Omega-3 fatty acid supplementation does not attenuate declines in skeletal muscle mitochondrial area in young, healthy females during immobilization

Abstract

Mitochondrial subcellular area influences function. Muscle disuse reduces mitochondrial content; however, its effect on mitochondrial subcellular location is unclear. Omega-3 fatty acid (n-3) attenuates declines in muscle mass and mitochondrial function during disuse; however, whether n-3 supplementation prevents the decline in mitochondrial content has not been examined. We investigated the effects of 2 weeks of leg immobilization followed by 2 weeks of remobilization on skeletal muscle mitochondrial content and subcellular localization with and without n-3 supplementation. Twenty healthy females supplemented with n-3 (2.97 g EPA and 2.03 g DHA) or control (isoenergetic sunflower oil) during 2 weeks of unilateral leg immobilization and 2 weeks of remobilization. Vastus lateralis biopsies were taken for electron microscopic analysis of mitochondrial content. Subsarcolemmal (SS) mitochondrial content decreased during immobilization (control: -9%, n-3: -66%, p = 0.009) and remained lower following recovery (control: -41%, n-3: -42%, p = 0.005). This effect was driven by the n-3 group (p < 0.02). Intermyofibrillar (IMF) mitochondrial content did not decline during immobilization, but was lower than baseline following recovery in the central (p = 0.01) IMF. The effects of leg immobilization on mitochondrial content differ by location, are not reversed with short-term recovery, and are influenced by n-3 supplementation.

Keywords: electron microscopy; mitochondria; muscle disuse; omega‐3 fatty acid; skeletal muscle.

FIGURE 1

A schematic diagram of experimental design.

FIGURE 2

Effects of immobilization and recovery on mitochondrial content. (a) Representative images prior to immobilization (D0, left), after 2‐weeks of immobilization (D14, middle), and after 2 weeks of recovery (D28, right)from a control individual; (b–d) box and whisker plots showing mitochondrial area density (%) in controls (n = 5) and omega‐3 FA (n = 7) supplemented individuals prior to immobilization (D0), after 2‐weeks of immobilization (D14) and after 2 weeks of recovery (D28) in the (B) (SSC) peripheral IMF and (d) central IMF. Comparisons made with 2‐way mixed model ANOVA; (e) correlational analyses showing the negative relationship between baseline SS mitochondrial content and the change in SS mitochondrial content induced by disuse. For panels b–d the line within the confines of the boxplot represents the median and the cross represents the mean, while the whiskers and borders of the boxplot represent the extreme values and interquartile range (IQR), respectively. AD, area density; FA, fatty acid; IMF, intermyofibrillar; SS, subsarcolemmal.

FIGURE 3

Box and whisker plots showing proportion of mitochondria (%) in contact with IMCL in the (a) SS, (b) peripheral IMF, and (c) central IMF regions prior to immobilization (D0), after 2 weeks of immobilization (D14) and after 2 weeks of recovery (D28) in controls (n = 5) and omega‐3 FA supplemented individuals (n = 7). Comparisons made with 2‐way mixed model ANOVA. Central IMF mitochondria‐IMCL apposition data (3C) was not able to be transformed for normal distribution. The line within the confines of the boxplot represents the median and the cross represents the mean, while the whiskers and borders of the boxplot represent the extreme values and interquartile range (IQR), respectively. AD, area density, FA, fatty acid, IMCL, intramyocellular lipid; IMF: Intermyofibrillar; SS, subsarcolemmal.

FIGURE 4

Representative blots and their accompanying box and whisker plots showing the content of autophagic proteins prior to immobilization (D0), after 2 weeks of immobilization (D14), and after 2 weeks of recovery (D28) in controls (n = 9) and omega‐3 FA supplemented individuals (n = 11). (a) SQSTM1; (b) BNIP3; and (c) LC3B‐I. Comparisons made with 2‐way mixed model ANOVA. The line within the confines of the boxplot represents the median, and the cross represents the mean, while the whiskers and borders of the boxplot represent the extreme values and interquartile range (IQR), respectively. Participants were staggered for supplementation, and all samples were run alongside a standard curve from a pool of all samples on each gel. D0, Day 0, prior to immobilization; D14, Day 14, post‐2 weeks of immobilization; D28, Day 28, post‐2 weeks of recovery; FA, Fatty acid; P5, 5 μg pooled sample, P10, 10 μg pooled sample, P20, 20 μg pooled sample. For full blots with marker ladder proteins, please see Figure S1A.

FIGURE 5

Representative blots and accompanying box and whisker plots showing content of intrinsic apoptotic proteins at baseline (D0), following 2‐weeks of immobilization (D14) and 2‐weeks of recovery (D28) in controls (n = 9) and omega‐3 FA supplemented individuals (n = 11). (a) procaspase‐3; (b) procaspase‐9; (c) Bcl‐2; (d) BAXand (e) BAX:Bcl‐2. Comparisons made with 2‐way mixed model ANOVA. Procaspase‐3 and BAX:Bcl‐2 data were transformed to achieve normality prior to ANOVA. The line inside the boxplot represents the median, and the cross represents the mean, while the whiskers and borders of the boxplot represent the extreme values and interquartile range (IQR), respectively. Participants were staggered for supplementation, and all samples were run alongside a standard curve from a pool of all samples on each gel. D0, Day 0, prior to immobilization; D14, Day 14, post‐2 weeks of immobilization; D28, Day 28, post‐2 weeks of recovery; FA, fatty acid; P5, 5 μg pooled sample; P10, 10 μg pooled sample; P20, 20 μg pooled sample. For full blots with marker ladder proteins, please see Figure S1B.

FIGURE 6

Representative blots and accompanying box and whisker plots showing protein content of mitochondrial fission and fusion proteins at baseline (D0), following 2‐weeks of immobilization (D14) and 2‐weeks of recovery (D28) in controls (n = 9) and omega‐3 FA supplemented individuals (n = 11). Comparisons made with 2‐way mixed model ANOVA. OPA1 data was not able to be transformed for normal distribution. (a) OPA1, (b) Fis1, and (c) ratio of OPA1 to Fis1. The line within the confines of the boxplot represents the median and the cross represents the mean, while the whiskers and borders of the boxplot represent the extreme values and interquartile range (IQR), respectively. Participants were staggered for supplementation, and all samples were run alongside a standard curve from a pool of all samples on each gel. D0, Day 0, prior to immobilization; D14, Day 14, post‐2 weeks of immobilization; D28, Day 28, post‐2 weeks of recovery; FA, fatty acid; P3, 3 μg pooled sample; P5, 5 μg pooled sample; P10, 10 μg pooled sample. For full blots with marker ladder proteins, please see Figure S1C.