Muscles Of Old Rats Research Articles

Resveratrol impinges on retrograde communication without inducing mitochondrial biogenesis in aged rat soleus muscle

The natural polyphenol resveratrol (RSV) might counteract the skeletal muscle age-related loss of muscle mass and strength/function partly acting on mitochondria. This work analysed the effects of a six-week administration of RSV (50 mg/kg/day) in the oxidative Soleus (Sol) skeletal muscle of old rats (27 months old). RSV effects on key mitochondrial biogenesis proteins led to un unchanged amount of SIRT1 protein and a marked decrease (60 %) in PGC-1α protein. In addition, Peroxyredoxin 3 (PRXIII) protein decreased by 50 %, which on overall suggested the absence of induction of mitochondrial biogenesis by RSV in old Sol. A novel direct correlation between PGC-1α and PRXIII proteins was demonstrated by correlation analysis in RSV and ad-libitum (AL) rats, supporting the reciprocally coordinated expression of the proteins. RSV supplementation led to an unexpected 50 % increase in the frequency of the oxidized base OH8dG in mtDNA. Furthermore, RSV supplementation induced a 50 % increase in the DRP1 protein of mitochondrial dynamics. In both rat groups an inverse correlation between PGC-1α and the frequency of OH8dG as well as an inverse correlation between PRXIII and the frequency of OH8dG were also found, suggestive of a relationship between oxidative damage to mtDNA and mitochondrial biogenesis activity. Such results may indicate that the antioxidant activity of RSV in aged Sol impinged on the oxidative fiber-specific, ROS-mediated, retrograde communication, thereby affecting the expression of SIRT1, PGC-1α and PRXIII, reducing the compensatory responses to the age-related mitochondrial oxidative stress and decline.

Associating Inulin with a Pea Protein Improves Fast-Twitch Skeletal Muscle Mass and Muscle Mitochondrial Activities in Old Rats.

Aging is associated with a decline in muscle mass and function, leading to increased risk for mobility limitations and frailty. Dietary interventions incorporating specific nutrients, such as pea proteins or inulin, have shown promise in attenuating age-related muscle loss. This study aimed to investigate the effect of pea proteins given with inulin on skeletal muscle in old rats. Old male rats (20 months old) were randomly assigned to one of two diet groups for 16 weeks: a 'PEA' group receiving a pea-protein-based diet, or a 'PEA + INU' group receiving the same pea protein-based diet supplemented with inulin. Both groups showed significant postprandial stimulation of muscle p70 S6 kinase phosphorylation rate after consumption of pea proteins. However, the PEA + INU rats showed significant preservation of muscle mass with time together with decreased MuRF1 transcript levels. In addition, inulin specifically increased PGC1-α expression and key mitochondrial enzyme activities in the plantaris muscle of the old rats. These findings suggest that dietary supplementation with pea proteins in combination with inulin has the potential to attenuate age-related muscle loss. Further research is warranted to explore the underlying mechanisms and determine the optimal dosage and duration of intervention for potential translation to human studies.

Effect of Eight Weeks of Resistance Training on PAX7 and MyoD in Fast and Slow-Twitch Skeletal Muscles of Old Rats

Background: Box 7 protein (PAX7) and myogenic differentiation (MyoD) proteins are the essential proteins to regulate the satellite cell and muscle hypertrophy. Objectives: We aimed to investigate the effect of resistance training (RT) on PAX7 and MyoD in soleus and extensor digitorum longus (EDL) muscles of old rats. Methods: In this experimental study, 12 old female rats were randomly divided into two groups including RT and control groups. The RT program was three days a week for eight weeks, in which the rats climbed a one-meter vertical ladder with 26 steps. The carried weights varied from 50% of body weight in the first week to 100% of body weight in the 8th week. Kolmogorov- Smirnov test and independent sample t test was used for statistical analysis (P≤0.05). Results: MyoD protein in RT group significantly increased in soleus and EDL muscles compared with the control group (P=0.001). Also, Pax7 significantly increased in the soleus muscle in the RT group compared with control group (P=0.002); nevertheless, there was no significant difference in Pax7 protein in the EDL muscle between the RT and control groups (P=0.10). Conclusion: RT can lead to proliferation and renewal of satellite cells in the soleus and EDL muscles by increasing PAX7 and MyoD proteins.

