1 Introduction

Given the heart’s high energy demand and function, along with its vital physiological role to the body, a prolonged and non-managed ischemia is detrimental with high risk of morbidity and mortality [1, 2]. Half a century ago, myocardial reperfusion following coronary blood flow obstruction emerged as a promising therapy to rescue the heart from ischemic damage. However, challenging reports emerged since 1970s contradicting the beneficial role of reperfusion on myocardial tissue recovery following ischemia, and highlighting the myocardial ischemia-reperfusion injury (I/RI) concept [3]. Multiple studies, thereafter, exposed the underlying mechanisms behind those findings. Hearse et al [4] were among the first group to report that sudden resumption of metabolic activity to energy-(and oxygen-) starved tissue resulted in a reoxygenation-dependent injury response independent of the hypoxic stress, commonly called “reperfusion injury”. I/RI development is multifactorial involving alterations in both mitochondrial and cellular homeostasis, including a shortage in ATP production, alterations in ion gradient homeostasis, excessive inflammation, Ca2+ handling dysregulation, and excessive ROS production. In fact, myocardial ROS surge following reperfusion was for long proposed to be the mediator of I/RI [5, 6]. Consistently, a large number of studies have intensively addressed the role of excessive ROS formation during I/R [7]. Of note, ROS is a well-known potent mediator of metabolic disruption, inflammation, necrosis, and cell death in multiple diseases including myocardial injuries [8]. In this chapter we emphasize the importance of ROS-mediated reperfusion injury, and highlight the promising mito-targeted antioxidant therapy. We also examine the paradoxal evidence supporting the beneficial effects of ROS bursts in pre- and postconditioning mechanisms.

2 From Permanent Occlusion to Reperfusion: The Bad, the Good, and the Ugly

2.1 The Bad: Myocardial Infarction

Coronary blood flow obstruction, commonly termed MI, is characterized by an inadequate blood flow and subsequent nutrient and oxygen deprivation to the affected area. The severity of MI is strongly dependent on the size of the area at risk, the duration of ischemia, and the presence or absence of comorbidities [9]. The onset of MI itself is characterized by multiple life-threatening pathologies, including ventricular fibrillation, atrio-ventricular block [10] and cardiogenic shock [11]. Following hospitalization and stabilization of potentially existing arrhythmias, non-reperfused MI patients undergo adverse remodeling of the myocardium with very poor prognosis and high risk of heart failure development and death. Based on the American Heart Association statistical report, an approximate number of 720,000 Americans are hospitalized either for a first time MI or coronary heart disease (CHD) events with a projection of a median survival of 8.4, 5.6, 7, and 5.5 years for ≥45 year old white males, white females, black males, and black females respectively. Additionally, sudden cardiac death accounts for 13.5% of death certificates with a relatively high lifetime risk for cardiac arrest survivors [12].

2.2 The Good: Reperfusion

Given the well-established positive correlation between the duration of ischemia and the extent of myocardial damage, coronary blood flow restoration was an inevitable solution. In the last two decades, researchers have conducted a multitude of studies and reported that the salvage of ischemic cells from inevitable death is only possible by revascularization. Thus, multiple interventions such as percutaneous coronary intervention (PCI), coronary artery bypass grafting (CABG), and pharmacological (thrombolysis) approaches to remove the occluding clot were developed and adopted [13]. Reperfusion has proven to limit the ischemic injury and subsequently the infarct size area. The importance of reperfusion therapy in MI patients was surveyed over the past 20 years and reported a continuous decline of 6-month mortality, along with a further 22% reduction in standardized mortality, from 2010 to 2015 following reperfusion therapy [14] .

2.3 The Ugly: Reperfusion Injury

Despite the perpetual improvement of multiple procedures to ensure a rapid, complete, effective, and permanent reopening of the acutely occluded coronary artery, numerous studies revealed that myocardium salvage following blood flow restoration is highly predisposed to another form of injury, known as reperfusion injury [11]. Aside from the reperfusion impact on cardiac remodeling, multiple pathological conditions are known to occur at the onset of blood flow restoration, including arrhythmias, myocardial stunning, and potential microvascular occlusion that could be life-threatening [15].

