First-line Screening of OXPHOS Deficiencies Using Microscale Oxygraphy in Human Skin Fibroblasts: A Preliminary Study.

The diagnosis of mitochondrial diseases is a real challenge because of the vast clinical and genetic heterogeneity. Classically, the clinical examination and genetic analysis must be completed by several biochemical assays to confirm the diagnosis of mitochondrial disease. Here, we tested the validity of microscale XF technology in measuring oxygen consumption in human skin fibroblasts isolated from 5 pediatric patients with heterogeneous mitochondrial disorders. We first set up the protocol conditions to allow the determination of respiratory parameters including respiration associated with ATP production, proton leak, maximal respiration, and spare respiratory capacity with reproducibility and repeatability. Maximum respiration and spare capacity were the only parameters decreased in patients irrespective of the type of OXPHOS deficiency. These results were confirmed by high-resolution oxygraphy, the reference method to measure cellular respiration. Given the fact that microscale XF technology allows fast, automated and standardized measurements, we propose to use microscale oxygraphy among the first-line methods to screen OXPHOS deficiencies.

A fast method for high resolution oxymetry study of skeletal muscle mitochondrial respiratory chain complexes.

High resolution oxymetry study (HROS) of skeletal muscle usually requires 90-120 min preparative phase (dissection, permeabilization and washing). This work reports on the suitability of a rapid muscle preparation which by-passes this long preparation. For a few seconds only, muscle biopsy from pigs is submitted to gentle homogenization at 8000 rotations per minute using an ultra-dispersor apparatus. Subsequent HROS is performed using FCCP instead of ADP, compounds crossing and not plasma membrane, respectively. This simplified procedure compares favorably with classical (permeabilized fibers) HROS in terms of respiratory chain complex activities. Mitochondria from cells undergoing ultradispersion were functionally preserved as attested by relative inefficacy of added cytochrome C (not crossing intact mitochondrial outer membrane) to stimulate mitochondrial respiration. Responsiveness of respiration to ADP (in the absence of FCCP) suggested that these intact mitochondria were outside cells disrupted by ultradispersion or within cells permeated by this procedure.

Comparison of fluid balance and hemodynamic and metabolic effects of sodium lactate versus sodium bicarbonate versus 0.9% NaCl in porcine endotoxic shock: a randomized, open-label, controlled study.

BACKGROUND: Sodium lactate has been shown to improve hemodynamics and avoid fluid overload. The objective of this study was to confirm a beneficial effect on fluid balance with sodium lactate infusion and to specify whether the advantage of lactate is related to a negative chloride balance, its particular metabolism, or simply its energy load. METHODS: This was an interventional, randomized, open-label, controlled experimental study. Fifteen female "large white" pigs (2 months old) were challenged with intravenous infusion of Escherichia coli endotoxin. Three groups of five animals were randomly assigned to receive different fluids: a treatment group received sodium lactate 11.2% (SL group); an isotonic control group received 0.9% NaCl (NC group); and a hypertonic control group, with the same amount of osmoles and sodium as the SL group, received sodium bicarbonate 8.4% (SB group). In order to provide the same energy load in the three groups, control groups were perfused with an equivalent energy supply. Statistical analysis was performed with non-parametric tests and the Dunn correction for multiple comparisons at p < 0.05. RESULTS: Fluid and chloride balance, hemodynamics, oxygenation markers, and microcirculatory parameters were measured over a 5-h period. Cumulative fluid balance was significantly lower in the SL group (550 (415-800) mL; median (interquartile range)) compared to the NC group (1100 (920-1640) mL, p = 0.01) and the SB group (935 (790-1220) mL, p = 0.03). Hemodynamics, cardiac efficiency, and microcirculation were significantly enhanced in the SL group, resulting in a significant improvement in oxygen delivery (SL group 417 (305-565) mL/min/m2 at 300 min versus the NC (207 (119-272) mL/min/m2, p = 0.01) and the SB (278, (211-315) mL/min/m2, p = 0.03) groups). Oxygenation markers (arterial oxygen partial pressure (PaO2)/inspired oxygen fraction (FiO2), mixed venous oxygen saturation (SvO2), and venoarterial carbon dioxide tension difference (Pv-aCO2) were enhanced with sodium lactate infusion. Chloride balance was equivalent in both hypertonic groups and significantly reduced compared to the NC group. CONCLUSION: Sodium lactate infusion improves fluid balance and hemodynamics. The advantage of lactate does not seem to be explained by its energy load or by the induced negative chloride balance with subsequent water movements.

