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Metformin partially blocks mitochondrial complex I and decreases superoxide production in human myocardium

Revascularization therapies are essential for treatment of acute coronary syndrome. Yet, besides rescuing the affected myocardium from excessive damage, reperfusion also inflicts additional injury, accounting for up to 50% of the final infarct size (1). The most likely culprit is excessive production of reactive oxygen species in mitochondria, primarily superoxide anion, and finding means to reduce this reperfusion-induced burst of radicals is of great clinical importance. A first-line medication for treatment of type II diabetes - metformin, besides reducing hyperglycemia, also exerts pleiotropic cardioprotective effects against the reperfusion injury (2). Although its mechanism of action is still not completely known, metformin was found to partially inhibit mitochondrial complex I (CI) and activate AMP-activated protein kinase, considered to be the key cardioprotective event. However, all studies have been performed in animal or cell culture models. Here, we investigated acute metformin effects on mitochondrial respiratory chain and superoxide production in human myocardium. Samples of left ventricular tissue (~5 mg) were obtained by needle biopsy from patients undergoing CABG surgery. They were homogenized in a respiration buffer and transferred into experimental vessels (Oroboros). Respiration was recorded as oxygen consumption rate using Clark- type electrode, in the presence of CI and CII substrates (pyruvate/malate/glutamate and succinate, respectively), and stimulated by ADP. Simultaneously, superoxide production was evaluated with Amplex Red detection system, using the appropriate fluorescent attachment. Recordings from each patient were performed in the presence of metformin (1mM) or control. Mitochondrial respiration driven by CI substrates was partially inhibited by metformin over the course of 60 minutes (Figure 1). On the other hand, no effect of metformin was observed on CII-linked respiration (51.96.6 pmolO2/s/mg vs. 46.711.8). Since some CI inhibitors increase superoxide production under conditions of ADP-stimulated electron flux, we measured if metformin produces this effect. However, no difference between control and treated samples was found. During cardiac ischemia/reperfusion, high protonmotive force and succinate accumulation occur in mitochondria, forcing the electrons to flux in reverse towards CI. These conditions produce large amounts of superoxide, also detected in vitro. The quantities of oxygen radicals were significantly diminished when heart tissue was treated with metformin (Figure 3). We demonstrate that metformin partly inhibits complex I, while not affecting complex II in human cardiac mitochondria. This inhibition of CI is associated with significantly decreased oxygen radical production during reverse electron flux, recognized as the greatest contributor to the reperfusion-induced injury (3). These effects of metformin could explain its cardioprotective effects in human heart. 1. D.M. Yellon, D.J. Hausenloy, Myocardial Reperfusion Injury, N Engl J Med. 357 (2007) 1121–1135. https://doi.org/10.1056/NEJMra071667 2. Higgins, L. ; Palee, S. ; Chattipakorn, S.C. ; Chattipakorn, N. Effects of metformin on the heart with ischaemia-reperfusion injury: Evidence of its benefits from in vitro, in vivo and clinical reports. Eur. J. Pharmacol. 2019, 858, 172489. 3. Chouchani ET, Pell VR, Gaude E, Aksentijević D, Sundier SY, Robb EL, Logan A, Nadtochiy SM, Ord ENJ, Smith AC, Eyassu F, Shirley R, Hu CH, Dare AJ, James AM, Rogatti S, Hartley RC, Eaton S, Costa ASH, Brookes PS, Davidson SM, Duchen MR, Saeb-Parsy K, Shattock MJ, Robinson AJ, Work LM, Frezza C, Krieg T, Murphy MP. Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS. Nature. 2014 Nov 20 ; 515(7527):431-435. doi: 10.1038/nature13909. Epub 2014 Nov 5. PMID: 25383517 ; PMCID: PMC4255242.

metformin ; mitochondria ; complex I ; human myocardium ; superoxide

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