molecular, pharmacological, and biochemical characterization of a plethora of ‘pro‐survival’ and ‘pro‐death’ molecules and their modulators, ‘cardioprotection’, especially in the clinical setup, still remains exasperatingly elusive 6,7. Such disillusionment can at least be partially attributed to the consideration of cell signaling and gene regulatory pathways in isolation, rather than in the context of the organismal and cellular milieu. Nevertheless, cardiac biologists over the years have accumulated a wealth of information regarding the role of reactive oxygen species and redox metabolism in cardiac injury as well as protection. However, how the respective signaling pathways ultimately integrate into either pro‐survival or pro‐death phenomenon is yet to be fully understood 8,9. The present review is aimed towards the compilation of a number of emerging concepts on redox‐metabolism in the ischemic myocardium, especially in the context of preconditioning.

Key messages

• Reactive oxygen‐nitrogen species play a nodal role in ischemia‐reperfusion injury as well as preconditioning of the myocardium.

• Regulated generation of certain reactive species may initiate specific cell signaling events with distinct consequences.

• Based on the type, amplitude, and duration of the reactive species generated, cardiac myocytes may opt for death or survival pathways.

Reactive oxygen/nitrogen species (ROS/RNS) as modulators of cellular functions

During the past several decades, our understanding of the roles of free radicals in cellular and organismal biology has painstakingly evolved. Although once it was believed that generation of free radicals/reactive oxygen species (ROS) are detrimental to the living organisms, subsequent studies have established that production of ROS to a limited extent may lead to distinctive cellular events like cell proliferation and differentiation 10–13. Free radicals are characterized by the presence of one or more unpaired electrons in the outermost orbital of their constituent atoms and thus are extremely reactive (and labile) as compared to their normal counterparts with paired electrons. Furthermore, certain compounds, namely hydrogen peroxide, are not free radicals per se, but in the presence of transition metals it generates highly active hydroxyl radicals (OH·) and thus is a ROS in specific biological contexts (14,15). ROS are generated in various cell types either under normal physiological conditions to mediate certain regulatory functions or under the influence of certain pathological stimuli with deleterious consequences 16. As an example, superoxide free radical (O₂·⁻) is produced by nicotinamide adenine dinucleotide phosphate (NADPH)‐oxidase complex in phagocytic cells for the purpose of killing invading microbes 17. Exogenous factors like cytokines and growth factors, environmental toxicants like tobacco smoke, ultra‐violet radiation, etc., and intracellular signal mediators like sphingolipids may also cause intracellular ROS generation, thereby influencing cellular events 18–20. In addition to ROS, a number of nitrogen‐containing free radicals have also emerged during the past decade as the key mediators of various pathophysiological events. Since the monumental discovery that nitric oxide (NO·) can act as an intracellular signal transducer, numerous studies have established it as a modulator of disparate cellular events such as dilatation of blood vessels, neurotransmission, inflammatory response, and regulation of gene expression 21,22. Also, nitric oxide can interact with other oxidants like superoxide radical (O₂·⁻), hydrogen peroxide (H₂O₂), or transition metal centers resulting in the generation of a number of highly reactive entities, collectively known as reactive nitrogen species (RNS) 23. Taken together, due to such close interrelationship between reactive oxygen and nitrogen species, their role(s) in biology are often considered in conjunction with one another.

