Proteins In Rat Skeletal Muscle Research Articles

Exercise-induced FKBP8-mediated Mitophagy Via AMPK Activation In Soleus Muscle Of Rats

Scientific Abstract PURPOSE: To investigate the effects of downhill running on mitochondrial structure/function and expression levels of mitophagy-related proteins in rat skeletal muscle, and whether AMP-activated protein kinase (AMPK) plays a regulatory role in this context. METHODS: Male Sprague-Dawley rats were divided into a control (C, n = 8) or an exercise (E, n = 56) group. Rats in the E group were exercised on a treadmill down a 16° incline at 16 m/min for 90 min, and were further divided into 0 h (E0), 12 h (E12), 24 h (E24), 48 h (E48) and 72 h (E72) post-exercise sub-groups. Rats in the E0 and E12 sub-groups received acute AMPK agonist AICAR treatment (500 mg/kg via I.P.) or saline (n = 8 each) 30 min before exercise. At each time point the soleus muscle was collected under full anesthesia. Mitochondrial ultrastructural changes in skeletal muscle were observed by using a transmission electron microscope. Protein expression levels of skeletal muscle p-AMPKα1/2(T172), total AMPKα, heat shock protein 60 (HSP60), cyclophilin D (CypD), mitochondrial FK506 binding protein 8 (FKBP8/FKBP38), microtubule-associated protein 1 light chain 3 (LC3) and cytosolic cytochrome C (Cyt C) were determined by using Western blot. LC3 co-localizations with mitochondria and FKBP8 were measured by the immunofluorescence technique. One-way and two-way ANOVAs were used for data analysis. RESULTS: After downhill running, skeletal muscle mitochondrial structure appeared to be abnormal and contained a large number of mitophagosomes; the expression levels of AMPK phosphorylation (E12 = 1.81 ± 0.54), FKBP8 (E12 = 3.11 ± 0.26), LC3 (E12 = 1.80 ± 0.12), CypD (E0 = 1.41 ± 0.26) and Cyt C (E0 = 2.31 ± 0.68) and the co-localization of FKBP8 and LC3 were significantly higher, whereas the expression levels of mitochondrial quantitative protein HSP60 (E12 = 0.62 ± 0.09) were significantly lower than those in the C group (p < 0.05 to p < 0.01). AICAR treatment enhanced AMPK phosphorylation in exercised muscle and the expression levels of mitochondrial FKBP8 and LC3 involved in mitophagy. CONCLUSION: A session of downhill running activated the AMPK phosphorylation and LC3 co-localizations with mitochondria and FKBP8, and induced mitophagy and mitochondrial damage within rat skeletal muscle. (Supported by Natural Science Foundation of China grant 31900842).

The effect of a bout of resistance exercise on skeletal muscle protein metabolism after severe fasting.

Resistance exercise (RE) activates the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway and increases muscle protein synthesis. Severe fasting induces 5′ adenosine monophosphate‐activated protein kinase (AMPK), which attenuates mTORC1 activation. However, the effect of RE on the response of mTORC1 signaling proteins after a period of severe fasting is unclear. We investigated the effect of RE on rat skeletal muscle protein metabolism after a period of severe fasting. We hypothesized that RE‐induced activation of mTORC1 signaling protein attenuates protein breakdown by autophagy. Male Sprague‐Dawley rats were divided into ordinary‐fed (C) and 72‐h fasting (F) groups. A bout of RE was replicated by percutaneous electrical stimulation in the right gastrocnemius muscle. The tuberous sclerosis complex 2 (TSC2) Ser1387 and autophagy marker of microtubule‐associated protein 1A/1B‐light chain 3‐II (LC3B‐II) expression of the F group increased twice that of the C group in sedentary state (P < 0.05). RE activated the mTORC1 signaling pathway in both groups (P < 0.05); however, in the F group, the magnitude of p70S6K (Thr389) phosphorylation was lower by 40% of that of the C group (P < 0.05). Protein synthesis after RE was increased by 50% from the level at sedentary state in the C group (P < 0.05), but not in the F. In the F group, the expression of LC3B‐II at 3 h after RE was decreased by almost 25% from the level at sedentary state (P < 0.05). Our results suggest that RE suppressed fasting‐induced autophagy but did not increase protein synthesis during severe fasting in rat skeletal muscle.

