A New Membrane Potential (ΔΨ)‐Independent Iron Indicator Selectively Detects Mitochondrial Chelatable Iron but Not Calcium in Living Cells

Abstract

Mitochondrial chelatable iron contributes to several injury processes, including ischemia/reperfusion injury, oxidative stress and the hepatotoxicity of acetaminophen. To measure mitochondrial chelatable iron in living cells and tissues, we synthesized a new fluorescent Fe2+ indicator, mitoferrofluor (MFeF), which has a reactive group that forms covalent adducts with mitochondrial proteins to allow retention of MFeF after subsequent mitochondrial depolarization (Hepatology 60 [Suppl], 812A). MFeF selectively accumulates in the polarized mitochondria of cultured rat hepatocytes. Here we assessed whether MFeF specifically reports mitochondrial iron independent of calcium. After loading cultured rat hepatocytes with 1 uM MFeF and 500 nM rhodamine 123 (Rh123) or 1 μM calcein‐AM for 30 min, confocal microscopy revealed red MFeF fluorescence that co‐localized with green mitochondrial Rh123. Membrane‐permeant Fe3+/8‐hydroxyquinoline(8‐HQ) (10–50 μM) progressively quenched mitochondrial MFeF fluorescence to ~80% within 15 min, but mitochondrial Rhodamine 123 was unchanged. Fe2+ specific calcein fluorescence in the cytosol was also quenched, confirming that Fe3+/8‐HQ undergoes rapid intracellular reduction to Fe2+. MFeF fluorescence increased when the lipophilic transition metal chelator pyridoxal isonicotinoyl hydrazone (PIH) was added to competitively bind mitochondrial chelatable iron. Importantly, MFeF was retained in mitochondria after mitochondrial de‐energization with CCCP (10 μM), oligomycin (10 μg/ml) and myxothiazol (10 μM), confirming mitochondrial retention of the covalently adducted MFeF. In hepatocytes loaded with MFeF, red fluorescence did not decrease after adding 2 μM of Ca2+‐mobilizing thapsigargin, whereas parallel experiments showed that Ca2+‐indicating mitochondrial Rhod‐2 fluorescence did increase, showing that MFeF is insensitive to physiologically relevant increases of mitochondrial Ca2+. In acetaminophen‐induced hepatotoxicity, progressive quenching of MFeF began at ~4 h after overdose acetaminophen (10 mM) in mouse hepatocytes, signifying increased mitochondrial chelatable Fe2+. Mitochondria then depolarized after ~10 h. Dipyridyl, a membrane‐permeable iron chelator, partially restored MFeF fluorescence. In conclusion, MFeF is a ΔΨ‐independent iron indicator that selectively detects mitochondrial chelatable Fe2+ but not Ca2+.Support or Funding InformationNIH grants: DK073336, AA021191 and T32 DK083262This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.