High-Resolution FluoRespirometry and OXPHOS Protocols for Human Cells, Permeabilized Fibers from Small Biopsies of Muscle, and Isolated Mitochondria

Abstract

Protocols for High-Resolution FluoRespirometry of intact cells, permeabilized cells, permeabilized muscle fibers, isolated mitochondria, and tissue homogenates offer sensitive diagnostic tests of integrated mitochondrial function using standard cell culture techniques, small needle biopsies of muscle, and mitochondrial preparation methods. Multiple substrate-uncoupler-inhibitor titration (SUIT) protocols for analysis of oxidative phosphorylation (OXPHOS) 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 control and electron transfer pathway states. 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 (CCCP, FCCP, DNP) to collapse the protonmotive force across the mitochondrial inner membrane and measure the electron transfer (ET) capacity (open-circuit operation of respiration). Intrinsic uncoupling and dyscoupling are evaluated as the flux control ratio between non-phosphorylating LEAK respiration (electron flow coupled to proton pumping to compensate for proton leaks) and ET capacity. If OXPHOS capacity (maximally ADP-stimulated O flux) is less than ET capacity, the phosphorylation pathway contributes to flux control. Physiological substrate combinations supporting the NADH and succinate pathway are required to reconstitute tricarboxylic acid cycle function. This supports maximum ET and OXPHOS capacities, due to the additive effect of multiple electron supply pathways converging at the Q-junction. ET pathways with electron entry separately through NADH (pyruvate and malate or glutamate and malate) or succinate (succinate and rotenone) restrict ET capacity and artificially enhance flux control upstream of the Q-cycle, providing diagnostic information on specific ET-pathway branches. O concentration is maintained above air saturation in protocols with permeabilized muscle fibers to avoid experimental O 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 O flux (systemic flux compensation) provide the unique experimental basis for high accuracy of quantitative results and quality control in High-Resolution FluoRespirometry.

Mitochondrial respiration variability and simulations in human skeletal muscle: The Gene SMART study

“…This means that it would be a challenge to observe true changes in mitochondrial capacity using the OROBOROS technology, since most studies do not report such large increases following exercise training (−9% to 20%). 18,19,32,[34][35][36][37][38] We acknowledge that some of the studies have observed significant changes in mitochondrial respiration without reaching the effect sizes we presented here. This implies that although such studies were significant, the sample sizes were too low to detect the magnitude of changes they reported at 80% power.…”

OXPHOS remodeling in high-grade prostate cancer involves mtDNA mutations and increased succinate oxidation

“…A high respiratory capacity is crucial for high malignant and invasive potential 45 . In contrast to tumors, human skeletal muscle and fibroblasts heavily rely on N-pathway capacity 46,47 and a similar compensatory mechanism is not known for mt-myopathies. Ultimately, our results suggest that the immediate net effect of deleterious mtDNA mutations is compensated and does not hamper cellular ATP homeostasis in primary human PCa.…”

Hippocampal mitochondrial dysfunction and psychiatric-relevant behavioral deficits in spinocerebellar ataxia 1 mouse model

“…Hippocampal-specific mitochondrial dysfunction in the young SCA1 mice. In order to investigate whether the brain abnormalities and behavioral deficits identified might be related to mitochondrial dysfunction, we employed high-resolution respirometry to measure mitochondrial respiration in the cerebellar and the hippocampal tissue of the SCA1 mice and their healthy littermates (N = 6 SCA1 and 5 WT mice; aged 11-13) applying the substrate-uncoupler-inhibitor titration (SUIT) protocol 31 (Fig. 7a) and citrate synthase activity measurements (Suppl.…”