Effects of clenbuterol administration on mitochondrial morphology and its regulatory proteins in rat skeletal muscle.

Clenbuterol induces a slow‐to‐fast fiber type transition in skeletal muscle. This muscle fiber transition decreased mitochondrial oxidative capacity and respiratory function. We hypothesized that the clenbuterol‐mediated reduction in oxidative capacity is associated with the alteration in mitochondrial morphology. To verify this hypothesis, we examined whether clenbuterol alters mitochondrial morphology and mitochondrial regulatory proteins in rat skeletal muscle. Clenbuterol was administered to rats via drinking water (30 mg/L) for 3 weeks. Myosin heavy chain (MHC) isoform composition, mitochondrial morphology, and fusion and fission regulatory protein levels in deep region and superficial region in tibialis anterior (TA) muscles were assessed. Clenbuterol induced the fiber type transition from slow to fast in both the regions of TA. The levels of optic atrophy protein 1, mitofusin 2, and mitochondrial fission 1, but not of dynamin‐related protein 1, significantly decreased in deep and superficial muscles after clenbuterol administration (P < 0.01). Also, observation using the transmission electron microscopy showed a decrease in mitochondrial volume (P < 0.05) and an increase in proportion of continuous or interacting mitochondria across Z‐lines (P < 0.05). We showed that clenbuterol administration induces a transition in the muscle fiber type composition toward fast phenotype and causes alterations in mitochondrial morphology with a concomitant decrease in mitochondrial fusion and fission regulatory protein levels. These mitochondrial morphological alterations may influence deleterious effects on skeletal muscle metabolism.

Downhill Running Acutely Elicits Mitophagy in Rat Soleus Muscle.

This study aimed to investigate the effects of downhill treadmill running on mitochondrial structure/function and expression levels of mitophagy-related proteins in rat skeletal muscle. A total of 48 male adult Sprague-Dawley rats were randomly divided into a control group (C, n = 8) and an exercise group (E, n = 40). Rats in the E group were exercised on a treadmill down a 16° decline at 16 m·min for 90 min and were further divided into 0 h (E0), 12 h (E12), 24 h (E24), 48 h (E48), and 72 h (E72) postexercise subgroups (n = 8 each). At each time point, the soleus muscle was collected under full anesthesia. Mitochondrial ultrastructural changes in skeletal muscle were observed by a transmission electron microscope. The content of quantitative enzyme citrate synthase and the activities of mitochondrial respiratory chain complex II and complex IV were measured by enzyme-linked immunosorbent assay. Protein expressions of skeletal muscle cytochrome c oxidase subunit 1 (COX1), PTEN-induced putative kinase 1 (PINK1), and mitochondrial Parkin microtubule-associated protein 1 light chain 3 (LC3) were determined by Western blot. Mitochondrial colocalizations with Parkin, ubiquitin (Ub), p62/sequestosome 1 (p62), and LC3 were measured by the immunofluorescence double labeling technique. After downhill treadmill running, the skeletal muscle mitochondrial structure changed dramatically, and a large amount of mitophagosomes were observed; the citrate synthase content and complex II activity were significantly lower (P < 0.05), whereas complex IV activity and COX1 protein level remained unchanged; the expression levels of PINK1, Parkin, Ub, p62, and LC3 were significantly higher than those in the C group (P < 0.05 or P < 0.01). A session of downhill treadmill running activated the PINK1/Parkin pathway and facilitated mitochondrial colocalizations with Ub, p62, and LC3, causing mitophagy and mitochondrial damage within the skeletal muscle.