3 Mechanisms of Cellular Cardiac Injury Following I/R

3.1 At the Onset of Ischemia

Following coronary artery clotting, cessation of cellular oxygen supply halts mitochondrial membrane polarization, reducing therefore adenosine triphosphate (ATP) formation and increasing mitochondrial ROS production [16]. Subsequently, reduced ATP-dependent Na+/K+ pump activity, leads to Na+ accumulation in the myocyte and lowered mitochondrial resting membrane potential. Na+ overload within the cell is counter-regulated by the reverse activity of Na+/Ca2+-exchanger (NCX) that pumps Na+ out in exchange for Ca2+, resulting eventually in intracellular and intra-mitochondrial Ca2+ overload. Concurrently with Ca2+ and Na+ overload, the absence of oxygen supply switches cellular metabolism to anaerobic glycolysis promoting lactate accumulation and cellular acidosis [17]. In summary, ischemia-induced accumulation of intracellular sodium, ROS, and calcium ions, favors, if sustained, the opening of the mitochondrial permeability transition pore (mPTP) [18]. This, together with ATP shortage, determines a loss of contractility, structural disorganization, and apoptotic, necroptotic, and necrotic cell death [19, 20]. However, the acidic conditions during ischemia prevent opening of the mPTP and subsequent cardiomyocyte death (Fig. 12.1a).

Fig. 12.1
figure 1

Schematization of the key components of acute myocardial ischemia reperfusion injury: (a) Loss of oxygen supply in ischemia leads to a loss of ATP production and a switch to anaerobic respiration, resulting in a drop in intracellular pH, accompanied with an increased intracellular and mitochondrial-derived ROS. The ATP consuming Na+-K+-pump ceases to function, leading to Na+ accumulation in the myocyte and the resting membrane potential is lowered. With the development of acidosis, the NHX further increases intracellular Na+ exacerbating Ca2+overload by forcing the NCX to manage, in a reverse mode the extrusion of Na+ and the influx of Ca2+ into the cell. The sarcolemmal L type voltage-gated Ca2+ (L-CC) are activated allowing more Ca2+entry as the resting membrane potential is low. Ca2+pump SERCA2 is now taken up the excess of Ca2+into the SR that releases it subsequently via RYR, leading to contraction and contracture. The acidic conditions during ischemia however prevent the opening of the mPTP and cardiomyocyte hypercontracture. (b) During reperfusion ATP production increases leading to the Na+-K+-pump reactivation, a slow restoration of both sodium gradient and NCX normal activity extruding the excess of cytosolic Ca2+. An excessive production of ROS accompanies reoxygenation, electron transport chain activation, and immune cells infiltration. ROS burst mediates myocardial reperfusion injury by inducing the opening of the mPTP, causing outer mitochondrial membrane permeabilization, apoptosis, necrosis, acting as a neutrophil and cytokines chemoattractant, mediating dysfunction of the SR and causing myofibril hypercontacture. Restoration of physiological pH Fig. 12.1 following reperfusion along with Ca2+ overload accentuates mPTP opening leading to an increased infract size, cellular dysfunction, and cell death. Ca 2+ calcium, Na + sodium, K + potassium, H + hydrogen, O 2 oxygen, SR sarcoplasmic reticulum, SERCA sarco/endoplasmic reticulum Ca2+-ATPase, ATP adenosine triphosphate, OxPHos oxidative phosphorylation, ROS reactive oxygen species, mPTP mitochondrial permeability transition pore, NCX 3Na+/1Ca2+-exchanger, NHX Na+-H+-exchanger, PMNs polymorphonuclear lukocytes, (+) stimulation, (−) inhibition, ↑ increase, ↓ decrease

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3.2 At the Onset of Reperfusion

Reperfusion is intended to restore ATP production and reactivate the Na+/K+ ATPase to slowly re-establish the sodium gradient, leading to normal cation fluxes and eventually extruding the excess cytosolic and mitochondrial Ca2+. However a massive mitochondrial ROS burst follows reoxygenation during reperfusion, which is further fueled by inflammation, increasing the risk of mPTP opening and cell death [18]. Additionally, persistent high intracellular Ca2+ levels observed during the early phase of reperfusion, increase the risk of a damaging myocardial hypercontracture that was otherwise inhibited during acidic ischemia (Fig. 12.1b). Besides, in the setting of ischemic–reperfusion injury, ROS burst is also responsible of the activation of protein kinase C delta (PKC-δ) stimulating its translocation to the mitochondria where it results in cytochrome c release, caspase 3 activation, and a decrease in the activity of pro-survival Akt, as well as poly (ADP-ribose) polymerase (PARP) cleavage in the nucleus. Pharmacological inhibition of PKC-δ is exploited in many therapeutic strategies like preconditioning [21]. In summary, reperfusion-induced cellular damage is largely dependent on ROS burst, Ca2+ overload, and mPTP opening [20].

