Heart failure is the most important clinical condition that is associated with the increased risk of sudden cardiac death (SCD) Citation[1] and with an increased level of reactive oxygen species (ROS) in the heart Citation[2,3]. Numerous clinical risk factors for atrial fibrillation including hypertension, pulmonary diseases, surgery and aging, are all associated with oxidative stress Citation[4].
The increased level of ROS associated with all major clinical risk factors for arrhythmia is indirect evidence that oxidative stress may be important in the genesis of both atrial and ventricular arrhythmias. Nevertheless, despite considerable evidence supporting the important role of oxidative stress in arrhythmia, clinical trials have failed to demonstrate the cardiovascular benefit of often-prescribed general antioxidants Citation[5,6]. A greater understanding of the biology of ROS production and mechanisms, by which ROS facilitate arrhythmia, is essential to designing an effective antioxidant therapy for the treatment of arrhythmias.
Cardiac sources of ROS
Reactive oxygen species such as superoxide (O2•-), hydrogen peroxide (H2O2), hydroxyl radical (•OH) and peroxynitrite (ONOO–) are highly reactive low-molecular-weight molecules containing oxygen. ROS react with proteins, lipids, DNA and other biomolecules resulting in loss of enzyme function, breaks in DNA strands, DNA mutations, lipid peroxidation, and other cellular and tissue damages.
Mitochondria, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and uncoupled nitric oxide synthase (NOS) are the major ROS production systems in the human heart. In the mitochondria, when electrons flow from nicotinamide adenine dinucleotide and flavin adenine dinucleotide to molecular oxygen via complex shuttle mechanisms, partially reduced semiquinone intermediates are produced and can react directly with oxygen (O2) to produce O2•-. Mitochondrial function differentially modulates NADPH oxidase (Nox) expression and activity; this finding highlights the existence of a functional interplay between NADPH oxidase and mitochondrial-triggered oxidative stress Citation[7,8].
Nox is an enzyme that uses NADPH to reduce molecular oxygen. NADPH oxidase is composed of a membrane-bound cytochrome b558 catalytic unit that consists of Nox, p22phox subunits, and multiple cytoplasmic accessory or signaling subunits Citation[9]. NADPH oxidase activity was first described in macrophages, and a number of NADPH oxidase isoforms (the Nox family) such as Nox1, -2 and -4 have been detected in the heart.
Uncoupled or dysfunctional NOSs are other important sources of ROS. They oxidize the terminal guanidine nitrogen atom of L-arginine by using electrons from NADPH to produce NO•. One important interaction of NO. is with superoxide, the product of which is peroxynitrite (ONOO-/ONOOH). The formation of ONOO-/ONOOH can, via the depletion of tetrahydrobiopterin, lead to NOS uncoupling that further reduces NO• production. Increased O2•- production reduces NO• signaling with detrimental effects on endothelial cell homeostasis Citation[10].
The function of cellular sources of oxidative stress is complex and there are positive feedback loops among the three major sources of cardiac ROS. An increase in ROS production by one of these sources can increase ROS production by the other sources.
Mechanisms of ROS-facilitated arrhythmia
Modification of membrane currents
In myocytes, the addition of H2O2 prolongs action potential duration and induces triggered activity via early afterdepolarization (EAD) and delayed after depolarization mechanisms Citation[11]. The perfusion of H2O2 into fibrotic rat and rabbit hearts in the Langendorff setting induces EAD and triggered activity and subsequent ventricular tachycardia (VT) and ventricular fibrillation (VF) Citation[12]. Treatment with H2O2 enhances the late Na+ current but decreases the overall Na+ current in isolated myocytes through the downregulation of SCN5A transcription Citation[13]. In an animal model of oxidative stress-mediated VT/VF, the arrhythmia was effectively suppressed by ranolazine, a late Na+ current blocker Citation[14]. These findings suggest the role of an increased late Na+ current in ROS-mediated EAD and arrhythmia. Although an increase in the late Na+ current may result in arrhythmia via an EAD mechanism, the reduction in the total Na+ current caused by ROS can reduce conduction velocity (CV) and provide a substrate for re-entry, or it can facilitate the genesis of arrhythmia via a Brugada syndrome-like effect. We have shown that the downregulation of cardiac sodium channels can be reversed by mitochondrial antioxidants Citation[15].
Reactive oxygen species also stimulate the L-type Ca2+ current, which results in abnormal intracellular calcium cycling in myocytes and facilitates EADs Citation[16]. In addition, hydroxyl radicals increase the open probability of cardiac ryanodine receptors, which control Ca2+ release from the sarcoplasmic reticulum to the cytoplasm Citation[17]. Even a brief exposure to hydroxyl radicals significantly decreases sarcoplasmic reticulum Ca2+ uptake, which leads to an increased Ca2+ level in myocytes during diastole Citation[18].
In summary, ROS-mediated reduction in the total Na+ current reduces CV and facilitates re-entry, and other ionic effects of ROS cause an increase in the inward current and intracellular Ca2+ level, facilitating EAD and delayed after depolarization.
Ca2+/CaM-dependent kinase II (CaMKII) expression is upregulated in patients with an increased susceptibility to arrhythmia Citation[19]. This mechanism may be due to the autophosphorylation of methionine residues resulting in sustained activation of CaMKII under pro-oxidant conditions Citation[20]. It appears that CaMKII mediates some of the arrhythmogenic effects of ROS such as an increase in the open probability of ryanodine receptor Citation[21], an increase in the late Na+ current Citation[22], and an increase in the L-type Ca2+ current Citation[23]. As CaMKII is a relatively indiscriminate kinase, it is possible that other mechanisms are involved in the genesis of arrhythmia by CaMKII activation.
