MRI is a clinical problem associated with procedures such as thrombolysis, angioplasty, and coronary bypass surgery, which are commonly used to re-establish the blood flow and minimize the damage to the heart due to severe myocardial ischemia. There are three main hypotheses which have been proposed to explain the pathogenesis of ischemia reperfusion (IR) injury: oxidative stress, iron mobilization, and Ca2+-overload[6,7]. All of these mechanisms are most likely related, but it is not known whether they operate simultaneously or one precedes the other (Figure 2).
Figure 2 Role of reactive oxygen species and iron mobilization in myocardial reperfusion injury and its clinical implications.
MVO: Microvascular obstruction; ONOO*: Peroxynitrite; NO: Nitric oxide; OH *: Radical hydroxyl; Fe: Iron; RyR: Ryanodine receptor channel; SERCA: Sarco/endoplasmic reticulum Ca2+-ATPase.
The level of myocardial tissue oxygenation increases following restoration of blood flow, which is initiated with a burst of ROS generation; these ROS are the major initiators of myocardial damage in MRI. Increased ROS production is mainly due to the activation of xanthine oxidase in endothelial cells, mitochondrial electron transport chain reactions in cardiomyocytes, and NADPH oxidase in inflammatory cells (Figure 1).
Oxidative stress occurs when there is an imbalance between the generation of ROS and the antioxidant defense systems in the body so that the latter becomes overwhelmed. ROS include hydrogen peroxide (H2O2), the superoxide radical anion, the hydroxyl radical (OH•), and peroxynitrite anion (ONOO-), and they have all been shown to increase with reperfusion (Figure 2). As a result of lipid peroxidation, oxidation of DNA and proteins and membrane damage may take place. This leads to alterations in membrane permeability and to modifications of protein structure and functional changes.
ROS sources: In pathophysiological conditions, there are many sources of ROS in myocardial tissue. The most important sources are NADPH oxidases (NOX), uncoupled eNOS, xanthine oxidases and the mitochondrion. NOX catalyzes the one electron reduction of O2 to generate super-oxide radical anion (O2•−), using NADPH as the source of electrons. This enzyme is largely present in the activated neutrophil, wherein it generates large amounts of toxic O2•− and other ROS important in bactericidal function. Pathogenic roles of NOX-derived ROS are also verified in human IR injury in vivo. It was recently reported that in isolated perfused murine hearts that NOX1 and/or NOX2 gene knock-out significantly attenuated MRI (by up to 50% of the final infarct size), thus demonstrating the crucial importance of this enzyme in MRI.
The NO synthases (NOS) are a family of enzymes that convert the amino acid L-arginine to L-citrulline and NO. Endothelial NOS (eNOS) plays a major role in the regulation of vascular function. The eNOS may become uncoupled in the absence of the NOS substrate L-arginine or the cofactor BH4. Uncoupled eNOS results in the production of O2•− instead of NO[16-18]. This perpetuates a vicious cycle because peroxynitrite, the reaction product of superoxide and NO, leads to further eNOS uncoupling. Furthermore, eNOS uncoupling may play a major role in MRI by increasing ROS production and limiting NO availability.
Xanthine oxidase is predominantly present in the vascular endothelium in the normal heart and generates O2•−, H2O2, and OH• as byproducts of its normal metabolic action. Under pathological conditions, such as tissue ischemia, xanthine dehydrogenase can be converted to Xanthine oxidase. In IR this enzyme catalyzes the formation of uric acid with the coproduction of O2•−. Superoxide release results in the recruitment and activation of neutrophils and their adherence to endothelial cells, which stimulates the formation of xanthine oxidase in the endothelium, with further O2•− production
Mitochondria are cellular organelles involved in energy production, so any injury that they may suffer could cause impairment of cellular energy that could lead, depending on the intensity of the injury, to apoptosis or different levels of cellular damage. During ischemia, due to the lack of oxygen, the electron transport chain cannot function correctly and therefore ROS are produced at high levels. Additionally, ROS may cause oxidative damage of mitochondrial DNA, impairing mitochondrial function. This damage performs a positive feedback on ROS production that, at the same time, perpetuates mitochondrial damage and ROS synthesis. Oxidative injury to the mitochondrial membrane can also occur, resulting in membrane depolarization and the uncoupling of oxidative phosphorylation, with altered cellular respiration. This can ultimately lead to mitochondrial damage, with release of cytochrome c, activation of caspases, and apoptosis.
