Remodeling pathway control of mitochondrial respiratory capacity by temperature in mouse heart: electron flow through the Q-junction in permeabilized fibers

Abstract

Fuel substrate supply and oxidative phosphorylation are key determinants of muscle performance. Numerous studies of mammalian mitochondria are carried out (i) with substrate supply that limits electron flow, and (ii) far below physiological temperature. To analyze potentially implicated biases, we studied mitochondrial respiratory control in permeabilized mouse myocardial fibers using high-resolution respirometry. The capacity of oxidative phosphorylation at 37 °C was nearly two-fold higher when fueled by physiological substrate combinations reconstituting tricarboxylic acid cycle function, compared with electron flow measured separately through NADH to Complex I or succinate to Complex II. The relative contribution of the NADH pathway to physiological respiratory capacity increased with a decrease in temperature from 37 to 25 °C. The apparent excess capacity of cytochrome c oxidase above physiological pathway capacity increased sharply under hypothermia due to limitation by NADH-linked dehydrogenases. This mechanism of mitochondrial respiratory control in the hypothermic mammalian heart is comparable to the pattern in ectotherm species, pointing towards NADH-linked mt-matrix dehydrogenases and the phosphorylation system rather than electron transfer complexes as the primary drivers of thermal sensitivity at low temperature. Delineating the link between stress and remodeling of oxidative phosphorylation is important for understanding metabolic perturbations in disease evolution and cardiac protection.

Figure 1

Mitochondrial pathways, substrate-uncoupler-inhibitor-titration (SUIT) protocols and respiration of permeabilized cardiac fibers. (a) Schematic representation of the electron transfer system (ETS) coupled to the phosphorylation system (ATP synthase, adenylate translocator and inorganic phosphate transporter). Electron flow from pyruvate&malate (PM) or glutamate&malate (GM) converges at the N-junction (NADH-cycle). Electrons converge at the Q-junction from Complex I (CI, NADH-ubiquinone oxidoreductase), Complex II (CII, succinate-ubiquinone oxidoreductase), glycerophosphate dehydrogenase Complex (CGpDH), electron-transferring flavoprotein Complex (CETF), dihydro-orotate dehydrogenase (DhoDH), sulfide-ubiquinone oxidoreductase (SQR), and choline dehydrogenase (not shown), followed by a linear downstream segment through Complexes III (CIII, ubiquinol-cytochrome c oxidoreductase) and CIV (cytochrome c oxidase), to the final electron acceptor oxygen. CI, CIII, and CIV are proton pumps generating an electrochemical potential difference across the inner mt-membrane. Coupling of the phosphorylation system with the ETS allows the proton potential to drive phosphorylation of ADP to ATP (coupled flow). Protonophores such as FCCP uncouple the ETS from ATP production. Rotenone, malonate and antimycin A are specific inhibitors of CI, CII and CIII, respectively, and were sequentially added at saturating concentrations. (b) Coupling/pathway control diagram illustrating the two protocols starting with either PM or GM (SUIT 1 and 2), convergent electron flow at the Q-junction in the NADH&succinate (NS) pathway, and azide titrations in the NS-pathway control state or single enzyme step of CIV. As&TM, Ascorbate&TMPD. (c) SUIT 2a with azide titration in the NS-pathway control state. (d) SUIT 2b with azide titration in the CIV single enzyme step as a basis of threshold plots.

