Health position paper and redox perspectives on reactive oxygen species as signals and targets of cardioprotection

Abstract

The present review summarizes the beneficial and detrimental roles of reactive oxygen species in myocardial ischemia/reperfusion injury and cardioprotection. In the first part, the continued need for cardioprotection beyond that by rapid reperfusion of acute myocardial infarction is emphasized. Then, pathomechanisms of myocardial ischemia/reperfusion to the myocardium and the coronary circulation and the different modes of cell death in myocardial infarction are characterized. Different mechanical and pharmacological interventions to protect the ischemic/reperfused myocardium in elective percutaneous coronary interventions and coronary artery bypass grafting, in acute myocardial infarction and in cardiotoxicity from cancer therapy are detailed. The second part keeps the focus on ROS providing a comprehensive overview of molecular and cellular mechanisms involved in ischemia/reperfusion injury. Starting from mitochondria as the main sources and targets of ROS in ischemic/reperfused myocardium, a complex network of cellular and extracellular processes is discussed, including relationships with Ca²⁺ homeostasis, thiol group redox balance, hydrogen sulfide modulation, cross-talk with NAPDH oxidases, exosomes, cytokines and growth factors. While mechanistic insights are needed to improve our current therapeutic approaches, advancements in knowledge of ROS-mediated processes indicate that detrimental facets of oxidative stress are opposed by ROS requirement for physiological and protective reactions. This inevitable contrast is likely to underlie unsuccessful clinical trials and limits the development of novel cardioprotective interventions simply based upon ROS removal.

Keywords: Cardioprotection; Infarct size; Ischemic conditioning; Mitochondrion; Myocardial infarction; Myocardial ischemia; Reperfusion.

Graphical abstract

Fig. 1

Time course of myocardial salvage. With short duration of ischemia, reperfusion alone salvages almost all myocardium at risk. With a long duration of ischemia and late reperfusion, there remains little myocardium to be salvaged. There is a narrow time window for cardioprotective interventions beyond reperfusion (encircled). The time scale as such is influenced by a number of intervening variables (inserted box). IRA, infarct-related artery. From [25].

Fig. 2

Inverse relationship between infarct size and ischemic myocardial blood flow. Open symbols depict infarct sizes in dog hearts following 24 (circles) or 48 h (squares) of coronary artery branch occlusion. Closed symbols are from published studies by Reimer and Jennings with 96 h (triangles) or 4 h (circles) of ischemia. Infarct size varied widely because of a high degree of variability in collateral flow among the canine hearts [329].

Fig. 3

Autophagic signaling cascade. The autophagic process is predominantly regulated by the Unc-51-like kinase-1 (ULK1) and is facilitated through phosphorylation via class III PI3K and Beclin-1. Mammalian target of rapamycin (mTOR) inhibits autophagy and is itself inhibited by adenosine monophosphate kinase (AMPK) and glycogen synthase kinase 3β (GSK3β). PRAS40 = proline-rich Akt (protein kinase B) substrate of 40 kDa. ATG13 = autophagy related gene protein 13.

Fig. 4

A: Apoptosis signaling. Apoptosis can be triggered by either via either extrinsic or intrinsic pathways. The former is typically triggered by the tumor necrosis factor receptor (TNFR), CD95/Fas or the tumor necrosis factor-related apoptosis inducing ligand receptor (TRAILR). The intrinsic pathway utilizes the tumor suppressing protein, p53, that may activate pro-apoptotic Bcl-2 family members, such as Bax and Bid. B: Ferroptosis signaling. Ferroptosis in myocardial ischemia-reperfusion is a consequence of ROS generation resulting from the Fenton reaction of ferric iron and mediated through down-regulation of glutathione peroxidase-4 (GPX4) during ischemia and reperfusion. The resulting lipid peroxidation leads to severe mitochondrial damage. C: Necroptosis signaling. Extrinsic activation of necroptotic signaling pathway occurs via similar receptors that also result in apoptosis: TNFR, Fas and TRAILR. However, the key difference is the recruitment of receptor-interacting serine/threonine-protein kinase (RIPK) RIPK1 and RIPK3 and facilitated through mixed lineage kinase domain like pseudo kinase (MLKL). D: Pyroptosis signaling. Pyroptosis is a response to damage associated molecular patterns (DAMPs) and pathogen associated molecular patterns (PAMPs) that signal to lead to the formation of the inflammasome that consists of sensor proteins and through association with apoptosis-associated spec-like protein containing a caspase activation and recruitment domain (ASC) to form a focus within the cell, and then recruit and activate caspase-1, ultimately leading to cytosolic perforation by gasdermin-D (GSDMD). Interestingly, apoptosis, through caspase-3, and necroptosis, through complex II (RIPK1/FADD/caspase-8), also lead to oligomerization of GSDM.

Fig. 5

Schematic presentation of mechanisms contributing to coronary microvascular injury with ischemia/reperfusion. From [113].