4 Myocardial ROS in I/R: Types and Sources

Compelling evidence pointing to the causal interconnection between oxidative stress and I/RI is well established [22]. Oxidative stress is a consequence of the imbalance between ROS production and antioxidant capacity, either because of heightened ROS release and/or an ineffective antioxidant system [23]. Under ischemic conditions, mitochondrial complexes I and III are primarily responsible of the conversion of molecular oxygen to unstable/reactive superoxide (O2 −) [24]. Cardiomyocytes, containing the highest number of mitochondria, consume a higher level of oxygen than any other cell and subsequently become major ROS producers [25]. As a result, heightened cellular ROS levels ultimately alter cellular homeostasis primarily by damaging proteins, lipids, and nucleic acids [26, 27]. In addition to local ROS production, immune cell infiltration into the myocardium following I/RI contributes substantially to increase ROS levels [28]. Upon reperfusion of the ischemic myocardium, inflammatory reaction is noticeably accelerated. Although inflammation is crucial for myocardial tissue healing, the re-establishment of blood flow to ischemic tissue accelerates and prolongs inflammatory response detrimentally. Among multiple immune cell infiltrations, neutrophils are considered the earliest and the most potent releaser of ROS, followed by macrophages [29]. Interestingly, clinical anti-neutrophil therapies did not succeed in slowing or preventing adverse myocardial remodeling post-MI [30]. These findings imply that local free radical outburst following reperfusion is potentially the main source of ROS-mediated injury during I/R. Xanthine oxidase (OX), nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, mitochondrial electron transport damage and uncoupling, uncoupled nitric oxidase synthase (NOS), and myeloperoxidase (MPO) are the major producers of ROS in reperfused ischemic myocardium [31] and will be discussed in this chapter.

4.1 Nitric Oxide Synthases (NOS)

One of the most studied sources of physiological and pathophysiological ROS are the three well-recognized isoforms of NOS enzymes known as neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) that normally produce NO during the oxidation of L-arginine to L-citrulline [32]. While eNOS and nNOS are known to be constitutively expressed in the myocardium, iNOS, although primarily induced in immune cells, is expressed in cardiomyocytes under ischemic conditions [33, 34]. Constitutive myocardial nitric oxide (NO) generation under physiological conditions is essential for physiologic cell signaling [35]. Blood flow and oxygen restoration following reperfusion significantly increase NOS activity and subsequent NO production [36]. Although NO has been reported to be protective against I/R-induced injury in different organs of experimental animals [37] and humans [38], the beneficial effects of NO activity are negated by increased O2 −-mediated peroxynitrite (ONOO−) generation following reperfusion (Table 12.1).

$$ \mathrm{NO}+{{\mathrm{O}}_2}^{-}={\mathrm{O}\mathrm{NOO}}^{-} $$

Contrarily to NO, ONOO− is very detrimental to proteins and lipids. ONOO– can negatively and irreversibly alter the structure and function of NOS by damaging its heme domain and oxidizing the tetrahydrobiopterin (BH4) cofactor [32, 39, 40] ultimately leading to NOS uncoupling an important source of I/R-induced ROS generation [32]. NOS function during I/R depends as well on its structural form; two cellular forms of constitutive NOS exist, the monomer and the homodimer forms. The monomer form is responsible of O2 − generation in small amounts; the shift towards excessive O2 − production depends on the homodimer/monomer ratio, intracellular L-arginine supply, and on BH4 oxidation [1].

Table 12.1 Potential sources of reactive oxygen species in the cardiac tissue exposed to ischemia and reperfusion
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A decrease in the local BH4/NOS ratio makes the balance, of a stoichiometric relationship between BH4 and eNOS, fall towards increased O2 − instead of NO [41, 42]. Uncoupled NOS, furthermore, produces more O2 − that acts as a positive feedback loop leading to further BH4 to BH2 oxidation and the propagation of NOS uncoupling. Besides the described loop, XO [43] and/or NADPH oxidase [44] play an important role in the I/R-induced reduction in BH4 levels by promoting O2 − generation.Also, an essential factor required for the synthesis of NO by eNOS is arginine, the nitrogen donor, and substrate for arginase I and II [45]. Increased arginase activity leads as well to increased production of O2 − by NOS, a mechanism called “arginine steal”. Finally, it was very early reported that myocardial eNOS actively produces NO during ischemia and reperfusion; however, parallel observations have shown that the enzyme is affected during ischemia. In fact, a prolonged ischemia is accompanied by intracellular acidosis that reversibly or irreversibly inhibits eNOS activity independently of the duration of acidosis [46] (Table 12.1).

