miRNA in the Regulation of Skeletal Muscle Adaptation to Acute Endurance Exercise in C57Bl/6J Male Mice

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNA species involved in post-transcriptional gene regulation. In vitro studies have identified a small number of skeletal muscle-specific miRNAs which play a crucial role in myoblast proliferation and differentiation. In skeletal muscle, an acute bout of endurance exercise results in the up-regulation of transcriptional networks that regulate mitochondrial biogenesis, glucose and fatty acid metabolism, and skeletal muscle remodelling. The purpose of this study was to assess the expressional profile of targeted miRNA species following an acute bout of endurance exercise and to determine relationships with previously established endurance exercise responsive transcriptional networks. C57Bl/6J wild-type male mice (N = 7/group) were randomly assigned to either sedentary or forced-endurance exercise (treadmill run @ 15 m/min for 90 min) group. The endurance exercise group was sacrificed three hours following a single bout of exercise. The expression of miR- 181, 1, 133, 23, and 107, all of which have been predicted to regulate transcription factors and co-activators involved in the adaptive response to exercise, was measured in quadriceps femoris muscle. Endurance exercise significantly increased the expression of miR-181, miR-1, and miR-107 by 37%, 40%, and 56%, respectively, and reduced miR-23 expression by 84% (P≤0.05 for all), with no change in miR-133. Importantly, decreased expression of miRNA-23, a putative negative regulator of PGC-1α was consistent with increased expression of PGC-1α mRNA and protein along with several downstream targets of PGC-1α including ALAS, CS, and cytochrome c mRNA. PDK4 protein content remains unaltered despite an increase in its putative negative regulator, miR-107, and PDK4 mRNA expression. mRNA expression of miRNA processing machinery (Drosha, Dicer, and DGCR8) remained unchanged. We conclude that miRNA-mediated post-transcriptional regulation is potentially involved in the complex regulatory networks that govern skeletal muscle adaptation to endurance exercise in C57Bl/6J male mice

Limb Immobilization Induces a Coordinate Down-Regulation of Mitochondrial and Other Metabolic Pathways in Men and Women

Advancements in animal models and cell culture techniques have been invaluable in the elucidation of the molecular mechanisms that regulate muscle atrophy. However, few studies have examined muscle atrophy in humans using modern experimental techniques. The purpose of this study was to examine changes in global gene transcription during immobilization-induced muscle atrophy in humans and then explore the effects of the most prominent transcriptional alterations on protein expression and function. Healthy men and women (N = 24) were subjected to two weeks of unilateral limb immobilization, with muscle biopsies obtained before, after 48 hours (48 H) and 14 days (14 D) of immobilization. Muscle cross sectional area (∼5%) and strength (10–20%) were significantly reduced in men and women (∼5% and 10–20%, respectively) after 14 D of immobilization. Micro-array analyses of total RNA extracted from biopsy samples at 48 H and 14 D uncovered 575 and 3,128 probes, respectively, which were significantly altered during immobilization. As a group, genes involved in mitochondrial bioenergetics and carbohydrate metabolism were predominant features at both 48 H and 14 D, with genes involved in protein synthesis and degradation significantly down-regulated and up-regulated, respectively, at 14 D of muscle atrophy. There was also a significant decrease in the protein content of mitochondrial cytochrome c oxidase, and the enzyme activity of cytochrome c oxidase and citrate synthase after 14 D of immobilization. Furthermore, protein ubiquitination was significantly increased at 48 H but not 14 D of immobilization. These results suggest that transcriptional and post-transcriptional suppression of mitochondrial processes is sustained throughout 14 D of immobilization, while protein ubiquitination plays an early but transient role in muscle atrophy following short-term immobilization in humans

PDK4 content and miR-107 expression following exercise.

<p>(A) PDK4 mRNA expression and (B) protein content, and (C) miR-107 expression in the <i>quadriceps</i> of C57Bl/6J mice (N = 7/group) 3-hour following an acute bout of END exercise vs. SED group. PDK4 mRNA expression, protein content and miR-107 expression are normalized to β-2 microglobulin, actin and <i>Rnu6</i>, respectively. Asterisks denote significant changes (P≤0.05).</p

Real-time PCR primer sequences.

<p>Real-time PCR primer sequences.</p

miR-1 and miR-181 expression following exercise.

<p>miR-1 and miR-181 expression in the <i>quadriceps</i> of C57Bl/6J mice (N = 7/group) 3-hour following an acute bout of END exercise vs. SED group. miR-1 and miR-181 expression are normalized to <i>Rnu6</i>. Asterisks denote significant changes (P≤0.05).</p

PGC-1α content and miR-23 expression following exercise.

<p>PGC-1α (A) mRNA expression and (B) protein content, and (C) miR-23 expression in the <i>quadriceps</i> of C57Bl/6J mice (N = 7/group) 3-hour following an acute bout of END exercise vs. SED group. (D) PGC-1α protein content negatively correlates (R = 0.62) with miR-23 content. PGC-1α mRNA expression, protein content and miR-23 expression are normalized to β-2 microglobulin, actin and <i>Rnu6</i>, respectively. Asterisks denote significant changes (P≤0.05).</p

Supplementation with α-lipoic acid, CoQ10, and vitamin E augments running performance and mitochondrial function in female mice.