Effects of Aging on Angiogenic and Muscle Growth-Related Factors in Naturally Aged Rat Skeletal Muscles.

BackgroundThis study explored the effects of aging on the expression of angiogenic and muscle protein synthesis factors, as well as the number of satellite cells affecting sarcopenia in naturally aged rat skeletal muscles.MethodsWe divided 16 Sprague-Dawley rats into young (12 weeks old, n=8) and old (24 months old, n=8) groups and compared muscle and body weight (BW) between them. We also analyzed the expression levels of angiogenic and muscle growth proteins in soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscles by western blotting and assessed the number of skeletal muscle satellite cells and myonuclei and mean fiber cross-sectional area (CSA) using by immunofluorescence staining.ResultsEDL/BW was significantly lower in old rats than in young rats (p=0.002). The vascular endothelial growth factor level in soleus muscles was significantly lower in old rats than in young rats (p=0.001). Hypoxia-inducible factor 1-alpha and fetal liver kinase 1 levels in EDL muscles were lower in old rats than in young rats (p=0.001). The mammalian target of rapamycin (mTOR), p70S6K, and 4E-BP1 levels were significantly lower in the soleus muscles of old rats than in those of young rats (p<0.01). Similarly, insulin growth factor-1, Akt, mTOR, and p70S6K levels were significantly lower in EDL muscles of old rats than in those of young rats (p<0.01). Additionally, myonuclei/fiber, Pax7/fiber, and mean fiber CSAs in both muscle types were significantly lower in old rats than in young rats (p<0.01).ConclusionThese data suggest different regulation of indices of angiogenic and muscle growth with aging in different muscle types.

Mechano‐therapy using high loads enhances regrowth of skeletal muscle in young, but induces muscle damage in old rats.

IntroductionOlder adults have an impaired ability to recover from skeletal muscle atrophy compared to young, predisposing them to loss of independence and poor quality of life. We have previously shown that mechano‐therapy in the form of cyclic, compressive loading (CCL) enhances regrowth of atrophied skeletal muscle in young adult rats, but not in old. Our preliminary data show that passive, transverse stiffness of muscles from old rats is elevated compared to young, potentially blunting the mechanical stimulus and subsequent growth response. In the current study, we hypothesized that an increased CCL load proportional to the higher tissue stiffness in old rats would enhance skeletal muscle regrowth when compared to atrophied muscles of old rats subjected to a lower load of CCL or ambulation alone.MethodsYoung (10 months; N = 6) and old (30 months; N = 6) adult male F344/Brown Norway rats underwent 14 days of hind limb suspension before being randomly assigned to one of the following: reloading for 7 days without CCL (RE); reloading with 4.5 N CCL (RE + CCL, 4.5 N); or reloading with 7.6 N CCL (RE + CCL, 7.6 N). Starting on day 0, four bouts of CCL were administered to the right gastrocnemius using a custom designed device capable of applying a precise load, with 48 hrs between bouts. Gastrocnemius muscle sections were analyzed for muscle fiber cross‐sectional area (CSA), satellite cell abundance (Pax7+/fiber), embryonic myosin heavy chain expression (eMyHC+/fiber) and centro‐nucleation using immunohistochemistry.ResultsMuscle fiber CSA was load dependent in young adult rats, but had no effect at either load in old rats. When compared to muscles from RE rats, 4.5 N CCL had no effect on satellite cell abundance regardless of age; however, satellite cell abundance was higher in both young and old adult muscle after 7.6 N CCL compared to RE with old rats having 2‐fold higher levels compared to RE. Regardless of load, CCL was not associated with differences in eMyHC expression or centrally located nuclei in young adult rat muscle, while muscles receiving 7.6 N CCL in old rats had a higher number of centrally located nuclei and were ~16‐fold higher in eMyHC+ fibers compared to RE.ConclusionsThese results show that CCL induces a load dependent anabolic response in young adult animals during muscle mass recovery following disuse, but has no regrowth effect on muscles from old animals at either load. Furthermore, using a higher CCL load, scaled for elevated transverse stiffness in aged muscle, induces damage in muscle of old but not young rats.Support or Funding InformationThis research was supported by NIH grant RO1AT009268 and R21AG042699.

