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.

Fig 1. Image processing result of a branched mitochondrion.

A centerline was extracted using a morphological thinning operation.

Fig 2. Representative Seahorse XFe24 output data for L4 N2 nematodes dosed with (A) DCCD, (B) FCCP, and (C) sodium azide.

Fig 3. Basal OCR is elevated in L4 drp-1 nematodes.

Statistical significance was analyzed via a one way ANOVA (main effect of strain, P<0.0001) (n = 31–45). Asterisks (*) denote statistical significance. Bars ± SEM.

Fig 4. Maximal and spare respiratory capacity in L4 nematodes.

(A) Treatment with FCCP caused a significant increase in OCR in all strains (two way ANOVA, main effects of strain, treatment and their interaction, P<0.0001 for all); however, L4 fzo-1, drp-1 and isp-1 had reduced maximal respiratory capacity compared to N2 nematodes (Student’s t-test, p = 0.03, p<0.0001, p = 0.01, respectively). (B) Spare respiratory capacity was reduced in fzo-1 and drp-1, compared to wild-type nematodes (one way ANOVA, P = 0.022). (n = 12–20). Asterisks (*) denote statistical significance. Bars ± SEM.

Fig 5. Proton leak in L4 nematodes.

(A) Sodium azide and DCCD caused significantly different reductions in OCR in N2, drp-1, pink-1, and pdr-1 nematodes (two way ANOVA, main effects of strain (P<0.0001), treatment (P<0.0001) and their interaction (p = 0.0002)), while fzo-1 and isp-1 responses were not significantly different. (B) L4 fzo-1 and isp-1 nematodes have reduce proton leak, while pink-1 C. elegans have increased leak (one way ANOVA, P<0.0001). (n = 8–12). Asterisks (*) denote statistical significance. Bars ± SEM.

Fig 6. Mitochondrial morphology in wild-type and mutant C. elegans.

Representative confocal images of mitochondrial morphology in wild-type (N2) (A-B) and mitochondrial mutant strains (fzo-1 (C-D), drp-1 (E-F), pink-1 (G-H), pdr-1 (I-J)) at L4. Left panel shows the sample at 63x, right panel shows a representative zoomed and cropped image from the z-stack (63x) used for image analysis.