Fig. 6

Schematic diagram of ischemic conditioning interventions and their impact on infarct size.

Fig. 7

Effect of remote ischemic preconditioning (RIPC) on left ventricular ejection fraction (LVEF) and mitochondrial structure in a pig model of anthracycline cardiotoxicity. Pigs received five biweekly intracoronary doxorubicin injections with RIPC of control applied before each injection. Serial cardiac magnetic resonance imaging studies were performed during 4 months follow-up. Anthracycline-induced systolic dysfunction was significantly attenuated in pigs receiving RIPC. At the ultrastructural level, mitochondria were fragmented in pigs receiving doxorubicin since subclinical stages (i.e. a time where cardiac function was still normal). At late stages (i.e. when severe cardiac dysfunction was present), mitochondria were severely damaged (electrodense and ultra-fragmented). Mitochondria from pigs undergoing RIPC before each anthracycline administration were much more preserved at every timepoint. Figure adapted from [299].

Fig. 8

The view in 1989 on pathomechanisms of I/R injury and the open questions on the role of ROS in it. From Ref. [327] with permission.

Fig. 9

Schematic diagram of ROS-generating systems in mitochondria. Big calcium-activated potassium channel (K_Ca); connexin 43 (Cx43); cyclophilin D (Cyp D); dicarboxylate carrier (DIC); mitochondrial calcium (Ca²⁺) uniporter (MCU); mitochondrial ATP-dependent potassium channel (K_ATP); mitochondrial permeability transition pore (MPTP); monoamino oxidase (MAO); p66shc, p66 Src homologous and collagen; reverse electron transport (RET); sarcolemmal monocarboxylate transporter 1 (MCT1); succinate dehydrogenase (SDH). Modified from [379].

Fig. 10

Superoxide Production by Complex I during Reperfusion Injury. (A) Operation of complex I in the forward direction oxidizing NADH in order to generate a protonmotive force (Δp) to be used to synthesize ATP. For forward electron transport to occur the difference in reduction potential between the NAD⁺/NADH and the Coenzyme Q (CoQ) pool across complex I (ΔEh) has to be sufficient to pump protons across the mitochondrial inner membrane against the Δp. As four protons are pumped for every two electrons that pass through complex I, 2ΔEh > 4Δp is the requirement for the forward reaction to occur. The red arrow in complex I indicates forward electron transport. SDH, succinate dehydrogenase. (B) RET by complex I. When the Δp is large and/or the ΔEh across complex I is low such that 4Δp > 2ΔEh, electrons can be driven backward from the CoQ pool onto the FMN of complex I, reducing the FMN which can donate a pair of electrons to NAD⁺ to form NADH, or pass one electron to oxygen to generate superoxide. The red arrow in complex I indicates RET. (C) The factors that favor RET at complex I during reperfusion. The condition to be met for RET to occur is that 4Δp > 2ΔEh. The rapid oxidation of the succinate that accumulates during ischemia favors reduction of the CoQ pool, thereby maintaining a large ΔEh. The reduced CoQ pool also favors proton pumping by complexes III and IV helping maintain a large Δp upon reperfusion. In addition, the degradation of adenine nucleotides during ischemia limits ADP availability upon reperfusion that would otherwise diminish Δp by stimulating ATP synthesis. From Ref. [367] with permission.