The Effect of Periodic Exercise and Resveratrol Supplementation on the Expression of Pparg Coactivator-1 Alpha and Pyruvate Dehydrogenase Kinase Genes in Gastrocnemius Muscle of Old Rats With Type 2 Diabetes

Aims: Type 2 diabetes is the result of complex interactions between genetic and environmental factors that affect fat and glucose metabolism. The purpose of this study was to determine the effect of periodic exercise and resveratrol supplement on the expression levels of Pparg Coactivator 1-Alpha (PGC-1α) and Pyruvate Dehydrogenase Kinase (PDK4) genes in gastrocnemius muscle of old rates with type 2 diabetes. Methods &amp; Materials: 42 male rats (mean age= 40-50 weeks; mean body weight= 250-300 g) were randomly divided into 6 groups: healthy-control, diabetic-control, Diabetic+Periodic Exercise, Diabetic+Supplement, Diabetic+Periodic Exercise+Supplement and Saline. The type 2 diabetes was induced by intraperitoneal injection of Streptozotocin (50 mg/kg body weight). The exercise protocol consisted of 10 sets of 1-min activities at 50% intensity and a 2-min rest period between sets, and each week the speed was increased by 2 meters per minute. The exercises were performed for eight weeks. Resveratrol supplement was injected intraperitoneally daily at a dose of 20 mg/kg body weight. The expressions of PDK4 and PGC-1α in the gastrocnemius muscle were measured by real time Polymerase Chain Reaction (PCR) method. Findings: highest expression level of PDK4 and PGC-1α genes in gastrocnemius muscle was observed in the diabetic group received both periodic exercise and Resveratrol supplement and the lowest level was reported in the diabetic-control and saline groups. Conclusion The combination of resveratrol supplementation and periodic exercise can have beneficial effects on PDK4 and PGC-1α expression levels in the gastrocnemius muscle of old rats with type 2 diabetes and reduce the risks of diabetes-related complications.

Testosterone is Key to Increase the Muscle Capillary Density of Old and Trained Rats

Introduction It is indicated that aging reduces muscular vascularity. Thus, strategies that aim to reverse or decrease the progression of these changes are suggested. Therefore, the present study aimed to analyze the blood capillary density of the plantaris muscle (PL) of aged rats submitted to strength training and exogenous testosterone use. Methods A total of 30 rats were divided equally into the following groups: EA: 20 months old; OLD: 24 months old; OLD + S: testosterone; ST: strength training; and ST + S: strength training + testosterone. Strength training was performed 3 times a week for 15 weeks. Testosterone administration was performed by the intraperitoneal method 2 times a week at a dose of 10mg/kg. All of the morphological parameters were analyzed using morphoquantitative techniques. Results A significant increase in the capillaries numerical density of the OLD + S and ST + S groups. The OLD + S and ST + S groups demonstrated a higher ratio of capillaries per myofiber than the EA, OLD and ST groups. The OLD and ST groups had a higher ratio than the EA group. The OLD + S, ST and ST + S groups presented a statistically significant increase in the cross-sectional area in the type I fibers when compared with the EA and OLD groups. However, the ST group showed a significant increase compared with the OLD group. In type II fibers, the ST and ST + S groups showed a greater cross-sectional area than the EA group. As for the mean cross-sectional area, the ST and ST + S groups presented significantly larger areas than the other groups. Conclusion Anabolic steroid administration is crucial to increase the blood capillary density of the plantaris muscle of old rats, independently of the effects of strength training.

Social isolation alters ultrasonic vocalizations but not thyroarytenoid neuromuscular junctions in old rats.

