Impact of Coenzyme Q10 Supplementation on Skeletal Muscle Respiration, Antioxidants, and the Muscle Proteome in Thoroughbred Horses

Abstract

Coenzyme Q10 (CoQ10) is an essential component of the mitochondrial electron transfer system and a potent antioxidant. The impact of CoQ10 supplementation on mitochondrial capacities and the muscle proteome is largely unknown. This study determined the effect of CoQ10 supplementation on muscle CoQ10 concentrations, antioxidant balance, the proteome, and mitochondrial respiratory capacities. In a randomized cross-over design, six Thoroughbred horses received 1600 mg/d CoQ10 or no supplement (control) for 30-d periods separated by a 60-d washout. Muscle samples were taken at the end of each period. Muscle CoQ10 and glutathione (GSH) concentrations were determined using mass spectrometry, antioxidant activities by fluorometry, mitochondrial enzyme activities and oxidative stress by colorimetry, and mitochondrial respiratory capacities by high-resolution respirometry. Data were analyzed using mixed linear models with period, supplementation, and period × supplementation as fixed effects and horse as a repeated effect. Proteomics was performed by tandem mass tag 11-plex analysis and permutation testing with FDR < 0.05. Concentrations of muscle CoQ10 (p = 0.07), GSH (p = 0.75), and malondialdehyde (p = 0.47), as well as activities of superoxide dismutase (p = 0.16) and catalase (p = 0.66), did not differ, whereas glutathione peroxidase activity (p = 0.003) was lower when horses received CoQ10 compared to no supplement. Intrinsic (relative to citrate synthase activity) electron transfer capacity with complex II (E_CII) was greater, and the contribution of complex I to maximal electron transfer capacity (FCR_PCI and FCR_PCIG) was lower when horses received CoQ10 with no impact of CoQ10 on mitochondrial volume density. Decreased expression of subunits in complexes I, III, and IV, as well as tricarboxylic acid cycle (TCA) enzymes, was noted in proteomics when horses received CoQ10. We conclude that with CoQ10 supplementation, decreased expression of TCA cycle enzymes that produce NADH and complex I subunits, which utilize NADH together with enhanced electron transfer capacity via complex II, supports an enhanced reliance on substrates supplying complex II during mitochondrial respiration.

Keywords: antioxidants; electron transport chain; high-resolution respirometry; mitochondria; reactive oxygen species.

Figure 1

Skeletal muscle CoQ10 concentrations did not differ significantly (p = 0.07) between control and CoQ10 treatments. Open circles represent horses on the CoQ10 supplement during the first supplementation period, and closed circles represent horses on the CoQ10 supplement during the second supplementation period. Open triangles represent horses on the control diet during the second supplementation period, and closed triangles represent horses on the control diet during the first supplementation period.

Figure 2

(A) CS activity and (B) integrative and (C) intrinsic CCO activities in the gluteus medius of fit Thoroughbred horses before and after 30 d supplementation of CoQ10 or control diet. Open circles represent horses on the CoQ10 supplement during the first supplementation period, and closed circles represent horses on the CoQ10 supplement during the second supplementation period. Open triangles represent horses on the control diet during the second supplementation period, and closed triangles represent horses on the control diet during the first supplementation period.

Figure 3

(A–F) Intrinsic (relative to CS activity) mitochondrial capacities in the gluteus medius of fit Thoroughbred horses before and after 30 d supplementation of CoQ10 or control diet. Open circles represent horses on the CoQ10 supplement during the first supplementation period, and closed circles represent horses on the CoQ10 supplement during the second supplementation period. Open triangles represent horses on the control diet during the second supplementation period, and closed triangles represent horses on the control diet during the first supplementation period. Within periods, different letters indicate significant treatment differences.

Figure 4

(A–E) Flux control ratios (FCR) of gluteus medius samples from fit Thoroughbred horses before and after 30 d supplementation of CoQ10 or control diet. Open circles represent horses on the CoQ10 supplement during the first supplementation period, and closed circles represent horses on the CoQ10 supplement during the second supplementation period. Open triangles represent horses on the control diet during the second supplementation period, and closed triangles represent horses on the control diet during the first supplementation period. # Regardless of time (period), control differs from CoQ10 (p ≤ 0.05). * Across treatments, period 1 differs from period 2 (p ≤ 0.05).

Figure 5

Skeletal muscle antioxidant activities and MDA concentrations relative to protein concentrations. (A) Activity of GPx in control and CoQ10 treatment groups. GPx activity was significantly lower in the CoQ10 supplemented horses (p = 0.003). (B) Activities of SOD in control and CoQ10 treatment groups were not significantly different with treatment (p = 0.17). (C) Activities of Cat in control and CoQ10 treatment groups were not significantly different with treatment (p = 0.66). (D) GSH concentrations in control and CoQ10 treatment groups were not significantly different (p = 0.75). (E) MDA concentrations in control and CoQ10 treatment groups were not significantly different (p = 0.47). Open circles represent horses on the CoQ10 supplement during the first supplementation period, and closed circles represent horses on the CoQ10 supplement during the second supplementation period. Open triangles represent horses on the control diet during the second supplementation period, and closed triangles represent horses on the control diet during the first supplementation period. ** p < 0.01.

Figure 6

Proteins with significantly decreased expression (red arrow) in the mitochondria of horses on CoQ10 compared to the control. Horses with CoQ10 supplementation had a downregulation of 2 subunits of complex I, 2 subunits of complex III, and 4 subunits of ATP synthase, NAD(P) transhydrogenase (NNT), malate dehydrogenase (MDH), 2-oxoglutarate dehydrogenase (OGDH), NADP dependent isocitrate dehydrogenase (IDH2), aconitase (ACO2), aspartate aminotransferase (AST), and the 2-oxoglutarate transport channel (OGC). Created with BioRender.com, accessed on 2 December 2022.