Eicosapentaenoic acid but not docosahexaenoic acid restores skeletal muscle mitochondrial oxidative capacity in old mice

Abstract

Mitochondrial dysfunction is often observed in aging skeletal muscle and is implicated in age-related declines in physical function. Early evidence suggests that dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) improve mitochondrial function. Here, we show that 10 weeks of dietary eicosapentaenoic acid (EPA) supplementation partially attenuated the age-related decline in mitochondrial function in mice, but this effect was not observed with docosahexaenoic acid (DHA). The improvement in mitochondrial function with EPA occurred in the absence of any changes in mitochondrial abundance or biogenesis, which was evaluated from RNA sequencing, large-scale proteomics, and direct measurements of muscle mitochondrial protein synthesis rates. We find that EPA improves muscle protein quality, specifically by decreasing mitochondrial protein carbamylation, a post-translational modification that is driven by inflammation. These results demonstrate that EPA attenuated the age-related loss of mitochondrial function and improved mitochondrial protein quality through a mechanism that is likely linked with anti-inflammatory properties of n-3 PUFAs. Furthermore, we demonstrate that EPA and DHA exert some common biological effects (anticoagulation, anti-inflammatory, reduced FXR/RXR activation), but also exhibit many distinct biological effects, a finding that underscores the importance of evaluating the therapeutic potential of individual n-3 PUFAs.

Keywords: aging; docosahexaenoic acid; eicosapentaenoic acid; mitochondria; omega 3; proteomics; sarcopenia.

Fig 1

Eicosapentaenoic acid restores muscle mitochondrial function in aged mice (A,B) Isolated mitochondria were respired with substrates targeting complex I (CI), complex I+II (CI+II), and complex II (CII). Mitochondrial membrane integrity was assessed by the addition of cytochrome c (Cyt c). Nonmitochondrial oxygen consumption was measured in the presence of antimycin A (AA). (C,D) Respiration rates were also measured using palmitoyl-carnitine substrates. Respiration rates were expressed per tissue wet (A,C) and mitochondrial protein content (B,D). Respiratory control ratio (RCR, state 3/state 4) and phosphorylation efficiency (ADP:O) were measured in isolated mitochondria (E,F). Bars represent means ± SEM. *, significant statistical differences from young control (P < 0.05, Tukey’s HSD). ^#, significant statistical differences from old control (P < 0.05, Tukey’s HSD). n = 8 per group.

Fig 2

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) do not restore age-related reductions in mitochondrial content of skeletal muscle volcano plots of differentially expressed muscle proteins in old vs. young control mice (A), EPA-treated old versus old control (B), and (C) DHA-treated old versus old control measured by mass spectrometry. The x-axis represents the log fold change (FC), while y-axis represents the –log P-value for each protein. Mitochondrial proteins are colored in red. Electron transport chain (ETC.) proteins are colored blue. Ingenuity pathway analysis was performed using mitochondrial proteins, and the top canonical pathways are given. Histogram bars represent P values for each pathway using either exclusively upregulated proteins (gray bars) or exclusively downregulated proteins (black bars). (D,E) Venn diagrams showing differentially expressed mitochondrial proteins (D) or all proteins (E).

Fig 3

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) do not stimulate mitochondrial biogenesis in aged skeletal muscle volcano plots of differentially expressed genes in old vs. young control mice (A), EPA-treated old versus old control (B), and (C) DHA-treated old versus old control measured by RNA sequencing. The x-axis represents the log fold change, while y-axis represents the –log P-value for each gene. Mitochondrial genes are colored in red. Ingenuity pathway analysis was performed using mitochondrial-related genes, and the top canonical pathways are given. Histogram bars represent p values for each pathway using either exclusively upregulated proteins (gray bars) or exclusively downregulated proteins (black bars). (D,E) Venn diagrams showing differentially expressed mitochondrial genes (D) or all genes (E).

Fig 4

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) do not stimulate protein synthesis in aged skeletal muscle fractional synthesis rates of mitochondrial proteins were measured in vivo from the rate of incorporation of ¹³C₆ phenylalanine and mass spectrometry (A). The overall mixed muscle protein synthesis rate was measured by long-term labeling with deuterium oxide (B). Bars represent means ± SEM. n = 8 per group.