Palmitate- and C6 ceramide-induced Tnnt3 pre-mRNA alternative splicing occurs in a PP2A dependent manner

BackgroundIn a previous study, we showed that consumption of diets enriched in saturated fatty acids causes changes in alternative splicing of pre-mRNAs encoding a number of proteins in rat skeletal muscle, including the one encoding skeletal muscle Troponin T (Tnnt3). However, whether saturated fatty acids act directly on muscle cells to modulate alternative pre-mRNA splicing was not assessed. Moreover, the signaling pathway through which saturated fatty acids act to promote changes in alternative splicing is unknown. Therefore, the objective of the present study was to characterize the signaling pathway through which saturated fatty acids act to modulate Tnnt3 alternative splicing.MethodsThe effects of treatment of L6 myotubes with saturated (palmitate), mono- (oleate), or polyunsaturated (linoleate) fatty acids on alternative splicing of pre-mRNA was assessed using Tnnt3 as a marker gene.ResultsPalmitate treatment caused a two-fold change (p < 0.05) in L6 myotube Tnnt3 alternative splicing whereas treatment with either oleate or linoleate had minimal effects compared to control myotubes. Treatment with a downstream metabolite of palmitate, ceramide, had effects similar to palmitate on Tnnt3 alternative splicing and inhibition of de novo ceramide biosynthesis blocked the palmitate-induced alternative splicing changes. The effects of palmitate and ceramide on Tnnt3 alternative splicing were accompanied by a 40–50% reduction in phosphorylation of Akt on S473. However, inhibition of de novo ceramide biosynthesis did not prevent palmitate-induced Akt dephosphorylation, suggesting that palmitate may act in an Akt-independent manner to modulate Tnnt3 alternative splicing. Instead, pre-treatment with okadaic acid at concentrations that selectively inhibit protein phosphatase 2A (PP2A) blocked both palmitate- and ceramide-induced changes in Tnnt3 alternative splicing, suggesting that palmitate and ceramide act through PP2A to modulate Tnnt3 alternative splicing.ConclusionsOverall, the data show that fatty acid saturation level and ceramides are important factors modulating alternative pre-mRNA splicing through activation of PP2A.

PO-242 Effects of tail suspension on the expression of FNDC5/Irisin protein in rat skeletal muscle

Objective Irisin is a myokine secreted by skeletal muscle,and it is a type I membrane protein factor encoded by the protein 5(FNDC5) gene after cleavage and modification of the type III fibronectin component.Dependence of peroxisome proliferator-activated receptor gamma coactivator (PGC-1α).In this study, the potential association between skeletal muscle atrophy and irisin was explored by detecting changes in rat soleus and gastrocnemius irisin-related proteins during unloading.&#x0D; Methods Twenty male 8-week rats were randomly divided into control group C (n=10) and suspension group T (n=10). The tail suspension system (TSS) was used to perform a 2-week tail suspension experiment on the T group. Two weeks after the tail suspension test, the weights of the rats and the wet weights of soleus and gastrocnemius muscles were measured. HE staining was performed under light microscope to observe the changes of muscle fiber area of skeletal muscle in each group. Western-blot was used to detect the protein expression of MURF1, PGC-1α and FNDC5 in soleus muscle and gastrocnemius muscle of each group.&#x0D; Results (1) The soleus muscle and gastrocnemius muscle mass in T group decreased by 28.6% (P&lt;0.05) and 25.8% (P&lt;0.01), respectively. (2) The cross-sectional area of soleus muscle and gastrocnemius muscle fiber in T group decreased by 20.5% (P&lt;0.01) and 25.2% (P&lt;0.05), respectively. (3) The MURF1 protein expression in the gastrocnemius muscle and soleus muscle in the T group was significantly higher than that in the C group (P&lt;0.01). (4) The expression of PGC-1α protein in gastrocnemius muscle and soleus muscle of T group was significantly lower than that in group C (P&lt;0.05). (5) The expression of FNDC5 protein in gastrocnemius muscle and soleus muscle in T group was significantly lower than that in group C (P&lt;0.05).&#x0D; Conclusions After sole tail suspension for two weeks, the soleus and gastrocnemius muscles of the rats were obviously atrophied, and soleus muscle atrophy was more obvious. Skeletal muscle atrophy may be related to increased expression of MURF1. The decrease of FNDC5/Irisin content may be related to the occurrence of skeletal muscle atrophy, and PGC-1α also may be involved in this process.

Effect of Aluminum Exposure on Glucose Metabolism and Its Mechanism in Rats.