Age-related muscle atrophy of the laryngeal muscles contributes to presbyphonia. Remodeling of the neuromuscular junction is one aspect underlying age-related muscle atrophy. Although muscle disuse has been shown to exacerbate age-related neuromuscular changes in the limb muscles, it is unknown if reduced vocal use has a similar effect in the laryngeal muscles. The objective of this study was to examine the use of social isolation as a novel method to reduce vocal use in old rats-and the impact of that reduced vocal use on ultrasonic vocalization acoustics and neuromuscular junction morphology in the thyroarytenoid muscle. Animal group comparison. Old F344/BN rats (31 months of age) were socially isolated (n = 8) or communally housed (n = 8) for 8 weeks. Effect of housing condition on ultrasonic vocalization acoustics was assessed by calculating the changes in vocalization fundamental frequency and amplitude from baseline to 8 weeks. Neuromuscular junction morphology was measured in the lateral and medial portions of the thyroarytenoid muscle at the conclusion of the experiment. Vocalization amplitude decreased by a mean of -4.4 dB (standard deviation [SD], 4.49) after social isolation, whereas amplitude increased by a mean of 5.7 dB (SD, 5.07) in the communally housed rats (P = 0.002). There was no significant difference in the change in fundamental frequency between groups. Furthermore, there were no group differences in any measure of neuromuscular junction morphology. These results suggest that neuromuscular junctions in the thyroarytenoid muscle of old rats are unaffected by 8 weeks of social isolation, despite functional changes in vocalizations. NA Laryngoscope, 129:E9-E14, 2019.

Mild aerobic training with blood flow restriction increases the hypertrophy index and MuSK in both slow and fast muscles of old rats: Role of PGC-1α

AimsExisting evidence emphasize the role of mitochondrial dysfunction in sarcopenia which is revealed as loss of skeletal muscle mass and neuromuscular junction remodeling. We assessed the effect of low-intensity aerobic training along with blood flow restriction on muscle hypertrophy index, muscle-specific kinase (MuSK), a pivotal protein of the neuromuscular junction and Peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) in aged male rats. Main methodsAnimals groups were control (CTL), sham (Sh), leg blood flow restriction (BFR), exercise (Ex), sham + exercise (Sh + Ex), and BFR plus exercise (BFR + Ex) groups. The exercise groups were trained with low intensity exercise for 10 weeks. 48 h after the last training session, animals were sacrificed under anesthesia. Soleus and EDL muscles were isolated, hypertrophy index was estimated and MuSK and PGC-1α were measured by western blot method. Key findingsHypertrophy index enhanced in soleus and Extensor digitorum longus (EDL) muscles of BFR + Ex group (P < 0.01 versus CTL and Sh groups, and P < 0.001 versus other groups). The MuSK protein of soleus and EDL muscles increased in BFR + Ex group (P < 0.01 and P < 0.001, respectively) in comparison with CTL and Sh groups. In BFR + Ex group, the PGC-1α protein increased in both soleus and EDL (P < 0.001 compared to other groups). Also the PGC-1α of soleus muscle was higher in Ex and Sh + Ex groups versus CTL and Sh groups (P < 0.05). SignificanceFindings suggest that low endurance exercise plus BFR improves the MuSK and hypertrophy index of both slow and fast muscles of elderly rats probably through the rise of PGC-1α expression.

Epigallocatechin-3-gallate increases autophagy signaling in resting and unloaded plantaris muscles but selectively suppresses autophagy protein abundance in reloaded muscles of aged rats

We have previously found that Epigallocatechin-3-gallate (EGCg), an abundant catechin in green tea, reduced apoptotic signaling and improved muscle recovery in response to reloading after hindlimb suspension (HS). In this study, we investigated if EGCg altered autophagy signaling in skeletal muscle of old rats in response to HS or reloading after HS. Fischer 344×Brown Norway inbred rats (age 34months) were given 1ml/day of purified EGCg (50mg/kg body weight), or the same sample volume of the vehicle by gavage. One group of animals received HS for 14days and the second group of rats received 14days of HS, then the HS was removed and they were allowed to recover by ambulating normally around the cage for two weeks. EGCg decreased a small number of autophagy genes in control muscles, but it increased the expression of other autophagy genes (e.g., ATG16L2, SNCA, TM9SF1, Pink1, PIM-2) and HS did not attenuate these increases. HS increased Beclin1, ATG7 and LC3-II/I protein abundance in hindlimb muscles. Relative to vehicle treatment, EGCg treatment had greater ATG12 protein abundance (35.8%, P<0.05), but decreased Beclin1 protein levels (−101.1%, P<0.05) after HS. However, in reloaded muscles, EGCg suppressed Beclin1 and LC3-II/I protein abundance as compared to vehicle treated muscles. EGCg appeared to “prime” autophagy signaling before and enhance autophagy gene expression and protein levels during unloading in muscles of aged rats, perhaps to improve the clearance of damaged organelles. However, EGCg suppressed autophagy signaling after reloading, potentially to increase the recovery of hindlimb muscles mass and function after loading is restored.