4.2 Monoamine Oxidase and p66shc

During I/R, mitochondria are responsible of the generation of hydrogen peroxide (H2O2) through serotonin oxidization via monoamine oxidase [47]. Serotonin accumulation, as well as increased monoamine oxidase activity, is noted during ischemia and substantially increased following reperfusion [48]. Moreover, mitochondria are also capable of H2O2 production using a novel pathway that involves the 66-kDa isoform of the growth factor adaptor protein, p66shc. Ischemic conditions are responsible of translocation of p66shc from the cytosol to the mitochondrial intermembrane space, allowing it to use reducing equivalents from the electron transport chain (ETC) via the oxidation of cytochrome c to make H2O2. This reaction acts in a vicious cycle to provide p66shc with increased substrate in the intermembrane space during ischemia [49].

4.3 Cellular Xanthine Dehydrogenase vs. Xanthine oxidase

During normoxic physiological conditions, xanthine dehydrogenase (XDH) catalyzes the oxidation of hypoxanthine to xanthine and then to uric acid by coupling the reaction with NAD+ reduction to yield NADH.

$$ \mathrm{hypoxanthine}+{\mathrm{NAD}}^{+}\overset{\mathrm{XDH}}{\to}\mathrm{xanthine}+\mathrm{NADH} $$
$$ \mathrm{xanthine}+{\mathrm{NAD}}^{+}\overset{\mathrm{XDH}}{\to}\mathrm{uric}\kern0.17em \mathrm{acid}+\mathrm{NADH} $$

However, ischemic context enhances the conversion of XDH to XO by the modification of a sulfhydryl group or by proteolytic cleavage. XO is a molybdo-flavoenzyme complex that controls, in addition to uric acid production, ROS generation through catalyzing the oxidation of hypoxanthine to xanthine.

$$ \mathrm{hypoxanthine}+{\mathrm{O}}_2\overset{\mathrm{XO}}{\to}\mathrm{xanthine}+{{\mathrm{O}}_2}^{-} $$
$$ \mathrm{xanthine}+{\mathrm{O}}_2\overset{\mathrm{XO}}{\to}\mathrm{uric}\kern0.17em \mathrm{acid}+{{\mathrm{O}}_2}^{-} $$

XO-derived ROS contribute to multiple pathologic conditions including I/RI (Table 12.1). The accumulation of XO following ischemia will increase O2 − formation. Besides, the oxygen burst at the onset of reperfusion drastically increases O2 − formation [50] .

4.4 Cellular Xanthine Dehydrogenase

Nevertheless, other mechanisms can explain the enhanced superoxide release, independently of XDH to XO conversion. In fact, XDH has an NADH oxidase activity in the presence of acidic conditions (pH 6.5) wherein NADH is oxidized rather than xanthine [33]. XDH is capable of generating superoxide at 4-times the rate of XO. Besides, XDH is the dominant isoform in the early reperfusion period and is most likely a more important source of superoxide than the XO isoform at the onset of the reperfusion.

figure a

Post-transcriptional regulation of XDH expression is reported during I/R, wherein the hypoxic and inflammatory environments are stimuli associated with increased XDH transcription [51]. On the other hand, XO activity is also regulated at the post-translational level. These modifications have been attributed to O2 tension that results in phosphorylation of the enzyme by p38 kinase [52]. In addition, along with hypoxic environment, the inflammatory context (mast cell degranulation and macrophage activation), which accompanies I/R, participates via multiple cytokines such as IL-1, IFN-γ, IL-6 and TNF-α to increase XDH/XO mRNA. Another feature of the XO capacity to produce ROS under ischemic conditions is its capacity to act as a nitrate/nitrite reductase (Table 12.1). This enzymatic reaction catalyzes the production of NO by one electron reduction of nitrite, and is optimal under anoxic/hypoxic and acidic conditions [53]. The generated NO, an important substrate for peroxynitrite generation, enhances the oxidative burst in the presence of an ischemia/inflammation loop in the ischemic heart [54] (Table 12.1). Finally, XO participates in leucocyte recruitment upon I/R, followed by neutrophil recruitment and XO-derived ROS secretion [55].

