Portal de Periódicos da CAPES

Mitochondrial Metabolomics Unravel the Primordial Trigger of Ischemia/Reperfusion Injury

2015; Elsevier BV; Volume: 148; Issue: 5 Linguagem: Inglês

10.1053/j.gastro.2015.03.041

1528-0012

Michal Heger, Megan J. Reiniers, Rowan F. van Golen,

Organ Transplantation Techniques and Outcomes

Chouchani ET, Pell RP, Gaude E, et al. Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS. Nature 2014;515:431–435. Ischemia/reperfusion (I/R) injury lies at the base of several prevalent and severe diseases, including myocardial infarction, stroke, and surgery-induced tissue injury (Nat Med 17:1391–1401). These diseases impose a considerable clinical and financial burden on health care systems globally. Unfortunately, there are no clinical interventions available to reduce the extent of I/R injury that occurs during contrived procedures, such as major liver resection and organ transplantation. The absence of effective intervention modalities is related to the fact that the pathophysiology of I/R injury (eg, reactive oxygen species [ROS] production, innate immune activation [Cytokine Growth Factor Rev 2012;23:69–84]) mainly becomes manifest during reperfusion. At this stage, however, much of the irreversible damage to tissue has already occurred. Nevertheless, most research has focused on the symptomatic treatment of I/R injury, which has proven of limited translational value. A more sensible approach would therefore be to intervene at the earliest stage of I/R injury, which is only possible when the "primordial trigger" of I/R damage is clear. In the November 5 issue of Nature, Chouchani et al (Nature 2014;515:431–435) demonstrate that all hallmarks of reperfusion injury can be traced back to a conserved mitochondrial response to ischemia involving accumulation of the electron transport chain substrate succinate. Based on earlier findings that overactivation of the electron transport chain and consequent mitochondrial ROS production are manifested at the onset of I/R injury (Nat Med 2013;19:753–759), the first set of experiments aimed to quantitate the concentration of mitochondrial metabolites in various organs subjected to ischemia. Using liquid chromatography-mass spectrometry, it was shown that only the complex II substrate succinate accumulated with ischemia time. This response not only proved selective for succinate, but was also observed in all tested organs (heart, liver, brain, and kidney), thus revealing a universal metabolic fingerprint of ischemia. Next, the authors pursued the source of succinate by forcing ischemic hearts to selectively respirate on 13C-containing mitochondrial substrates ex vivo, which revealed that succinate buildup could only be achieved with 13C-aspartate supplementation, but not with conventional citric acid cycle substrates, such as glucose or fatty acids. Inasmuch as succinate cannot be formed from aspartate when the citric acid cycle functions normally, in silico metabolic flux modeling was used to explain the ex vivo findings. Computational modeling indicated that reversal of the enzyme succinate dehydrogenase (electron transport chain complex II) drives succinate formation under ischemic conditions, which in turn results from excessive fumarate production owing to ischemia-induced purine catabolism (Science 1971;171:397–400) and overactivation of the aspartate-malate shuttle (Cardiovasc Res 2011;91:382–391). The significance of these findings was corroborated in I/R-subjected primary rat cardiomyocytes, which rapidly consumed succinate stores during the first minutes of reperfusion to fuel a cytotoxic burst in mitochondrial ROS production from the electron transport chain complex I by reverse electron transport (Biochem J 2009;417:1–13). This acute surge in ROS production could be suppressed by preventing succinate accumulation with the succinate dehydrogenase inhibitor dimethyl malonate (DMM), thereby confirming the previously modeled scenarios. More important, the in vitro results were leveraged to the in vivo situation because intravenous DMM administration suppressed ROS production and reduced infarct sizes by ±50% in murine models of myocardial and cerebral I/R injury. The study therefore not only unveiled the 'primordial trigger' of I/R injury, but also provided a potential avenue for effective clinical interventions. The newly discovered biochemical basis of I/R injury will, or at least should, alter the focus of I/R research(ers) in several ways. The difference between conventional I/R research and the study by Chouchani et al (Nature 2014;515:431–435) is that the latter primarily concentrated on the ischemic phase, which is often overlooked in conventional I/R research. This underexposure mainly stems from the traditional view that ischemia-induced tissue injury and loss of function is an arbitrary consequence of anoxia that can be controlled only by limiting ischemia times or by suppressing the oxygen demand of ischemic tissue through, for example, hypothermia (Hepatology 2015;61:395–399). Targeted therapies for ischemic injury per se have therefore never reached the limelight, whereas a seemingly endless list of interventions that reduce reperfusion injury have been developed, albeit to little clinical avail. The persistent absence of clinical applicability does not relate necessarily to the general difficulty of translating