Fig. 2: Mtfp1 is required for bioenergetic efficiency in cardiac mitochondria.

a Substrates from fatty acid oxidation (mustard) and glycolysis (purple, blue) are metabolized in the TCA cycle which fuels the electron transport chain (ETC) complexes located in the inner mitochondrial membrane by providing NADH and FADH to complexes I (purple) and II (blue), respectively. Complexes I, III and IV extrude protons from matrix into the intermembrane space creating an electrochemical gradient driving the phosphorylation of ADP at the ATP synthase (complex V). The electron flow is limited by the availability of oxygen, a terminal acceptor of electrons at the complex IV (cytochrome oxidase). Uncoupling proteins such as ANT promote a proton leak, playing an important role in regulation of membrane potential and oxidative phosphorylation efficiency. Specific inhibitors of complex I (rotenone), complex V (oligomycin), and ANT (carboxyatractyloside, CATR). Figure created with BioRender. b Oxygen consumption rates (JO₂) of cardiac mitochondria from WT (n = 5) and cMKO (n = 5) male mice at 18 weeks measuring complex I driven respiration (left) in presence of pyruvate, malate, glutamate (PGM) and ADP followed by the addition of rotenone and succinate to assess complex II driven respiration (middle) and antimycin A, carbonyl cyanide m-chlorophenyl hydrazine (CCCP), and N,N,N′,N′-Tetramethyl-p-phenylenediamine (TMPD) and ascorbate to measure complex IV driven respiration (right). Data represent mean ± SD; unpaired Student’s t-test, *p < 0.05. c Oxygen consumption rates (JO₂) of cardiac mitochondria from WT (n = 4) and cMKO (n = 5) female mice at 34 weeks measuring complex I driven respiration (left) in presence of pyruvate, malate, glutamate (PGM) and ADP followed by the addition of rotenone and succinate to assess complex II driven respiration (middle) and antimycin A, carbonyl cyanide m-chlorophenyl hydrazine (CCCP), and N,N,N′,N′-Tetramethyl-p-phenylenediamine (TMPD) and ascorbate to measure complex IV driven respiration (right). Data represent mean ± SD; unpaired Student’s t-test, *p < 0.05. d Oxygen consumption rates (JO₂) of cardiac mitochondria isolated from WT and cMKO female mice between 8–10 weeks (left). Respiration was measured in presence of pyruvate, malate, and glutamate (PGM) (state 2) followed by the addition of ADP (state 3), Oligomycin (Omy- state 4) (WT n = 9, cMKO n = 9) and Carboxyatractyloside (CATR) (WT n = 3, cMKO n = 3). Data represent mean ± SD. Multiple t-test, state 2 ***p < 0.001, state 4 *p < 0.05. Respiratory control ratios (RCR) of state 3:2 (middle: JO₂ ADP/PGM) and RCR of state 3:4 (right: JO₂ ADP/Omy). Data represent mean ± SD; 2-tailed unpaired Student’s t-test, *p < 0.05, ***p < 0.001. e Mitochondrial membrane potential (ΔΨ) measured by quenching of Rhodamine 123 (RH123) fluorescence in cardiac mitochondria isolated from WT and cMKO female mice between 8-10 weeks. ΔΨ was measured in presence of pyruvate, malate, and glutamate (PGM) (state 2) followed by the addition of ADP (state 3) and Oligomycin (state 4) (WT n = 12, cMKO n = 12) and Carboxyatractyloside (CATR) (WT n = 3, cMKO n = 3). Data represent mean ± SD; Multiple t-test, ***p < 0.001,****p < 0.0001. f Representative BN-PAGE immunoblot analysis of cardiac OXPHOS complexes isolated from WT and cMKO male mice at 8–10 weeks using the indicated antibodies, repeated on biological replicates WT (n = 4) and cMKO (n = 3) samples (see Fig. S2c, left) with similar results. g Equal amounts of protein extracted from WT (n = 5) and cMKO (n = 5) hearts of male mice between 8–10 weeks were separated by SDS–PAGE and immunoblotted with the indicated antibodies and quantified by densitometry using VINCULIN as a loading control. Data represent mean ± SD. h Oxygen consumption rates measured by high-resolution respirometry (left; JO₂) and mitochondrial membrane potential (right; ΔΨ) measured by quenching of Rhodamine 123 (RH123) fluorescence in cardiac mitochondria of WT (n = 4) and cMKO (n = 4) female mice between 8-10 week of age. JO₂ and ΔΨ were measured in presence of pyruvate, malate, and glutamate (PGM, state 2) followed by the addition of ADP (state 3) and Carboxyatractyloside (CATR) (state 4). Data represent mean ± SD; Multiple t-test, *p < 0.05, ***p < 0.001. i Respiratory control ratio (RCR) of state 3:4 (ADP/CATR) between WT (n = 4) and cMKO (n = 4) calculated from h. Data represent mean ± SD. j Oxygen consumption rates measured by high-resolution respirometry (left; JO₂) and mitochondrial membrane potential (right; ΔΨ) measured by quenching of Rhodamine 123 (RH123) fluorescence in cardiac mitochondria of WT and cMKO female mice between 8–10 week of age. JO₂ and ΔΨ were measured by adding pyruvate, malate, and glutamate (PGM, state 2) after the pre-treatment (WT n = 3, cMKO n = 3) or not of mitochondria (WT n = 8, cMKO n = 8) with Carboxyatractyloside (CATR). Data represent mean ± SD; Multiple t-test, *p < 0.05, ***p < 0.001. k Oxygen consumption rates measured by high-resolution respirometry (left; JO₂) and mitochondrial membrane potential (right; ΔΨ) measured by quenching of Rhodamine 123 (RH123) fluorescence in cardiac mitochondria of WT and cMKO female mice between 8-10 week of age. JO₂ and ΔΨ were measured by addition of malate and palmitoyl-carnitine (PC, state 2) after the pre-treatment (WT n = 4, cMKO n = 4) or not (WT n = 6, cMKO n = 6) of mitochondria with Carboxyatractyloside (CATR). Data represent mean ± SD; Multiple t-test, **p < 0.01.