Intracellular generation of ROS/RNS

Mitochondria and peroxisomes are the two major intracellular sources of ROS generation. The mitochondrial electron transport chain (complex I to IV) is a highly structured assembly of redox proteins dedicated to the generation of a proton gradient (ΔpH) and a membrane potential (ΔΨm) across the inner mitochondrial membrane resulting in ATP synthesis. When these two values are sufficiently high, electron transport is inhibited by a feedback mechanism, leading to an accumulation of electrons which then leak out and concomitantly bind to oxygen, thereby generating O₂·⁻. Amongst four electron transport complexes, complex I and III are considered to be the primary producers of O₂·⁻24–26. Although O₂·⁻ is highly reactive, it is instantaneously converted into hydrogen peroxide by superoxide dismutase (Mn‐SOD in the mitochondria and Cu/Zn‐SOD in the cytosol). Thereafter, H₂O₂ is converted to oxygen and water by catalase. However, under certain circumstances, especially due to excess generation, O₂·⁻ may escape this attenuation process thereby producing other ROS causing oxidative injuries 27. Like ROS, a number of RNS can also be generated by various intracellular pathways. As an example, NO· reacts with O₂·⁻ by a diffusion‐controlled reaction generating peroxynitrite (ONOO⁻) which is of potential importance in certain cell types such as macrophages and endothelial cells which simultaneously produce O₂·⁻ and NO· 28. Peroxynitrite can cause oxidation and nitration of proteins (at thiols, methionine, and tyrosine) as well as lipid peroxidation 29. Also upon protonation, peroxynitrite produces ONOOH, which decays either via production of NO₂· (nitrogen dioxide radical) and OH·, or NO₃⁻ and H⁺23. Potential protective roles of nitrite and nitrate anions (and nitric oxide) in ischemia‐reperfusion setup have been documented 30. Also, excessive generation of NO₂· may lead to the formation of N₂O₃, a potent nitrosating agent 31. Generation of other reactive nitrogen species such as nitroxyl radical (HNO), especially in the context of ischemic brain, has also been described 32,33. As described in the following sections, and summarized in Figure 1, many of these ROS/RNS have been implicated in cell signaling, especially when generated at a lower doses, while their excessive generation often leads to the oxidative modifications of cellular proteins (such as formation of carbonyls, oxidation of methionines/cysteines and cross‐linking of polypeptides) with deleterious consequences 34–37.

Figure 1. Generation and effects of intracellular ROS/RNS: Superoxide (O₂⋅⁻) and nitric oxide (NO·) generated from various intracellular sources are identified by oval‐shaped boxes. Those two radicals can be converted into various other ROS/RNS by mutually interactive pathways as described in the text. Depending on type, combination, amplitude, and duration of those radicals the respective signals are finally integrated into an output‐like preconditioning effect (physiological) or apoptosis (pathological).

Redox equilibrium and cell fate

During evolution, while aerobic organisms acquired the ability to harness energy by reducing atmospheric oxygen, a plethora of other redox‐sensitive molecules carrying out diverse cellular functions has also emerged. Whenever ROS/RNS is generated in living cells, it reacts with lipids, proteins, carbohydrates, and nucleic acids, and the outcome depends upon the cell type and the nature, intracellular localization, amplitude, and the life span of the reactive species 38–43. Furthermore, since redox homeostasis is an essentiality of normal cellular functions, nature has also evolved an extensive network of antioxidant defense systems attenuating various ROS/RNS. Antioxidant enzymes like superoxide dismutase (converts O₂·⁻ into H₂O₂), catalase (converts H₂O₂ into water), glutathione peroxidase (converts H₂O₂ into water), thioredoxin‐thioredoxin reductase, glutaredoxin‐glutaredoxin reductase, and small molecules like vitamin E and C thus play a major role in maintaining the redox equilibrium 44–46. Thus, an intricate network of enzymes (and other biomolecules) dedicated to the generation, utilization, and diminution of ROS/RNS maintains intracellular redox, and any perturbation of redox‐status has distinctive effects 43,47–50. Emerging evidences suggest that while general oxidative modifications of proteins have deleterious effects (on both structure and functions), controlled oxidation might have regulatory consequences 51–53. Thus, based upon the reactive species and its targets, the effects can be divergent 54–58.

