Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial genome acquisition restores respiratory function and tumorigenic potential of cancer cells without mitochondrial DNA

We report that tumor cells devoid of their mitochondrial genome (mtDNA) show delayed tumor growth and that tumor formation is associated with acquisition of mtDNA from host cells. This leads to partial recovery of mitochondrial function in cells derived from primary tumors grown from cells without mtDNA and a shorter lag in tumor growth. Cell lines from circulating tumor cells showed further recovery of mitochondrial respiration and an intermediate lag to tumor growth, while cells from lung metastases exhibited full restoration of respiratory function and no lag in tumor growth. Stepwise assembly of mitochondrial respiratory supercomplexes was correlated with acquisition of respiratory function. Our findings indicate horizontal transfer of mtDNA from host cells in the tumor microenvironment to tumor cells with compromised respiratory function to re-establish respiration and tumor-initiating efficacy. These results suggest a novel pathophysiological process for overcoming mtDNA damage and support the notion of high plasticity of malignant cells

MicroRNA-126 suppresses mesothelioma malignancy by targeting IRS1 and interfering with the mitochondrial function

MiR126 was found to be frequently lost in many types of cancer, including malignant mesothelioma (MM), which represents one of the most challenging neoplastic diseases. In this study, we investigated the potential tumor suppressor function of MiR126 in MM cells. The effect of MiR126 was examined in response to oxidative stress, aberrant mitochondrial function induced by inhibition of complex I, mitochondrial DNA (mtDNA) depletion, and hypoxia

Characterisation of mesothelioma-initiating cells and their susceptibility to anti-cancer agents

Malignant mesothelioma (MM) is an aggressive type of tumour causing high mortality. One reason for this paradigm may be the existence of a subpopulation of tumour-initiating cells (TICs) that endow MM with drug resistance and recurrence. The objective of this study was to identify and characterise a TIC subpopulation in MM cells, using spheroid cultures, mesospheres, as a model of MM TICs. Mesospheres, typified by the stemness markers CD24, ABCG2 and OCT4, initiated tumours in immunodeficient mice more efficiently than adherent cells. CD24 knock-down cells lost the sphere-forming capacity and featured lower tumorigenicity. Upon serial transplantation, mesospheres were gradually more efficiently tumrigenic with increased level of stem cell markers. We also show that mesospheres feature mitochondrial and metabolic properties similar to those of normal and cancer stem cells. Finally, we show that mesothelioma-initiating cells are highly susceptible to mitochondrially targeted vitamin E succinate. This study documents that mesospheres can be used as a plausible model of mesothelioma-initiating cells and that they can be utilised in the search for efficient agents against MM

Mesospheres derived from different tumour generations show different mitochondrial features.

<p>Sphere cells representing individual generations were evaluated for respiration using the routine protocol (A) and the protocol for permeabilised cells (B) by means of the Oxygraph 2k high resolution respirometer. The symbols GM_L, GM_P, GMS_P, GMS_E and S(Rot)_E represent routine respiration, respiration via complex I, respiration via complex I+II, uncoupled respiration and uncoupled respiration via CII, respectively. (C) Mitochondrial mass was evaluated using MitoTracker Green as detailed in the Methods section. Superoxide was evaluated using the fluorescent probe MitoSOX (D), glucose uptake using the fluorescent glucose analogue 2-NBDG (E), ATP levels by a luciferase-based assay (F), ΔΨ_m using the fluorescent probe TMRM (G), lactate levels using a commercial kit (H), for SDH (I) and SQR (J) activities using enzymatic assays, and hand-held cells counter for cell size (K). (L) TEM was performed on individual generation sphere cells as detailed in the Methods section. Data in panels A-K are mean values ±S.D., and are derived from three individual experiments. The symbol ‘*’ denotes statistically significant differences with <i>p</i><0.05. Images in panel L are representative of three independent experiments. The white bar in panel K in the upper images represents 500 nm, in the lower images 200 nm.</p

CD24 supports initiation and progression of mesotheliomas.

<p>(A) Mock-transfected and CD24^- Ist-Mes-2 cells (10⁶ per animal) were grafted subcutaneously into NOD/SCID mice and tumour formation followed using USI. (B) CD24^- and mock-transfected Ist-Mes-2 cell-derived tumours were evaluated for CD24, CD47, EpCAM and Oct3/4 by western blotting using anti-actin IgG as a loading control, with panel C documenting densitometric evaluation of the blots. (D) A representative image of a mouse with CD24^- cell-derived tumour and parental cell-derived tumour is shown. (E) Representative USI images of a tumour derived from mock-transfected and CD24^- cells acquired on different days are shown, with the yellow arrows indicating the position of the tumours. Data in panel A are mean values ±S.E.M., and are derived from four animals. The symbol ‘*’ denotes statistically significant differences with <i>p</i><0.05. Images in panel B are representative of two independent experiments with the densitometric evaluation showing average data with differences less than 10%.</p

Mesospheres form tumours in serial transplantations.

<p>(A) Ist-Mes-2 sphere cells (generation 1, G1) were subcutaneously grafted into Balb-c/nude mice at 10⁶ cells per animal. When tumours reached about 2,000 mm³, the mice were sacrificed, tumours excised and malignant cells grew <i>in vitro</i> as a cell line. The adherent cells were converted into spheres (G1) and these were grafted into Balb-c/nude mice to form tumours that were used for generation 2 (G2) spheres. This procedure was repeated two more times to derive G3 and G4 spheres. The inset in panel A shows the lag to tumour appearance following cell grafting for individual sphere cell generations. Cells of individual generations were evaluated for stemness markers as shown using qPCR (B) and WB (panel C shows the blots, panel D their densitometric evaluations) and for CS activity (E). (F) Tumours derived from adherent cells and spheres of individual generations were excised, paraffin-embedded and stained for the MM marker mesothelin (upper images show staining with the exclusion of the primary IgG) and with H&E. Data shown in panel A are derived from 5 animals and are mean values ± S.E.M, data in panels B and E are mean values from three independent experiments ± S.D. Images in panel C are representative of two individual experiments and their densitometric evaluations in panel D represent mean values with differences lower than 10%. Images in panel F were obtained using one tumour for each condition (generation). The symbol ‘*’ denotes statistically significant differences with <i>p</i><0.05. Images are representative of three different tumours.</p

Effect of CD24 knock-down on mitochondrial function.

<p>Parental and CD24^- adherent and sphere Ist-Mes-2, non-permeabilised cells were evaluated for routine respiration using the Oxygraph instrument (A). Panel B shows respiration related to the maximum respiratory capacity of the cells (ETC). The symbols in panel A stand for: R, routine, L, leak, E, ETC, netR, R-L, ROX, residual respiration. Parental, mock-transfected, CD24^- adherent and sphere Ist-Mes-2 cells were evaluated for ΔΨ_m using TMRM (C), superoxide generation using MitoSOX (D), lactate production using a commercial kit (E), citrate synthase (CS) activity (F), relative SDH (G) and SQR activity (H), glucose uptake (I) and ATP level (J). Panel K documents the level of <i>PCG1α</i>mRNA in parental, mock-transfected and CD24^- adherent and sphere Ist-Mes-2 cells. Data shown are mean values ±S.D., and are derived from three individual experiments. The symbol ‘*’ denotes statistically significant differences with <i>p</i><0.05.</p