Epigallocatechin-3-gallate improves plantaris muscle recovery after disuse in aged rats.
ABSTRACT: 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 34 months) received either EGCg (50 mg/kg body weight), or water daily by gavage. One group of animals received HLS for 14 days and a second group of rats received 14 days of HLS, then the HLS was removed and they recovered from this forced disuse for 2 weeks. Animals that received EGCg over the HLS followed by 14 days 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±113.8 ?m(2)) vs. vehicle treated animals (1953.0±41.9 ?m(2)). 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.
Project description:?-Hydroxy-?-methylbutyrate (HMB) is a leucine metabolite shown to reduce protein catabolism in disease states and promote skeletal muscle hypertrophy in response to loading exercise. In this study, we evaluated the efficacy of HMB to reduce muscle wasting and promote muscle recovery following disuse in aged animals. Fisher 344×Brown Norway rats, 34 mo of age, were randomly assigned to receive either Ca-HMB (340 mg/kg body wt) or the water vehicle by gavage (n = 32/group). The animals received either 14 days of hindlimb suspension (HS, n = 8/diet group) or 14 days of unloading followed by 14 days of reloading (R; n = 8/diet group). Nonsuspended control animals were compared with suspended animals after 14 days of HS (n = 8) or after R (n = 8). HMB treatment prevented the decline in maximal in vivo isometric force output after 2 wk of recovery from hindlimb unloading. The HMB-treated animals had significantly greater plantaris and soleus fiber cross-sectional area compared with the vehicle-treated animals. HMB decreased the amount of TUNEL-positive nuclei in reloaded plantaris muscles (5.1% vs. 1.6%, P < 0.05) and soleus muscles (3.9% vs. 1.8%, P < 0.05). Although HMB did not significantly alter Bcl-2 protein abundance compared with vehicle treatment, HMB decreased Bax protein abundance following R, by 40% and 14% (P < 0.05) in plantaris and soleus muscles, respectively. Cleaved caspase-3 was reduced by 12% and 9% (P < 0.05) in HMB-treated reloaded plantaris and soleus muscles, compared with vehicle-treated animals. HMB reduced cleaved caspase-9 by 14% and 30% (P < 0.05) in reloaded plantaris and soleus muscles, respectively, compared with vehicle-treated animals. Although, HMB was unable to prevent unloading-induced atrophy, it attenuated the decrease in fiber area in fast and slow muscles after HS and R. HMB's ability to protect against muscle loss may be due in part to putative inhibition of myonuclear apoptosis via regulation of mitochondrial-associated caspase signaling.
Project description:Aging is associated with poor skeletal muscle regenerative ability following extended periods of hospitalization and other forms of muscular disuse. Resveratrol (3,5,4'-trihydroxystilbene) is a natural phytoalexin which has been shown in skeletal muscle to improve oxidative stress levels in muscles of aged rats. As muscle disuse and reloading after disuse increases oxidative stress, we hypothesized that resveratrol supplementation would improve muscle regeneration after disuse. A total of thirty-six male Fisher 344 × Brown Norway rats (32 mo.) were treated with either a water vehicle or resveratrol via oral gavage. The animals received hindlimb suspension for 14 days. Thereafter, they were either sacrificed or allowed an additional 14 day period of cage ambulation during reloading. A total of six rats from the vehicle and the resveratrol treated groups were used for the hindlimb suspension and recovery protocols. Furthermore, two groups of 6 vehicle treated animals maintained normal ambulation throughout the experiment, and were used as control animals for the hindlimb suspension and reloading groups. The data show that resveratrol supplementation was unable to attenuate the decreases in plantaris muscle wet weight during hindlimb suspension but it improved muscle mass during reloading after hindlimb suspension. Although resveratrol did not prevent fiber atrophy during the period of disuse, it increased the fiber cross sectional area of type IIA and IIB fibers in response to reloading after hindlimb suspension. There was a modest enhancement of myogenic precursor cell proliferation in resveratrol-treated muscles after reloading, but this failed to reach statistical significance. The resveratrol-associated improvement in type II fiber size and muscle mass recovery after disuse may have been due to decreases in the abundance of pro-apoptotic proteins Bax, cleaved caspase 3 and cleaved caspase 9 in reloaded muscles. Resveratrol appears to have modest therapeutic benefits for improving muscle mass after disuse in aging.