ROS/RNS as second messengers

Although the intracellular generation of ROS/RNS and its role in oxidative/nitrosative modifications of proteins have been documented for several decades, their potential role in intracellular signaling became apparent following the discovery of nitric oxide as a transducer of specific signals 21. Subsequent years have seen a paradigm shift when other reactive species emerged as the potential mediators of intracellular communications 59. Increasing evidence suggests that ROS/RNS can cause transient and reversible modifications (oxidative/nitrosative) of proteins, especially when momentary generated, resulting in loss‐gain of functions, thereby acting as second messengers 59,60. Second messengers are generated upon receptor stimulation, are of short lifespan, and act upon specific targets. In that context, while ROS/RNS are also generated upon receptor stimulation and are very transient in nature, their specificity of action (except that of nitric oxide) has often been a subject of intense debate 59,61. As an example, while a highly reactive species like OH·⁻ is unlikely to have any specificity, others like O₂· and H₂O₂ are highly diffusible, less reactive, and therefore might act in a specific manner 62,63. Generation of ROS/RNS has been attributed to the modulation of a number of signaling/gene regulatory modules including MAP kinase, PI3 kinase/Akt, transcription factors AP‐1 and NFκB 64–67. However, only a few direct targets of oxidative/nitrosative modifications have been identified as yet, and those are primarily the cysteine residues present in the catalytic sites of the respective proteins 59,68,69. Noticeably, a number of nonsignaling regulatory modules like ryanodine receptor, sarco/endoplasmic Ca²⁺ pumps (SERCA) have also been identified as the targets of oxidative modifications in the context of cardiovascular biology. Ryanodine receptor contains a large number of cysteine residues, and redox‐modifications of at least some of those modulate its function 70,71. Nitric oxide (NO) can also form S‐nitroso derivatives of some of those cysteine residues and thereby play a role in modulating its functions 72. Similarly, peroxynitrite inhibits SERCA activity by oxidative modifications thereby affecting calcium transients 73.

Thioredoxin/glutaredoxin systems and redox‐homeostasis

Although the side chains of various amino acids are amenable to oxidative and nitrosative modifications, those of cysteine thiols have drawn significant attention 74. Many of the cysteine oxidations are reversible and might constitute a highly evolved mechanism of activation‐inactivation of cognate proteins like that by phosphorylation‐dephosphorylation 75,76. Oxidative modifications of cysteines also play a critical role in pathobiology in general and cardiovascular biology in particular 77. Emerging evidence suggests that a group of enzymes and low molecular weight peptides, namely thioredoxin and glutaredoxin oxidoreductases, plays a key role in restoring protein thiols and thereby maintaining redox homeostasis. Although these proteins are functionally equivalent to free radical scavengers, their role(s) are much wider in the context of metabolism, signaling, and gene regulatory events 78–80. Thioredoxin is a general disulphide reductase with a low redox potential and thus capable of acting upon a wide range of oxidized proteins including thioredoxin peroxidase, which is a ROS scavenger. In mammals, there are two thioredoxins, namely Trx1, found in cytosol and the nucleus, while Trx2 is mitochondrial 81,82. Trx1 can also be secreted out and act as a chemokine. Heart failure patients show elevated levels of plasma thioredoxin as an adaptive response towards increased oxidative stress 83. Thioredoxin also provides protection against oxidative injuries caused by adriamycin and reperfusion 84. Protective effects of thioredoxin are mediated via its interaction with a diverse family of regulatory molecules like apoptotic signal regulating kinase 1 (ASK‐1), vitamin D3‐upregulated protein‐1 (VDUP‐1), gene regulatory proteins NFkB and AP‐1. Noticeably, thioredoxin can attenuate ASK‐1 function both in a redox‐dependent and ‐independent manner 85,86. VDUP‐1 exerts its pro‐apoptotic effect by the suppression of thioredoxin activity 87,88. Thioredoxin has also been attributed to the increased expression of antioxidant genes like Mn‐SOD and heme oxygenase‐1 89. Our laboratory has demonstrated that in ex vivo working heart, the thioredoxin level decreases upon ischemia, while preconditioning upregulates its expression 90,91. Treatment with thioredoxin inhibitor cis‐diammine‐dichloroplatinum abolishes cardioprotection by preconditioning. Transgenic mouse hearts overexpressing thioredoxin‐1 shows significant improvement in postischemic ventricular recovery and reduced myocardial infarct size, further supporting its nodal role in cardioprotection 90,91.