4.5 Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase

The Noxs are a family of 7 isoforms expressed in multiple types of cells, including vascular endothelial cells, smooth muscle cells, fibroblasts, cardiomyocytes, and polymorphonuclear leukocytes (PMNs). Nox isoforms are known as Nox-1 to Nox-5 and dual oxidases (Duox)-1 and -2 [56, 57]. Nox/Duox are considered the major source of ROS in multiple pathological conditions including cardiac remodeling post-MI [58,59,60] (Table 12.1). The contribution of Nox enzymes to reperfusion injury is documented by multiple studies reporting both an increased expression and/or activity of Nox in ischemic tissue and attenuation of I/R induced injury following Nox inhibition [61, 62]. A large amount of data reported the involvement of multiple factors in the activation of Noxs in I/RI. For example, studies confirmed that hypoxia inhibitory factor-1α (HIF-1α) activation, evoked by the hypoxic state that accompanies ischemia, promote production and activation of Noxs [63]. Among all Nox isoforms, Nox2 is one of the most widely expressed in cardiac cells and, therefore, a prominent ROS producer in myocardial I/R (Table 12.1). The activation of XO and the resulting increase in ROS and intracellular Ca2+ levels have been reported to be indispensable for Nox2 activation under ischemic conditions. The stimulation of PKC by XO-generated ROS also contributes to ischemic-evoked Nox2 activation. Furthermore, the inhibition of XO halts ischemic-induced upregulation of HIF-1α proving that Nox2 activation by XO is essential for HIF-1α activation under ischemic conditions [64]. Of note, both the activation of the complement system and increased generation of angiotensin II are also associated with an increase in Nox activity in cardiac post-ischemic tissue [61, 65].

4.6 Mitochondrial ETC ROS Production

Mitochondria constitute 33% of the total cardiac myocyte cell volume, highlighting their fundamental role in cardiac function and the high energy demand of the myocardium. The mitochondrial ETC complex is comprised of a series of multi-subunit complexes (complexes I–IV) located in the inner mitochondrial membrane (IMM) and coupled to mobile carriers such as coenzyme Q and cytochrome c. The complexes and cytochrome c contain redox groups (Fe-S clusters and/or heme) that allow for the transfer of electrons along the components of the ETC, generating a proton electrochemical gradient, ultimately promoting ATP production via ATPase [25, 66]. Mitochondria are considered a normal source of ROS that play a crucial role as cell signaling intermediates in order to maintain cellular homeostasis. Under normal physiological conditions, ETC reduces oxygen to water using more than 97% of the entire electron flux through mitochondria. The remaining 2–3% of electrons consistently leak from ETC to form O2 −. In addition to its important role in signaling, physiological production of O2 − plays a critical role in multiple crucial cell functions such as metabolism, proliferation, and apoptosis [67].

Following ischemia, the decrease in mitochondrial respiration as well as ATP production, along with complex I/III alterations, increases NADH:NAD+ ratio and reduces flavin mononucleotide prosthetic group within the NADH dehydrogenase component of complex I. These changes increase the leakage of electrons that form O2 − via univalent reduction of O2 and subsequent ROS production beyond physiological levels [24, 25, 68, 69]. Although reduced cytochrome c controls mitochondrial ROS levels by scavenging O2 −, persistent ischemia increases the oxidized state of cytochrome c contributing further to mitochondrial damage and the accumulation of O2 − (Table 12.1).

Upon reperfusion, oxygen burst into an already stunned mitochondria drastically increase ROS production to a much higher extent than during ischemia. Additional sites within complex I may contribute to ROS generation. Mitochondrial increase in superoxide production is normally accompanied by an increase in H2O2 formation through MnSOD activity within the mitochondrial matrix [70]. Superoxide dismutase enzymes contain either copper, manganese, or nickel metal centers that are reduced or oxidized to convert cellular O2 − into H2O2 (Table 12.1) [71, 72]. H2O2 interaction with NO also increases formation of ONOO−. Of note, ROS is able to freely spread within the mitochondrial network mainly through the mPTP and inner membrane ion channels, centralizing therefore cellular damage [25, 73].

5 ROS Mediated Adverse Effects in I/R

ROS production during the ischemic, reperfusion, and remodeling phases contribute to cardiac injury post-MI. The extent of injury, however, varies based on the size of the affected myocardium, the magnitude of ROS reactions, and the severity of cardiomyocyte damage. Uncontrolled sustained ROS burst causes modification and denaturation of a multitude of structural and functional molecules leading to irreversible tissue damage. The effect of each ROS, however, depends on its type. OH- for instance acts instantly right after generation. O2 − and NO− radicals on the other hand, are of much lesser reactivity, more specific, and can mediate radical reactions on sites that are distant form their site of production. In the absence of appropriate ROS scavengers, sustained ROS production triggers oxidative vicious cycles that could permanently damage the cells. Of the well-known ROS-mediated cellular damage, lipid peroxidation, protein denaturation, mitochondrial, and DNA damage constitute the basis behind those effects.

