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Impairment of pH gradient and membrane potential mediates redox dysfunction in the mitochondria of the post-ischemic heart

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An Erratum to this article was published on 27 June 2017

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Abstract

The mitochondrial electrochemical gradient (Δp), which comprises the pH gradient (ΔpH) and the membrane potential (ΔΨ), is crucial in controlling energy transduction. During myocardial ischemia and reperfusion (IR), mitochondrial dysfunction mediates superoxide (·O2 −) and H2O2 overproduction leading to oxidative injury. However, the role of ΔpH and ΔΨ in post-ischemic injury is not fully established. Here we studied mitochondria from the risk region of rat hearts subjected to 30 min of coronary ligation and 24 h of reperfusion in vivo. In the presence of glutamate, malate and ADP, normal mitochondria (mitochondria of non-ischemic region, NR) exhibited a heightened state 3 oxygen consumption rate (OCR) and reduced ·O2 − and H2O2 production when compared to state 2 conditions. Oligomycin (increases ΔpH by inhibiting ATP synthase) increased ·O2 − and H2O2 production in normal mitochondria, but not significantly in the mitochondria of the risk region (IR mitochondria or post-ischemic mitochondria), indicating that normal mitochondrial ·O2 − and H2O2 generation is dependent on ΔpH and that IR impaired the ΔpH of normal mitochondria. Conversely, nigericin (dissipates ΔpH) dramatically reduced ·O2 − and H2O2 generation by normal mitochondria under state 4 conditions, and this nigericin quenching effect was less pronounced in IR mitochondria. Nigericin also increased mitochondrial OCR, and predisposed normal mitochondria to a more oxidized redox status assessed by increased oxidation of cyclic hydroxylamine, CM-H. IR mitochondria, although more oxidized than normal mitochondria, were not responsive to nigericin-induced CM-H oxidation, which is consistent with the result that IR induced ΔpH impairment in normal mitochondria. Valinomycin, a K+ ionophore used to dissipate ΔΨ, drastically diminished ·O2 − and H2O2 generation by normal mitochondria, but less pronounced effect on IR mitochondria under state 4 conditions, indicating that ΔΨ also contributed to ·O2 − generation by normal mitochondria and that IR mediated ΔΨ impairment. However, there was no significant difference in valinomycin-induced CM-H oxidation between normal and IR mitochondria. In conclusion, under normal conditions the proton backpressure imposed by ΔpH restricts electron flow, controls a limited amount of ·O2 − generation, and results in a more reduced myocardium; however, IR causes ΔpH impairment and prompts a more oxidized myocardium.

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Change history

  • 27 June 2017

    An erratum to this article has been published.

Abbreviations

IR:

Myocardial ischemia and reperfusion

Δp :

Electrochemical gradient

ΔpH:

pH gradient or proton gradient

ΔΨ:

Membrane potential

·O2 − :

Superoxide anion radical

EPR:

Electron paramagnetic resonance

OCR:

Oxygen consumption rate

ETC:

Electron transport chain

ETA:

Electron transfer activity

ROS:

Reactive oxygen species

RCR:

Respiratory control ratio

TMRM:

Tetramethylrhodamine methyl ester CM-H, 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine

FCCP:

Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone

Complex I:

NADH-ubiquinone reductase

Complex II:

Succinate-ubiquinone reductase

Complex III:

Ubiquinol-cytochrome c reductase

Complex IV:

Cytochrome c oxidase

DCPIP:

Dichlorophenyl indophenol

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

DMPO:

5,5-Dimethyl-1-pyrroline N-oxide

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Acknowledgements

This work was supported by National Institutes of Health Grant HL83237 (to Y.-R C) and HL115114 (to WMC).

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Authors and Affiliations

  1. Department of Integrative Medical Sciences, College of Medicine, Northeast Ohio Medical University, 4209 State Route 44, PO Box 95, Rootstown, OH, 44272, USA

    Patrick T. Kang, Chwen-Lih Chen, Paul Lin, William M. Chilian & Yeong-Renn Chen

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Correspondence to Yeong-Renn Chen.

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Additional information

The original version of this article was revised: Figures 7 and 9 were interchanged.

On page 9 the word “oligomycin” was changed to “valinomycin” in the sentence “The effects of nigericin and valinomycin on the Δψ of normal mitochondria were highlighted in Fig. 7c, d.”

An erratum to this article is available at https://doi.org/10.1007/s00395-017-0632-3.

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Kang, P.T., Chen, CL., Lin, P. et al. Impairment of pH gradient and membrane potential mediates redox dysfunction in the mitochondria of the post-ischemic heart. Basic Res Cardiol 112, 36 (2017). https://doi.org/10.1007/s00395-017-0626-1

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