measurement-mitochondrial-oxygen-consumption-permeabilized-fibers

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

Université de Moncton

The oxidative phosphorylation (OxPhos) pathway is responsible for most aerobic ATP production and is the only metabolic pathway with proteins encoded by both nuclear and mitochondrial genomes. In studies examining mitonuclear interactions among distant populations within a species or across species, the interactions between these two genomes can affect metabolism, growth, and fitness, depending on the environment. However, there is little data on whether these interactions impact natural populations within a single species. In an admixed Fundulus heteroclitus population with northern and southern mitochondrial haplotypes, there are significant differences in allele frequencies associated with mitochondrial haplotype. In this study, we investigate how mitochondrial haplotype and any associated nuclear differences affect six OxPhos parameters within a population. The data demonstrate significant OxPhos functional differences between the two mitochondrial genotypes. These differences are most apparent when individuals are acclimated to high temperatures with the southern mitochondrial genotype having a large acute response and the northern mitochondrial genotype having little, if any acute response. Furthermore, acute temperature effects and the relative contribution of Complex I and II depend on acclimation temperature: when individuals are acclimated to 12°C, the relative contribution of Complex I increases with higher acute temperatures, whereas at 28°C acclimation, the relative contribution of Complex I is unaffected by acute temperature change. These data demonstrate a complex gene by environmental interaction affecting the OxPhos pathway.

Gene by environmental interactions affecting oxidative phosphorylation and thermal sensitivity.

Although the mitochondrial metabolism responses to warm acclimation have been widely studied in fish, the time course of this process is less understood. Here, we characterized the changes of rainbow trout () cardiac mitochondrial metabolism during acute warming from 10 to 16°C, and during the subsequent warm acclimation for 39 days. We repeatedly measured mitochondrial oxygen consumption in cardiac permeabilized fibers and the functional integrity of mitochondria (i.e. mitochondrial coupling and cytochromeeffect) at two assay temperatures (10 and 16°C), as well as the activities of citrate synthase (CS) and lactate dehydrogenase (LDH) at room temperature. LDH and CS activities significantly increased between day 0 (10°C acclimated fish) and day 1 (acute warming to 16°C) while mitochondrial oxygen consumption measured at respectivetemperatures did not change. Enzymatic activities and mitochondrial oxygen consumption rates significantly decreased by day 2, and remained stable during warm acclimation (days 2-39). The decrease in rates of oxygen between day 0 and day 1 coincided with an increased cytochromeeffect and a decreased mitochondrial coupling, suggesting a structural/functional impairment of mitochondria during acute warming. We suggest that after 2 days of warm acclimation, a new homeostasis is reached, which may involve the removal of dysfunctional mitochondria. Interestingly, from day 2 onwards, there was a lack of differences in mitochondrial oxygen consumption rates between the assay temperatures, suggesting that warm acclimation reduces the acute thermal sensitivity of mitochondria. This study provides significant knowledge on the thermal sensitivity of cardiac mitochondria that is essential to delineate the contribution of cellular processes to warm acclimation.

Dynamic changes in cardiac mitochondrial metabolism during warm acclimation in rainbow trout.

Anadromy is a distinctive life-history strategy in fishes that has evolved independently many times. In an evolutionary context, the benefits of anadromy for a species or population must outweigh the costs and risks associated with the habitat switch. The migration of fish across the freshwater-ocean boundary coincides with potentially energetically costly osmoregulatory modifications occurring at numerous levels of biological organization. By integrating whole animal and sub-cellular metabolic measurements, this study presents significant findings demonstrating how an anadromous salmonid (i.e. rainbow trout, Oncorhynchus mykiss) is able to transform from a hyper- to hypo-osmoregulatory state without incurring significant increases in whole animal oxygen consumption rate. Instead, underlying metabolic mechanisms that fuel the osmoregulatory machinery at the organ level (i.e. intestine) are modulated, as mitochondrial coupling and anaerobic metabolism are increased to satisfy the elevated energetic demands. This may have positive implications for the relative fitness of the migrating individual, as aerobic capacity may be maintained for locomotion (i.e. foraging and predator avoidance) and growth. Furthermore, the ability to modulate mitochondrial metabolism in order to maintain osmotic balance suggests that mitochondria of anadromous fish may have been a key target for natural selection, driving species adaptations to different aquatic environments.

Increased mitochondrial coupling and anaerobic capacity minimizes aerobic costs of trout in the sea.

The study of glycolysis, the TCA cycle, and oxidative phosphorylation in animals has yielded a wealth of information about bioenergetics. Less is known about how animals use fuels other than glucose and less characterized enzymes that are also used to provide electrons to the electron transport system. It has become clear that bioenergetic flexibility is employed by a wide variety of animals in order to successfully grow, maintain cells, and reproduce, and has contributed to the exploitation of new environments and ecological niches through evolution. In most cases, the discovery of these "alternative" fuels and non-classical pathways is relatively recent, but is starting to call into question long believed paradigms about the diversity of animal bioenergetics. We present several specific examples of these "alternatives" and the animals that use them and present some implications for animal mitochondrial physiology research.

"Alternative" fuels contributing to mitochondrial electron transport: Importance of non-classical pathways in the diversity of animal metabolism.

The small heat shock protein (sHsp) Hsp22 from Drosophila melanogaster (DmHsp22) is part of the family of sHsps in this diptera. This sHsp is characterized by its presence in the mitochondrial matrix as well as by its preferential expression during ageing. Although DmHsp22 has been demonstrated to be an efficient in vitro chaperone, its function within mitochondria in vivo remains largely unknown. Thus, determining its protein-interaction network (interactome) in the mitochondrial matrix would help to shed light on its function(s). In the present study we combined immunoaffinity conjugation (IAC) with mass spectroscopy analysis of mitochondria from HeLa cells transfected with DmHsp22 in non-heat shock condition and after heat shock (HS). 60 common DmHsp22-binding mitochondrial partners were detected in two independent IACs. Immunoblotting was used to validate interaction between DmHsp22 and two members of the mitochondrial chaperone machinery; Hsp60 and Hsp70. Among the partners of DmHsp22, several ATP synthase subunits were found. Moreover, we showed that expression of DmHsp22 in transiently transfected HeLa cells increased maximal mitochondrial oxygen consumption capacity and ATP contents, providing a mechanistic link between DmHsp22 and mitochondrial functions.

Identification of proteins interacting with the mitochondrial small heat shock protein Hsp22 of Drosophila melanogaster: Implication in mitochondrial homeostasis.

Article	Year
Gene by environmental interactions affecting oxidative phosphorylation and thermal sensitivity. American journal of physiology. Regulatory, integrative and comparative physiology\| PubMed ID: 27225945	2016
Dynamic changes in cardiac mitochondrial metabolism during warm acclimation in rainbow trout. The Journal of experimental biology\| PubMed ID: 28202582	2017
Increased mitochondrial coupling and anaerobic capacity minimizes aerobic costs of trout in the sea. Scientific reports\| PubMed ID: 28361996	2017
"Alternative" fuels contributing to mitochondrial electron transport: Importance of non-classical pathways in the diversity of animal metabolism. Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology\| PubMed ID: 29155008	2017
Identification of proteins interacting with the mitochondrial small heat shock protein Hsp22 of Drosophila melanogaster: Implication in mitochondrial homeostasis. PloS one\| PubMed ID: 29509794	2018

Nicolas Pichaud

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