Like thioredoxin, the glutaredoxin system comprising glutaredoxin, glutathione, and glutathione reductase also plays a key role in maintaining intracellular redox equilibrium. In mammals, there are two glutaredoxin genes, namely Grx 1 and Grx 2. In human, while Grx1 is cytosolic and is involved in the reduction of various enzymes, Grx2 has two splice variants localized in the nucleus and the mitochondrion 92. The mitochondrial electron transport chain is a major source of ROS that leads to a reduced GSH/GSSG ratio and glutathionylation of various proteins including the NADH‐binding pockets of complex I (thereby further increasing the ROS generation). Grx 2 plays a major role in regenerating key mitochondrial proteins by deglutathionylation 93. In agreement, ablation of Grx 2 by RNAi results in increased susceptibility towards cell death by inducers of ROS like doxorubicin 94. In a recent study, decrease in glutaredoxin (and reduced glutathione), in conjunction with increased oxidant levels, has been documented in cardiac interfibrillar (but not in subsarcolemmal) mitochondria of old rats 95. Also, in cardiac myoblast cell line H9c2, 17 beta‐estradiol prevents oxidative stress‐induced apoptosis by glutathione/glutaredoxin‐dependent redox modulation of Akt activity 96.

Ischemia‐reperfusion injury is caused by the generation of ROS/RNS

It has long been documented that restoration of blood flow into the ischemic myocardium leads to ventricular arrhythmias, contractile dysfunction, myocardial stunning, and myocyte death 97–101. Subsequent studies established a cause‐and‐effect relationship between oxidative stress and ischemia‐reperfusion injury 102–104. The advent of 1) more sensitive assays for ROS/RNS generation, both in tissues and in cultured cells 105,106; 2) molecular cloning, overexpression, and targeted deletion of genes of various antioxidant enzymes 62,107–109; and 3) availability of various redox‐responsive markers 26,71 have unequivocally established the deleterious role of ROS/RNS in ischemia‐reperfusion injury. Simultaneously, increasing evidence such as generation of free radicals in conjunction with the accumulation of oxidized proteins in the mitochondria had established it as the primary source of ROS generation during ischemic injury 110–116. During ischemia, diminished oxygen supply inhibits the TCA cycle and oxidative phosphorylation while increasing the free fatty acid level, NADH/NAD⁺ ratio, and glycolysis, thereby leading to acidosis with deleterious consequences 117. Decrease in respiration also leads to reduced electron transport in the mitochondria, causing one electron leakage followed by the reduction of O₂·⁻ to O₂·⁻. Under normal circumstances, O₂⁻· would be acted upon by superoxide dismutase, converting it into H₂O₂. However, in the reperfused myocardium, due to acidosis and reducing environment, ferric and ferrous ions are released from metalloproteins which in turn catalyse (Fenton reaction) the generation of highly reactive hydroxyl radical from O₂·⁻ and H₂O₂118. Under ischemic conditions, enzymes like xanthine oxidase also reduce hydrogen peroxide into hydroxyl radical 119, while upon reperfusion, due to concurrent generation of NO and superoxide ions, highly reactive peroxynitrite is formed 120. Also during reperfusion, increased intracellular H⁺ stimulate the Na⁺/H⁺ exchanger, resulting in increased intracellular Na⁺ which in turn stimulate Na⁺/Ca²⁺ exchanger, ultimately causing an increase in intracellular (and intramitochondrial) Ca²⁺ with detrimental effects like mitochondrial swelling, opening of mitochondrial permeability transition pores (PTP), and apoptosis 121,122. Since intracellular Ca²⁺ regulates a plethora of cellular enzymes like kinases, phosphatases, endonucleases, and proteases, an elevation in Ca²⁺ level adversely affects other cellular functions as well 123.