Project description:Spinal cord injury (SCI) leads to rapid muscle atrophy due to paralysis/paresis and subsequent disuse. SS-31 is a mitochondrial-targeting peptide that has shown efficacy in protecting skeletal muscle mass and function in non-SCI models of muscle wasting. We aimed to determine if SS-31 could prevent muscle loss after SCI. Male C57BL/6 mice aged 9 weeks underwent sham surgery or 50 kdyne contusion SCI and were administered daily injections of vehicle or 5 mg/kg SS-31 for 14 d. Both SCI groups had sustained losses in body mass compared to Sham animals and ~10% reductions in gastrocnemius, plantaris and tibialis anterior muscle mass after SCI with no clear effect of SS-31. Measurements of protein synthesis in the soleus and plantaris were similar among all groups. mRNA expression of atrophy-associated proinflammatory cytokines was also similar among all groups. There was elevation in MYH7 mRNA and a statistical reduction in MYH2 mRNA expression in the SCI+SS-31 animals compared to Sham animals. There was an SCI-induced reduction in mRNA expression of the E3 ligase FBXO32 (MAFbx), but no effect of SS-31. In summary, a 50 kdyne contusion SCI was able to reduce body mass but was not associated with substantial muscle atrophy or alterations in gene expression profiles associated with muscle health and function 14 d post-injury. SS-31 was not associated with protection against SCI-related changes in body or muscle mass, protein synthesis or gene expression in hindlimb muscles.
Project description:Skeletal muscle atrophy is a physiological response to disuse, aging, and disease. We compared changes in muscle mass and the transcriptome profile after short-term immobilization in a divergent model of high and low responders to endurance training to identify biological processes associated with the early atrophy response. Female rats selectively bred for high response to endurance training (HRT) and low response to endurance training (LRT; n = 6/group; generation 19) underwent 3 day hindlimb cast immobilization to compare atrophy of plantaris and soleus muscles with line-matched controls (n = 6/group). RNA sequencing was utilized to identify Gene Ontology Biological Processes with differential gene set enrichment. Aerobic training performed prior to the intervention showed HRT improved running distance (+60.6 ± 29.6%), while LRT were unchanged (-0.3 ± 13.3%). Soleus atrophy was greater in LRT vs. HRT (-9.0 ±8.8 vs. 6.2 ±8.2%; P<0.05) and there was a similar trend in plantaris (-16.4 ±5.6% vs. -8.5 ±7.4%; P = 0.064). A total of 140 and 118 biological processes were differentially enriched in plantaris and soleus muscles, respectively. Soleus muscle exhibited divergent LRT and HRT responses in processes including autophagy and immune response. In plantaris, processes associated with protein ubiquitination, as well as the atrogenes (Trim63 and Fbxo32), were more positively enriched in LRT. Overall, LRT demonstrate exacerbated atrophy compared to HRT, associated with differential gene enrichments of biological processes. This indicates that genetic factors that result in divergent adaptations to endurance exercise, may also regulate biological processes associated with short-term muscle unloading.
Project description:Recovery from burn injury is associated with stimulated whole-body protein turnover. Since skeletal muscle and liver are the tissues most likely to influence whole-body measurements, we studied protein kinetics in soleus and plantaris muscles as well as liver 3 days after a 3 s burn on one hindlimb of the rat. Muscles from both the burned and unburned limbs of burned rats were compared with those of uninjured controls to distinguish between local and systemic factors involved. The following measurements were performed: (1) fractional growth rate of the tissue protein pool, determined from tissue protein content on days 2, 3 and 4; (2) fractional protein-synthetic rate, measured by [14C]tyrosine constant infusion on day 3; (3) fractional protein-degradation rate, calculated from the difference between the rates of protein synthesis and growth. Protein growth by soleus and plantaris muscles of control rats and unburned limb of burned rats was not paralleled by those in the burned limb, which showed progressive atrophy between 2 and 4 days post-burn (P less than 0.005). Protein synthesis by soleus but not plantaris muscle in the unburned limb of burned rats was enhanced by 62% (P less than 0.04) above control. Protein synthesis by burned-limb soleus and plantaris muscles was elevated by 114% (P less than 0.001) and 67% (P less than 0.02) respectively above control. Protein degradation by both soleus and plantaris muscles in the unburned limb of burned rats did not differ from control. In contrast, that of soleus and plantaris muscles in the burned limb was stimulated by 230% (P less than 0.001) and 164% (P less than 0.001) respectively compared with controls. Protein turnover of soleus muscles in both control and burned rats was more rapid than in corresponding plantaris muscles. Liver protein mass exhibited steady growth in control rats, but remained unchanged in burned animals between 2 and 4 days post-burn. Liver protein synthesis in burned rats was elevated by 56% (P less than 0.01) and protein breakdown was stimulated by 61% (P less than 0.002) above those of controls. The data indicate that both local and systemic factors influence tissue protein turnover in animals recovering from a single-hindlimb scald.
