Abstract
Fully-activated Na+/H+ exchanger-1 (NHE1) generates the cardiomyocyte's largest trans-membrane extrusion of H+ ions for an equimolar influx of Na+ ions. This has the desirable effect of clearing excess intracellular acidity, but comes at a large energetic premium because the exchanged Na+ ions must ultimately be extruded by the sodium pump, a process that consumes the majority of the heart's non-contractile ATP. We hypothesize that the state of NHE1 activation depends on metabolic resources, which become limiting in periods of myocardial hypoxia. To test this functionally, NHE1 activity was measured in response to in vitro and in vivo hypoxic treatments. NHE1 flux was interrogated as a function of intracellular pH by fluorescence imaging of rodent ventricular myocytes loaded with pH-sensitive dyes BCECF or cSNARF1. Anoxic superfusates promptly inhibited NHE1, tracking the time-course of mitochondrial depolarization. Mass spectrometry of NHE1 immuno-precipitated from Langendorff-perfused anoxic hearts identified Tyr-581 dephosphorylation and Tyr-561 phosphorylation. The latter residue is part of the domain that interacts with phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane lipid that becomes depleted under metabolic inhibition. Tyr-561 phosphorylation is expected to electrostatically weaken this activatory interaction. To test if a period of hypoxia produces a persistent inhibition of NHE1, measurements under normoxia were performed on myocytes that had been incubated in 2% O2 for 4 h. NHE1 activity remained inhibited, but the effect was ablated in the presence of Dasatinib, an inhibitor of Abl/Src-family tyrosine kinases. Chronic tissue hypoxia in vivo, attained in a mouse model of anemic hypoxia, also resulted in persistently slower NHE1. In summary, we show that NHE1 responds to oxygen, a physiologically-relevant metabolic regulator, ostensibly to divert ATP for contraction. We describe a novel mechanism of NHE1 inhibition that may be relevant in cardiac disorders featuring altered oxygen metabolism, such as myocardial ischemia and reperfusion injury.
Highlights
Na+/H+ exchanger-1 (NHE1), a secondary active transporter coded by the SLC9A1 gene [1], has an established role in regulating the intracellular pH of cardiac myocytes [2, 3]
The pHi recovery in anoxia was attributable to NHE1, as it was wholly inhibited by NHE1-selective blocker cariporide (30 μM)
The results of this study demonstrate a functional coupling between oxygen tension and the ionic flux carried by NHE1 in the heart
Summary
Na+/H+ exchanger-1 (NHE1), a secondary active transporter coded by the SLC9A1 gene [1], has an established role in regulating the intracellular pH (pHi) of cardiac myocytes [2, 3]. Whilst the NHE1 transport cycle does not hydrolyze ATP directly, it incurs an energetic cost because the imported Na+ ions must eventually be extruded by the Na+/K+ ATPase (“sodium pump”). Calculations performed for the beating heart suggest that two-thirds of cardiac ATP turnover is attributable to cross-bridge cycling [5, 6], and much of the remaining consumption is linked to primary active transport, of which the sodium pump accounts for half of the demand [7]. In guinea-pig and rat beating hearts, the time-averaged flux carried by the sodium pump is 2–3 mM/min [8], which balances sarcolemmal Na+ influx carried by a number of pathways, including NHE1. Maximal activation of NHE1 at low pHi can evoke fluxes as large as >20 mM/min, which could deplete cellular ATP, if the mitochondrial supply pipeline is restricted
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