Enhancement of Skeletal Muscle in Aged Rats Following High-Intensity Stretch-Shortening Contraction Training.

Exercise is the most accessible, efficacious, and multifactorial intervention to improve health and treat chronic disease. High-intensity resistance exercise, in particular, also maximizes skeletal muscle size and strength-outcomes crucial at advanced age. However, such training is capable of inducing muscle maladaptation when misapplied at old age. Therefore, characterization of parameters (e.g., mode and frequency) that foster adaptation is an active research area. To address this issue, we utilized a rodent model that allowed training at maximal intensity in terms of muscle activation and tested the hypothesis that muscles of old rats adapt to stretch-shortening contraction (SSC) training, provided the training frequency is sufficiently low. At termination of training, normalized muscle mass (i.e., muscle mass divided by tibia length) and muscle quality (isometric force divided by normalized muscle mass) were determined. For young rats, normalized muscle mass increased by ∼20% regardless of training frequency. No difference was observed for muscle quality values after 2 days versus 3 days per week training (0.65 ± 0.09 N/mg/mm vs. 0.59 ± 0.05 N/mg/mm, respectively). For old rats following 3 days per week training, normalized muscle mass was unaltered and muscle quality was 30% lower than young levels. Following 2 days per week training at old age, normalized muscle mass increased by 17% and muscle quality was restored to young levels. To investigate this enhanced response, oxidative stress was assessed by lipid peroxidation quantification. For young rats, lipid peroxidation levels were unaltered by training. With aging, baseline levels of lipid peroxidation increased by 1.5-fold. For old rats, only 2 days per week training decreased lipid peroxidation to levels indistinguishable from young values. These results imply that, appropriately scheduled high-intensity SSC training at old age is capable of restoring muscle to a younger phenotype in terms of lipid peroxidation levels and muscle quality.

Desensitized morphological and cytokine response after stretch-shortening muscle contractions as a feature of aging in rats

Recovery from contraction-induced injury is impaired with aging. At a young age, the secondary response several days following contraction-induced injury consists of edema, inflammatory cell infiltration, and segmental muscle fiber degeneration to aid in the clearance of damaged tissue and repair. This morphological response has not been wholly established at advanced age. Our aim was to characterize muscle fiber morphology 3 and 10days following stretch-shortening contractions (SSCs) varying in repetition number (i.e. 0, 30, 80, and 150) for young and old rats. For muscles of young rats, muscle fiber degeneration was overt at 3days exclusively after 80 or 150 SSCs and returned significantly closer to control values by 10days. For muscles of old rats, no such responses were observed. Transcriptional microarray analysis at 3days demonstrated that muscles of young rats differentially expressed up to 2144 genes while muscles of old rats differentially expressed 47 genes. Bioinformatic analysis indicated that cellular movement was a major biological process over-represented with genes that were significantly altered by SSCs especially for young rats. Protein levels in muscle for various cytokines and chemokines, key inflammatory factors for cell movement, increased 3- to 50-fold following high-repetition SSCs for young rats with no change for old rats. This age-related differential response was insightful given that for control (i.e. 0 SSCs) conditions, protein levels of circulatory cytokines/chemokines were increased with age. The results demonstrate ongoing systemic low-grade inflammatory signaling and subsequent desensitization of the cytokine/chemokine and morphological response to contraction-induced injury with aging — features which accompany age-related impairment in muscle recovery.

Correction of Mitochondrial Enzyme Activities in the Skeletal Muscles of Old Rats in Response to Addition of Olive Oil to the Ration.

Activities of mitochondrial electron transport chain enzymes NADH-CoQ oxidoreductase (complex I), cytochrome C-oxidase (complex IV), and citrate synthase were measured by spectrophotometry in m. quadriceps femoris homogenate from old rats receiving olive oil with the ration. Reduced activities of complexes I and IV in old animals were restored to the level of young animals after 6-week consumption of olive oil. Activity of citrate synthase did not change with age. Positive effect of olive oil on fatty-acid composition of the muscle tissue in old animals was demonstrated. The content of summary monounsaturated fatty acids, reduced with aging, and of summary polyunsaturated ones, increasing with age, were restored in old rats to the levels virtually not differing from the levels of young animals.