RNS sources: The ROS are not solely responsible for free radical damage. Reactive nitrogen species (RNS), mainly peroxynitrite anions (ONOO-), also generate RNS-damage, thus producing nitrosative stress. Peroxynitrite results from the interaction between NO and the superoxide anion, and NO is synthesized mainly by nitric oxide synthases which have two isoforms in the cardiomyocyte: endothelial (eNOS) and inducible (iNOS). Oxidative and nitrosative damage causes the uncoupling of both NOS isoforms, resulting in the enhanced synthesis of O2•−.
Evidence supports the view that nitrosative stress plays an important role in the pathogenesis of MRI. While NO itself is not harmful, some of the reaction products (mainly OH•) resulting from high ONOO- formation in the cell are highly cytotoxic substances. The production of O2•− is increased during reperfusion, which interacts with NO and leads to the formation of ONOO-, thus triggering the previously described phenomenon. Peroxynitrite not only causes structural damage by attacking macromolecules, but it also leads to myocardial functional impairment. The general view about the mechanisms that lead to nitrosative stress is that IR can induce iNOS expression and that the resulting high concentrations of NO can lead to cardiac injury. The drop in NO concentration occurring during cardiac IR plays an important role in triggering the transcription nuclear factor kappaB (NF-κB) leading to activation and successive induction of iNOS expression during the reperfusion phase[29-31]. Figure 1 shows a diagram of ROS and RNS sources in myocardial tissue.
Oxidative stress modifies phospholipids and proteins leading to lipid peroxidation and thiol-group oxidation; these changes are considered to alter membrane permeability and configuration in addition to producing functional modifications of various cellular proteins. Oxidative stress may result in cellular defects including a depression in the sarcolemma Ca2+-pump ATPase that leads to a decreased Ca2+-efflux, and a depression in (Na + K)-ATPase activity that, in turn, leads to an increased Ca2+-influx. Oxidative stress has also been reported to depress the sarcoplasmic reticulum Ca2+-pump ATPase (SERCA) and thus inhibit Ca2+ sequestration from the cytoplasm in cardiomyocytes. The depression in Ca2+-regulatory mechanism by ROS ultimately results in intracellular Ca2+ ([Ca2+]i) overload and cell death. In addition, an increase in [Ca2+]i during ischemia induces the conversion of xanthine dehydrogenase to xanthine oxidase and subsequently results in increased production of O2•−.
Recently it has been shown that the function of the channel ryanodine receptor (RyR) is controlled by ROS. It has been demonstrated that NADPH oxidase and the RyR channel could be located adjacent to each other in the T-tubules of cardiomyocytes. Thus, the increase in ROS production after myocardial reperfusion could lead to an increase in RyR channel function, resulting in an intracellular calcium overload, thereby causing activation of pro-apoptotic intracellular pathways, necrosis, and electromechanical alteration. All these mechanisms are summarized in Figure 3.
Figure 3 Central role of calcium in the electro-mechanical dissociation of cardiomyocyte after myocardial reperfusion.
RyR: Ryanodine receptor channel; SERCA: Sarco / endoplasmic reticulum Ca2+-ATPase; mPTP: Mitochondrial permeability transition pore; Ca: Calcium; ROS: Reactive oxygen species.
Redox-sensitive signaling pathways: Not only do ROS exert their actions by directly modifying organic molecules, but ROS are also involved in the regulation of the expression of several genes. NF-ĸB and AP-1, both of which can experience ROS-mediated activation, stimulate the transcription of several protein mediators, for example, proinflammatory cytokines that activate several cell death pathways. The role of cytokines, chemokines, leukocytes, and acute-phase proteins such as high-sensitivity C-reactive protein in the pathogenesis of MRI has been reported in several studies[49,50]. Oxidative stress, ROS and inflammation are linked in a way that is very difficult to dissect. These phenomena have important molecular bridges that are activated in the presence of ROS, leading to the activation of multiple mechanisms that cause heart tissue remodeling and therefore enhance the susceptibility to rhythm disorders. Among those molecules, the most studied has been the transcriptional factor NF-ĸB, a factor that responds to changes of the cellular oxidative state, ischemia-reperfusion, and inflammatory molecules. When NF-ĸB is activated, for example in the presence of ROS by phosphorylation of its inhibitory cofactor (Iĸ-B), it bonds to a DNA response element and promotes the transcription of genes involved in inflammatory and pro-fibrotic response, for example IL-6, which transforms growth factors TGF-β and TNF-α. Those molecules act in various tissues, but particularly in the heart, producing extracellular matrix remodeling and fibrosis (structural remodeling), which changes the electrophysiological properties of the heart. Several studies have associated NF-ĸB activation with cardiac dysfunction, ventricular hypertrophy, and maladaptive cardiac growth (Figure 2).