5.1 DNA Oxidation

OH-mediated hydrogen extraction interferes with cellular DNA, causing purine and/or pyrimidine direct modification and/or fragmentation producing a plethora of DNA lethal lesions [74]. These lesions can induce mutagenesis, crosslinks between DNA strands and proteins, stand breaks, which affect thereafter DNA replication and transcription [75] and ultimately promote a pro-apoptotic and pro-necrotic effect (Table 12.2).

Table 12.2 ROS targets following I/RI
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5.2 Lipid Peroxidation

Lipid peroxidation is a typical 3 phase oxidative reaction that occurs abundantly during I/R. The alkenes, unsaturated fatty acids and major component of biological membrane’s phospholipid bilayers, are very susceptible to hydrogen extraction by ROS. The generated carbon-centered and peroxyl radicals constitute the initial phase of ROS attack followed by an amplification phase also known as the propagation phase [76]. Lipid peroxidation continues with additional similar abstractions until two radical species combine in a termination phase. Reactive aldehydes such as malondialdehyde (MDA), 4-hydroxynonenal, and isoprostane are major end products of this classical oxidation cascade and are known to increase during I/R [77]. Lipid peroxidation byproducts are bioactive and well-involved in the adverse remodeling of I/R. 15-F2t-isoprostane, for example, is reported to induce a dose-dependent vasoconstriction in coronary arteries, promoting therefore cardiac dysfunction following I/R [78] (Table 12.2).

5.3 Protein Oxidation

ROS-mediated activation of necrotic and pro-apoptotic proteins determines the severity and the extent of infarct size [79]. For instance, ROS can modify cellular proteins via oxidation and nitration, impairing subsequent myocardial contraction and promoting myocardial stunning following I/R [80]. Similarly to what is observed with lipids, hydrogen extraction by OH- is a key player in the initiation phase of the oxidative attack on proteins by affecting amino-acid functional groups [81]. Denaturation of proteins by ROS oxidation reactions is due to the cleavage of peptide bonds, functional group cross-linking, and by hydrophobicity alterations of amino acids on protein surfaces [81] (Table 12.2).

Proteins with signaling roles, such as kinases and phosphatases, can also be oxidized by ONOO−, affecting therefore their signaling capacities and impact [82]. By regulating mitogen-activated protein kinases (MAPKs), ROS contribute to cellular responses to mitogens, inflammatory cytokines, and (un)physiological stimuli [83]. Activation of p38 can have either pro- or anti-apoptotic effects, and is exaggerated during I/R. Also, p38 has been reported to play a role in regulating mitochondrial ROS levels and intracellular signaling pathways, as well as controlling mitochondrial events associated with development of I/R-associated damage (Table 12.2). Other signaling pathways that have also been shown to be involved in this regulation include: the RAF-MEK pathway that, in contrast, prevents mitochondrial accumulation of ROS/Ca2+ and cell death [83], and the PI-3 kinase (PI3K)/protein kinase C (PKC/AKT) pathway that has a protective role against cellular I/R-induced cell death.

ROS are implicated as well in inflammatory signaling, not only by fueling the pro-inflammatory response in a self-perpetuating manner, but also by regulating the process of high-mobility box 1 (HMGB1) protein release that occurs especially in response to cellular damage. HMGB1 is an agonist for Toll-like Receptors (TLRs). Accordingly, TLR4-mediated NFκB activation is recruited for oxidative stress-activated intracellular signaling pathways [84].

Two additional developmental pathways also figure among the most important pathways in this context: the Wnt/s-catenin signaling that is activated by ROS [84] and NOTCH signaling that suppresses ROS production [85]. Nevertheless, this sort of crosslink between intracellular signaling and regulation of mitochondrial ROS production has been demonstrated for p53 [86], protein kinase A (PKA) [87], rapidly accelerated fibrosarcoma (RAF) kinase, protein kinase B (Akt/PKB), and B-cell lymphoma 2 (Bcl-2) [83]. The tyrosine kinase pathway plays a role via p66shc, which acts as a redox enzyme that generates mitochondrial ROS through oxidation of cytochrome c [49]. In addition, oxidative stress leads to alterations in the activation state of different PKCs. This activation provides a protective role in the context of preconditioning by activating the specific PKC-ɛ isoform [88]. However, activation of PKCδ isoform increases, in a positive-loop manner, ROS generation. Activation of PKCδ by ROS regulates the expression and function of apoptosis-related proteins, and represents a target for caspases leading to cellular death [88].

