Karro 2019 MiP2019

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Niina Karro
Respiration of permeabilized cardiomyocytes from mice: no sex differences, but substrate-dependent changes in the apparent ADPaffinity.

Link: MiP2019

Karro N, Laasmaa M, Vendelin M, Birkedal R (2019)

Event: MiP2019

COST Action MitoEAGLE

In recent years there has been an increased focus on sex differences in cardiac physiology and pathology. It is recommended for future research that sex differences are taken into account [1]. This is particularly important in studies of transgenic mice, where underlying sex differences may lead to sex specific outcomes of genetic modifications. C57BL/6 mice are commonly used as background for transgenic mouse strains. This study assessed whether isolated, permeabilized cardiomyocytes from male and female C57BL/6 mice differ in terms of their respiration with multiple substrates and overall intracellular diffusion restriction estimated by the apparent ADP-affinity of respiration.

To perform this study, we used isolated, permeabilized cardiomyocytes from 10 male and 10 female C57BL/6 mice. Cardiomyocytes were isolated using a method described previously [2]. Measurements of oxygen consumption were carried out using a Strathkelvin RC 650 Respirometer (Strathkelvin Instruments Limited, UK). Using respirometry, we recorded 1) the activities of respiratory complexes I, II and IV, 2) the respiration rate with substrates fuelling either complex I, II, or I + II, and 3) the apparent ADP-affinity with substrates fuelling complex I and I + II. ADP-titrations were carried out in the presence of 5 mM glutamate and 2 mM malate only (ADPGM-titration) and in the presence of 5 mM glutamate, 2 mM malate, 5 mM pyruvate and 15 mM succinate (ADPGMPS-titration). For most experiments, 10 μM cytochrome c was added at the end of the ADP-titration to test the intactness of the outer mitochondrial membrane. The respiration rates were normalized to protein content and citrate synthase (CS) activity, these measurements were performed in triplicate and the results averaged.

We found no sex differences in CS activity (a marker of mitochondrial content) normalized to protein content or in any of the respiration measurements. This suggests that cardiomyocytes from male and female mice do not differ in terms of mitochondrial respiratory capacity and apparent ADP-affinity. Pyruvate modestly lowered the respiration rate, when added to succinate, glutamate and malate. This may be explained by intramitochondrial compartmentalization caused by the formation of supercomplexes and their association with specific dehydrogenases. Our data illustrate well how the coupling efficiency of respiration varies with the substrates. When comparing the ADPGM-titration and ADPGMPS-titration, the coupling efficiency with GM was slightly, but significantly higher than that with GMPS. To our knowledge, we show for the first time that the apparent ADP-affinity was substrate-dependent. This suggests that substrates may change or regulate intracellular barriers in cardiomyocytes.


Bioblast editor: Plangger M, Tindle-Solomon L


Labels: MiParea: Respiration, Comparative MiP;environmental MiP, Gender 


Organism: Mouse  Tissue;cell: Heart  Preparation: Permeabilized cells 





Affiliations

Lab Systems Biology, Dept Cybernetics, Tallinn Univ Technology, Tallinn, Estonia. - niina@sysbio.ioc.ee

References

  1. Ventura-Clapier R, Dworatzek E, Seeland U, Kararigas G, Arnal JF, Brunelleschi S, Carpenter TC, Erdmann J, Franconi F, Giannetta E, Glezerman M, Hofmann SM, Junien C, Katai M, Kublickiene K, König IR, Majdic G, Malorni W, Mieth C, Miller VM, Reynolds RM, Shimokawa H, Tannenbaum C, D'Ursi AM, Regitz-Zagrosek V (2017) Sex in basic research: concepts in the cardiovascular field. Cardiovasc Res 113:711–24.
  2. Branovets J, Sepp M, Kotlyarova S, Jepihhina N, Sokolova N, Aksentijevic D, Lygate CA, Neubauer S (2013) Unchanged mitochondrial organization and compartmentation of high-energy phosphates in creatine-deficient GAMT−/− mouse hearts. Am J Physiol Heart Circ Physiol 305:H506–20.