The effects of aluminum (Al) exposure on glucose metabolism and its mechanism were investigated. A total of 30 healthy Wistar male rats were randomly divided into two groups: control (GC) and experimental (GE). The GC group received intraperitoneal normal saline. The GE was established by intraperitoneal injected AlCl3 solution at 10mg/kg for 30days. Fasting blood glucose (FBG) and serum levels of insulin (FINS) were measured. The insulin resistance index (HOMA-IR) and pancreatic β cell function index (HOMA-β) were calculated and analyzed with homeostasis model assessment (HOMA). Pancreatic tissue was taken for pathological examination. Glucose transporter 4 (GLUT4) expression in skeletal muscle was detected by quantitative PCR and Western blot. Levels of FBG and HOMA-IR in GE were higher than those in GC at day 10 and 20 (P < 0.05). FINS in GE were higher than those in GC at day 10 and 20, and lower than those in GC at day 30 (P < 0.05). HOMA-β in GE was lower than that of GC at every time point (P < 0.05). Pathology showed that pancreatic damage changed more profoundly with prolongation of time in GE. Expression levels of GLUT4 mRNA and protein in rat skeletal muscle in GE were significantly lower than those in GC (P < 0.05). The results suggested that Al exposure affected glucose metabolism through pancreatic damage and reduction of GLUT4 expression.

Combined effects of resistance training and calorie restriction on mitochondrial fusion and fission proteins in rat skeletal muscle.

Recent studies have demonstrated that resistance exercise leads not only to muscle hypertrophy, but it also improves mitochondrial function. Because calorie restriction (CR) has been suggested as a way to induce mitochondrial biogenesis, we examined the effects of resistance training with or without CR on muscle weight and key mitochondrial parameters in rat skeletal muscle. Four weeks of resistance training (thrice/wk) resulted in increased gastrocnemius muscle weight by 14% in rats fed ad libitum (AL). The degree of muscle-weight increase via resistance training was lower in rats with CR (7.4%). CR showed no effect on phosphorylation of mammalian target of rapamycin (mTOR) signaling proteins rpS6 and ULK1. Our results revealed that CR resulted in elevated levels of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) protein, a known master regulator of mitochondrial biogenesis. Resistance training alone also resulted in increased PGC-1α levels in skeletal muscle. The magnitude of the increase in PGC-1α was similar in rats in both the CR and AL groups. Moreover, we found that resistance training with CR resulted in elevated levels of proteins involved in mitochondrial fusion (Opa1 and Mfn1), and oxidative phosphorylation, whereas there was no effect of CR on the fission-regulatory proteins Fis1 and Drp1. These results indicate that CR attenuates resistance training-induced muscle hypertrophy, and that it may enhance mitochondrial adaptations in skeletal muscle.

Heat stress acutely activates insulin-independent glucose transport and 5'-AMP-activated protein kinase prior to an increase in HSP72 protein in rat skeletal muscle.

Heat stress (HS) stimulates heat shock protein (HSP) 72 mRNA expression, and the period after an increase in HSP72 protein is characterized by enhanced glucose metabolism in skeletal muscle. We have hypothesized that, prior to an increase in the level of HSP72 protein, HS activates glucose metabolism by acutely stimulating 5′-AMP-activated protein kinase (AMPK). Rat epitrochlearis muscle was isolated and incubated either with or without HS (42°C) for 10 and 30 min. HS for 30 min led to an increase in the level of Hspa1a and Hspa1b mRNA but did not change the amount of HSP72 protein. However, HS for both 10 and 30 min led to a significant increase in the rate of 3-O-methyl-d-glucose (3MG) transport, and the stimulatory effect of 3MG transport was completely blocked by cytochalasin B. HS-stimulated 3MG transport was also inhibited by dorsomorphin but not by wortmannin. HS led to a decrease in the concentration of ATP, phosphocreatine, and glycogen, to an increase in the level of phosphorylation of AMPKα Thr172, and to an increase in the activity of both AMPKα1 and AMPKα2. HS did not affect the phosphorylation status of insulin receptor signaling or Ca2+/calmodulin-dependent protein kinase II. These results suggest that HS acts as a rapid stimulator of insulin-independent glucose transport, at least in part by stimulating AMPK via decreased energy status. Although further research is warranted, heat treatment of skeletal muscle might be a promising method to promote glucose metabolism acutely.

