Cardiolipin fatty acid remodeling regulates mitochondrial function by modifying the electron entry point in the respiratory chain

Abstract

Modifications of cardiolipin (CL) levels or compositions are associated with changes in mitochondrial function in a wide range of pathologies. We have made the discovery that acetaminophen remodels CL fatty acids composition from tetralinoleoyl to linoleoyltrioleoyl-CL, a remodeling that is associated with decreased mitochondrial respiration. Our data show that CL remodeling causes a shift in electron entry from complex II to the β-oxidation electron transfer flavoprotein quinone oxidoreductase (ETF/QOR) pathway. These data demonstrate that electron entry in the respiratory chain is regulated by CL fatty acid composition and provide proof-of-concept that pharmacological intervention can be used to modify CL composition.

Keywords: Acetaminophen; Cardiolipin; Electron entry point; Electron transport chain; Mitochondria.

Figure 1. ApAP modifies cardiolipin composition in MPCs

L₄CL (A) and LO₃CL (B) levels and L₄CL/LO₃CL ratio (C) measured in untreated MPCs and in MPCs treated with 100 μM of ApAP for 2 hours or for indicated time (n=6) or with 200 μM of DHA for 2 hours (D) (n=3). L₄CL and LO₃CL levels are expressed as the ratio to the internal standard, M₄CL. Statistical significance was analyzed by Mann-Whitney test (error bars indicate SEM).

Figure 2. ApAP decreases mitochondrial respiration in MPCs without altering ATP production

Oxygen consumption rate (A) and ATP level (B) in untreated MPCs and in MPCs treated with 100 μM of ApAP for 2 hours (n=6). ATP production by mitochondria isolated from untreated and treated MPCs (C) (n=3). Superoxide concentration (D) measured in untreated MPCs and in MPCs treated with 100 μM of ApAP for 2 hours using fluorescent dyes (MitoSox) (n=7). Statistical significance was analyzed by Mann-Whitney test (error bars indicate SEM).

Figure 3

Electron transport chain in the mitochondria showing the 3 electron entry points at complex I (1), complex II (2) and the ETF/QOR complex (3) (modified from Hoffman and Brookes, 2009).

Figure 4. ApAP shifts electron entry point from complex II to ETF/QOR in MPCs

The different electron entry points were assessed by measuring oxygen consumption rate in MiRO5 supplemented with 2 mM of ADP and specifics substrates and inhibitors. O₂ consumption rate evaluated in untreated permeabilized MPCs and in permeabilized MPCs treated with 100 μM of ApAP for 2 hours incubated in presence of 10 mM glutamate, 5 mM malate and 2 mM malonate (A) or in presence of 10 mM succinate and 1 μM rotenone (B) or in presence of 2 mM malonate, 1 μM rotenone and 20 μM palmitoylcarnitine (C). Ratio between complex II and ETF/QOR-supported respiration measured in untreated MPCs and in MPCs treated with 100 μM of ApAP for 2 hours (D). Statistical significance was analyzed by Mann-Whitney test (n=4, error bars indicate SEM).

Figure 5. Addition of CL prevents modifications of oxygen consumption induced by ApAP

Mitochondrial L₄CL/LO₃CL ratio measured in untreated MPCs and in MPCs treated with 100 μM of ApAP for 2 hours preincubated with CL:DOPC liposomes (A). Oxygen consumption rate in permeabilized MPCs treated or not with 100 μM of ApAP for 2 hours before and after 45 minutes treatment with CL:DOPC liposomes or DOPC liposomes (n=4) (B). O₂ consumption rates evaluated in untreated MPCs and in MPCs treated with 100 μM of ApAP for 2 hours after 45 minutes of treatment with CL-liposomes in presence of 10 mM glutamate, 5 mM malate and 2 mM malonate (C) or in presence of 10 mM succinate and 1 μM rotenone (D) or in presence of 2 mM malonate, 1 μM rotenone and 20 μM palmitoylcarnitine (E) (n=5). Ratio between complex II and ETF/QOR-supported respiration measured in untreated MPCs and in MPCs treated with 100 μM of ApAP for 2 hours after 45 minutes treatment with CL:DOPC liposomes (F).Statistical significance was analyzed by Mann-Whitney test (error bars indicate SEM).