Mechanisms for independent and combined effects of calorie restriction and acute exercise on insulin-stimulated glucose uptake by skeletal muscle of old rats.

Either calorie restriction [CR; consuming 60-65% of ad libitum (AL) intake] or acute exercise can independently improve insulin sensitivity in old age, but their combined effects on muscle insulin signaling and glucose uptake have previously been unknown. Accordingly, we assessed the independent and combined effects of CR (beginning at 14 wk old) and acute exercise (3-4 h postexercise) on insulin signaling and glucose uptake in insulin-stimulated epitrochlearis muscles from 30-mo-old rats. Either CR alone or exercise alone vs. AL sedentary controls induced greater insulin-stimulated glucose uptake. Combined CR and exercise vs. either treatment alone caused an additional increase in insulin-stimulated glucose uptake. Either CR or exercise alone vs. AL sedentary controls increased Akt Ser(473) and Akt Thr(308) phosphorylation. Combined CR and exercise further elevated Akt phosphorylation on both sites. CR alone, but not exercise alone, vs. AL sedentary controls significantly increased Akt substrate of 160 kDa (AS160) Ser(588) and Thr(642) phosphorylation. Combined CR and exercise did not further enhance AS160 phosphorylation. Exercise alone, but not CR alone, modestly increased GLUT4 abundance. Combined CR and exercise did not further elevate GLUT4 content. These results suggest that CR or acute exercise independently increases insulin-stimulated glucose uptake via overlapping (greater Akt phosphorylation) and distinct (greater AS160 phosphorylation for CR, greater GLUT4 for exercise) mechanisms. Our working hypothesis is that greater insulin-stimulated glucose uptake in the combined CR and exercise group vs. CR or exercise alone relies on greater Akt activation, leading to greater phosphorylation of one or more Akt substrates other than AS160.

P65 Inhibition of cystathionine gamma-lyase increases endothelium-dependent relaxation of aortic smooth muscles in old rats

Purpose One of the reasons of increasing the risk of cardiovascular diseases during aging is a dysfunction of endothelium. At the same time we can see more and more publications about vascular protection of hydrogen sulfide (H2S). Cystathionine gamma-lyase (CSE) is the main enzyme for H2S synthesis in blood vessels. Methods The segments of aorta from adult and old rats were used. Activation of isolated samples was carried out by phenylephrine (Phe). Acetylcholine (Ach)-mediated vasorelaxation in the aortic vessel were investigated. The activity of CSE was inhibited with propargylglycine (PPG) (incubation 30 min). H2S was determined by N,N-dimethyl-p-phenylenediamine method, NO de novo synthesis – by NOs activity and nitrite determination. Results The experiments on isolated segments of aorta have shown that the effect of Ach endothelium-dependent vasodilator results in less profound relaxation of aortic smooth muscles (SM) in old animals (12.4%) compared to adult ones (66.3%). PPG addition resulted in growing aortic SM relaxation amplitude from 12.4% to 49.5% (p NO 2 - pools. Also increased the activity of cNOS and decreased the activity of iNOS (p Conclusions Therefore, we can state that use of PPG has led to growing production of two powerful vasodilators – H2S and NO and to the restoration of endothelium-dependent aortic SM vasorelaxation in old animals.

Epigallocatechin-3-gallate improves plantaris muscle recovery after disuse in aged rats

Aging exacerbates muscle loss and slows the recovery of muscle mass and function after disuse. In this study we investigated the potential that epigallocatechin-3-gallate (EGCg), an abundant catechin in green tea, would reduce signaling for apoptosis and promote skeletal muscle recovery in the fast plantaris muscle and the slow soleus muscle after hindlimb suspension (HLS) in senescent animals. Fischer 344×Brown Norway inbred rats (age 34months) received either EGCg (50mg/kg body weight), or water daily by gavage. One group of animals received HLS for 14days and a second group of rats received 14days of HLS, then the HLS was removed and they recovered from this forced disuse for 2weeks. Animals that received EGCg over the HLS followed by 14days of recovery, had a 14% greater plantaris muscle weight (p<0.05) as compared to the animals treated with the vehicle over this same period. Plantaris fiber area was greater after recovery in EGCg (2715.2±113.8μm2) vs. vehicle treated animals (1953.0±41.9μm2). In addition, activation of myogenic progenitor cells was improved with EGCg over vehicle treatment (7.5% vs. 6.2%) in the recovery animals. Compared to vehicle treatment, the apoptotic index was lower (0.24% vs. 0.52%), and the abundance of pro-apoptotic proteins Bax (−22%), and FADD (−77%) was lower in EGCg treated plantaris muscles after recovery. While EGCg did not prevent unloading-induced atrophy, it improved muscle recovery after the atrophic stimulus in fast plantaris muscles. However, this effect was muscle specific because EGCg had no major impact in reversing HLS-induced atrophy in the slow soleus muscle of old rats.

