Abstract
Background: Mitochondria generate energy in form of ATP in mature eukaryotic cells. The components of the electron transport chain (ETC) are expressed at embryonic (E) day 9.5 in mouse hearts, but ETC activity and oxidative phosphorylation (OXPHOS) are uncoupled, because the permeability transition pore (PTP) is open. In mouse hearts the mPTP closes around E11.5. Opening and closing of the PTP is regulated by cyclophilin D (CypD), but the regulatory mechanism are not understood. Hypothesis: Maturation of ETC activity is regulated by acetylated CypD. Methods: Cardiac tissue homogenates or isolated heart mitochondria from mice, ranging in age from E 9.5 to adult, were used to measure oxygen consumption and the calcium retention capacity. The expression of proteins of the ETC and their assembly into supercomplexes was followed by denaturing and native electrophoresis. The expression of CypD and its acetylation status were assessed by western blotting and densitometry. The enzymatic activity of the ETC complexes and CypD was measured using spectrophotometry. Results: In the heart of mouse embryos at E 9.5 mitochondrial ETC activity and OXPHOS are not coupled (respiratory control ratio (RCR) 1.48 ± 0.17, n=9). Addition of 1 μM cyclosporin A, an inhibitor of CypD and the PTP acutely increases the RCR to 3.69 ± 0.59 (n=5). At E13.5, the end of the embryonic period in the mouse, OXPHOS is coupled and not significantly different from the adult heart. The enzymatic activity of the ETC complexes I, II, III and V increases significantly from E9.5 to the adult heart and the assembly of mitochondrial supercomplexes begins at about E13.5. The total expression of CypD increases as the mouse heart matures, but the enzymatic activity of CypD decreases. In addition, the ratio of acetylated CypD to the total expressed CypD decreased from 1.1 ± 0.16 (n=3) at E9.5 to 0.54 ± 0.06 (n=3) in the adult heart. Conclusion: The activity of acetylated CypD regulates maturation of mitochondrial ETC activity in the developing mouse heart.
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