A whole blood approach improves speed and accuracy when measuring mitochondrial respiration in intact avian blood cells

Nord, Andreas; Chamkha, Imen; Elmér, Eskil

doi:10.1096/fj.202201749r

Cited by 4 publications

(2 citation statements)

References 47 publications

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“…We measured mitochondrial respiration in whole blood using an Oxygraph O2k high-resolution respirometer (Oroboros Instruments, Innsbruck, Austria), following the protocol described in Nord, Chamkha & Elmér. 49 To test how rest-phase hypothermia affected mitochondrial function, we ran duplicate samples from the same individual simultaneously in each of two thermal states: a representative normothermic daytime bird body temperature (41°C; henceforth "normothermia") and a hypothermic night-time body temperature in the lower range of the measured natural variation (35°C; henceforth "hypothermia") 50 using two respirometers. These were calibrated at assay temperature (i.e., 35 or 41°C) daily, and we changed instrument/assay temperature combinations between days to avoid confounding experimental results by any variation pertaining to the specific instruments.…”

Section: Blood Sampling and Mitochondrial Measurement In Whole Bloodmentioning

confidence: 99%

Plasticity of mitochondrial function safeguards phosphorylating respiration during in vitro simulation of rest‐phase hypothermia

et al. 2023

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Many animals downregulate body temperature to save energy when resting (rest‐phase hypothermia). Small birds that winter at high latitudes have comparatively limited capacity for hypothermia and so pay large energy costs for thermoregulation during cold nights. Available evidence suggests this process is fueled by adenosine triphosphate (ATP)‐dependent mechanisms. Most ATP is produced by oxidative phosphorylation in the mitochondria, but mitochondrial respiration may be lower during hypothermia because of the temperature dependence of biological processes. This can create conflict between increased organismal ATP demand and a lower mitochondrial capacity to provide it. We studied this in blood cell mitochondria of wild great tits (Parus major) by simulating rest‐phase hypothermia via a 6°C reduction in assay temperature in vitro. The birds had spent the night preceding the experiment in thermoneutrality or in temperatures representing mild or very cold winter nights, but night temperatures never affected mitochondrial respiration. However, across temperature groups, endogenous respiration was 14% lower in hypothermia. This did not reflect general thermal suppression of mitochondrial function because phosphorylating respiration was unaffected by thermal state. Instead, hypothermia was associated with a threefold reduction of leak respiration, from 17% in normothermia to 4% in hypothermia. Thus, the coupling of total respiration to ATP production was 96% in hypothermia, compared to 83% in normothermia. Our study shows that the thermal insensitivity of phosphorylation combined with short‐term plasticity of leak respiration may safeguard ATP production when endogenous respiration is suppressed. This casts new light on the process by which small birds endure harsh winter cold and warrants future tests across tissues in vivo.

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Section: Blood Sampling and Mitochondrial Measurement In Whole Bloodmentioning

confidence: 99%

Plasticity of mitochondrial function safeguards phosphorylating respiration during in vitro simulation of rest‐phase hypothermia

et al. 2023

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“…The sample was contained in 2mL K2-EDTA (dipotassium ethylenediaminetetraacetic acid) tubes (BD Vacutainer ® , Franklin Lakes, NJ, USA) that were kept at 10-12°C until further processing. Thirty minutes to 2 h later, we measured the mitochondrial metabolism in intact blood cells based on whole blood measurement at 41°C (a representative daytime body temperature for a passerine bird; Prinzinger et al, 1991) using high-resolution respirometers (Oxygraph O2k high-resolution respirometers, Oroboros Instruments, Innsbruck, Austria) following Nord et al, 2023. After mixing, 50µL whole blood was added to 1.95 mL of respiration medium (MiRO5: 0.5mM EGTA, 3mM MgCl2, 60mM K-lactobionate, 20mM taurine, 10mM KH2PO4, 20mM HEPES, 110mM sucrose, 1g/L free-fatty-acid bovine serum albumin, pH 7.1) in the respirometer chambers. We first measured baseline respiration rate, which is directly related to the oxygen consumption on endogenous substrates (i.e., ROUTINE respiration).…”

Section: Mitochondrial Measurementsmentioning

confidence: 99%

Permeabilization status affects the relationship between basal metabolic rate and mitochondrial respiration in great tit blood cells

Thoral

Garcia-Diaz

Persson

et al. 2023

Preprint

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Although mitochondrial respiration is believed to explain a substantial part of the variation in whole-animal basal (BMR) or resting metabolic rate (RMR), few studies have addressed the relationship between organismal and cellular metabolism and how this may vary in environments where individual demands for energy differ. We investigated the relationship between whole-individual metabolic rate, measured in temperatures ranging thermoneutrality to far below thermoneutrality, and mitochondrial respiration of intact or permeabilized blood cells in two separate studies on wild great tits (Parus major L.). Our results show that, in permeabilized cells, there are significant positive relationships between BMR or RMR and several mitochondrial traits, including phosphorylating respiration rate through both complexes I and II (i.e., OXPHOS respiration). However, surprisingly, the LEAK respiration (i.e., basal respiration that mainly counteract for proton leakage) was not related to BMR or RMR. When measurements were performed using intact blood cells, BMR was positively related to ROUTINE respiration (i.e., mitochondrial respiration on endogenous substrates) in one of the two studies, but no other mitochondrial traits could explain variation in BMR or RMR in any thermal environment. These studies seem to show that the level of activation of mitochondrial metabolism as well as the permeabilization status of blood cells play a primary role on the extent to which blood metabolism might explain variations in the whole-individual metabolic rate.

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Development in the cold renders bird mitochondria more susceptible to heat stress

Correia,

Thoral,

Persson

et al. 2024

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Research on birds suggests that extreme weather events during development may have long-lasting consequences on form and function. The underlying cellular mechanisms mediating such phenotypic effects are poorly studied. We raised Japanese quail in warm (30oC) or cold (10oC) temperatures from hatching until adulthood, and then measured mitochondrial metabolism in intact blood cells at representative normothermic body temperature (41oC) and a hyperthermic temperature (45oC) that quail commonly attain when heat stressed. To investigate whether any developmental effects were reversible, half of the cold- and warm-acclimated birds were assigned to a common garden (20oC) 3 weeks before the measurements. Across groups, hyperthermia was associated with increased proton leak, but decreases in both phosphorylating respiration (where ATP is produced) and working capacity of the mitochondria. Cold-acclimated birds were more strongly affected by heat stress: the increase in proton leak was 1.6-fold higher, and the decrease in phosphorylating capacity during endogenous respiration was 1.7-fold greater, compared to warm-acclimated birds. These differences did not remain in the common-garden birds. Our study suggests that developmental cold acclimation is traded off against heat tolerance at the level of cellular metabolism, with implications for our understanding of avian responses to climate change.

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A whole blood approach improves speed and accuracy when measuring mitochondrial respiration in intact avian blood cells

Cited by 4 publications

References 47 publications

Plasticity of mitochondrial function safeguards phosphorylating respiration during in vitro simulation of rest‐phase hypothermia

Plasticity of mitochondrial function safeguards phosphorylating respiration during in vitro simulation of rest‐phase hypothermia

Permeabilization status affects the relationship between basal metabolic rate and mitochondrial respiration in great tit blood cells

Development in the cold renders bird mitochondria more susceptible to heat stress

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