Cardoso 2018 MiPschool Tromso E2

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Luiza Helena Cardoso
Concomitant respiration and ATP production measurements to analyse P»/O2 ratios at physiological normoxia.

Link: MitoEAGLE

Cardoso LHD, Iglesias-Gonzalez J, Gnaiger E (2018)

Event: MiPschool Tromso-Bergen 2018

COST Action MitoEAGLE

Most of the studies regarding mitochondrial respiration are performed in hyperoxic conditions in comparison to the physiological O2 concentrations in the cell. However, there are evidences that mitochondrial phosphorylation efficiency increases in lower partial O2 pressures (pO2) [1]. The mitochondrial respiration, and especially coupling control ratios, have been broadly used as parameters to assess mitochondrial function and efficiency. Nevertheless, those methods allow to analyse the O2 consumed coupled to the phosphorylation by ATP synthase in OXPHOS state, but not the ATP production itself. A method to measure ATP production using a Mg2+-sensible fluorescent probe (Magnesium Green, MgG), exploiting the differential binding of ADP and ATP to Mg2+ has been developed and can be used concomitantly to respiration assays in the Oroboros O2k FluoRespirometer [2,3]. Therefore, we aim to further develop the use of these two methods combined to analyse P»/O2 ratios in different samples, and especially in physiological normoxic conditions. This will allow to explore the contribution of different pathway control states (e.g. NADH, succinate and glycerophosphate pathways) at physiological intracellular normoxia.

In this context, we intend to use mouse heart isolated mitochondria to first analyse whether the P»/O2 ratios differ when measured at physiological normoxia and at the conditions typically used in most of these assays, performed at ambient O2 levels that lead to hyperoxic conditions. Physiological normoxia will be obtained by injecting N2 in the chambers of the O2k FluoRespirometer in order to set-up the oxygen partial pressure desired.

By assessing mitochondrial O2 consumption under physiological normoxia in parallel with ATP production using MgG, as well as membrane potential using safranin, and ROS production using the probe for H2O2 Amplex UltraRed, we expect to provide a more comprehensive view of the mitochondria physiology. This can be further applied to study the impact of ischemia-reperfusion injury on cardiac muscle mitochondria, which occurs in diseases such as myocardial infarction and also in transplantation, having a great impact on public health.


O2k-Network Lab: AT Innsbruck Oroboros, AT Innsbruck Gnaiger E


Labels: MiParea: Respiration, Instruments;methods 





HRR: Oxygraph-2k, O2k-Fluorometer  Event: E2 


Affiliations

Cardoso LHD (1,2), Iglesias-Gonzalez J (1,2), Gnaiger E (1,2)

  1. Oroboros Instruments, Innsbruck, Austria
  2. Daniel Swarovski Research Lab, Dept Visceral, Transplant and Thoracic Surgery, Medical Univ Innsbruck, Austria

References

  1. [1] Gnaiger 2000 Proc Natl Acad Sci U S A
  2. [2] Chinopoulos 2009 Biophys J
  3. [3] Chinopoulos 2014 Methods Enzymol