MitoCLox: A Novel Mitochondria-Targeted Fluorescent Probe for Tracing Lipid Peroxidation

Abstract

Peroxidation of cardiolipin (CL) in the inner mitochondrial membrane plays a key role in the development of various pathologies and, probably, aging. The four fatty acid tails of CL are usually polyunsaturated, which makes CL particularly sensitive to peroxidation. Peroxidation of CL is involved in the initiation of apoptosis, as well as in some other important cellular signaling chains. However, the studies of CL peroxidation are strongly limited by the lack of methods for its tracing in living cells. We have synthesized a new mitochondria-targeted fluorescent probe sensitive to lipid peroxidation (dubbed MitoCLox), where the BODIPY fluorophore, carrying a diene-containing moiety (as in the C11-BODIPY (581/591) probe), is conjugated with a triphenylphosphonium cation (TPP+) via a long flexible linker that contains two amide bonds. The oxidation of MitoCLox could be measured either as a decrease of absorbance at 588 nm or as an increase of fluorescence in the ratiometric mode at 520/590 nm (emission). In CL-containing liposomes, MitoCLox oxidation was induced by cytochrome c and developed in parallel with cardiolipin oxidation. TPP+-based mitochondria-targeted antioxidant SkQ1, in its reduced form, inhibited oxidation of MitoCLox concurrently with the peroxidation of cardiolipin. Molecular dynamic simulations of MitoCLox in a cardiolipin-containing membrane showed affinity of positively charged MitoCLox to negatively charged CL molecules; the oxidizable diene moiety of MitoCLox resided on the same depth as the cardiolipin lipid peroxides. We suggest that MitoCLox could be used for monitoring CL oxidation in vivo and, owing to its flexible linker, also serve as a platform for producing peroxidation sensors with affinity to particular lipids.

Novel Fluorescent Mitochondria-Targeted Probe MitoCLox Reports Lipid Peroxidation in Response to Oxidative Stress In Vivo

“…The earlier findings demonstrated that CL (a) was selectively oxidized under conditions compatible to those of our experiments and (b) could be protected against oxidation by cationic mitochondria-targeted antioxidants [44][45][46]. We suggest that MitoCLox most likely preferably reports on the oxidation of CL, in agreement with results of molecular dynamic modeling that predicted a particular affinity of MitoCLox to CL [29]. Specifically, the application of MitoCLox revealed that the oxidation of lipids took place immediately after addition of hydrogen peroxide and preceded the drop in MMP.…”

“…In all tested cases, MitoCLox was reliable in reporting LPO. The data obtained are fully consistent with the behavior of MitoCLox in the model liposome system [29].…”

“…Chemicals. MitoCLox was synthesized as described in [29]. SkQ1 was synthesized, as described in [31].…”

“…Elsewhere, we have described MitoCLox, a new mitochondria-targeted fluorescence probe for tracing cardiolipin (CL) oxidation [29]. In MitoCLox, similar to the previously introduced MitoPerOx [30], a BODIPY (581/591) fluorophore is linked with a triphenylphosphonium cation (TPP + ).…”

“…It was shown that MitoCLox can report CL oxidation in liposomes but did not react with organic hydroperoxides even in the presence of ferric ions [29]. Based on results of molecular dynamic simulations, it was suggested that Mito-CLox and its derivatives, owing to the positive charge(s), could be selectively sensitive to oxidation of cardiolipin, the dominant negatively charged phospholipid in the inner mitochondrial membrane [29].…”