Regulation of oxidative phosphorylation complex activity: effects of tissue-specific metabolic stress within an allometric series and acute changes in workload

Abstract

The concentration of mitochondrial oxidative phosphorylation complexes (MOPCs) is tuned to the maximum energy conversion requirements of a given tissue; however, whether the activity of MOPCs is altered in response to acute changes in energy conversion demand is unclear. We hypothesized that MOPCs activity is modulated by tissue metabolic stress to maintain the energy-metabolism homeostasis. Metabolic stress was defined as the observed energy conversion rate/maximum energy conversion rate. The maximum energy conversion rate was assumed to be proportional to the concentration of MOPCs, as determined with optical spectroscopy, gel electrophoresis, and mass spectrometry. The resting metabolic stress of the heart and liver across the range of resting metabolic rates within an allometric series (mouse, rabbit, and pig) was determined from MPOCs content and literature respiratory values. The metabolic stress of the liver was high and nearly constant across the allometric series due to the proportional increase in MOPCs content with resting metabolic rate. In contrast, the MOPCs content of the heart was essentially constant in the allometric series, resulting in an increasing metabolic stress with decreasing animal size. The MOPCs activity was determined in native gels, with an emphasis on Complex V. Extracted MOPCs enzyme activity was proportional to resting metabolic stress across tissues and species. Complex V activity was also shown to be acutely modulated by changes in metabolic stress in the heart, in vivo and in vitro. The modulation of extracted MOPCs activity suggests that persistent posttranslational modifications (PTMs) alter MOPCs activity both chronically and acutely, specifically in the heart. Protein phosphorylation of Complex V was correlated with activity inhibition under several conditions, suggesting that protein phosphorylation may contribute to activity modulation with energy metabolic stress. These data are consistent with the notion that metabolic stress modulates MOPCs activity in the heart.

Fig. 1.

Native PAGE activity assay characterization. A: blue native (BN)-PAGE Complex IV activity assay characterization. B: BN-PAGE Complex V activity assay characterization. C: clear native (CN)-PAGE Complex IV activity assay characterization. D: CN-PAGE Complex V activity assay characterization. For A–D: 1) Coomassie-stained native gel; 2) gel activity assay at 10, 20, 40, and 60 min; 3) a plot of activity relative to protein concentration; and 4) a plot of activity as a function of time for the 25-, 50-, and 75-μg lanes. E: CN-PAGE Complex V activity assay characterization with ethanol control (E1)or 50 μM oligomycin (E2). Left, paired Coomassie-stained CN-PAGE gel; right, Complex V activity data at 10, 20, 40, and 60 min.

Fig. 2.

Transmural Complex V activity. In 3 pigs, transmural tissue samples from the outer, middle, and inner regions of the left ventricle were assayed for Complex V activity. A: raw CN-PAGE data; B: quantitative data.

Fig. 3.

Mouse (A) and pig (B) where heart proteins are labeled red and liver green. The β subunit of Complex V is shown as an inset, since its color saturated at the window level necessary to reveal a majority of proteins.

Fig. 4.

Mitochondrial oxidative phophorylation complex (MOPC) activity in the heart and liver and as an allometric series. BN-PAGE activities of Complexes I, IV, and V in the heart and liver in mouse (A) and pig (B) are shown, where the signal intensity is proportional to Complex activity. C: quantitative liver/heart enzyme activity per mole of Complex (n = 6). Complex IV and V activity as a function of allomtery are shown in the heart (D) and liver (E), with normalized activities referenced to pig (n = 4) (F). G: plot adapted from Hoppeler et al. (36) where the resting (circles) and maximum (triangles) cardiac heart rates are shown as a function of animal size.

Fig. 5.

Complex IV and Complex V activity from the pig heart and liver. Total tissue biopsies, 3 from each tissue, were taken from anesthesized pigs, processed, and analyzed by CN-PAGE. A: representative CN-PAGE images from this series, with the quantitative data presented in B. A comparison of Complex IV (C) and Complex V (D) activity from isolated mitochondria pig heart mitochondria and total tissue biopsies is also shown.

Fig. 6.

Complex V activity from hearts at different metabolic stresses. First column of panels is from the pig heart, in vivo. A: Coomassie-stained bands from Complex V. B: Complex V activity in control (predobutamine), dobutamine, and postdobutamine. C: quantitative summary of data. Second column is from the perfused rabbit heart. D: Coomassie-stained bands from Complex V. E: Complex V activity after KCl infusion, Control, and dobutamine infusion. F: quantitative summary.

Fig. 7.

Complex V subunits and in situ ³²P labeling. A: one-dimensional gel electrophoresis of isolated Complex V. B: Western blot for IF₁ protein. C: 2-dimensional gel electrophoresis of isolated Complex V stained with Coomassie blue. Zoomed in autoradiograms of ³²P-labeled heart and liver Complex V subunits resolved by 2D gels are the following: β (D) α (E), d-chain (F), and γ (G).

Fig. 8.

Effect of phosphatases and Ca²⁺ on the ³²P-labeling and activity of Complex V. A: ³²P-labeled Complex V incubated with difference phosphatases. B: effect of λ-phosphatase on pig heart and liver Complex V activity. Effect of 650 nM free Ca²⁺ on the ³²P-labeling (C) and activity of Complex V (D) in heart and liver mitochondria (Ca²⁺ incubation done in intact mitochondria, before Complex V isolation). E: quantitative summary.

Fig. 9.

Volume of mitochondria per heart muscle mass across an allometric series. These data were adapted from Hoppeler et al. (36) and reveals that mitochondrial volume is fixed at ∼21% in cardiac cells across species. Benson (13) found that 14% of the wet weight of the heart is noncollagen protein. This corresponds to 140 mg of cellular protein per gram of heart wet weight. With the use of the volume fraction of 21% and assuming a similar density of protein across different cell compartments, a value of ∼30 mg of mitochondria protein per gram of heart is calculated. Since there is ∼1 nmole cyto a/mg mitochondria protein (see results), the data from Hoppeler et al. (36) predicts a cyto a content of ∼30 nmole cyto a/gram of heart, which is in good agreement with the values presented in Table 2.