Gap junctional remodeling
Gap junctions are low-resistance channels among myocytes that form a syncytium through which electrical signals flow. Gap junctions are formed from molecules called connexins Citation[24]. A significant reduction in, or lack of connexin43 (Cx43), results in slow CV and VT/VF Citation[25]. In an animal model of myocardial infarction, an increase in the level of phosphorylated (Tyr-416) c-Src tyrosine kinase (the active form of c-Src) results in the downregulation of Cx43 via the competition between phosphorylated c-Src and Cx43 for a binding site at zonula occludens-1, which is an intercalated disk scaffolding protein Citation[26]. Tyrosine phosphorylation of Cx43 by c-Src also impairs gap junction function Citation[27]. Treatment with H2O2 activates c-Src in cardiac myocytes Citation[28]. Some studies support the existence of a positive feedback loop between ROS and c-Src, in which an increase in the level of ROS upregulates c-Src and increases oxidative stress even further Citation[29].
We observed an elevated level of c-Src, a reduction in the total amount of Cx43, a significant reduction in gap junction conduction and VT/VF in an animal model of angiotensin II activation and oxidative stress Citation[30–32].
Antioxidant therapy strategies
In general, antioxidant therapeutic agents can be designed against three categories of targets: the sources of ROS production, ROS molecules and the key signaling molecules that mediate the arrhythmogenic effect of ROS.
The NADPH oxidase inhibitor apocynin may prevent the arrhythmogenic effect of ROS under certain conditions. The effectiveness of NOS inhibitors such as NG-nitro-L-arginine methyl ester (L-NAME) in treating arrhythmias has been tested in various experiments. In a model of occlusion–reperfusion arrhythmia in cats, it was shown that repeated injections of L-NAME decreased the incidence of occlusion arrhythmias by 40%, eliminated reperfusion-induced VT/VF and reduced the latency of occlusion arrhythmias Citation[33]. We observed that Mito-Tempo, a mitochondria-targeted antioxidant, prevented VT/VF and SCD in a RAS-activation animal model Citation[32].
When designing an antioxidant agent that inhibits sources of ROS, several important factors must be considered. Various sources of ROS can be activated under different pathologic conditions; therefore, an antioxidant against a specific source of ROS may be effective in the prevention of arrhythmia only under certain conditions. Also, because of positive feedback loops among the sources of cardiac ROS, the simultaneous targeting of several important sources of ROS may prove to be an effective therapy.
Mitochondria, which occupy more than 30% of the myocyte volume, are probably the most important source of cardiac ROS Citation[34]. Thus, mitochondria-targeted antioxidants may be the most promising antioxidant therapy against a single source of ROS. Mitochondria-targeted antioxidants such as Mito-Tempo do not decrease ROS production from mitochondria; instead, they concentrate the antioxidant in the mitochondria. This means that ROS generation is compartmentalized in the cell, and the concentration of ROS is higher in mitochondria than in other intracellular organelles or spaces.
A different therapeutic approach for the suppression of ROS involves the use of oxygen-radical scavengers such as vitamin E and vitamin C, which neutralize ROS molecules. Administration of vitamin C may reduce the incidence of postoperative atrial fibrillation Citation[35]. Nevertheless, most studies demonstrated that oxygen-radical scavengers are either not very effective in the treatment of cardiovascular disorders or produce only a modest benefit Citation[5,6]. The lack of benefit may occur because ROS react more readily with the more abundant biological substrates. Antioxidants that do not target the intracellular compartments with the highest ROS concentration may not effectively inhibit oxidative stress. In addition, these scavengers may need to neutralize many types of ROS.
A third therapeutic strategy may target signals that are downstream from ROS. For example, CaMKII inhibition may prevent many of the ROS-mediated effects on Ca2+ and Na+ channels or on the promotion of the fibrosis that causes arrhythmia. Ranolazine, a late Na+ current blocker, may inhibit some of the arrhythmogenic effects of ROS. We demonstrated that the inhibition of c-Src tyrosine kinase prevents the effects of angiotensin II and ROS on Cx43 remodeling and SCD Citation[31]. Thus, c-Src may be another example of antiarrhythmic therapeutic targets that are probably downstream from oxidative stress.
Conclusion & future directions
There is considerable evidence that oxidative stress has an important role in many cardiovascular disorders, including arrhythmia. Designing an effective antioxidant therapy requires a better understanding of the complex biology of oxidative stress.
It seems that mitochondria generate a great portion of the ROS found in myocytes. Therefore, mitochondria-targeted antioxidants may prove to be the most effective therapeutic intervention for arrhythmia. Because there are positive feedback loops among various sources of ROS, the simultaneous inhibition of a few sources of ROS generation may be required to attain better control of oxidative stress. Finally, the identification of targets downstream from ROS that cause arrhythmia, such as c-Src and CaMKII, may yield effective antiarrhythmic therapies, even though those treatments will not reduce the ROS level directly.
Financial & competing interests disclosure
Ali A Sovari is funded by the American Heart Association Midwest Affiliate Postdoctoral Fellowship Award (grant no. AHAA10POST4450037). Samuel C Dudley is funded by the National Institute of Health (grant no. RO1 RO1HL085558, PO1 HL058000). Samuel C Dudley has the following pending patents: Prevention of sudden death by modulation of Src family (application no. 13/032,629); Modulating mitochondrial reactive oxygen species to increase cardiac sodium current and mitigate sudden death (application no. CIP 12/929786); Oxidative stress markers predict atrial fibrillation (application no. 11/882,627). Ali A Sovari and Samuel C Dudley have the pending patent: Mitochondria anti-oxidants for prevention of sudden death by raising connexin43 levels (application no. 61/503,096). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
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