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

  • Patrick T. Kang
  • Chwen-Lih Chen
  • Paul Lin
  • William M. Chilian
  • Yeong-Renn ChenEmail author
Original Contribution

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.

Keywords

Myocardial ischemia and reperfusion Mitochondria Redox dysfunction pH gradient Membrane potential 

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

Notes

Acknowledgements

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

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Supplementary material

395_2017_626_MOESM1_ESM.pdf (2.3 mb)
Supplementary material 1 (PDF 2387 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  1. 1.Department of Integrative Medical Sciences, College of MedicineNortheast Ohio Medical UniversityRootstownUSA

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