Exposure to low-to-moderate ROS levels should trigger a survival response and reinforce ROS scavengers of the antioxidant defense system to elicit a cardioprotective effect for myocardial reperfusion. The molecular mechanism responsible for this adaptive change involves enhanced antioxidant activity achieved by up-regulating several housekeeping genes partly under the control of Nrf2 (nuclear factor-erythroid 2-related factor 2); Nrf2 is normally sequestered in the cytosol by Keap. Upon oxidative stimulation, Nrf2 oxidizes or covalently modifies Keap1 thiol groups, which dissociate from Keap1 and undergo nuclear translocation. In the nucleus, Nrf2 binds to antioxidant response elements in target gene promoters, which increase the expression of antioxidant enzymes. It has been demonstrated that the constitutive levels/activities of a number of important antioxidants and phase 2 enzymes, such as CAT, GSH-Px, glutathione reductase, glutathione transferase, NADPH-quinone oxidoreductase 1, and heme oxygenase-1 in primary cardiomyocytes are dependent on Nrf2 status. In addition, Nrf2 diminishes the susceptibility of cardiomyocytes to injury elicited by oxidants and electrophilic species, making the Nrf2 signaling pathway an important mechanism for myocardial cytoprotection. It is of interest to note that ROS levels could be responsible for the activation of NF-κB and/or Nrf2 pathways.
Clinical implications: Myocardial damage caused by ischemia-reperfusion events are mainly associated with four clinical conditions: lethal reperfusion, myocardial stunning, no-reflow phenomenon, and reperfusion arrhythmias (Figure 2).
Lethal reperfusion is a paradoxical type of MRI caused by the restoration of coronary blood flow after an ischemic episode. It is defined as the death of cardiomyocytes that were viable immediately before myocardial reperfusion. Its main manifestation is as an increased infarct size due to reperfusion, a condition mainly associated with AMI. In the late fifties, it was suggested that myocardial reperfusion contributes part of the histological damage associated with ischemia-reperfusion models. However, for decades it was very complex to determine the precise evolution of necrosis along the transition from ischemia to reperfusion in myocardial tissue. Nowadays, the harmful effects of myocardial reperfusion damage, also known as lethal reperfusion injury, are considered to involve myocardial cell death derived from the restoration of blood flow subsequent to an ischemic process, and to act through mechanisms strongly associated with oxidative stress.
Reperfusion arrhythmias clinically represent a major comorbidity of AMI with an 88.7% occurrence rate in certain small clinical trials with continuous monitoring. In addition, postoperative atrial fibrillation (POAF), the most common reperfusion arrhythmia associated with cardiac surgeries, has an incidence ranging between 20%-40%. Myocardial stunning, despite being a reversible damage, is the cause of an impaired ventricular function that leads to increased morbidity. It is derived from a short-term ischemia-reperfusion process that was first reported in the early 1930s. Myocardial stunning is present to a greater or lesser extent in all survivors of AMI. In the late 1980s evidence began to appear suggesting an important role of oxidative stress in the development of myocardial stunning, proposing that the main injury pathway could be an altered calcium homeostasis associated with sarcoplasmic reticulum damage. More recently, clinical studies have strengthened this hypothesis, and it has been reported in animal models that interventions aimed to improve antioxidant defenses attenuate myocardial stunning[64,65].
The no-reflow phenomenon is an impaired myocardial perfusion of a specific segment of the coronary system that is not associated with an angiographic occlusion of the respective vessel. Vascular and endothelial damage can occur after the reperfusion of previously blocked coronary circulation. It can be exhibited as a microvascular dysfunction after restoring the flow during either angioplasty or thrombolysis, thus leading to the development of the no-reflow phenomenon. The presence of coronary microvascular dysfunction and this phenomenon are associated with larger infarct size, lower left ventricular ejection fraction, adverse left ventricular remodeling in the remote stage of myocardial infarction, and increased incidences of heart failure and death, compared with patients without no-reflow phenomenon. Some studies using animal models showed that antioxidant strategies are able to reduce this phenomenon[69-71], and this data is consistent with a small clinical trial finding that antioxidant depletion is associated with no-reflow phenomenon in AMI. In addition, recent research in rabbits shows that the suppression of the oxidative stress-sensitive transcription factor NF-ĸB, a key mediator of inflammation in cardiovascular systems, reduces myocardial no-reflow phenomenon.
Recently, our group has reported major clinical benefits with the use of antioxidants in pathologies associated with myocardial reperfusion, such as POAF and AMI. With regard to POAF, we documented a significant decrease in the incidence of this arrhythmia in patients undergoing cardiac surgery with extracorporeal circulation after administration of ascorbate, alpha-tocopherol, and omega-3 polyunsaturated fatty acids, which was accompanied by a significant decrease in oxidative stress biomarkers in auricular tissue and peripheral blood.