Assessing mitochondrial dysfunction in cells

Abstract

Assessing mitochondrial dysfunction requires definition of the dysfunction to be investigated. Usually, it is the ability of the mitochondria to make ATP appropriately in response to energy demands. Where other functions are of interest, tailored solutions are required. Dysfunction can be assessed in isolated mitochondria, in cells or in vivo, with different balances between precise experimental control and physiological relevance. There are many methods to measure mitochondrial function and dysfunction in these systems. Generally, measurements of fluxes give more information about the ability to make ATP than do measurements of intermediates and potentials. For isolated mitochondria, the best assay is mitochondrial respiratory control: the increase in respiration rate in response to ADP. For intact cells, the best assay is the equivalent measurement of cell respiratory control, which reports the rate of ATP production, the proton leak rate, the coupling efficiency, the maximum respiratory rate, the respiratory control ratio and the spare respiratory capacity. Measurements of membrane potential provide useful additional information. Measurement of both respiration and potential during appropriate titrations enables the identification of the primary sites of effectors and the distribution of control, allowing deeper quantitative analyses. Many other measurements in current use can be more problematic, as discussed in the present review.

Figure 1. The proton circuit across the mitochondrial inner membrane and an equivalent electrical circuit

(A) The primary, ATP-generating, proton circuit is shown as bold lines/boxes and the pathway of electron flow as a dashed vertical line. Secondary pathways of proton re-entry include metabolite transport [represented in the present Figure by the phosphate carrier, the transhydrogenase for the reduction of NADP⁺ to NADPH, and the endogenous and UCP (uncoupling protein)-mediated proton leaks]. Ca²⁺ cycles between the uniport and the Na⁺/Ca²⁺ antiport, and a sodium circuit links the proton and calcium circuits. (B) The equivalent electrical circuit modelling the primary proton circuit. The resistances associated with each ‘complex’ reflect the observation that membrane potential drops as the proton current drawn increases.

Figure 2. Modular kinetic analysis and modular control analysis

(A) Three modules of mitochondrial energy metabolism in isolated mitochondria or in intact cells connected by pmf. ‘Substrate oxidation’ consists of all reactions involved in substrate uptake, metabolism and electron transport, and generates pmf. ‘ATP turnover’ consists of all reactions involved in phosphorylation of ADP to ATP and the export and turnover of ATP in the extramitochondrial space, and consumes pmf. ‘Proton leak’ consists of all other reactions that consume pmf. The proton current through the system, measured as respiration rate, is generated by substrate oxidation, flows through pmf and is then divided between proton leak and ATP turnover. (B) Modular kinetic analysis of the three modules in mitochondria from A549.B2 human lung carcinoma cells. The three curves with filled symbols show the kinetic responses of the rates of each of the three modules in (A) to their common intermediate, pmf. Succinate (4 mM) was present as the substrate. The response of substrate oxidation rate to pmf was determined by titration of proton leak with FCCP; the response of proton leak to pmf was determined by titration of substrate oxidation with malonate, and the response of ATP turnover plus proton leak to pmf was determined by titration of state 3 respiration with malonate (the response of ATP turnover alone can be calculated by subtraction of the proton leak curve). Filled symbols, control; open symbols, plus 25 nM myxothiazol. In the presence of myxothiazol, the kinetics of substrate oxidation were significantly different at membrane potentials above 98 mV. Redrawn from [33] © the Biochemical Society. (C) Flux control coefficients of the three modules over respiration rate in rat liver mitochondria at different rates of oxidative phosphorylation (varied by titration with hexokinase in the presence of ATP), calculated from titrations similar to those in (B). The flux control coefficients describe quantitatively how strongly each of the modules in (A) controls the respiration rate under each defined condition. Redrawn from [107] © the Biochemical Society; data from [21].

Figure 3. Cell respiratory control

(A) Representative cell respiratory control experiment. Rat cortical neurones in the presence of glucose were exposed sequentially to oligomycin (oligo), FCCP and rotenone/myxothiazol. Non-mitochondrial respiration after the final addition (e) was subtracted from the other values. a, basal respiration; b, oligomycin-insensitive (leak) respiration; c, oligomycin-sensitive (ATP turnover) respiration; d, maximal respiration in the presence of FCCP. Derived parameters: coupling efficiency (c/a); respiratory control ratio (d/b); spare respiratory capacity (d−a). (B) Mouse cortical synaptosomes respiring on glucose in the presence of varying concentrations of the pyruvate transport inhibitor α-cyanocinnamate (CCIN). Note that inhibition is only seen when respiratory control is relieved by the protonophore, indicating an effect upstream of the proton circuit. (C) Synaptosomes respiring on glucose plus pyruvate; effect of the Na⁺ channel activator veratridine (Vt). Note the oligomycin-sensitive increase in basal respiration, indicating increased ATP utilization, and the unexpected decrease in maximal respiration. (D) Effect of supplementing glucose in the medium with pyruvate. Note that respiration is increased at all stages, showing that enhanced substrate supply exerts some control over basal and oligomycin-insensitive respiration (mediated by a slight mitochondrial hyperpolarization), although the major enhancement is seen in the presence of FCCP. Rates in (B–D) are expressed as a percentage of basal respiration in the presence of glucose. Adapted from [66].

Figure 4. Mitochondrial membrane potential changes in situ

(A) TMRM fluorescence (in quench mode) from a single cerebellar granule neuron exposed to glutamate plus glycine (Glut/gly) to activate NMDA (N-methyl-D-aspartate) receptors. Where indicated, oligomycin and FCCP were added. (B) Values for Δψ_p and Δψ_m input to a spreadsheet simulating Nernst equilibria across both membranes, differential equilibration times across plasma (slow) and mitochondrial (fast) membranes and matrix quenching at a critical concentration. (C) Simulated trace generated from (B). Note that the small initial increase indicates a slight (5 mV) mitochondrial depolarization; the plasma membrane depolarization resulting from receptor activation causes a slow decrease in fluorescence; the 10 mV hyperpolarization during the oligomycin ‘null-point test’ indicates that the mitochondria were still generating ATP, and the spike with FCCP confirms that the experiment was performed in quench mode. Adapted from [80]. min/div, min per time division shown.