Sumbalova 2016 Abstract Mito Xmas Meeting Innsbruck

Methods for evaluation of mitochondrial membrane potential with multi-sensor high-resolution respirometry: potentiometry vs fluorometry.

Link:

Sumbalova Z, Krumschnabel G, Gnaiger E (2016)

Event: Mito Xmas Meeting 2016 Innsbruck AT

Under physiological conditions mitochondrial (mt) membrane potential (mt) is maintained within a healthy range compatible with major mt functions [1]. In pathophysiological states, elevated values of mt may be associated with increased production of reactive oxygen species, while diminished mt values compromise mt ATP generation and Ca2+ retention capacity. Absolute values of mt depend on available substrates and the prevailing coupling state of a mt preparation [2]. Details of the interrelationship between respiratory states and mt can be studied using high-resolution respirometry (HRR) combined with potentiometric or fluorometric detection of mt.

Methods for measurement of mt depend on the addition of a reporter ion. For the potentiometric approach, the tetraphenylphosphonium ion (TPP+) is frequently used, which can be readily detected using an ion sensitive electrode system (OROBOROS ISE-Module). For fluorometric detection of mt, fluorescent dyes Safranin [3] or TMRM [4] can be applied with detection of their fluorescence by the OROBOROS O2k-Fluo LED2-Module. Unfortunately, at commonly applied concentrations of 1.5 - 2 µM all these probes interfere with mt respiration to some extent. Inhibition by TPP+ was found to be the lowest (< 3%) among these three dyes in mouse brain mitochondria. TPP+ could be successfully used for the simultaneous measurement of respiration and mt with NADH-linked substrates (N) and N combined with succinate (NS) [5], whereas limited sensitivity of potentiometric method at low mt rendered it unsuitable in combination with succinate (S). In comparison, fluorometric methods using TMRM or Safranin appeared more sensitive in the range of low mt, but at the cost of considerable inhibition of mt respiration particularly when employed with N-linked substrates (~30 %).

From the potentiometric TPP+ experiments absolute values of mt [mV] can be calculated, since the signal of the TPP+ electrode corresponds to the concentration of free TPP+ outside the mitochondria. In contrast, the fluorescence signal obtained with Safranin or TMRM consists of a mixture of the signal from free and bound probe and thus cannot be considered as a defined dye concentration convertible to mV. For future applications a method can be established for transformation of the fluorescence signal to mt for each type of mitochondria and protein concentration, which has to be kept constant in any set of experiments.

Both fluorometric and potentiometric measurements can be a valuable tool to distinguish differences in mt between experimental groups. Optimization of the experimental approach is possible by selecting the fluorometric or potentiometric approach and corresponding dye according to the specific questions to be addressed and the tissue- and species-specific mitochondrial properties.

• O2k-Network Lab: AT Innsbruck OROBOROS

Labels: MiParea: Respiration 

Regulation: mt-Membrane potential 

HRR: Oxygraph-2k, O2k-Fluorometer, TPP  Event: Poster 

Affiliations

Sumbalova Z(1,2), Krumschnabel G(1), Gnaiger E(1,3)

1. OROBOROS INSTRUMENTS, Innsbruck, Austria
2. Pharmacobiochemical Laboratory, Faculty of Medicine, Comenius University Bratislava, Slovakia
3. Department of Visceral, Transplant and Thoracic Surgery, Daniel Swarovski Research Laboratory, Medical University Innsbruck, Austria

References

1. Nicholls DG (2006) Simultaneous monitoring of ionophore- and inhibitor-mediated plasma and mitochondrial membrane potential changes in cultured neurons. J Biol Chem 281:14864-74.
2. Sumbalova Z, Fasching M, Gnaiger E (2011) Substrate control in mitochondrial respiration and regulation of mitochondrial membrane potential. Abstract Mitochondrial Medicine Chicago. (http://wiki.oroboros.at/index.php/Sumbalova_2011_Abstract_Mitochondrial_Medicine)
3. Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81.
4. Scaduto RC Jr, Grotyohann LW (1999) Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. Biophys J 76:469-77.
5. Sumbalova Z, Fasching M, Gnaiger E (2012) Evaluation of mitochondrial respiration and membrane potential in mouse brain homogenate. Mitochondr Physiol Network 17.12:61(http://wiki.oroboros.at/index.php/Sumbalova_2012_Abstract_Bioblast)

Support

Action Austria-Slovakia (SZ) and K-Regio project MitoFit (GE).