High-Resolution Respirometry: OXPHOS Protocols for Human Cells and Permeabilized Fibers from Small Biopsies of Human Muscle

Part of the Methods in Molecular Biology book series (MIMB, volume 810)

Abstract

Protocols for high-resolution respirometry (HRR) of intact cells, permeabilized cells, and permeabilized muscle fibers offer sensitive diagnostic tests of integrated mitochondrial function using standard cell culture techniques and small needle biopsies of muscle. Multiple substrate–uncoupler–inhibitor titration (SUIT) protocols for analysis of oxidative phosphorylation improve our understanding of mitochondrial respiratory control and the pathophysiology of mitochondrial diseases. Respiratory states are defined in functional terms to account for the network of metabolic interactions in complex SUIT protocols with stepwise modulation of coupling and substrate control. A regulated degree of intrinsic uncoupling is a hallmark of oxidative phosphorylation, whereas pathological and toxicological dyscoupling is evaluated as a mitochondrial defect. The noncoupled state of maximum respiration is experimentally induced by titration of established uncouplers (FCCP, DNP) to collapse the proton gradient across the mitochondrial inner membrane and measure the capacity of the electron transfer system (ETS, open-circuit operation of respiration). Intrinsic uncoupling and dyscoupling are evaluated as the flux control ratio between nonphosphorylating LEAK respiration (electron flow coupled to proton pumping to compensate for proton leaks) and ETS capacity. If OXPHOS capacity (maximally ADP-stimulated oxygen flux) is less than ETS capacity, the phosphorylation system contributes to flux control. Physiological Complex I + II substrate combinations are required to reconstitute TCA cycle function. This supports maximum ETS and OXPHOS capacities, due to the additive effect of multiple electron supply pathways converging at the Q-junction. Substrate control with electron entry separately through Complex I (pyruvate + malate or glutamate + malate) or Complex II (succinate + rotenone) restricts ETS capacity and artificially enhances flux control upstream of the Q-cycle, providing diagnostic information on specific branches of the ETS. Oxygen levels are maintained above air saturation in protocols with permeabilized muscle fibers to avoid experimental oxygen limitation of respiration. Standardized two-point calibration of the polarographic oxygen sensor (static sensor calibration), calibration of the sensor response time (dynamic sensor calibration), and evaluation of instrumental background oxygen flux (systemic flux compensation) provide the unique experimental basis for high accuracy of quantitative results and quality control in HRR.

Key words

Substrate–uncoupler–inhibitor titration Human vastus lateralis Needle biopsy HEK HPMC HUVEC Fibroblasts Routine respiration Oxidative phosphorylation Q-junction Pyruvate Glutamate Malate Succinate Leak Coupling control Uncoupling Oxygraph Oxygen flux Residual oxygen consumption Instrumental background 

Abbreviations

CCP

Coupling control protocol

E

Electron transfer system capacity

FCR

Flux control ratio

HRR

High-resolution respirometry

L

LEAK respiration

mt

Mitochondrial

O2k

Oxygraph-2k

P

OXPHOS capacity

POS

Polarographic oxygen sensor

R

ROUTINE respiration

ROX

Residual oxygen consumption

SUIT

Substrate–uncoupler–inhibitor titration

Ww

Wet weight

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.D.Swarovski Research Laboratory, Department of General and Transplant SurgeryMedical University of InnsbruckInnsbruckAustria

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