O(2)-sensing signal cascade: clamping of O(2) respiration, reduced ATP utilization, and inducible fumarate respiration

Abstract

These studies explore the consequences of activating the prolyl hydroxylase (PHD) O(2)-sensing pathway in spontaneously twitching neonatal cardiomyocytes. Full activation of the PHD pathway was achieved using the broad-spectrum PHD inhibitor (PHI) dimethyloxaloylglycine (DMOG). PHI treatment of cardiomyocytes caused an 85% decrease in O(2) consumption and a 300% increase in lactic acid production under basal conditions. This indicates a approximately 75% decrease in ATP turnover rate, inasmuch as the increased ATP generation by glycolysis is inadequate to compensate for the lower respiration. To determine the extent to which decreased ATP turnover underlies the suppressed O(2) consumption, mitochondria were uncoupled with 2,4-dinitrophenol. We were surprised to find that 2,4-dinitrophenol failed to increase O(2) consumption by PHI-treated cells, indicating that electron transport chain activity, rather than ATP turnover rate, limits respiration in PHI-treated cardiomyocytes. Silencing of hypoxia-inducible factor-1alpha (HIF-1alpha) expression restored the ability of uncoupled PHI-treated myocytes to increase O(2) consumption; however, basal O(2) uptake rates remained low because of the unabated suppression of cellular ATP consumption. Thus it appears that respiration is actively "clamped" through an HIF-dependent mechanism, whereas HIF-independent mechanisms are responsible for downregulation of ATP consumption. In addition, we find that PHD pathway activation enables mitochondria to utilize fumarate as a terminal electron acceptor when cytochrome c oxidase is inactive. The source of fumarate for this unusual respiration is derived from aspartate via the purine nucleotide cycle. In sum, these studies show that the O(2)-sensing pathway is sufficient to actively "clamp" O(2) consumption and independently suppress cellular ATP consumption. The PHD pathway also enables the mitochondria to utilize fumarate for respiration.

Fig. 1.

Activation of the prolyl hydroxylase (PHD) O₂ sensor reduces ATP turnover by downregulating contractility. A: O₂ consumption and lactic acid rates were used to estimate ATP turnover in twitching neonatal mouse myocytes after treatment with PHD inhibitor (PHI) dimethyloxaloylglycine (DMOG, 250 μM) for 22 h. B: 1 mM butanedione monoxime (BDM) was added 15 min before O₂ consumption and lactic acid measurements in control cells and myocytes pretreated with DMOG for 22 h. Values are means ± SE (n = 6–9). *Significantly different from corresponding control (P < 0.05, by t-test).

Fig. 2.

Active suppression of respiration by the PHD O₂ sensor. A: neonatal cardiomyocytes were pretreated with DMSO (vehicle) or the PHI DMOG (250 μM) for 22 h before O₂ consumption measurements. 2,4-Dinitrophenol (DNP, 1 μM) was injected to measure uncoupled respiration. B: octanoic acid (OA) was added to standard glucose-containing buffer 35 min before injection of DNP. Values are means ± SE (n = 9). Every PHI-treated respiration value was significantly lower than corresponding vehicle control values (P < 0.05, by ANOVA followed by Bonferroni's post hoc analysis).

Fig. 3.

Active, but not passive, suppression of respiration is dependent on hypoxia-inducible factor-1α (HIF-1α). A: coupled and uncoupled O₂ consumption measurements in neonatal myocytes in which HIF-1α expression was silenced. Myocytes were inoculated with adenovirus (adeno, 50 plaque-forming units/cell) that expresses short hairpin RNA targeting HIF-1α or control green fluorescent protein (GFP) 30 h before an additional 22 h of DMOG or vehicle treatment. O₂ consumption is normalized to basal consumption rates (100%). Values are means ± SE (n = 14). *Significant difference between DNP- and PHI-treated cells in noninfected or GFP-infected groups; #significant difference between PHI-treated cells and respective controls (P < 0.05, by ANOVA followed by Bonferroni's post hoc analysis of individual comparisons). B: nonnormalized basal O₂ uptake rates for myocytes treated as described in A. Values are means ± SE (n = 9). *Significantly different from corresponding control (P < 0.05, by t-test).

Fig. 4.

