Garcia-Roves 2022 Abstract Bioblast

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4.4. Β«10+5Β»
Pablo Garcia-Roves
Gama-Perez P, Cardoso L, Komlodi K, Bosch N, Gnaiger E, Garcia-Roves Pablo M (2022) Tuning the assessment of coenzyme Q redox state and respiration in permeabilized skeletal muscle fibers.
Bioblast 2022: BEC Inaugural Conference. In: Β»Watch the presentationΒ«

Link: Bioblast 2022: BEC Inaugural Conference

Gama Perez Pau, Cardoso Luiza HD, Komlodi Timea, Bosch Nuria, Gnaiger Erich, Garcia-Roves Pablo Miguel (2022)

Event: Bioblast 2022

Metabolic plasticity in skeletal muscle facilitates the response of this tissue to a myriad of physiological and pathological conditions. In this sense, mitochondria play a critical role and accordingly, the assessment of its content and functionality represents a breakthrough to understand this adaptive response. Mitochondrial function in skeletal muscle was initially explored by inferential combinations of oxygen kinetics and enzymatic assays in many different biopsy specimens. Nonetheless, these initial approaches have been replaced by more sensitive and reliable methods that allow direct measurements of oxygen consumption (e.g., high-resolution respirometry, HRR). Using polarographic oxygen sensors, HRR offers a detailed real-time assessment of respiration and, by the titration of different substrates and compounds, can provide relevant information about the function of specific components of the electron transfer system. In addition, the growing popularization of this methodology has been paralleled by mechanical and chemical permeabilization as the preferred choice for tissue preparation, since it offers critical advantages over commonly used alternatives (i.e., mitochondrial isolation). Essentially, the amount of tissue needed to perform HRR in permeabilized fiber (pfi) is significantly reduced, all mitochondrial populations (subsarcolemmal and intermyofibrilar) are equally represented, and mitochondrial network is preserved. Thus, HRR in pfi represents a suitable and increasingly widespread option to address skeletal muscle bioenergetics.
Beyond HRR, over the years additional instrumentation has been implemented to explore other complementary readouts that enhance our understanding of mitochondrial bioenergetics. Accordingly, HRR in combination with fluorescence or potentiometric sensors allows reliable measurements of membrane potential, H2O2 and ATP production in a wide range of biological samples. Additionally, in an innovative effort to expand our exploratory capacity, the novel Oroboros NextGen-O2k Q-Module has been recently developed for a simultaneous real-time monitoring of oxygen consumption and Q-redox state using a three-electrode system, and a short-chain coenzyme Q mimetic (CoQ2) as a probe. This setup has already been successfully used to determine the Q-reduced fraction in isolated brain and heart mitochondria (Komlodi 2021). Hence, the main purpose of this study is to address the feasibility to simultaneously evaluate these complementary readouts in permeabilized muscle fibers.

Since we have demonstrated that reported CoQ2 concentrations in isolated mitochondria can negatively impact respirometry measurements in pfi, we first aimed to optimize the CoQ2 concentration for a proper sensitivity and permissibility. We observed that in the presence of pfi and absence of respiratory substrates, CoQ2 was fully oxidized, and could not be further oxidized upon addition of the CI inhibitor rotenone. This was followed by application of a substrate-uncoupler-inhibitor titration (SUIT) protocol, which permits to assess respiration and reduced Q-fraction in the NADH- and succinate-linked pathways in the LEAK, OXPHOS, and electron transfer (ET) coupling control states. We observed that the Q-pool became partially reduced upon addition of the respiratory substrates, pyruvate and malate, and even further reduced when succinate was employed. Addition of ADP in the presence of pyruvate and malate led to partial oxidation of the Q-pool, however, this was not pronounced. As expected for skeletal muscle, addition of uncoupler to reach ET-capacity did not affect either respiration or the reduced Q-fraction. In conclusion, these results demonstrate that it is possible to use the Q-Module to expand the evaluation of skeletal muscle mitochondrial physiology.

  1. KomlΓ³di T, Cardoso LHD, Doerrier C, Moore AL, Rich PR, Gnaiger E (2021) Coupling and pathway control of coenzyme Q redox state and respiration in isolated mitochondria.

β€’ Keywords: Coenzyme Q, permeabilized muscle fibers, high-resolution respirometry, skeletal muscle, electron transfer system, Q-junction

β€’ O2k-Network Lab: AT Innsbruck Oroboros, ES Barcelona Garcia-Roves PM, ES Barcelona IDIBAPS Hospital Clinic, HU Budapest Tretter L

Affiliations and support

Gama-Perez P1, Cardoso L2, Komlodi T2,3, Bosch N1, Gnaiger E2, Garcia-Roves PM1,4*
  1. Dept Physiological Sciences, Faculty of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain - [email protected]
  2. Oroboros Instruments, Innsbruck, Austria
  3. Department of Biochemistry, Semmelweis University, Budapest, Hungary
  4. Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain
This work was part of the Oroboros NextGen-O2k project, with funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement nΒΊ 859770.

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