Ischemic preconditioning, an enigmatic phenomenon

Ischemic preconditioning, a phenomenon where brief repetitive cycles of ischemia‐reperfusion render the myocardium more resistant towards subsequent ischemic insult, was first documented in the mid‐80s 3. Thereafter, numerous physiological agonists like norepinephrine, angiotensin II, bradykinin, acetylcholine, cytokines, opioid peptides, and diffusible molecules like adenosine and nitric oxide 124–128, pharmacological/chemical agents like peroxynitrite, hydrogen sulfide, and volatile anesthetics have been also identified to have preconditioning effects 129–131. However, it is believed that there are subtle differences in the mechanism by which each mediates its cytoprotective effects 9,132. A number of signaling kinases (namely PKC, ERK, Src, PI3 kinase‐Akt, p70S6 kinase), gene regulatory proteins (namely AP‐1, NFκB, STAT), mitochondrial permeability transition pores, K_ATP, Cl⁻, and the inward rectifier K⁺ channels are some of the mediators of preconditioning effects 124,125,127,133–145. Ischemic preconditioning occurs in two phases: first it is observed within a few minutes of brief ischemia‐reperfusion and stays for several hours (classical or early preconditioning); while the second phase starts after 12–24 hours and stays for 3–4 days (second window of protection or late preconditioning). It is thus believed that while the first phase of preconditioning is primarily achieved through the calibration of biochemical parameters (such as minimizing the usage of ATP), late preconditioning is more of a competent state of the myocardium achieved through a wider alteration of cellular proteome 146,147. During the early phase of preconditioning, certain biochemical changes in the intracellular milieu, such as altered pH, oxygen tension, redox equilibrium, etc., are the possible contributors towards preconditioning effects 148,149. Preconditioning has also been observed in other tissues like brain and presumably involves conserved pathways rather than a mechanism solely dedicated to cardioprotection 150.

Amongst various types of pharmacological preconditioning, that induced by adenosine has been extensively investigated and adenosine receptor (AR) subtypes 1 and 3 are primarily attributed towards this process 151,152. While transgenic mice overexpressing A1 AR are more tolerant to ischemic injury, those having deletion of either both or single alleles are refractile towards preconditioning effects and are susceptible to postischemic injury 124. Transgenic mice overexpressing A3 AR also show less ATP depletion following ischemia, reduced infarct size following ischemia‐reperfusion, and better postischemic recoveries 153,154. Intriguingly, A3 AR gene knockout mice showed improved postischemic recovery as compared to their normal counterparts 155. Adenosine receptors are members of the G protein coupled receptor (GPCR) superfamily and, upon stimulation, activate pro‐survival signaling kinases like ERK, p38, PKC, and Akt mediating its beneficial effects 156. In addition, rhoA‐phospholipase D1, mitochondrial permeability transition pore, catalase, and superoxide dismutase have been attributed to the preconditioning induced by adenosine 152,157,158. Nevertheless, certain discrepancies in the contributions of the downstream mediators have also been reported. For example, while Lasley et al. 159 used open chest anesthetized rat and demonstrated the