Inhibition of mitochondrial respiration mediates apoptosis induced by the anti-tumoral alkaloid lamellarin D.

Lamellarin D (Lam D), a marine alkaloid, exhibits a potent cytotoxicity against many different tumors. The pro-apoptotic function of Lam D has been attributed to its direct induction of mitochondrial permeability transition (MPT). This study was undertaken to explore the mechanisms through which Lam D promotes changes in mitochondrial function and as a result apoptosis. The use of eight Lam derivatives provides useful structure-apoptosis relationships. We demonstrate that Lam D and structural analogues induce apoptosis of cancer cells by acting directly on mitochondria inducing reduction of mitochondrial membrane potential, swelling and cytochrome c release. Cyclosporin A, a well-known inhibitor of MPT, completely prevents mitochondrial signs of apoptosis. The drug decreases calcium uptake by mitochondria but not by microsomes indicating that Lam D-dependent permeability is specific to mitochondrial membranes. In addition, upon Lam D exposure, a rapid decline of mitochondrial respiration and ATP synthesis occurs in isolated mitochondria as well as in intact cells. Evaluation of the site of action of Lam D on the electron-transport chain revealed that the activity of respiratory chain complex III is reduced by a half. To determine whether Lam D could induce MPT-dependent apoptosis by inhibiting mitochondrial respiration, we generated respiration-deficient cells (rho0) derived from human melanoma cells. In comparison to parental cells, rho0 cells are totally resistant to the induction of MPT-dependent apoptosis by Lam D. Our results indicate that functional mitochondria are required for Lam D-induced apoptosis. Inhibition of mitochondrial respiration is responsible for MPT-dependent apoptosis of cancer cells induced by Lam-D.

Galectin-3 modulates epithelial cell adaptation to stress at the ER-mitochondria interface.

Cellular stress response contributes to epithelial defense in adaptation to environment changes. Galectins play a pivotal role in the regulation of this response in malignant cells. However, precise underlying mechanisms are largely unknown. Here we demonstrate that Galectin-3, a pro and anti-apoptotic lectin, is required for setting up a correct cellular response to stress by orchestrating several effects. First, Galectin-3 constitutes a key post-transcriptional regulator of stress-related mRNA regulons coordinating the cell metabolism, the mTORC1 complex or the unfolded protein response (UPR). Moreover, we demonstrated the presence of Galectin-3 with mitochondria-associated membranes (MAM), and its interaction with proteins located at the ER or mitochondrial membranes. There Galectin-3 prevents the activation and recruitment at the mitochondria of the regulator of mitochondria fission DRP-1. Accordingly, loss of Galectin-3 impairs mitochondrial morphology, with more fragmented and round mitochondria, and dynamics both in normal and cancer epithelial cells in basal conditions. Importantly, Galectin-3 deficient cells also display changes of the activity of the mitochondrial respiratory chain complexes, of the mTORC1/S6RP/4EBP1 translation pathway and reactive oxygen species levels. Regarding the ER, Galectin-3 did not modify the activities of the 3 branches of the UPR in basal conditions. However, Galectin-3 favours an adaptative UPR following ER stress induction by Thapsigargin treatment. Altogether, at the ER-mitochondria interface, Galectin-3 coordinates the functioning of the ER and mitochondria, preserves the integrity of mitochondrial network and modulates the ER stress response.