Figure 2

Respiration of permeabilized cardiac fibers (J _O2, per wet weight), and flux control ratios normalized for NS-ETS capacity. (a) Oxygen consumption measured at 37 °C in SUIT 2 protocol: GM (N-LEAK respiration; GM_L), ADP (N-OXPHOS capacity; GM_P), cytochrome c (integrity of outer mt-membrane), pyruvate (N-OXPHOS capacity; PGM_P), succinate (NS-OXPHOS capacity; PGMS_P), FCCP (NS-ETS capacity; PGMS_E), rotenone (Rot; inhibition of CI, S-ETS capacity; S_E), malonic acid and antimycin A (Mna and Ama; less than 2% residual oxygen consumption, ROX). (b,c) ROX-corrected respiration at 37 °C and 4 °C: LEAK respiration and OXPHOS capacity with PM (SUIT 1, open boxes, n = 7 and 6 at 37 and 4 °C) or GM (SUIT 2, filled boxes, n = 16 and 8 at 37 and 4 °C). Note the switch of the flux control pattern for N-OXPHOS capacity (PM_P vs. GM_P). The two SUIT protocols merge at state PGM_P, but results are shown separately (not significantly different). Flux control ratios are normalized relative to median PGMS_E of the combined protocols. Box plots indicate the minimum, 25^th percentile, median, 75^th percentile, and maximum. *Significant differences between the two SUIT protocols for the same state. For abbreviations see Fig. 1.

Figure 3

Respiration of permeabilized cardiac fibers (J _O2, per wet weight), and flux control ratios, FCR, normalized for CIV activity at different temperatures. N_L and N_P with PM (SUIT 1; n = 4–7) or GM (SUIT 2; n = 5–16); N_P with PGM, NS_P and NS_E with PGMS (both protocols combined; n = 9–23); normalized relative to median CIV_E (n = 4–16). *Significant differences between the two SUIT protocols for the same state. For box plots and abbreviations see Figs 1 and 2.

Figure 4

Effect of temperature on mitochondrial respiration. (a) Mass-specific oxygen flux (J _O2) in respiratory states PGMS_P (filled boxes) and CIV_E0 extrapolated from the threshold plots in Fig. 5 (empty boxes). (b) Respiratory flux (j _O2) relative to a simple temperature reference model: the reference flux is defined at 37 °C as 1.0 and at other temperatures as 1.0 if Q ₁₀ is constant at 2.0 (horizontal dashed line). Non-linear deviations from standard conditions (full and dotted lines) are obtained when Q ₁₀ differs from 2.0 (Q ₁₀ shown by numbers) and is constant throughout the entire temperature range. (c) j _O2 relative to standard temperature correction of flux at 37 °C (Q ₁₀ = 2.0; horizontal lines): CIV_E0 (● extrapolated from the threshold plots, Fig. 5), ETS capacity (■ PGMS_E), and LEAK respiration (▲ pooled GM_L and PM_L). Vertical bars show deviations of experimental results (means, n = 5 to 14) from the theoretical line for a Q ₁₀ of 2.0. The dotted trend line illustrates the change of temperature sensitivity for ETS, particularly at 4 °C. For box plots and abbreviations see Figs 1 and 2.

Figure 5

Azide titration and Complex IV threshold in permeabilized cardiac fibers at 40 to 4 °C. (a to e) Effect of azide titration on relative NS-pathway ETS capacity (PGMS_E; circles, dashed line: linear interpolations) and velocity of the single enzyme cytochrome c oxidase (CIV_E; squares, solid line: hyperbolic fit). (f to j) Threshold plots of relative NS-pathway flux as a function of relative inhibition of CIV at identical azide concentrations. Data up to the threshold of inhibition are shown by open symbols. The CIV_E0/NS_E flux ratio is calculated as the intercept at zero CIV inhibition of a linear regression through the data above the inflection point (closed symbols; r ² ≥ 0.99). CIV_E0/NS_E values are listed in the graphs as medians (min to max). The apparent excess capacity of CIV is j _ExCIV = CIV_E0/NS_E − 1. Triangles on Y axes show medians of relative CIV activities measured directly with ascorbate and TMPD (CIV_E/NS_E = 3.0 at 4 °C; not shown). The threshold of inhibition is at the intercept between the linear regression and the extrapolated line drawn from the control to the first inhibited flux (dotted vertical lines). Circles are means ± SD (n = 4–5).