Exercise protects against doxorubicin-induced markers of autophagy signaling in skeletal muscle

Doxorubicin (DOX) is an effective antitumor agent used in cancer treatment. Unfortunately, DOX is also toxic to skeletal muscle and can result in significant muscle wasting. The cellular mechanism(s) by which DOX induces toxicity in skeletal muscle fibers remains unclear. Nonetheless, DOX-induced toxicity is associated with increased generation of reactive oxygen species, oxidative damage, and activation of the calpain and caspase-3 proteolytic systems within muscle fibers. It is currently unknown if autophagy, a proteolytic system that can be triggered by oxidative stress, is activated in skeletal muscles following DOX treatment. Therefore, we tested the hypothesis that systemic administration of DOX leads to increased expression of autophagy markers in the rat soleus muscle. Our results reveal that DOX administration results in increased muscle mRNA levels and/or protein abundance of several important autophagy proteins, including: Beclin-1, Atg12, Atg7, LC3, LC3II-to-LCI ratio, and cathepsin L. Furthermore, given that endurance exercise increases skeletal muscle antioxidant capacity and protects muscle against DOX-induced oxidative stress, we performed additional experiments to determine whether exercise training before DOX administration would attenuate DOX-induced increases in expression of autophagy genes. Our results clearly show that exercise can protect skeletal muscle from DOX-induced expression of autophagy genes. Collectively, our findings indicate that DOX administration increases the expression of autophagy genes in skeletal muscle, and that exercise can protect skeletal muscle against DOX-induced activation of autophagy.     

Evidence for mitochondrial thioesterase 1 as a peroxisome proliferator-activated receptor-alpha-regulated gene in cardiac and skeletal muscle

The physiological role of mitochondrial thioesterase 1 (MTE1) is unknown. It was proposed that MTE1 promotes fatty acid (FA) oxidation (FAO) by acting in concert with uncoupling protein (UCP)3. We previously showed that ucp3 is a peroxisome proliferator-activated receptor-alpha (PPAR alpha)-regulated gene, allowing induction when FA availability increases. On the assumption that UCP3 and MTE1 act in partnership to increase FAO, we hypothesized that mte1 is also a PPAR alpha-regulated gene in cardiac and skeletal muscle. Using real-time RT-PCR, we characterized mte1 gene expression in rat heart and soleus muscles. Messenger RNA encoding for mte1 was 3.2-fold higher in heart than in soleus muscle. Cardiac mte1 mRNA exhibited modest diurnal variation, with 1.4-fold higher levels during dark phase. In contrast, skeletal muscle mte1 mRNA remained relatively constant over the course of the day. High-fat feeding, fasting, and streptozotocin-induced diabetes, interventions that increase FA availability, muscle PPAR alpha activity, and muscle FAO rates, increased mte1 mRNA in heart and soleus muscle. Conversely, pressure overload and hypoxia, interventions that decrease cardiac PPAR alpha activity and FAO rates, repressed cardiac mte1 expression. Specific activation of PPAR alpha in vivo through WY-14643 administration rapidly induced mte1 mRNA in cardiac and skeletal muscle. WY-14643 also induced mte1 mRNA in isolated adult rat cardiomyocytes dose dependently. Expression of mte1 was markedly lower in hearts and soleus muscles isolated from PPAR alpha-null mice. Alterations in cardiac and skeletal muscle ucp3 expression mirrored that of mte1 in all models investigated. In conclusion, mte1, like ucp3, is a PPAR alpha-regulated gene in cardiac and skeletal muscle.     

Temporal and spatial patterns of gene expression in skeletal muscles in response to swim training in adult zebrafish (Danio rerio)

In adult zebrafish, 4 weeks of exercise training is known to induce an increase in mitochondrial enzymes such as citrate synthase (CS) when determined in mixed (red and white) muscle. However, this remodeling is not accompanied by changes in PGC-1α mRNA, a potent inducer of mitochondrial biogenesis in mammals. To further understand this response, we examined absolute and relative changes in red muscle area by histochemistry after 4 weeks of swim training. We also examined fiber-type specific responses in the expression of metabolic genes and putative regulators in red and white muscle of adult zebrafish at 1 and 8 weeks of training and in recovery from a single bout of exercise. Total red muscle area was unaltered after 4 weeks of training. The mRNA expression of CS was unaffected in red muscle, while it was increased in white muscle after 1 week of training and remained elevated at 8 weeks of training, suggesting an increase in oxidative capacity of this fiber type. In contrast, PGC-1α mRNA was elevated in both muscles only after 1 week of training. In both muscles, an acute bout of exercise rapidly (within 0–2 h post-exercise) induced PGC-1α mRNA and a delayed (24 h) increase in CS mRNA post-exercise. These results suggest complex temporal and spatial adaptive molecular responses to exercise in the skeletal muscles of zebrafish.     