Effect of electrical stimulation-induced resistance exercise on mitochondrial fission and fusion proteins in rat skeletal muscle.

It is well known that resistance exercise increases muscle protein synthesis and muscle strength. However, little is known about the effect of resistance exercise on mitochondrial dynamics, which is coupled with mitochondrial function. In skeletal muscle, mitochondria exist as dynamic networks that are continuously remodeling through fusion and fission. The purpose of this study was to investigate the effect of acute and chronic resistance exercise, which induces muscle hypertrophy, on the expression of proteins related to mitochondrial dynamics in rat skeletal muscle. Resistance exercise consisted of maximum isometric contraction, which was induced by percutaneous electrical stimulation of the gastrocnemius muscle. Our results revealed no change in levels of proteins that regulate mitochondrial fission (Fis1 and Drp1) or fusion (Opa1, Mfn1, and Mfn2) over the 24-h period following acute resistance exercise. Phosphorylation of Drp1 at Ser616 was increased immediately after exercise (P < 0.01). Four weeks of resistance training (3 times/week) increased Mfn1 (P < 0.01), Mfn2 (P < 0.05), and Opa1 (P < 0.01) protein levels without altering mitochondrial oxidative phosphorylation proteins. These observations suggest that resistance exercise has little effect on mitochondrial biogenesis but alters the expression of proteins involved in mitochondrial fusion and fission, which may contribute to mitochondrial quality control and improved mitochondrial function.

Reflections on the use of protease inhibitor cocktails for preserving protein in tissue lysates

Commercially‐available protease inhibitor cocktails (PI) are routinely included in tissue lysates to insure that there is no loss of protein integrity of tissue samples. In view of the paucity of published research, our study investigated whether such inclusion was efficacious. We examined the effects of four protease inhibitor cocktails on rat skeletal muscle protein lysates. Total protein band analysis was performed after polyacrylamide gel electrophoresis (PAGE); in addition, myosin light chain (MLC) banding was compared after Western blot. Our results showed that, although most of the tested PI's do not appear to alter the protein content or band pattern (from 10 to 250kD) of the samples, anomalies were evident. One PI significantly reduced the measured amount of MLC. In another experiment, muscle (biceps femoris) lysates were treated with protease (trypsin) and incubated at 37°C for one hour. The enzyme treatment eliminated all of the protein bands (including MLC). In trypsin‐treated lysates containing PI's, most protein bands were rescued from hydrolysis to varying degrees. Restoration of MLC bands varied with the PI. Our conclusion from these studies is precautionary; we recommend that investigators reflect on their routine use of any PI. (Support from SURE Program at MSMC and Pace Scholarly Research)

Exercise training‐induced adaptations associated with increases in skeletal muscle glycogen content

Chronic exercise training results in numerous skeletal muscle adaptations, including increases in insulin sensitivity and glycogen content. To understand the mechanism leading to increased muscle glycogen, we studied the effects of exercise training on glycogen regulatory proteins in rat skeletal muscle. Female Sprague Dawley rats performed voluntary wheel running for 1, 4 or 7 weeks. After 7 weeks of training, insulin-stimulated glucose uptake was increased in epitrochlearis muscle. As compared with sedentary control rats, muscle glycogen did not change after 1 week of training, but increased significantly after 4 and 7 weeks. The increases in muscle glycogen were accompanied by elevated glycogen synthase activity and protein expression. To assess the regulation of glycogen synthase, we examined its major activator, protein phosphatase 1 (PP1), and its major deactivator, glycogen synthase kinase (GSK)-3. Consistent with glycogen synthase activity, PP1 activity was unchanged after 1 week of training but significantly increased after 4 and 7 weeks of training. Protein expression of R(GL)(G(M)), another regulatory PP1 subunit, significantly decreased after 4 and 7 weeks of training. Unlike PP1 activity, GSK-3 phosphorylation did not follow the pattern of glycogen synthase activity. The ~ 40% decrease in GSK-3α phosphorylation after 1 week of exercise training persisted until 7 weeks, and may function as a negative feedback mechanism in response to elevated glycogen. Our findings suggest that exercise training-induced increases in muscle glycogen content could be regulated by multiple mechanisms, including enhanced insulin sensitivity, glycogen synthase expression, allosteric activation of glycogen synthase, and PP1 activity.

