Free radicals in the heart: friend or foe?

In 1973, David Hearse published his famous ‘oxygen paradox’ paper in which he noted that reoxygenation of isolated rat hearts following anoxia resulted in abrupt killing of heart cells, as demonstrated by a massive release of cardiac enzymes [1]. The source of the injury was not identified, but among the possible mechanisms listed was the release of free radicals. In mitochondria, molecular oxygen undergoes a four-electron reduction to water as the metabolic substrate is oxidized. Sequentially adding electrons to oxygen produces, in order, superoxide radical, hydrogen peroxide, hydroxyl radical and, finally, water. Radicals have unpaired electrons in their outer shell so they are potent oxidizing or reducing agents against any molecule they happen to contact, making them very toxic in biological systems. While hydrogen peroxide is technically not a radical it is still very reactive. Collectively, these three intermediate molecules are often referred to as reactive oxygen species (ROS). Following Hearse’s paper there was an explosion of studies exploring the effects of free radicals on the ischemic heart and other organs. At the time of writing, a PubMed search for the terms ‘myocardial ischemia’ and ‘free radical’ produced more than 4300 hits.

In 1994 Griendling et al. showed that ROS are not just general toxins, but they also act as second messengers to mediate the constriction of blood vessels via angiotensin II [2]. They found that angiotensin II activates NADPH oxidase, an enzyme known to cause ROS production in neutrophils, to generate ROS locally in the blood vessels. A similar process of redox signaling mediates angiotensin-induced remodeling in the heart [3]. ROS were seen to be involved in discrete signal transduction pathways, albeit still associated with pathological processes.

Most of today’s cardiologists have grown up in a culture in which free radicals have been touted as dangerous insurgents that should be eliminated whenever possible. But not all redox signaling in the heart is detrimental. For several decades scientists have been searching for an intervention to make the heart resistant to cell death during ischemia/reperfusion. Surprisingly, free radical scavengers were not very effective in that regard [4]. However, in 1986 it was noted that preconditioning the heart with a few minutes of transient ischemia causes it to become very resistant to ischemia-induced infarction for approximately 1–2 h thereafter [5]. We now know that preconditioning’s protection is triggered by the release of adenosine, bradykinin and opioid during the preconditioning ischemia. Their receptors couple to signal transduction pathways that ultimately inhibit the formation of mitochondrial permeability transition pores when the deeply ischemic heart is reperfused. These pores destroy the myocyte’s mitochondria and hence contribute to the infarction process. Apparently, a large number of cells are killed by this mechanism because infarcts in preconditioned hearts are typically less than half the size of those in nonpreconditioned hearts undergoing an identical ischemic insult.

The signaling by which these receptors trigger the protected state is most unusual. Preconditioning’s protection requires opening of ATP-sensitive potassium channels in the mitochondria (mitoKATP) [6]. Diazoxide, a mitoKATP opener, pharmacologically preconditions the heart, but its protection is completely blocked when administered along with the free radical scavenger N-acetyl cysteine [7]. This indicated that redox signaling was involved in preconditioning’s protection. We found that opening mitoKATP causes mitochondria to produce ROS, which then act as second messengers to activate protein kinase C (PKC), an enzyme needed for the protective effects of preconditioning [8]. For the first time, ROS and redox signaling were discovered to actually do something beneficial for the heart. Using rabbit myocytes we showed that the ROS came from mitochondria and that any maneuver that caused potassium ions to enter the mitochondria would cause ROS production [9].

Redox signaling is still not completely understood, but it is believed that ROS react with thiol groups on their target proteins to modulate them. The putative target in preconditioning’s redox signaling is PKC. Hearts can be preconditioned by simply infusing free radicals into the coronary arteries and that protection can be blocked by a PKC antagonist [10]. Furthermore, Korichneva et al. found that ROS can activate PKC in vitro by reacting with thiol groups associated with the zinc finger region of the molecule [11].

The source of ROS during ischemic preconditioning appears to be the electron transport chain in mitochondria. Blocking it with myxothiazol abrogates all ROS production from mitoKATP openers [12]. Recently, it was reported that isolated mitochondria can be made resistant to transition pore formation by treatment with mitoKATP openers or potassium ionophores. This protection is both PKC- and ROS-dependent, suggesting that the entire process, including ROS generation, resides within the mitochondria [13]. However, this does not explain why NADPH oxidase-deficient mice cannot be preconditioned [14].

Several investigators have reported that hydrogen peroxide [15] and superoxide [16] production begins very soon after the onset of myocardial ischemia/anoxia and their production ceases with reoxygenation as would occur in preconditioning. This would suggest that signaling occurs during the occlusion phase of preconditioning. The cell-permeant scavenger mercaptopropionyl glycine (MPG) very effectively blocks the protective effects of preconditioning [8]. MPG scavenges hydroxyl radicals and peroxynitrite but not superoxide or hydrogen peroxide [17]. We used MPG to test whether the ROS that triggers protection is made during the ischemic phase or the reperfusion phase of the preconditioning protocol [18]. Having MPG present in the tissue during the coronary occlusion had no effect, while reperfusing the tissue with MPG in the perfusate completely blocked protection. Reperfusing with hypoxic perfusate actually made infarcts bigger. Clearly, protective redox signaling occurs when oxygen is reintroduced into the heart. That explains why an occlusion followed by reperfusion is required to precondition the heart. Ischemia releases autacoids, which populate the receptors and open mitoKATP, while reperfusion supplies the oxygen substrate for ROS formation.

While preconditioning demonstrates that the heart can be made resistant to infarction, the need for pretreatment severely limits its clinical usefulness. Recently, it was found that postconditioning the heart with short cycles of coronary reperfusion/occlusion applied immediately following a prolonged ischemic insult also protects against infarction [19]. Postconditioning’s mechanism appears to be very similar to that seen in preconditioning since signal transduction pathways also prevent transition pore formation [20]. Since postconditioning is applied during the toxic free radical burst following a prolonged ischemic insult it seemed unlikely that meaningful redox signals could occur under such conditions. However, when Penna et al. included a ROS scavenger in the reperfusate, it did not protect the untreated hearts and actually abolished protection in postconditioned hearts [21]. Thus, protective redox signaling can still occur during the height of the free radical burst seen when the deeply ischemic heart is reperfused. It is not understood why the massive ROS burst that occurs at reperfusion does not protect the ischemic heart when it is reperfused without a postconditioning protocol, but perhaps under these conditions the ROS burst does not include the molecular species responsible for the signaling. Including MPG in the reperfusate at the end of a 30-min occlusion blocks protection in a heart that has been ischemically preconditioned as well [22]. As might be expected, protection from a direct activator of PKC given at reperfusion was unaffected by a ROS scavenger.

It is suspected that many patients with acute myocardial infarction actually benefit from preconditioning, be it from antecedent angina prior to thrombosis or from the many drugs, such as opioids, that they receive. In such patients, antioxidant therapy would obviously be contraindicated. Similar inadvertent preconditioning also likely occurs in experimental animals undergoing infarction. This Dr Jeckyll/Mr Hyde nature of ROS in the heart may explain the very divergent findings obtained with scavengers tested for cardioprotective abilities.

Financial & competing interests disclosure

The authors have no 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. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.