The nuclear receptor, Nor-1, markedly increases type II oxidative muscle fibers and resistance to fatigue

Nuclear hormone receptors (NR) have been implicated as regulators of lipid and carbohydrate metabolism. The orphan NR4A subgroup has emerged as regulators of metabolic function. Targeted silencing of neuron-derived orphan receptor 1 (Nor-1)/NR4A3 in skeletal muscle cells suggested that this NR was necessary for oxidative metabolism in vitro. To investigate the in vivo role of Nor-1, we have developed a mouse model with preferential expression of activated Nor-1 in skeletal muscle. In skeletal muscle, this resulted in a marked increase in: 1) myoglobin expression, 2) mitochondrial DNA and density, 3) oxidative enzyme staining, and 4) genes/proteins encoding subunits of electron transport chain complexes. This was associated with significantly increased type IIA and IIX myosin heavy chain mRNA and proteins and decreased type IIB myosin heavy chain mRNA and protein. The contractile protein/fiber type remodeling driving the acquisition of the oxidative type II phenotype was associated with 1) the significantly increased expression of myocyte-specific enhancer factor 2C, and phospho-histone deacetylase 5, and 2) predominantly cytoplasmic HDAC5 staining in the Tg-Nor-1 mice. Moreover, the Nor-1 transgenic line displayed significant improvements in glucose tolerance, oxygen consumption, and running endurance (in the absence of increased insulin sensitivity), consistent with increased oxidative capacity of skeletal muscle. We conclude that skeletal muscle fiber type is not only regulated by exercise-sensitive calcineurin-induced signaling cascade but also by NR signaling pathways that operate at the nexus that coordinates muscle performance and metabolic capacity in this major mass tissue.     

The effect of chronic ethanol ingestion on protein metabolism in type-I- and type-II-fibre-rich skeletal muscles of the rat.

1. The effects of chronic ethanol feeding on muscles containing a predominance of either Type I (aerobic, slow-twitch) or Type II (anaerobic, fast-twitch) fibres were studied. Male Wistar rats, weighing approx. 90 g or 280 g, were pair-fed on a nutritionally complete liquid diet containing 36% of total energy as ethanol, or isovolumetric amounts of the same diet in which ethanol was replaced by isoenergetic glucose. After 6 weeks feeding, fractional rates of protein synthesis were measured with a flooding dose of L-[4-(3)H]-phenylalanine and muscles were analysed for protein, RNA and DNA. 2. Ethanol feeding decreased muscle weight, protein, RNA and DNA contents in both small and large rats. Type-II-fibre-rich muscles showed greater changes than did Type-I-fibre-rich muscles. Changes in protein paralleled decreases in DNA. 3. The capacity for protein synthesis (RNA/protein), fractional rates of protein synthesis and absolute rates of protein synthesis were decreased by ethanol feeding in both small and large rats. The amounts of protein synthesized relative to RNA and DNA were also decreased. Changes were less marked in Type-I than in Type-II-fibre-rich muscles. Loss of protein, RNA and DNA was greater in small rats, but protein synthesis was more markedly affected in large rats. 4. It was concluded that chronic ethanol feeding adversely affects protein metabolism in skeletal muscle. Fibre composition and animal size are also important factors in determining the pattern of response.     

Bed rest reduces metabolic protein content and abolishes exercise-induced mRNA responses in human skeletal muscle

The aim was to test the hypothesis that 7 days of bed rest reduces mitochondrial number and expression and activity of oxidative proteins in human skeletal muscle but that exercise-induced intracellular signaling as well as mRNA and microRNA (miR) responses are maintained after bed rest. Twelve young, healthy male subjects completed 7 days of bed rest with vastus lateralis muscle biopsies taken before and after bed rest. In addition, muscle biopsies were obtained from six of the subjects prior to, immediately after, and 3 h after 45 min of one-legged knee extensor exercise performed before and after bed rest. Maximal oxygen uptake decreased by 4%, and exercise endurance decreased nonsignificantly, by 11%, by bed rest. Bed rest reduced skeletal muscle mitochondrial DNA/nuclear DNA content 15%, hexokinase II and sirtuin 1 protein content ～45%, 3-hydroxyacyl-CoA dehydrogenase and citrate synthase activity ～8%, and miR-1 and miR-133a content ～10%. However, cytochrome c and vascular endothelial growth factor (VEGF) protein content as well as capillarization did not change significantly with bed rest. Acute exercise increased AMP-activated protein kinase phosphorylation, peroxisome proliferator activated receptor-γ coactivator-1α, and VEGF mRNA content in skeletal muscle before bed rest, but the responses were abolished after bed rest. The present findings indicate that only 7 days of physical inactivity reduces skeletal muscle metabolic capacity as well as abolishes exercise-induced adaptive gene responses, likely reflecting an interference with the ability of skeletal muscle to adapt to exercise.