Mitochondrial Diagnostics: A Multiplexed Assay Platform for Comprehensive Assessment of Mitochondrial Energy Fluxes

Abstract

Chronic metabolic diseases have been linked to molecular signatures of mitochondrial dysfunction. Nonetheless, molecular remodeling of the transcriptome, proteome, and/or metabolome does not necessarily translate to functional consequences that confer physiologic phenotypes. The work here aims to bridge the gap between molecular and functional phenomics by developing and validating a multiplexed assay platform for comprehensive assessment of mitochondrial energy transduction. The diagnostic power of the platform stems from a modified version of the creatine kinase energetic clamp technique, performed in parallel with multiplexed analyses of dehydrogenase activities and ATP synthesis rates. Together, these assays provide diagnostic coverage of the mitochondrial network at a level approaching that gained by molecular "-omics" technologies. Application of the platform to a comparison of skeletal muscle versus heart mitochondria reveals mechanistic insights into tissue-specific distinctions in energy transfer efficiency. This platform opens exciting opportunities to unravel the connection between mitochondrial bioenergetics and human disease.

Keywords: bioenergetics; creatine kinase clamp; diagnostics; heart; methods; mitochondria; skeletal muscle.

Figure 1.. Mitochondrial Energy Transfer

(A–D) Simplified model (A) and detailed depictions (B–D) of mitochondrial energy transfer and the corresponding control nodes. (B) Upper left box shows several common mechanisms of metabolite exchange in the inner mitochondrial membrane. “IN” and “OUT” refer to substrate uptake into, or export from the matrix, respectively. The enzymes (color-filled boxes) activated under each substrate condition are depicted within distinct segments. Black filled, primary (1°) NAD-linked dehydrogenase; gray filled, secondary (2°) NAD-linked dehydrogenases; blue filled, FAD-linked dehydrogenases; white filled, nondehydrogenase enzymes. (C) The difference in redox potentials between NADH/NAD⁺ and H₂O/O₂ (780 mV - (-280 mV) = 1,060 mV) provides energy for pumping of protons from the matrix to the inner-membrane space during the process of electron transport. (D) depicts the increase in ATP free energy as the ATP:ADP ratio is displaced from equilibrium during the process of mitochondrial energy transfer.

Figure 2.. Assessment of Mitochondrial Respiratory Conductance via the CK Clamp

(A) Changes in ADP, ATP, creatine (Cr), and phosphocreatine (PCr) and the estimated free energy of ATP hydrolysis (ΔG_ATP) during a typical CK clamp experiment. The transition from a high to low energy charge, and thus low to high energy demand, mimics the transition from rest to exercise. (B–D) Representative JO₂ trace (B) and linear relationship between JO₂ and ΔG_ATP during a PCr titration experiment in skeletal muscle mitochondria energized with G/M (C) or G/M versus Oct/M (D). (E) Representative PCr titration experiments in skeletal muscle mitochondria demonstrating the linear range of the relationship between JO2 and ΔG_ATP.

Figure 3.. Respiratory Conductance Workflow Localizes Distinct Sites of Resistance Imposed by Specific Inhibitors

(A–H) Relationship between mitochondrial oxygen consumption (JO₂) and ATP free energy (ΔG_ATP) in heart mitochondria energized with either Pyr/Mal (A–D) or Oct/M (E–H) performed in the presence of DMSO and (A–D) UK5099 or (E–H) oligomycin and rotenone. In (E), the JO₂ rates depicted to the right of the dashed line, at ΔG_ATP = 0, represent maximal, uncoupled JO₂ induced via addition of 1 mM FCCP. (B and F) Slope of the relationships between JO₂ and ΔG_ATP depicted in (A) and (B), respectively. (C and G) Mitochondrial Δ and (D and H) NAD(P)H/NAD(P)⁺ redox state measured under identical conditions to those displayed in (A) and (E). Data are mean ± SEM. Differences between DMSO and each inhibitor (UK5099, Rot, Oligo) were analyzed by paired t tests. *p < 0.05, **p < 0.001, and ***p < 0.0001. (E, G, and H) The asterisks (*) above the data points correspond to the DMSO versus Oligo comparison, while those below correspond to DMSO versus Rot. n = 3–4/ group, where n represents 3–4 biological replicates (i.e., mitochondrial preparations).