Activation of enzymes including MMPs and caplains is also pronounced following increased ROS production and pH restoration, and is capable subsequently of degrading crucial functional proteins, such as myosin light chain [89], α-actinin [90], and cardiac troponin [91, 92] (Table 12.2). During ischemia, oxidative stress is also broadly responsible for Na+/H+ exchanger (NHX) activation, a mechanism that attempts to restore intracellular pH by increasing cellular Na+ levels. The Na+/Ca2+ exchanger (NCX) is thus activated, leading to intracellular accumulation of Ca2+ and a state of mitochondrial Ca2+ overload and depolarization. This phenomenon is exacerbated upon reperfusion with mitochondrial calcium uniporter (CaU) exaggerated opening. ROS-mediated alterations of anion exchanger function leads to pH recovery. Excessive ROS, however, favor opening of the mPTP (Fig. 12.1b) [93], leading to mitochondrial matrix swelling and loss of MOM. This result is fatal due to the pro-apoptotic molecules that are released from the mitochondrial intermembrane space (IMS). Additionally, another type of Ca2+ permeable cationic channel is affected by increased ROS production during I/R. The transient receptor potential melastatin 2 (TRPM2) is in fact a ROS sensor [94]. Oxidative stress-mediated activation of TRPM2 results in mitochondrial Na+ and Ca2+ overload, which leads to a disrupted mitochondrial membrane, cytochrome c release, PARP-1 cleavage via induction of caspase-8 activation, and finally apoptotic cell death [95].

It is worthwhile to point out that radical reactions are of a semi-random nature, so they do not necessarily yield irreversible cell damage. For example, the magnitude of the oxidative attack on membranes, proteins, or DNA may not be enough to have an adverse effect on their functions. Besides, if the damaged protein is not of critical functional relevance, normal cell processes, such as phospholipid and protein turnover, can remove the altered biomolecule and the cell will survive. Understanding the contribution of ROS to the development of I/RI may identify additional targets for therapeutic interference. That being said, the understanding of aberrant signaling in this particular pathological condition holds the promise for novel therapeutic approaches that specifically target the regulation of mitochondrial function (Table 12.3).

Table 12.3 Summary of selected clinical trials using general ROS scavengers therapeutics for MI
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6 General and Mitochondria Targeted Antioxidants Reduce I /R Injury

Mitochondrial Ca2+ overload and overexuberant mitochondrial ROS burst, constitute the hallmark of I/R-mediated cardiac cell injury [96, 97]. A massive burst of ROS following reperfusion localizes in mitochondrial regions that progress to swelling and eventually stimulates opening of the mPTP [98, 99]. mPTP opening is directly linked to mitochondrial DNA rearrangement and fragmentation, a complete disrupted mitochondrial structure and function (Table 12.2), followed by mitochondrial perforation and lysis [18]. Mitochondria-targeted antioxidant therapy has the ability to salvage I/R-assaulted cardiomyocytes more so than general antioxidants (Table 12.4) at different levels including: (1) preventing excessive detrimental cellular ROS production that is largely and mainly produced by mitochondria with I/R, (2) promoting low and beneficial ROS signaling through protein kinase Cε and its downstream substrates, and (3) preventing harmful ROS signaling through protein kinase Cδ and its downstream effectors. Examples of protective therapies targeting mitochondrial ROS are detailed in Table 12.4.

Table 12.4 Different mitochondrial ROS targeting strategies
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7 The Paradoxal Cardioprotective Effects of ROS