involvement of both ERK and p38 kinase in mediating cardioprotection by A1 AR, Button et al. 160 used adult ventricular strips and hypoxia‐reoxygenation protocol and observed that cardioprotection by A1, A2A, and A3 ARs does not require ERK and PI3 kinases. Similarly, while Germack et al. 151 used neonatal cardiac myocytes and hypoxia‐reoxygenation protocol demonstrating that A2A AR does not have any role in preventing cell death, Button et al. (adult ventricular strips and hypoxia‐reoxygenation protocol) demonstrated its involvement in preconditioning 160. Discrepant observations were also made with reference to other mediators further downstream. While Guo et al. 161 used iNOS‐/‐ mouse and demonstrated its essentiality in delayed preconditioning by A1 AR, Lasley et al. 159 used a pharmacological inhibitor of iNOS (1400 W) and demonstrated its nonessentiality for the same. Opioids are also involved in cardioprotection during ischemia, and experimental evidence suggests that endogenous opioids released from the myocytes act in an autocrine fashion providing their beneficial effects 125. Studies with rabbit and rat heart indicate that amongst three major opioid peptides (endorphins, dynorphins, and enkephalins), enkephalin and the δ1‐receptor are the primary mediators of the protective effects 125,162,163. In addition, recent studies have also appreciated the involvement of κ receptor in this process 164,165. Like adenosine receptors, opioid receptors also belong to the GPCR superfamily (Gi) and mediate their effect via PKC‐δ, PI3 kinase, tyrosine kinase, ERK, PKA and 12‐lipoxigenase 166–170 which modulate their downstream targets like iNOS, sarcolemmal K_ATP and mitochondrial K_ATP channels 161,164. Two other GPCR agonists, Ang II and norepinephrine, have also been attributed to preconditioning. And depending upon their concentration, both could be beneficial as well as detrimental for the heart 171–173. Such disparate behavior is attributed to their ability to initiate concentration‐dependent differential signaling affecting downstream kinases (like p38 MAPK, JNK, and Akt), other regulatory proteins (like IGF‐IR, EGF‐R, and HO‐1), and downstream transcription factors (like NFkB, Nrf2, and AP‐1) 173–175. NE‐mediated preconditioning is also mediated by PI3 kinase, PKC, PKA and p38 MAP kinase 176–178. Ischemic stimuli also releases bradykinin, primarily from the endothelium, that acts upon the bradykinin receptors (B2) on myocytes and provide preconditioning effects via PKC, PKG, Akt, and nonkinase effectors like iNOS and ATP‐sensitive K⁺ channel 128,141,179–181. Although the physiological and pharmacological stimuli described above provide cardioprotection, it is likely that there are subtle differences in their mechanisms of action 9,132. Like in ischemia, reperfusion of the hypoxic myocardium also has deleterious effects (myocyte loss) and has been investigated both in whole heart and in isolated myocytes 182,183. Hypoxic injury also involves generation of ROS, reduced glutathione levels, decreased Na⁺/Ca²⁺ exchanger activity and calcium overload 184,185. Studies with cultured myocytes has also demonstrated that during hypoxia, the generation of ROS from the mitochondrial electron transport chain leads to the activation of p38 MAP kinase and JNK 186,187. Finally, nitric oxide, K_ATP channel, ROS, and gene regulatory protein HIF‐1α have been attributed to the hypoxic preconditioning 188,189.