Antioxidant supplements are widely consumed by the general public; however, their effects of on exercise performance are controversial. The aim of this study was to examine the effects of an antioxidant cocktail (α-lipoic acid, vitamin E and coenzyme Q10) on exercise performance, muscle function and training adaptations in mice. C57Bl/J6 mice were placed on antioxidant supplement or placebo-control diets (n = 36/group) and divided into trained (8 wks treadmill running) (n = 12/group) and untrained groups (n = 24/group). Antioxidant supplementation had no effect on the running performance of trained mice nor did it affect training adaptations; however, untrained female mice that received antioxidants performed significantly better than placebo-control mice (p ≤ 0.05). Furthermore, antioxidant-supplemented females (untrained) showed elevated respiratory capacity in freshly excised muscle fibers (quadriceps femoris) (p ≤ 0.05), reduced oxidative damage to muscle proteins (p ≤ 0.05), and increased expression of mitochondrial proteins (p ≤ 0.05) compared to placebo-controls. These changes were attributed to increased expression of proliferator-activated receptor gamma coactivator 1α (PGC-1α) (p ≤ 0.05) via activation of AMP-activated protein kinase (AMPK) (p ≤ 0.05) by antioxidant supplementation. Overall, these results indicate that this antioxidant supplement exerts gender specific effects; augmenting performance and mitochondrial function in untrained females, but does not attenuate training adaptations

Elevated Mitochondrial Oxidative Stress Impairs Metabolic Adaptations to Exercise in Skeletal Muscle

<div><p>Mitochondrial oxidative stress is a complex phenomenon that is inherently tied to energy provision and is implicated in many metabolic disorders. Exercise training increases mitochondrial oxidative capacity in skeletal muscle yet it remains unclear if oxidative stress plays a role in regulating these adaptations. We demonstrate that the chronic elevation in mitochondrial oxidative stress present in <i>Sod2</i>^<i>+/-</i> mice impairs the functional and biochemical mitochondrial adaptations to exercise. Following exercise training <i>Sod2</i>^<i>+/-</i> mice fail to increase maximal work capacity, mitochondrial enzyme activity and mtDNA copy number, despite a normal augmentation of mitochondrial proteins. Additionally, exercised <i>Sod2</i>^<i>+/-</i> mice cannot compensate for their higher amount of basal mitochondrial oxidative damage and exhibit poor electron transport chain complex assembly that accounts for their compromised adaptation. Overall, these results demonstrate that chronic skeletal muscle mitochondrial oxidative stress does not impact exercise induced mitochondrial biogenesis, but impairs the resulting mitochondrial protein function and can limit metabolic plasticity.</p> </div

Mitochondrial complex assembly worsens in <i>Sod2</i>^<i>+/-</i> mice subjected to exercise.

<p>Lauryl-maltoside solubilized mitochondria were separated using two-dimensional Blue-Native PAGE in order to visualize native migration of each of four electron transport chain complexes. A lack of intermediate species in assembling the native complex is visualized as a shorter horizontal migration, while unassembled complexes are evident as tails or very broad bands. Note the divergent response to exercise between the two genotypes. Samples were run simultaneously on the same native gel. Image frames are identically sized for each complex and were acquired from the same film exposure. This experiment was repeated twice using different samples from each of the four groups in order to confirm that this was a reproducible observation.</p

Mitochondrial function is compromised with exercise training in <i>Sod2</i>^<i>+/-</i> mice without influencing organelle biogenesis.

<p>Total protein lysates of tibialis anterior muscle were probed by immunoblotting for individual complex subunits (I: NDUFB8, II: SDHB, III: UQCRC2, IV: COXI, V: ATP5A) or assayed for enzyme activity. (A) The protein expression of mitochondrial proteins determined by densitometry relative to LDHA, (B) representative immunoblots of mitochondrial proteins and (C) enzyme activities of citrate synthase (CS), complex I+III, and complex IV (CIV) per unit of protein (<i>n</i> = 8-10). Data are expressed relative to the Sod2^+/+ SED group. Mitochondrial respiration rates in saponin permeabilized muscle fibers from quadriceps femoris muscle using (D) glutamate and malate without adenylates to indicate a respiratory leak state, (E) glutamate, malate, succinate and ADP to measure maximal coupled oxidative phosphorylation capacity and (F) respiratory coupling ratio of ADP stimulated and non-stimulated respiration using glutamate and malate as substrates (<i>n</i> = 4-5). (G) UCP3 and (H) ANT1 protein expression in isolated mitochondrial lysates from quadriceps femoris muscle relative to VDAC and (I) representative immunoblots for each group (<i>n</i> = 8-10). All data are mean±SE. *Indicates a significant difference (<i>p</i> < 0.05) from the indicated group as determined by 2-way ANOVA. <b><i>#</i></b>Indicates a main effect of exercise training (<i>p</i> < 0.05). <b><i>^†</i></b>Indicates a main effect of genotype (<i>p</i> < 0.05). NS, non-significant.</p