Activating the PHD pathway in quiescent adult feline myocytes suppresses uncoupled respiration but does not affect basal O₂ consumption. A: O₂ consumption rates in adult myocytes treated with DMOG (PHI) or vehicle (control) for 22 h. The uncoupler DNP was added where indicated. Some cultures where treated with 250 μM OA in addition to 1 mM glucose as metabolic substrate. Values are means ± SE (n = 8). *Significant difference between DNP and corresponding control (P < 0.05, by ANOVA followed by Bonferroni's post hoc analysis of individual comparisons). B: O₂ consumption of DMOG (PHI) and untreated (control) myocytes 10 min before (basal) and 10 min after BDM treatments. No significant differences were found between treatment groups.

Fig. 5.

Activation of PHD O₂-sensing pathway results in succinate accumulation during metabolic inhibition: evidence of fumarate respiration. A: representative HPLC of carboxylic acids extracted from mouse neonatal myocyte cultures pretreated with vehicle or DMOG for 24 h. Cultures were metabolically inhibited with 2 mM KCN and 10 mM 2-deoxyglucose (2-DG) in glucose-free medium that contained ¹³C isotopomer-labeled asparagine (1 mM) for 1 h before extraction and derivatization of carboxylic acids with 4-N,N-dimethylaminopyridine. Citric acid (CA) is also shown. Values are absorbance units at 450-nm wavelength. Inset: mass spectrum (MS) of the peak demonstrating isotopomer labeling of succinic acid (SA). B: levels of selected citric acid cycle intermediates extracted from untreated and DMOG-pretreated cultures after 1 h of metabolic inhibition. Values are means ± SE (n = 3). Values were calculated using 2-methylsuccinic acid as an internal standard. Pyruvic/oxaloacetic acid (Pyr/OAA) resolved as a single peak, whereas citric acid was not detected (ND) in DMOG-treated cultures under these conditions. *Significantly different from corresponding vehicle control (P < 0.05, by Student's t-test).

Fig. 6.

Aspartate aminotransferase inhibitors do not affect the ability of DMOG-treated cultures to maintain mitochondrial membrane potential (ΔΨ_mito) using fumarate respiration in metabolic inhibition (MI) conditions. Cultures were treated as described in Fig. 5 legend with cyanide and 2-DG for 2 h in the presence of 1 mM aminooxyacetate or 1 mM l-vinyl glycine. JC-1 was loaded during the final 15 min of MI. ΔΨ_mito is expressed as ratio of 590-nm to 530-nm fluorescence as quantified with a fluorescent plate reader after JC-1 staining (n = 6) under respective conditions. The mitochondrial uncoupler carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone was used a positive control (not shown). Values are means ± SE (n = 8). *Significantly different from corresponding control (P < 0.05, by t-test).

Fig. 7.

Activation of the PHD O₂-sensing pathway enhances myocyte NH₃ production, and purine nucleotide cycle (PNC) inhibitors reduce ability of DMOG-treated cultures to maintain ΔΨ_mito in MI conditions: evidence that enhanced PNC activity provides the fumarate necessary for anaerobic respiration. A: NH₃ production in myocytes that were pretreated with vehicle or DMOG for 22 h and subjected to 0.5 h of MI. B: cultures were treated as described in A with DMOG and subjected to 2 h of MI. The PNC inhibitors 5-aminoimidazole-4-carboxamide-1β-d-ribofuranoside (AICAR) and hadacidin were included with MI conditions in the indicated cultures. Ability of cells to maintain mitochondrial polarization in these conditions was assessed with JC-1. Values are means ± SE (n = 6). *Significantly different from corresponding non-DMOG-treated control; #significantly lower than corresponding DMOG-treated control (P < 0.05, by t-test).

Fig. 8.

Inhibition of PNC activity with hadacidin abolishes cytoprotection conferred by DMOG treatment. Myocytes were pretreated with vehicle or DMOG for 22 h, subjected to 3 h of MI (with or without hadacidin), and stained with acridine orange (fluoresces green, indicating live cells) and ethidium bromide (fluoresces orange, indicating dead cells). A: representative fluorescent images of myocytes. B: quantification of data generated from cultures treated with DMOG and subjected to 3 h of MI. CN, cyanide; 2-DG, 2-deoxyglucose. Values are means ± SE of an average of 12 randomly selected fields of view in 3 independent samples. *Significantly different from corresponding non-DMOG-treated control (P < 0.05, by t-test).