Pre- and postconditioning are manipulations during which short periods or bouts of ischemia are applied by occluding and opening the coronary artery, prior or subsequent to, permanent occlusion [100]. Pharmacological and interventional ischemia pre- and postconditioning has gained immense attention due to its protective effects on cardiac remodeling prior to or following reperfusion [101, 102]. This protection is, however, impeded with application of antioxidants. In fact, unlike excessive and sustained ROS burst that is now proven to be detrimental, low levels of ROS are protective (Table 12.4). A growing body of recent evidence has established that generation of ROS at low levels can serve as a signal mediating physiologic responses. The protective role of preconditioning on the myocardium was first described in 1986 by Murry et al., as a slower ATP depletion rate and smaller infarct size in the heart treated with brief episodes of I/R before prolonged occlusion, followed by reperfusion [101, 102]. Mitochondrial pathways play an important role in promoting the activation of cell survival programs following preconditioning via ROS signaling-dependent mechanisms [103]. A good example of cardioprotective roles of reliable amounts of ROS is the metabolic vasodilator effect of H2O2, produced by myocardial mitochondria. H2O2 serves as a mediator that couples oxygen consumption to coronary blood flow by acting as an activator of redox- and 4-aminopyridine-sensitive voltage-dependent potassium (Kv) channels in smooth muscle cells [100]. In addition, H2O2 that derives from complexes I and III in the endothelial mitochondria’s electron transport chain is capable of triggering calcium activated potassium (BKCa) channels in order to enhance acetylcholine- and flow-induced coronary vasodilation [104, 105]. More recently, several methods of preconditioning have been developed including ischemic preconditioning (IPC), exercise preconditioning, and pharmacological preconditioning [106,107,108]. The opening of mitochondrial ATP-sensitive K+ (mitoKATP) channel is one of the most important mechanisms activated by preconditioning stimuli (Table 12.4). This activation allows potassium to flow into mitochondria leading to depolarization and matrix alkalization. Subsequently, an increase in ROS production activates downstream survival signaling events through PKC, preventing mPTP opening [106, 109]. Additionally, the generation of mild matrix swelling improves ATP synthesis and fatty acid oxidation, conditioning the cells to any potential ischemic injury [109].

Ischemic postconditioning on the other hand was first introduced by Zhao et al. in 2003. This term refers to brief periods of ischemia alternating with brief periods of reflow applied at the onset of reperfusion following sustained ischemia. The timing of post-conditioning interference is crucial given that reperfusion injuries occurs only within several minutes following blood reflow [110]. The basics of preconditioning- and postconditioning-mediated protection are very similar [110, 111]. In fact, similar to preconditioning, the mitoKATP/ROS/PKC axis pathway constitutes the basis of postconditioning protective therapy [110]. However, the degree of protection largely depends on the timing of axes activation following reperfusion [112]. Of note, both pre- and postconditioning share an important effect that underlines their cardioprotective efficacy. In fact, the associated prolongation of cellular acidosis that takes place initially during early reperfusion after ischemia favors inhibition of mPTP opening for a few minutes following reperfusion. Pre- and postconditioning released ROS take advantage of delayed protective pH normalization to induce activation of cell survival programs. Therefore, following pH normalization, an arsenal of downstream effectors that prevent mPTP opening is boosted, to preserve mitochondrial and cellular integrity [113].

Several other signaling pathways are implicated in the infarct-sparing effect of pre- or postconditioning [114]. The Reperfusion Injury Salvage Kinases (RISK) pathway involves the activation of two signaling pathways consisting of pro-survival kinases ERK1/2 and Akt that converge on mitochondria to decrease mPTP opening [115]. The Survivor Activating Factor Enhancement (SAFE) pathway involves the induction of JAK-STAT3 signaling. The relative contribution of the RISK and SAFE pathways to cardiac protection varies with the experimental ischemic protocol, as well as species. Some studies have linked SAFE signaling to the initiation of the RISK pathway, although the mechanism is not defined [115]. Both the RISK and SAFE pathways are activated by ROS.

8 Conclusion and Future Direction

Reperfusion of the coronary circulation is necessary to prevent irreversible loss of the myocardium. Yet reperfusion causes further harm to the heart via the generation of ROS, which invariably leads to heart failure and shortened lifespan. These ROS target phospholipids of the cell membrane, various structural, transport, and signaling proteins, and DNA, which may then act synergistically to further ROS generation and damage the heart. MMPs, caspases, and calpains are activated as well, further exacerbating structural damage. Much progress has been made in identifying the sources of ROS, which include NOXs, MAO, uncoupled NOSs, p66shc, xanthine dehydrogenase/oxidase, and mitochondria. Paradoxically, lower levels of ROS may activate a number of signaling mechanisms that tamp down excessive ROS generation by mitochondria as initially revealed in preconditioning experiments. The targets of this manipulation include both direct effects on mitochondria, as well as the upregulation of protective proteins at later time points. For practical reasons, direct preconditioning strategies have little if any translational potential. However, complementary approaches, such as exercise-induced preconditioning and ischemic postconditioning, offer clinical promise. Pharmacological manipulations that specifically target mitochondrial complexes that generate ROS during reperfusion are gaining interest as therapies. Although much progress has been realized in the last decade in understanding the source and implications of ROS as foe in I/R-mediated injury to the heart, the upcoming decade should result in the practical application of therapeutic strategies that are based on the revelation of mechanisms defined by the protective actions of ROS in the myocardium.