Fig. 11

Regulation of mitochondrial respiration and redox state by ion handling. The Krebs cycle is stimulated by Ca²⁺ that enters mitochondria via the mitochondrial Ca²⁺ uniporter (MCU) and is exported by the mitochondrial Na⁺/Ca²⁺-exchanger (NCLX). The Krebs cycle produces NADH, which donates electrons to the electron transport chain (ETC). Sequential redox reactions along the ETC establish a proton gradient (ΔpH) across the inner mitochondrial membrane (IMM) which together with the electrical potential (ΔΨ_m) constitutes the proton motive force (Δμ_H), which is harnessed by the F₁/F_o-ATP synthase (ATPase) to regenerate ATP via oxidative phosphorylation of ADP. During respiration, superoxide (O₂⁻) is generated at complexes I and III, which are dismutated to hydrogen peroxide (H₂O₂) by the Mn²⁺-dependent superoxide dismutase (MnSOD). H₂O₂ is then eliminated by glutathione peroxidase (GPX) and the thioredoxin/peroxiredoxin system (not shown). GPX is regenerated by reduced glutathione (GSH), which in turn is reduced by the glutathione reductase (GR), which uses NADPH that is produced by NADP⁺-dependent isocitrate dehydrogenase (IDP_m) and the nicotinamide nucleotide transhydrogenase (NNT). Reactive oxygen species (ROS) from NADPH oxidases (Nox) 2 and 4, but also xanthine/xanthine oxidase (XO), nitric oxide synthase (NOS) or other mitochondria (Mitos) can activate redox-sensitive ion channels in the IMM, such as the permeability transition pore (PTP), the inner mitochondrial membrane anion channel (IMAC) or the ATP-sensitive K⁺-channel (K_ATP). Opening of these channels dissipates ΔΨ_m, requiring accelerated electron flux along the ETC to maintain ΔΨ_m. This oxidizes NADH and (via reverse-mode NNT) NADPH and thereby, the antioxidative capacity, limiting H₂O₂ elimination. ROS can leave mitochondria through the IMAC or PTP and trigger ROS release from neighboring mitochondria. Depending on the concentrations and durations of ROS elevations, ROS can serve protective roles, such as ischemic preconditioning, longevity and/or protein quality control, but at higher concentrations can deteriorate excitation-contraction coupling and induce epigenetic signaling, apoptosis and/or necrosis. When ΔΨ_m (transiently or permanently) dissipates, ATP production ceases, which activates sarcolemmal K_ATP channels, making the cell inexcitable. Heterogeneities of ΔΨ_m in different cardiac myocytes within the myocardium resemble “metabolic sinks” which can induce re-entry mechanisms to induce arrhythmias. In heart failure, elevated cytosolic [Na⁺]_i accelerates mitochondrial Ca²⁺ extrusion, which can be ameliorated by inhibiting the NCLX with CGP-37157 (CGP) or lowering [Na⁺]_i by inhibitors of the Na⁺/H⁺-exchanger (NHE), of late Na⁺ current (i.e., ranolazine) and as observed for Sodium/Glucose Co-transporter 2 (SGLT2)-inhibitors via inhibiting NHE and/or late Na⁺ current. CsA, cyclosporine A. GSSG, oxidized glutathione. CaMKII, Ca²⁺/calmodulin-dependent protein kinase II; HDAC4, histone deacetylase 4; EC coupling, excitation-contraction coupling. Modified from [398].

Fig. 12

Schematic illustration of the effects of H₂S in different heart diseases and the molecular mechanisms underlying H₂S-induced cardioprotection. From Ref. [492] with permission.

Fig. 13

Roles of NADPH oxidases (NOX) in physiology and pathophysiology. Both NOX2 and NOX4, the main NOX isoforms in cardiomyocytes, can regulate intermediary metabolism in response to a variety of stresses. Several mechanisms are involved, including activation of transcription factors or in the case of NOX4 location at the MAM, targeted ROS signaling to influence mitochondrial function and cell viability. ATF4, activating transcription factor 4; NRF2, nuclear factor erythroid factor 2-related factor 2; HIF1α, hypoxia-inducible factor 1-alpha; ISR, integrated stress response. From Ref. [516] under the Creative Commons license.

Fig. 14

Crosstalk between different sources of RONS: mitochondria, NADPH oxidase (NOX), xanthine oxidase (XO) and uncoupled NOS. XO originates from oxidative stress-mediated conversion of the xanthine dehydrogenase via oxidation of critical thiols in cysteine535/992. NOS (mainly eNOS) are uncoupled upon oxidative depletion of BH4, S-glutathionylation (-SSG), adverse phosphorylation by protein kinase C (PKC) and other redox switches [535]. Mitochondrial O₂^•‾/H₂O₂ formation is triggered by oxidative stress from all ROS sources (including other damaged/activated mitochondria) via redox-activation of PKC, mitogen-activated protein kinases (MAPK), other kinase pathways and potential involvement of redox-sensitive mtK_ATP with subsequent p66^Shc, monoamine oxidase (MAO), respiratory complex activation or impairment of mitochondrial antioxidant defense [352]. Mitochondrial O₂^•‾/H₂O₂ is released to the cytosol via mitochondrial pores and channels (e.g. redox-sensitive mPTP, inner membrane anion channel (IMAC) or aquaporins) or by diffusion due to increased mitochondrial permeability under pro-inflammatory conditions. In the cytosol these species (along with released calcium) cause activation of redox-sensitive PKC and tyrosine kinases (cSrc) with subsequent activation of NOX and amplification of the cellular oxidative stress [410]. Adapted from Ref. [352] with permission.

Fig. 15

Effects and sources of exosomes. (upper part) Some of the major effects that have been reported of exosomes that are relevant to the ischemic heart, and the cell types that have been reported to be involved in the effect. See text for details. (lower part) The different potential sources of exosomes discussed in this review. Although each type of cell has certain unique characteristics, the exosomes they produce are notable for the consistent array of effects they induce. From Ref. [553] with permission.

Fig. 16

ROS and RNS are commonly thought to induce injury by oxidizing (i.e., damaging) the cellular fabric, including proteins, lipids and nucleotides. There is significant pre-clinical evidence that oxidants are damaging based on protection by antioxidants, but this has not translated though to human studies with large-scale clinical trials often showing no benefit or sometimes adverse outcomes. This could be because antioxidants scavenge oxidants that otherwise initiate protective signaling events. As electron donors, antioxidants have the potential to fuel oxidants generation as shown by the dotted line, which adds further complexity. Another consideration is that oxidized antioxidants can exert biological actions via their pro-oxidant chemistry.