A persistent increase in insulin-stimulated glucose uptake by both fast-twitch and slow-twitch skeletal muscles after a single exercise session by old rats

Exercise has been demonstrated to enhance subsequent insulin-stimulated glucose uptake (GU) by predominantly type II (fast-twitch) muscle of old rats, but previous research has not evaluated exercise effects on GU by type I (slow-twitch) muscle from old rats. Accordingly, we studied male Fischer 344/Brown Norway rats (24 months old) and determined GU (0, 100, 200, and 5,000 μU/ml insulin) of isolated soleus (predominantly type I) and epitrochlearis (predominantly type II) muscles after one exercise session. Epitrochlearis (100, 200, and 5,000 μU/ml insulin) and soleus (100 and 200 μU/ml insulin) GU were greater at 3-h postexercise vs. age-matched sedentary controls. Insulin receptor tyrosine phosphorylation (Tyr1162/1163) was unaltered by exercise in either muscle. Akt phosphorylation (pAkt) was greater for exercised vs. sedentary rats in the epitrochlearis (Ser473 and Thr308 with 100 and 200 μU/ml, respectively) and soleus (Ser473 with 200 μU/ml). AS160 phosphorylation (pAS160) was greater for exercised vs. sedentary rats in the epitrochlearis (Thr642 with 100 μU/ml), but not the soleus. Exercised vs. sedentary rats did not differ for total protein abundance of insulin receptor, Akt, AS160, or GLUT4 in either muscle. These results demonstrate that both predominantly type I and type II muscles from old rats are susceptible to exercise-induced improvement in insulin-mediated GU by mechanisms that are independent of enhanced insulin receptor tyrosine phosphorylation or altered abundance of important signaling proteins or GLUT4. Exercise-induced elevation in pAkt, and possibly pAS160, may contribute to this effect in the epitrochlearis of old rats, but other mechanisms are likely important for the soleus.

Ageing prolongs inflammatory marker expression in regenerating rat skeletal muscles after injury

BackgroundSome of the most serious consequences of normal ageing relate to its effects on skeletal muscle, particularly significant wasting and associated weakness, termed "sarcopenia". The underlying mechanisms of sarcopenia have yet to be elucidated completely but an altered muscle inflammatory response after injury is a likely contributing factor. In this study we investigated age-related changes in the expression of numerous inflammatory markers linked to successful muscle regeneration.MethodsRight extensor digitorum longus (EDL) muscles from young (3 month), adult (12 month) and old (24 month) male F344 rats were injected with bupivacaine hydrochloride to cause complete muscle fibre degeneration, then excised 12, 24, 36, and 72 hours later (n = 5/age group/time point). We used qRT-PCR to quantify the mRNA expression levels of the inflammatory markers TNFα, IFNγ, IL1, IL18, IL6, and CD18 as well as regenerative markers MyoD and myogenin.ResultsInflammatory markers were all increased significantly in all age groups after myotoxic injury. There was a trend for expression of inflammatory markers to be higher in uninjured muscles of old rats, especially at 72 hours post injury where the expression levels of several markers was significantly higher in old compared with young and adult rats. There was also a decrease in the expression of regenerative markers in old rats at 72 hours post injury.ConclusionOur findings identify a prolonged inflammatory signature in injured muscles from old compared with young and adult rats together with a blunted expression of key markers of regeneration in muscles of old rats. Importantly, our findings identify potential targets for future therapeutic strategies for improving the regenerative capacity of skeletal muscle during ageing.