experimental findings, but mainly reflects that tackling a problem at its roots should always deserve priority over symptomatic treatment. By dissociating from conventional approaches, Chouchani et al have highlighted the "I" in I/R injury by explicitly linking the origin of reperfusion injury to a coordinated metabolic response to ischemia. Although succinate accumulation was observed in various ischemic organs, several reasons make that these findings are particularly relevant for hepatic I/R injury. First, the extent of succinate accumulation was more pronounced in ischemic livers (±20-fold increase) than in other organs (≤4-fold increase), which likely stems from the disproportionally high metabolic activity of hepatocytes (Free Radic Biol Med 2011;51:700–712). Because succinate consumption directly fueled mitochondrial ROS production during reperfusion, the extent of succinate formation under hypoxic conditions suggests that the liver chiefly abides by the I/R injury mechanisms unveiled by Chouchani et al. More important, liver surgery is among the rare situations in which ischemia is used as a contrived procedure that takes place under direct supervision of a physician. In sharp contrast with other ischemic events, such as myocardial and brain ischemia, patients undergoing liver resection or transplantation can be prepared pharmacologically for I/R (eg, with DMM), with the aim of minimizing hepatic succinate buildup and consequent tissue injury. Because not all clinical manifestations of I/R injury offer an opportunity to act preemptively, targeting mitochondrial ROS production immediately downstream of succinate consumption may be the second best option to lessen I/R injury. This claim is substantiated by the large body of evidence showing that the genetic overexpression of antioxidant enzymes (Hum Gene Ther 2001;12:2167–2177) or the targeted detoxification of mitochondrial ROS (Free Radic Biol Med 2012;53:1123–1138) attenuates hepatic I/R injury. However, it should be noted that the clinical use of antioxidant-based therapies remains controversial owing to the disappointing results reported in large clinical trials (JAMA 2013;310:1178–1179). Although it might be tempting to question the relevance of oxidative stress in I/R pathologies based on these data, it should be kept in mind that there are important pharmacologic and pharmacodynamic barriers associated with the current antioxidant therapies (eg, α-tocopherol). Considering that markers of oxidative injury (eg, malondialdehyde) have been found in clinical liver I/R samples (World J Surg 2007;31:2039–2043), the disappointing efficacy of current antioxidant therapies calls for the development and use of more refined antioxidant compounds than for a drastic change of strategy (Free Radic Biol Med 2013;66:20–23). Although succinate-mediated ROS production may emerge as a leading candidate for intervention, the ROS-independent immunostimulatory role of succinate that was recently reported also deserves closer attention (Nature 2013;496:238–242; Trends Cell Biol 2014;24:313–320). The effects of succinate in this study were limited to macrophages, which in response to cell-surface Toll-like receptor 4 stimulation with lipopolysaccharide utilized glutamine to increase the intracellular succinate stores. Cytosolic succinate in turn stabilized the transcription factor hypoxia-inducible factor 1α, ultimately leading to an increase in interleukin 1β production. The high succinate levels additionally induced an increase in lysine succinylation, which is a recently discovered posttranslational protein modification that is not yet fully understood on a functional level (Nat Rev Mol Cell Biol 2014;15:536–550). Whereas these data help to interpret the earlier postulations that hypoxia-inducible factors (Gastroenterology 2010;138:1143–1154.e1–2) and interleukin 1β (Hum Gene Ther 2011;22:853–864) mediate hepatic I/R injury, they also expose several dots that still need to be connected experimentally. The most important study that needs to be performed next is to determine whether succinate accumulates in clinical settings of I/R. If yes, a need will arise for a new class of intervention modalities that aim to prevent I/R injury by targeting the mitochondrial response to ischemia (eg, the use of complex II inhibitors). For ischemic events that do not allow preventive measures, novel antioxidant therapies that neutralize the succinate-induced mitochondrial ROS burst during early reperfusion should be devised. In addition to the ROS-related consequences of succinate, it also remains to be shown whether leukocytes accumulate succinate under the sterile inflammatory conditions observed during I/R and whether protein succinylation is related functionally to I/R injury. With respect to the liver, the relevance of the succinate pathway should also be investigated in the presence of parenchymal liver disease (eg, cholestasis, steatosis), because these conditions not only exacerbate hepatic I/R injury, but do so by already derailing mitochondrial metabolism before ischemia (Antioxid Redox Signal 2012;17:1109–1123; Antioxid Redox Signal 2013;21:1119–1142). The lengthy 'bucket list' mainly reflects the broad implications of the findings reported by Chouchani et al (Nature 2014;515:431–435), which may ultimately bring forth a clinically effective treatment of I/R injury.