O2-sensing signal cascade: clamping of O2 respiration, reduced ATP utilization, and inducible fumarate respiration

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Abstract
These studies explore the consequences of activating the prolyl hydroxylase (PHD) O2-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 O2 consumption and a 300% increase in lactic acid production under basal conditions. This indicates a ∼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 O2 consumption, mitochondria were uncoupled with 2,4-dinitrophenol. We were surprised to find that 2,4-dinitrophenol failed to increase O2 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-1α (HIF-1α) expression restored the ability of uncoupled PHI-treated myocytes to increase O2 consumption; however, basal O2 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 O2-sensing pathway is sufficient to actively “clamp” O2 consumption and independently suppress cellular ATP consumption. The PHD pathway also enables the mitochondria to utilize fumarate for respiration.

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