ROS/RNS and ischemic preconditioning: a paradoxical paradigm

In spite of significant progress in our understanding of the biochemical and molecular events associated with ischemic injury and cardioprotection, the precise mechanism by which cardiac myocytes decide to survive or to die is yet to be deciphered 6. Furthermore, studies done over the past decade indicate that the mechanisms of cardioprotection by different pharmacological agents might be divergent, at least partially 9. It is thus likely that the vulnerability of the myocardium towards ischemia‐reperfusion insult vis‐à‐vis its attainment of preconditioned status is defined by a distinctive cellular proteome rather than by the presence or absence of a set of ‘protective’ or ‘detrimental’ molecules. In this context, studies done by various laboratories have documented that ROS generation plays a nodal role in mediating both ischemic injury and cardioprotection 8. Such paradoxical behavior of ROS can be explained by its disparate regulatory potential. As discussed in the previous sections, reactive oxygen species are well suited as second messengers involved in the activation and/or inactivation of ERK, p38, JNK and PI3 kinases, affecting disparate cellular events 190–197. Since intracellular ROS can be generated at multiple sites (like NADPH oxidase, mitochondrial complex I and III) while being simultaneously attenuated by a battery of antioxidant enzymes 198, the net ROS threshold in a particular cellular context decides whether it will be deleterious or beneficial 172. Emerging evidence also suggests that once specific redox‐responsive signaling pathways are activated, the cognate signal is transmitted to the nucleus initiating discrete gene expression programs 172,199,200. In this context, mammalian gene regulatory protein Redox factor‐1 (Ref‐1) has been a paradigm of redox‐sensitive gene regulatory events. Ref‐1 has two distinct functions, while its C‐terminus functions as a DNA repair (oxidative and radiation‐damaged) enzyme, its N‐terminal region contains a redox regulatory domain involved in the reductive modifications of a battery of transcription factors like activator protein‐1 (AP‐1), nuclear factor κB (NFκB), p53, hypoxia inducible factor‐1 (HIF‐1) and thyroid transcription factor‐1 (TTF‐1) (Figure 2) 201–204. In response to oxidative stimuli, thioredoxin translocates to the nucleus, interacts with Ref‐1 which in turn transmits the signal by the reduction of specific cysteine residues to its downstream targets 205. Two cysteine residues (at positions 65 and 93) of Ref‐1 mediate this process by acting as redox switches (sulfhydryl switches). Protein kinase C can also modulate Ref‐1 activity, further diversifying its regulatory potential 206. Ref‐1 thus acts as a coupler of upstream redox events to a plethora of transcription factors and the DNA repair apparatus further downstream 207. Although it generally believed that Ref‐1 is a mediator of pro‐survival events, evidence in support of its pro‐apoptotic function also exists 208–210. The feasibility of both pro‐ and anti‐apoptotic functions of Ref‐1 can be explained from such dual functions of its targets, namely AP‐1, NFκB, and HIF‐1alpha 211–214, while the activation of p53 by Ref‐1 supports its pro‐apoptotic potential 215. The diversity of Ref‐1 functions is still being uncovered, and emerging evidence suggests its role in redox‐independent cellular events as well 216,217. Taken together, Ref‐1 is a bona fide candidate for playing a nodal role in regulating redox events leading to both ischemic injury and cardioprotection 205. Another gene regulatory protein, namely Nrf‐2 (NF‐E2‐related factor 2), has also drawn considerable attention for its role in regulating oxidative events. Nrf‐2 binds the DNA sequence known as antioxidant response element (ARE), commonly found in the regulatory regions of antioxidant and phase II detoxifying enzymes such as glutathione S‐transferase, NAD(P)H, quinone oxidoreductase 1, and heme‐oxygenase‐1. Nrf‐2 is uniquely characterized by it inducibility by a variety of ROS/RNS, cellular lipid oxidation products, and xenobiotics 218,219. Under normal conditions, Nrf‐2 is sequestered in the cytoplasm by the cysteine‐rich protein Keap‐1 (Kelch‐like ECH‐associated protein 1). In the presence of ROS/RNS, cysteine residues of Keap‐1 are modified, leading to a conformational change followed by its dissociation, and proteasomal degradation. Nrf‐2 then translocates to the nucleus and activates cognate genes eliciting an antioxidant and detoxification response 220. The mechanism of Keap‐1 activation by various stimuli is an active area of research and is less understood in the context of ROS/RNS generated under ischemia‐reperfusion 220. Nevertheless, emerging evidence suggests a key role of Nrf‐2 in mediating redox signaling under ischemia‐reperfusion injury as well as cardiac preconditioning 221.

Figure 2. Activation of Ref‐1 by thioredoxin: schematic presentation of interconversion of thioredoxin systems in conjunction with the activation of Ref‐1, followed by that of AP‐1, NFκB and p53.

Summary and conclusion

In the post genome era, our knowledge of various biological phenomena has achieved a new dimension due to the use of integrated approaches such as gene annotation, proteomics, microarray analysis, etc., and it is now expanding at an unprecedented pace 9. With the available information, any biological event can thus be considered in a wholesome manner rather than in isolation. In that context, our perception of the role(s) of ROS/RNS in modulating cellular events has also taken a new direction, where it can be either beneficial or detrimental based upon its nature, modes of generation, and its mutual interaction with other cellular constituents in a given context. However, how those ROS/RNS (same or different) can convert a death signal into a survival signal is an emerging theme not only in the context of the biology of ischemia‐reperfusion in specific, but also in the context of the pathophysiology of various diseases in general. It is thus expected that with the identification of more and more redox‐responsive biomolecules and the understanding of their specific roles in respective cellular events, coming years will see further consolidation of our knowledge of the intricacies of cell signaling and gene expression associated with the unique phenomena of ischemia‐reperfusion and cardioprotection.