Translocation of iron from lysosomes to mitochondria during ischemia predisposes to injury after reperfusion in rat hepatocytes

Abstract

The mitochondrial permeability transition (MPT) initiated by reactive oxygen species (ROS) plays an essential role in ischemia-reperfusion (IR) injury. Iron is a critical catalyst for ROS formation, and intracellular chelatable iron promotes oxidative injury-induced and MPT-dependent cell death in hepatocytes. Accordingly, our aim was to investigate the role of chelatable iron in IR-induced ROS generation, MPT formation, and cell death in primary rat hepatocytes. To simulate IR, overnight-cultured hepatocytes were incubated anoxically at pH 6.2 for 4h and reoxygenated at pH 7.4. Chelatable Fe(2+), ROS, and mitochondrial membrane potential were monitored by confocal fluorescence microscopy of calcein, chloromethyldichlorofluorescein, and tetramethylrhodamine methyl ester, respectively. Cell killing was assessed by propidium iodide fluorimetry. Ischemia caused progressive quenching of cytosolic calcein by more than 90%, signifying increased chelatable Fe(2+). Desferal and starch-desferal 1h before ischemia suppressed calcein quenching. Ischemia also induced quenching and dequenching of calcein loaded into mitochondria and lysosomes, respectively. Desferal, starch-desferal, and the inhibitor of the mitochondrial Ca(2+) uniporter (MCU), Ru360, suppressed mitochondrial calcein quenching during ischemia. Desferal, starch-desferal, and Ru360 before ischemia also decreased mitochondrial ROS formation, MPT opening, and cell killing after reperfusion. These results indicate that lysosomes release chelatable Fe(2+) during ischemia, which is taken up into mitochondria by MCU. Increased mitochondrial iron then predisposes to ROS-dependent MPT opening and cell killing after reperfusion.

Keywords: Cell death; Free radicals; IR; Iron; Ischemia; KRH; Krebs–Ringer–Hepes; LTR; LysoTracker Red; Lysosome; MCU; MPT; Mitochondria; OH(•); PI; ROS; RhDex; TMRM; V-ATPase; chloromethyldichlorofluorescein; chloromethyldihydrodichlorofluorescein diacetate; cmDCF; cmH(2)DCF-DA; hydroxyl radical; ischemia–reperfusion; mitochondrial Ca(2+) uniporter; mitochondrial membrane potential; mitochondrial permeability transition; propidium iodide; reactive oxygen species; rhodamine–dextran; tetramethylrhodamine methyl ester; vacuolar proton-pumping ATPase; ΔΨ.

Fig. 1

Inhibition of ischemia-induced quenching of cytosolic calcein by desferal and starch–desferal. Hepatocytes were coloaded with calcein-AM, TMRM, and PI and subjected to ischemia. In (A), confocal images were collected after 3–240 min. In (B) and (C), hepatocytes were preincubated with desferal or starch–desferal 1 h before ischemia. In (D) and (E), average calcein and mitochondrial TMRM fluorescence of individual hepatocytes after background subtraction was quantified at 3–240 min as the percentage of fluorescence after 3 min. *p < 0.01 vs other groups (n = 3–5 hepatocytes from at least three independent experiments per group).

Fig. 2

Prevention of ischemia-induced quenching of mitochondrial calcein by desferal and starch–desferal. RhDex-labeled hepatocytes were loaded with calcein-AM by cold ester loading/warm incubation and subjected to ischemia. Hepatocytes were preincubated with (A) vehicle, (B) desferal, or (C) starch–desferal. In (D) and (E), average mitochondrial and lysosomal calcein fluorescence was determined. *p < 0.01 vs other groups (n = 3–5 hepatocytes from at least three independent experiments per group).

Fig. 3

Suppression of mitochondrial calcein quenching during ischemia by Ru360. In (A) and (B), hepatocytes were loaded with fluorophores as described for Figs. 1 and 2. Cells were treated with Ru360 1 h before ischemia.

Fig. 4

Inhibition of mitochondrial ROS generation and cell death after reperfusion by desferal, starch–desferal, and Ru360. Hepatocytes were subjected to IR as described for Fig. 1 except in the absence of calcein. CmH₂-DCF-DA was added 30 min before and continuously after reperfusion. Hepatocytes were pretreated with (A) vehicle, (B) desferal, (C) starch–desferal, or (D) Ru360. Arrows, PI-labeled nuclei; 3 ×, fluorescence intensity increased by factor of 3.

Fig. 5

Protection by desferal, starch–desferal, and Ru360 against reperfusion injury. Hepatocytes were loaded with fluorophores and subjected to ischemia. During the last 15 min of ischemia, cells were reloaded with calcein-AM (0.5 μM). Images were collected after reperfusion (A) without or (B–D) with pretreatment with (B) desferal, (C) starch–desferal, and (D) Ru360. Arrows, PI-labeled nuclei.

Fig. 6

Protection by desferal, starch–desferal, and Ru360 against reactive oxygen species generation and cell killing after ischemia–reperfusion. Hepatocytes were subjected to IR. Some cells were pretreated with desferal, starch–desferal, or Ru360. In (A), cmH₂DCF-DA (10 μM) was added 30 min before and continuously after reperfusion, and cmDCF formation was measured with a plate reader. In (B), hepatocytes were incubated with PI (30 μM), and cell killing was measured with a plate reader. *p < 0.01 vs other groups.

Fig. 7

Two-hit model of vulnerability to ischemia–reperfusion injury. During ischemia, ATP depletion inhibits V-ATPase of lysosomes/endosomes. Consequently, lysosomal pH increases, leading to Fe²⁺ release into the cytosol. Mitochondria then accumulate some this iron via MCU. Translocation of iron from lysosomes into mitochondria represents a first hit predisposing to injury. The second hit occurs after reperfusion when mitochondrial respiration leads to formation of H₂O₂, which reacts with Fe²⁺ to form toxic OH^•, leading to the MPT, bioenergetic failure, and cell death. In liver, fructose supports glycolytic ATP formation to sustain V-ATPase activity during anoxia. Desferal (Desf) and starch–desferal (St-Desf) chelate iron and prevent OH^• formation, with starch–desferal acting specifically within lysosomes. Ru360 blocks mitochondrial iron uptake via MCU. Thus, Ru360 suppresses iron-catalyzed Fenton chemistry in the mitochondrial matrix but not the cytosol.