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. 2024 Feb 9;27(3):109157. doi: 10.1016/j.isci.2024.109157

Figure 3.

Figure 3

Deletion of CypD alters embryonic ETC, OXPHOS, and citrate synthase activity throughout development

(A) Schematic of ETC activity and electron flux (e−); c/Cytc: cytochrome c, ETC complexes: I, II, III, IV and V, ubiquinone: q.

(B–G) Assays of ETC Cx I, II, and V activity in embryonic heart homogenates or fetal and postnatal isolated heart mitochondria (left and right side of panels, respectively) were performed in a spectrophotometer and represent: NADH oxidase (B, NADH->NAD+ in Cx I), NADH:Ubiquinone dehydrogenase (DH) (C, electron flux from NADH through Cx I to q), NADH:Cytochrome c DH (D, electron flux from NADH through Cx I to Cytc), Succinate:Ubiquinone DH (E, electron flux from succinate to q via Cx II), Succinate:Cytc DH (F, electron flux from succinate to Cytc), and ATP hydrolase (G, reverse reaction of ATP synthase/Cx V).

(H) Citrate synthase activity in embryonic homogenates (left) and isolated mitochondria from fetal to adult hearts (right).

(I) Citrate synthase protein expression in homogenates from embryonic to adult hearts by immunoblotting and normalized to protein loading (au, arbitrary units).

(J–N) OXPHOS activity was measured in a Clark oxygen electrode in homogenates from hearts throughout development.

(J–L) Substrate-mediated oxygen consumption (V0, succinate for E9.5-E11.5, malate/glutamate E13.5-A), maximal oxygen consumption (Vmax, substrate +ADP), and respiratory control ratio (RCR, = Vmax/V0, a measure of coupling electron transport to ATP generation) values at each age and genotype.

(M and N) Composite RCR data (M) and representative traces (N) of oxygen consumption in WT (+/−1 μm CsA) and CypD KO E9.5 heart homogenates; arrows in N denote the addition of 10 mM succinate (Suc), 1 mM ADP, and 0.1 mM atractyloside (ATR). V0 was measured after adding succinate, Vmax was measured after adding ADP, and rates of oxygen consumption are denoted by yellow bars. Data is presented as Mean ± SEM and analyzed by one-way ANOVA with Tukey’s or Sidak’s multiple comparison test to compare data between WT and CypD KO hearts (red stars) at each age and also between successive ages of the same genotype (WT-underlined black stars, CypD KO-underlined gray stars). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. N: (B–H) 3–12 (I) 3, and (J-M) 6–15 samples per age/genotype.