Both short intense and prolonged moderate in vitro stimulation reduce the mRNA expression of calcium-regulatory proteins in rat skeletal muscle

Sarcoplasmic and t-tubule membrane proteins regulating sarcoplasmic Ca(2+) concentration exhibit fibre-type-dependent isoform expression, and play central roles in muscle contraction and relaxation. The purpose of this study was to evaluate the effects of in vitro electrical stimulation on the mRNA expression of components involved in Ca(2+) regulation in oxidative and glycolytic skeletal muscle. The mRNA level of Ca(2+)-ATPase (SERCA1, 2), calsequestrin (CASQ1, 2), ryanodine receptor (RyR1), and dihydropyridine receptor (Cacna1) was assessed in rat extensor digitorum longus (EDL) and soleus (SOL) muscles at 4 h of recovery following in vitro stimulations (either short intensive (SHO) 60 Hz, 5 min, or prolonged moderate (PRO) 20 Hz, 40 min). Stimulation induced acute regulation of the mRNA level of Ca(2+)-regulating proteins in a manner that does not follow typical fibre-type-specific transitions. In general, stimulation decreased mRNA content of all proteins studied. Most prominent down-regulation was observed for Cacna1 (26 and 32 % after SHO and PRO, respectively, in SOL; 19 % after SHO in EDL). SERCA1, SERCA2, CASQ1, CASQ2, and RyR1 mRNA content also decreased significantly in both muscles relative to resting control. Of notice is that hexokinase II mRNA content was increased in EDL and unchanged in SOL underlining the specificity of the down-regulation of mRNA of Ca(2+) regulatory proteins. The results demonstrate contraction-induced down-regulation of mRNAs for the main components of Ca(2+)-regulating system in skeletal muscle. The down-regulation of both isoforms of SERCA and CASQ after a single electrical stimulation session suggests that adaptations to repeated stimulation involve further regulatory mechanisms in addition to acute mRNA responses.

The effect of acute exercise on the expression of mitochondria functiom and content protein in rat skeletal muscle

The effect of acute exercise on the expression of mitochondria functiom and content protein in rat skeletal muscle

The effects of β-adrenergic stimulation and exercise on NR4A3 protein expression in rat skeletal muscle

β-Adrenergic stimulation and exercise up-regulate the mRNA expression of nuclear receptor NR4A3, which is involved in the regulation of glucose and fatty acid utilization genes in skeletal muscle. The objective of our study was to examine the effects of β-adrenergic stimulation and exercise on the expression of NR4A3 protein in rat skeletal muscle. A single subcutaneous injection of clenbuterol, which is a β2-adrenergic receptor (β2-AR) agonist, increased NR4A3 mRNA and protein expression in the fast-twitch glycolytic triceps muscle. On the other hand, an acute 3-h session of either treadmill running or swimming did not increase the NR4A3 protein level in the exercised muscle, although both treadmill running and swimming increased NR4A3 mRNA. Finally, loss of postural contractile activity because of hindlimb immobilization reduced NR4A3 mRNA and protein in the slow-twitch oxidative soleus muscle. These results suggest that: β-adrenergic stimulation up-regulates not only NR4A3 mRNA but also NR4A3 protein in fast-twitch glycolytic muscle; exercise may increase NR4A3 mRNA but not NR4A3 protein in skeletal muscle; and local postural contractile activity plays a crucial role in maintaining NR4A3 protein expression level in postural muscle.