Effects of treadmill exercise and training frequency on anabolic signaling pathways in the skeletal muscle of aged rats

Physical exercise is the most effective intervention against sarcopenia of aging; however, the cellular and molecular mechanisms mediating training-induced adaptations are not yet completely understood. Furthermore, it is unclear whether exercise training initiated late in life affects myocyte anabolic signaling in a dose-dependent manner. Hence, we sought to investigate the effects of treadmill exercise and training frequency on anabolic pathways, including insulin signaling, in the skeletal muscle of old rats. Aged (14–16-month-old) male Wistar rats were trained on a treadmill for 3 (EX3) or 5 days/week (EX5) during 8 weeks and compared with age-matched sedentary controls (SED). Four-month-old rats were used as young controls (YC). Protein expression levels of insulin receptor (IR), insulin receptor substrate 1 (IRS-1), activated (phosphorylated) mammalian target of rapamycin (p-mTOR) and glucose transporter GLUT4 were determined in quadriceps muscle extracts via immunoblotting. Mitochondrial cytochrome c oxidase (COX) activity was assessed by histochemical staining, while electron microscopy was employed to quantify the sarcomere volume (V src). Body weight (BW) increased, whereas muscle weight (MW) and V src decreased with age. EX5, but not EX3 increased MW and V src, without affecting BW. The expression of IR and GLUT4 was higher in SED rats relative to the YC group. Conversely, protein levels of IRS-1 and p-mTOR as well as COX activity were reduced in advanced age. Compared with SED rats, EX3 animals displayed reduced IR expression and increased IRS-1 levels and COX activity. The expression of GLUT 4 and p-mTOR was unaffected by EX3. EX5 up-regulated IRS-1 and p-mTOR expression and COX activity, while decreasing GLUT4 levels, with no effect on IR expression. In summary, substantial impairments in muscle anabolic pathways, including insulin signaling, were detected in aged sedentary rats. These changes were ameliorated by exercise training, concomitant with improvements in muscle trophism. Benefits were more evident in rats trained for 5 days/week, suggesting that physical exercise initiated late in life affects anabolic signaling in a dose-dependent manner.

Effects Of Leucine Supplementation In The Protein Synthesis Signalling Pathways Of Soleus And Edl Muscle In Young And Old Rats

PURPOSE: The objective of this study was to carry out a comparative phosphoproteome analysis of soleus and EDL muscles of young (6 months) and old rats (22 months) supplemented with low dose and high dose of leucine. METHODS: Food-deprived (18h) rats (both groups) were assigned randomly to one of three treatments: control (saline), low dose leucine (0.135g/kg) or high dose of leucine (1.35g/kg) by oral gavage. Thirty min after the administration of the amino acid, the rats were killed by decapitation and EDL and soleus muscles were collected and stored at -80°C. The proteins were extracted in lysis buffer using a cell disruptor and phosphorylated proteins were separated using a phosphoprotein purification kit. Differential analysis was carried out using SDS-PAGE and the ImageJ software. The proteins were excised and in-gel digested with trypsin. Mass spectrometry was performed using a Capillary LC-Q-TOF - MS/MS. Mascot Daemon was used to process spectra and identify proteins using the IPIrat 2010 database. An identification was considered as valid based on the MASCOT algorithm threshold given for each protein search (p < 0.05). RESULTS: Eighteen protein spots were identified and 9 of these were differentially expressed. Calsequestrin 1, ATP synthase subunit alfpha, alpha-crystallin B chain, myosin regulatory light chain 2, troponin and hemoglobin subunit beta -1 were induced in muscles of old rats given high doses of leucine, while acta 1 actin, troponin I and carbonic anhydrase 3 were induced in two conditions: young muscles given low doses of leucine and old muscles given high doses of leucine. CONCLUSION: The phosphorylation profile of young muscles given low doses of leucine was partially similar to the profile of old muscles given high doses of leucine. On the other hand, there was no detectable change in the phosphorylation profile of young muscles given high doses or old muscles given low doses of leucine when compared to the controls, indicating that the response may be dose-dependent. The role of leucine in inducing the phosphorylation of these proteins is still unknown and is currently under investigation. Furthermore, western-blot and histochemistry analysis are being performed. Supported by FAPESP: 2009/52022-2 and Laboratório Nacional de Luz Síncrotron.