Sumbalova 2016 Abstract Mito Xmas Meeting Innsbruck

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

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Sumbalova Z, Krumschnabel G, Gnaiger E (2016)

Event: Mito Xmas Meeting 2016 Innsbruck AT

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

Methods for measurement of ΔΨmt 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-selective electrode system (Oroboros ISE-Module). For fluorometric detection of ΔΨmt, 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 ΔΨmt with NADH-linked substrates (N) and N combined with succinate (NS) [5], whereas limited sensitivity of potentiometric method at low ΔΨmt rendered it unsuitable in combination with succinate (S). In comparison, fluorometric methods using TMRM or Safranin appeared more sensitive in the range of low ΔΨmt, 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 ΔΨmt [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 ΔΨmt 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 ΔΨmt 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, SK Bratislava Sumbalova Z


Labels: MiParea: Respiration, Instruments;methods 


Organism: Mouse  Tissue;cell: Nervous system 


Coupling state: LEAK, OXPHOS, ET  Pathway: F, N, S, NS, ROX  HRR: Oxygraph-2k, O2k-Fluorometer, TPP, Theory  Event: A2, Oral  Labelled by author 

Affiliations

Sumbalova Z(1,2), Krumschnabel G(1), Gnaiger E(1,3)
  1. Oroboros Instruments, Innsbruck, Austria
  2. Pharmacobiochemical Lab, Fac Medicine, Comenius Univ Bratislava, Slovakia
  3. Dept Visceral, Transplant and Thoracic Surgery, D. Swarovski Research Lab, Medical Univ 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 K-Regio MitoFit (GE).