The effects of eccentric contraction on myofibrillar proteins in rat skeletal muscle

The present study investigated the effects of eccentric muscle contractions (ECC) on the content of myofibrillar proteins (my-proteins) and the catalytic activity of myofibrillar ATPase (my-ATPase) in skeletal muscles. Rat extensor digitorum longus and tibialis anterior muscles were exposed to 200-repeated ECC or isometric contractions (ISC) and used for measures of force output and for biochemical analyses, respectively. Whereas in ISC-treated muscles, full restoration of tetanic force was attained after 2days of recovery, force developed by ECC-treated muscles remained depressed (P<0.05) after 6days. The total my-protein content and the relative content of myosin heavy chain (MHC) in total my-proteins were unaltered during 4days of recovery after ECC, but fell (P<0.05) to 55.9 and 63.4% after 6days of recovery, respectively. my-ATPase activity expressed on a my-protein weight basis was unaltered immediately after ECC. However, it decreased (P<0.05) to 75.3, 45.3, and 49.3% after 2, 4 and 6days of recovery, respectively. Total maximal calpain activity measured at 5mM Ca(2+) was significantly augmented (P<0.05) after 2days of recovery, reaching a level of threefold higher after 6days. These alterations were specific for ECC and not observed for ISC. These results suggest that depressions in my-ATPase activity contribute to ECC-induced decreases in force and power which can take a number of days to recover.

Aging and Endurance Training Alters Mitochondrial Fusion and Fission Dynamics in Rat Skeletal Muscle

Mitochondria undergo constant changes in shape, localization and number regulated by dynamic fusion, fission and motility events. In mammalian cells mitochondrial fusion is largely mediated by mitofusin (Mfn1 and 2) whereas fission is controlled mainly by Fis1 and the dynamin-related protein 1 (Drp1). PURPOSE: To investigate how aging and exercise training affect the gene expression of mitochondrial fusion and fission proteins in rat skeletal muscle. METHODS: Male Sprague-Dawley rats were divided into 6 groups (N=8): young control (YC), young trained (YT); middle-age control (MC), middle-age trained (MT); old control (OC) and old trained (OT). Trained rats ran on treadmill (64%VO2max) at 15 m/min, 5% grade for 45 min/day, 5 days/wk for 12 wks. Rats were killed at 5, 15 and 20 mo of age. Soleus and vastus lateralis (VL) muscles were harvested 48 h after last training bout at rest. RESULTS: Mfn1 mRNA level measured by RT-PCR was decreased in OC vs. YC (P<0.05) in VL, whereas Mfn1 protein content measured by Western blot was lower in OC (P<0.05) in soleus. Mfn2 mRNA level was lowered with age in both muscles (P<0.05). Training increased both Mfn1and Mfn2 mRNA levels (P<0.01) in soleus and VL, but Mfn1/2 protein content was unaltered. Both soleus and VL showed higher Fis1 mRNA levels in MC and OC vs. YC (P<0.01), and elevated mRNA with training was observed in all age groups (P<0.01). Fis1 protein content was also elevated with aging and training in soleus (P<0.01) and VL (P<0.05). Increased Drp1 mRNA and protein levels were observed in OC vs. YC (P<0,01) in both muscles, and with training (P<0.01). Subsarcolemma and intramyofibrial mitochondrial density was not affected by age but increased with training in soleu and VL (P<0.01). CONCLUSION: Mitochondrial fusion protein expression is decreased whereas fission protein expression is elevated with aging in rat skeletal muscle. Endurance training stimulates both fusion and fission proteins expression suggesting a dynamic mitochondrial turnover and biogenesis.

The Effects of Hindlimb Suspension on Proteins Essential to Cholesterol Metabolism in Rat Skeletal Muscle

