Mitochondrial Morphology and Fundamental Parameters of the Mitochondrial Respiratory Chain Are Altered in Caenorhabditis elegans Strains Deficient in Mitochondrial Dynamics and Homeostasis Processes

Abstract

Mitochondrial dysfunction has been linked to myriad human diseases and toxicant exposures, highlighting the need for assays capable of rapidly assessing mitochondrial health in vivo. Here, using the Seahorse XFe24 Analyzer and the pharmacological inhibitors dicyclohexylcarbodiimide and oligomycin (ATP-synthase inhibitors), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (mitochondrial uncoupler) and sodium azide (cytochrome c oxidase inhibitor), we measured the fundamental parameters of mitochondrial respiratory chain function: basal oxygen consumption, ATP-linked respiration, maximal respiratory capacity, spare respiratory capacity and proton leak in the model organism Caenhorhabditis elegans. Since mutations in mitochondrial homeostasis genes cause mitochondrial dysfunction and have been linked to human disease, we measured mitochondrial respiratory function in mitochondrial fission (drp-1)-, fusion (fzo-1)-, mitophagy (pdr-1, pink-1)-, and electron transport chain complex III (isp-1)-deficient C. elegans. All showed altered function, but the nature of the alterations varied between the tested strains. We report increased basal oxygen consumption in drp-1; reduced maximal respiration in drp-1, fzo-1, and isp-1; reduced spare respiratory capacity in drp-1 and fzo-1; reduced proton leak in fzo-1 and isp-1; and increased proton leak in pink-1 nematodes. As mitochondrial morphology can play a role in mitochondrial energetics, we also quantified the mitochondrial aspect ratio for each mutant strain using a novel method, and for the first time report increased aspect ratios in pdr-1- and pink-1-deficient nematodes.

Mitochondrial Function and Cell Size: An Allometric Relationship

“…Proton leakage has been shown to be partly dependent on mitochondrial inner membrane area [48], suggesting that highly connected mitochondria would have less leakage. However, a recent study in C. elegans has reported opposite effects, as proton leakage was reduced by deletion of mitochondrial fusion proteins [49], suggesting 25 that proton leakage is low in highly fragmented mitochondria. Furthermore, we have not observed strong nonlinear proton leakage under unperturbed conditions and inhibition of DRP1 actually increases leakage in the larger cells [10].…”

A screening-based platform for the assessment of cellular respiration in Caenorhabditis elegans

“…Nevertheless, C. elegans has traditionally been considered as a poor candidate for drug-related assays due to the relatively inefficient uptake of compounds caused by impermeability of the cuticle to non-water soluble compounds45–47. Consequently, it does not come as a surprise that, except for FCCP, the compounds typically used in cultured cells are inefficient in acutely inhibiting specific aspects of mitochondrial function in C. elegans (Supplementary Figure 1)28 . However, there are alternative compounds, such as the complex IV/V inhibitor sodium azide, which can be used to help decipher aspects of mitochondrial respiration in C. elegans 28,48 (Figure 1b).…”

“…Consequently, it does not come as a surprise that, except for FCCP, the compounds typically used in cultured cells are inefficient in acutely inhibiting specific aspects of mitochondrial function in C. elegans (Supplementary Figure 1)28 . However, there are alternative compounds, such as the complex IV/V inhibitor sodium azide, which can be used to help decipher aspects of mitochondrial respiration in C. elegans 28,48 (Figure 1b). …”

“…Although initially developed for cultured or isolated cells, XF respirometers can also be used for whole organisms such as Caenorhabditis elegans, when taking some important adjustments into account25–28. C. elegans is often used as a model organism to study mitochondria in the context of longevity29.…”

Effects of reduced mitochondrial DNA content on secondary mitochondrial toxicant exposure in Caenorhabditis elegans

“…The fundamental parameters of the mitochondrial respiratory chain (basal oxygen consumption rate (OCR), maximal OCR, ATP-linked respiration, spare respiratory capacity (SRC), and proton leak) were measured following 4 and 8 days of exposure to EtBr using the Seahorse XF e 24 Bioanalyzer (Seahorse Bioscience, Massachusetts, USA) as previously described [32, 33]. Briefly, 50 nematodes were loaded into each well of a Seahorse utility plate and 8 basal OCR readings were taken.…”