Figure 4.. Respiratory Control in Mitochondria Isolated from Heart Compared to Skeletal Muscle

(A–C) Changes in mitochondrial (A) JO₂, (B) Δ Ψ, and (C) NAD(P)H/NAD(P)⁺ redox state as a function of ATP free energy (ΔG_ATP) and substrate: G/M, Pyr/M, Oct/M, and Succ/Rot. (D) The linear range of JO₂, located to the left of the dashed line in (A), was used to calculate the corresponding slopes. (E–G) Relationship between mitochondrial JO₂ and Δ in the presence of (E) Pyr/M, (F) Oct/M, and (G) Succ/Rot. Data are mean ± SEM. Tissue-specific differences in mitochondrial phenotypes were analyzed by Student’s t test. *p < 0.05, **p < 0.001, and ***p < 0.0001. n = 4–7 individual mice/group.

Figure 5.. Thioredoxin Reductase Inhibition Reveals Stepwise Increases in Electron Leak during Transition from High to Low Energetic Demands

(A) Relationship between mitochondrial oxygen consumption and ATP free energy (ΔG_ATP) in heart mitochondria (0.1 mg/mL) energized with Oct/M performed in the presence of DMSO or auranofin (AF) (0.1 mM). (B) Representative trace showing H₂O₂ emission from skeletal muscle mitochondria energized with Pyr/M under increasing ATP free energies performed in the absence (AF) or presence (+AF) of AF. (C) Quantification of the data depicted in (B). (D and E) Electron leak, expressed as a percentage of total flux (JH₂O₂/JO₂ = % Electron Leak), measured in isolated mitochondria prepared from (D) skeletal muscle or (E) heart. Mitochondria were energized with G/M, Pyr/M, or Oct/M and assayed under near-state 4 conditions followed by exposure to ATP free energies of 12.95, 13.95, and 14.49 kcal/mol corresponding to PCr concentrations of 1, 6, and 15 mM. (F) Difference in absolute JH₂O₂ emitting potential between skeletal muscle and heart for each experimental condition expressed as linear fold chance (FC) (Hrt/SkM). Data are mean ± SEM. Differences between groups were analyzed by Student’s t tests in (C) and (F), and one-way ANOVA with Tukey’s multiple comparison in (D) and (E). *p < 0.05, **p < 0.001, and ***p < 0.0001. n = 4–7/group, where n represents 4–7 biological replicates (i.e., mitochondrial preparations).

Figure 6.. Comprehensive Assessment of Dehydrogenase Activities and ATP Synthase

(A) Representative traces depicting NADH auto-fluorescence during JNADH assays of various dehydrogenase enzymes. Hadha activity is assessed via NADH oxidation and thus plotted on the right y axis. Mitochondrial concentrations were aΔΨ usted to obtain linearity over time: MDH (0.005 mg/mL), PDH, AKGDH, and Hadha (0.025 mg/mL), GDH (0.15 mg/mL), and BCKDH (0.3 mg/mL). (B) Representative traces depicting NADPH auto-fluorescence during JNADPH assays of various dehydrogenases. NADPH production from GDH was assessed in the absence (GDH) and presence (GDP_ADP) of ADP (2 mM). IDH2 is plotted on the right y axis. Mitochondrial concentrations were as follows: IDH2, ME (0.025 mg/mL), GDH (0.15 mg/mL). (C) JNADPH from GDH in both skeletal muscle and heart mitochondria recorded in the absence and presence of ADP (2 mM). (D) HADHA activity in isolated mitochondria prepared from skeletal muscle and heart. (E) Rates of NADH and NADPH production from all dehydrogenases studies in skeletal muscle and heart mitochondria. (F) GOT2 activity in mitochondrial lysates from skeletal muscle and heart. (G) CV activity in mitochondrial lysates prepared from skeletal muscle and heart. (H) Quantified rates of ATP synthesis (JATP) from skeletal muscle and heart mitochondria energized with G/M, Pyr/M, Oct/M, and Succ/R assayed in the context of a hexokinase ADP (0.2 mM) clamp. (I) Relationship between mitochondrial JO₂ versus ATP free energies (ΔG_ATP) in mitochondria energized with G/M in the absence and presence of 0.6 mM CaCl₂ (free Ca⁺~500 nM). (J) Calculated slopes from the data depicted in (I). (K) Mitochondria JO₂ plotted against Δ in the presence of G/M and CaCl₂. Data are mean ± SEM. Differences between tissues were analyzed by Student’s t tests. *p < 0.05, **p < 0.001, and ***p < 0.0001. n = 3–7 individual mice/group.

Figure 7.. Mitochondrial Diagnostics Tree

Diagram summarizing how this functional assay platform can be used to diagnose distinct sites of resistance within the mitochondrial energy transduction pathway. Primary respiratory phenotypes are depicted in the gray-filled ovals. Listed below each phenotype are candidate sites of regulation and potential strategies for validation, assuming Pyr/M was the substrate. Expected diagnostic results for several common respiratory inhibitors are listed.