Melatonin rescues cell respiration impaired by hypoxia/reoxygenation in aortic endothelial cells and affects the mitochondrial bioenergetics targeting the F1FO-ATPase

Abstract

Melatonin is evaluated as a potential molecular therapy to counteract mitochondrial dysfunction caused by hypoxia/reoxygenation (H/R) in aortic endothelial cells (pAECs). The mitochondrial permeability transition pore (mPTP) opening undergoes a desensitizing action coupled with a reduction of superoxide anion production in mitochondria treated with melatonin. The effect on mPTP has been attributed to the direct interaction of melatonin with the hydrophilic F₁ domain of Ca²⁺-activated F₁F_O-ATPase. Mutual exclusion analysis highlights an overlapping binding site between melatonin and the specific F₁ inhibitor NBD-Cl. The results are corroborated by melatonin inhibition of ATPase activity of the purified F₁ domain in the presence of Ca²⁺, but not in the presence of natural cofactor Mg²⁺. Moreover, the impairment of bioenergetics parameters in pAECs metabolism and the increase of oxidative stress arising by H/R injury have been rescued in cells protected by melatonin treatment.

Keywords: F(1)F(O)-ATPase; H/R injury; Melatonin; Mitochondrial dysfunction; Mitochondrial permeability transition pore; ROS production.

Graphical abstract

Fig. 1

Effect of melatonin on mitochondrial Mg²⁺- and Ca²⁺-activated F₁F_O-ATPase activities and OXPHOS. Melatonin titration curve on mitochondrial (A) Mg²⁺- and (B) Ca²⁺-activated F₁F_O-ATPase activities at increasing melatonin concentrations. Mg²⁺-activated F₁F_O-ATPase activities were evaluated at increasing concentrations of melatonin (1–25 mM) at different temperatures (20–25 - 30–37 °C) (C, D). Arrhenius diagram in the absence (E) and 10 mM melatonin (F). Tm indicates the temperature of the discontinuity (break) point of the diagram; Ea₁ red and Ea₂ blue indicate the activation energies above and below Tm, respectively. Melatonin effect on selected oxidative phosphorylation parameters: State 3, State 4o, State 3u and State3/State 4o ratio. G) Pyruvate/malate and (H) Succinate-stimulated mitochondrial oxidative phosphorylation without (CTR, ) and with 1 mM () or 10 mM () melatonin. Data represent the mean ± SD (vertical bars) from at least three independent experiments carried out on different mitochondrial preparations. Statistical analysis was performed by Dunnett's test on each group vs the control (0 mM melatonin) (D). ∗ Indicate significantly different (∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001), ns indicate no significantly different.

Fig. 2

Evaluation of the inhibition mechanism and interaction site of melatonin. A) Inhibition mechanism of melatonin on mitochondrial Ca²⁺-activated F₁F_O-ATPase. Dixon (1/V y-axis) and Cornish–Bowden (S/V y-axis) plots were obtained at 1 mM (○) or 3 mM (□) ATP. Multiple inhibitor analysis using Dixon plots to evaluate the melatonin inhibition on mitochondrial Ca²⁺-activated F₁F_O-ATPase. The activity was assessed in the absence (○) or presence of 7 μM NBD-Cl (□) (B); in the absence (○) or presence of 0.25 μM DCCD (□) (C). Effect of melatonin on the F₁ domain (D, E). The activities of F₁-ATPase activated by (D) Ca²⁺ and (E) Mg²⁺ were evaluated in the absence or presence of inhibitors: 3 μg/mL oligomycin; 7 mM or 10 mM melatonin, or 75 μM NBD-Cl. All points represent the mean ± SD (vertical bars) of three separate experiments performed on different mitochondrial preparations. Statistical analysis was performed by Dunnett's test on each group vs the control (CTR) (D, E). ∗ Indicate significantly different (∗∗∗∗P < 0.0001), ns indicate no significant difference.

Fig. 3

Evaluation of mPTP opening and ROS production on isolated mitochondria. A) Representative curves of four experiments of calcium retention capacity (CRC). CRC was monitored in response to successive pulses of 10 μM CaCl₂ (shown by arrows), in the absence (CTR-black line) and in the presence of the inhibitor 1 mM MgADP (red line) and 10 mM melatonin (green line). B) Evaluation of superoxide anion production in mitochondria energized with pyruvate plus malate as substrates for the first oxidative phosphorylation site (complex I), and with succinate (C) as substrate for the second oxidative phosphorylation site (complex II). The red line indicates the presence of 10 mM melatonin. Graphs labeled AA indicate the preliminary addition of 1 μM antimycin A to mitochondrial respiration to trigger superoxide anion stimulation. Experiments were performed in triplicate on three separate mitochondrial preparations. All points represent the mean ± SD (vertical bars) of three separate experiments performed on different mitochondrial preparations.

Fig. 4

Effect of melatonin on pAECs under H/R condition. A) Representative images of pAECs treated with 0, 1, 5, 10, or 25 mM of melatonin. B) Effect of melatonin on H/R injury model. Representative images of pAECs after H/R injury in the absence or presence of 1 mM melatonin. Each bar represents the mean ± SD of three independent experiments. Scale bar (−) 100 μm. Statistical analysis was performed by one-way ANOVA, post hoc Dunnet comparison test between each Melatonin treatment vs the control (CTR) group. ∗ Indicate significantly different (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). C) Effect of melatonin on pAECs metabolism subjected to H/R injury. Mitochondrial respiration profile was obtained from oxygen consumption rate (OCR) in normoxia, without () and with () 1 mM melatonin and in H/R, without () and with () 1 mM Melatonin under basal respiration conditions and after addition of 1.5 μM oligomycin (olig), 1.0 μM FCCP and a mixture of 0.5 μM rotenone plus antimycin A (Rot + AA). Modulator injections are shown with arrows. D) Mitochondrial parameters (basal respiration, proton leak, maximal respiration, spare respiratory capacity and ATP production) in normoxia and H/R without () or in the presence () of 1 mM melatonin. E) Evaluation of superoxide anion production in pAECs in the Normoxia or H/R injury without () or in the presence () of 1 mM melatonin. Each bar represents the mean ± SD of four (D) and three (E) independent experiments. Statistical analysis was performed by Dunnett's test on each group vs the control (0 mM melatonin). ∗ Indicate significantly different (∗P < 0.05, ∗∗P < 0.01), ns indicate no significant difference.