IntroductionHindlimb suspension (HS) results in significant deleterious effects on numerous physiological systems, including atrophy of weight‐bearing muscles. However, HS and its effects on proteins essential for cholesterol metabolism have not been investigated in unloaded skeletal muscle. Cholesterol serves as a critical constituent of all cell membranes, including skeletal muscle. The purpose of this study was to examine the effects of 28 days of HS on sterol regulatory element‐binding protein 2 (SREBP‐2), which is a transcriptional factor necessary for endogenous cholesterol synthesis. We hypothesized that HS causes a downregulation of this protein in rat skeletal muscle.MethodsTwelve male Sprague‐Dawley rats, 6 months of age, were hindlimb suspended for 28 days (HS; n=5) or served as cage controls (CC; n=7). After 4 weeks, plantarflexor muscles were harvested, weighed and analyzed by Western Blotting for SREBP‐2.ResultsSoleus muscle mass and soleus muscle mass to body mass were significantly different between groups (HS: 97.0 ± 14.2 mg and 0. 22 ± 0. 03 mg/g; CC: 244.3 ± 38.1 mg and 0.49 ± 0. 06 mg/g, P &lt; 0.05). However, mature SREBP‐2 was not significantly different between groups (HS: 0.66 ± 0.31 arbitrary units (AU) and CC: 1.06 ± 0.39 AU, P=0.09).ConclusionAfter 28 days, HS resulted in significant decreases in soleus muscle mass, but not mature SREBP‐2, as compared to cage controls.

Contraction-induced changes in TNFα and Akt-mediated signalling are associated with increased myofibrillar protein in rat skeletal muscle

Resistance training results in skeletal muscle hypertrophy, but the molecular signalling mechanisms responsible for this altered phenotype are incompletely understood. We used a resistance training (RT) protocol consisting of three sessions [day 1 (d1), day 3 (d3), day 5 (d5)] separated by 48 h recovery (squat exercise, 4 sets x 10 repetitions, 3 min recovery) to determine early signalling responses to RT in rodent skeletal muscle. Six animals per group were killed 3 h after each resistance training session and 24 and 48 h after the last training session (d5). There was a robust increase in TNFalpha protein expression, and IKK(Ser180/181) and p38MAPK(Thr180/Tyr182) phosphorylation on d1 (P < 0.05), which abated with subsequent RT, returning to control levels by d5 for TNFalpha and IKK(Ser180/181). There was a trend for a decrease in MuRF-1 protein expression, 48 h following d5 of training (P = 0.08). Notably, muscle myofibrillar protein concentration was elevated compared to control 24 and 48 h following RT (P < 0.05). Akt(Ser473) and mTOR(Ser2448) phosphorylation were unchanged throughout RT. Phosphorylation of p70S6k(Thr389) increased 3 h post-exercise on d1, d3 and d5 (P < 0.05), whilst phosphorylation of S6(Ser235/236) increased on d1 and d3 (P < 0.05). Our results show a rapid attenuation of inflammatory signalling with repeated bouts of resistance exercise, concomitant with summation in translation initiation signalling in skeletal muscle. Indeed, the cumulative effect of these signalling events was associated with myofibrillar protein accretion, which likely contributes to the early adaptations in response to resistance training overload in the skeletal muscle.

DIGE analysis of rat skeletal muscle proteins using nonionic detergent phase extraction of young adult versus aged gastrocnemius tissue

Contractile weakness and loss of muscle mass are critical features of the aging process in mammalians. Age-related fibre wasting has a profound effect on muscle metabolism, fibre type distribution and the overall physiological integrity of the neuromuscular system. This study has used mass spectrometry-based proteomics to investigate the fate of the aging rat muscle proteome. Using nonionic detergent phase extraction, this report shows that the aged gastrocnemius muscle exhibits a generally perturbed protein expression pattern in both the detergent-extracted fraction and the aqueous protein complement from senescent muscle tissue. In the detergent-extracted fraction, the expression of ATP synthase, isocitrate dehydrogenase, enolase, tropomyosin and beta-actin was increased. Different isoforms of creatine kinase and prohibitin showed differential changes. In the aqueous fraction, malate dehydrogenase, sulfotransferase, triosephosphate isomerase, aldolase, cofilin-2 and lactate dehydrogenase showed increased levels. Interestingly, differential effects on dissimilar 2-D spots of the same protein species were shown for Cu/Zn superoxide dismutase, albumin, annexin A4 and phosphoglycolate phosphatase. Mitochondrial Hsp60, Hsp71 and nucleoside diphosphate kinase B exhibited a reduced abundance in aged muscle. The majority of altered proteins were found to be involved in mitochondrial metabolism, glycolysis, metabolic transportation, regulatory processes, the cellular stress response, detoxification mechanisms and muscle contraction.