Project description:Impaired recovery of aged muscle following a disuse event is an unresolved issue facing the older adult population. Although investigations in young animals have suggested that rapid regrowth of skeletal muscle following a disuse event entails a coordinated involvement of skeletal muscle macrophages, this phenomenon has not yet been thoroughly tested as an explanation for impaired muscle recovery in aging. To examine this hypothesis, young (4-5 mo) and old (24-26 mo) male mice were examined as controls following 2 wk of hindlimb unloading (HU) and following 4 (RL4) and 7 (RL7) days of reloading after HU. Muscles were harvested to assess muscle weight, myofiber-specifc cross-sectional area, and skeletal muscle macrophages via immunofluorescence. Flow cytometry was used on gastrocnemius and soleus muscle (at RL4) single-cell suspensions to immunophenotype skeletal muscle macrophages. Our data demonstrated impaired muscle regrowth in aged compared with young mice following disuse, which was characterized by divergent muscle macrophage polarization patterns and muscle-specifc macrophage abundance. During reloading, young mice exhibited the classical increase in M1-like (MHC II+CD206-) macrophages that preceeded the increase in percentage of M2-like macrophages (MHC II-CD206+); however, old mice did not demonstrate this pattern. Also, at RL4, the soleus demonstrated reduced macrophage abundance with aging. Together, these data suggest that dysregulated macrophage phenotype patterns in aged muscle during recovery from disuse may be related to impaired muscle growth. Further investigation is needed to determine whether the dysregulated macrophage response in the old during regrowth from disuse is related to a reduced ability to recruit or activate specific immune cells.
Project description:Purpose: The goal of this study was to determine the gene expression changes that occur over 7 days in parralyzed muscle in response to isometric contraction elicited by electrical stimulation initiated 4 months after spinal cord injury and to compare such changes to those observed in a normal muscle subjected to overload. Methods: Electrical stimulation of the soleus and plantaris muscle was stimulated in female rats with complete transection of the spinal cord at the interspace between the 9th and 10th thoracic vertebrae. Stimulation was begun 16 weeks after spinal cord transection and produced near-isometric contraction of soleus, plantaris and tibialis anterior. Muscle was analyzed at 1, 2 and 7 days after starting exercise with electrical stimulation. To provide a baseline reference for gene expression at 16 weeks after spinal cord injury, muscle was also analysed from an additional group of spinal cord transected animals. One additional group of animals with a sham-spinal cord injury was included to provide information about gene expression in neurologically intact animals of similar age. In parallel studies, rats underwent bilateral gastrocnemius ablation to overload soleus and plantaris, or a sham ablation as a control. Muscle was analyzed at 1, 3 and 7 days after gastrocnemius ablation or sham-ablation. Gene expression was determined using Affymetrix Rat Exon microarrays. For each group of animals, microarray analysis was performed for soleus muscle for each of 3 separate animals, using one array per animal. Control sammples for the spinal cord injured groups included a group of animals with a Sham-spinal cord injury, and a group of spinal cord injured animals that did not get electrical stimulation. The comparator for determining fold-change expression values was the spinal cord injured group that did not receive electrical stimulation. For each day after gastrocnemius ablation, a control was included that received all procedures needed for this ablation except cutting the distal insertion of the gastrocnemius into the Achilles tendon to control for effects of the surgery on gene expression.
Project description:Groups of young rats (100 g body wt.) were starved from 23:00 to 11:00 h. The animals were then infused intravenously with diluent or insulin at three different doses to achieve plasma insulin concentrations of 20, 50 and 150 microunits/ml. Before the start of the infusion, animals received a single intravenous injection of indomethacin (250 micrograms) or diluent. After 20 min of infusion, the rats were injected with a large amount of labelled phenylalanine and were killed 10 min later. Insulin produced a dose-dependent decrease in plasma glucose and a dose-dependent rise in protein synthesis in cardiac, gastrocnemius, plantaris and soleus muscles. Protein synthesis in the liver was unaffected by insulin. Indomethacin had no effect on plasma glucose concentrations, but blocked the insulin-induced rise in protein synthesis in cardiac, gastrocnemius and plantaris, but not in soleus muscle. The hormone also increased the plasma concentration of prostaglandin E2 and of prostaglandins F2 alpha and E2 in gastrocnemius and plantaris muscle. The results show close similarities to previous observations with isolated rabbit muscles in vitro and suggest that the involvement of arachidonic acid metabolism in the action of insulin on protein synthesis is of physiological significance.
Project description:The effect of glucagon on the rate of muscle protein synthesis was examined in vivo and in the isolated perfused rat hemicorpus. An inhibition of protein synthesis in skeletal muscles from overnight-fasted rats at various plasma concentrations of glucagon was demonstrated in vivo. The plantaris muscle (Type II, fibre-rich) was more sensitive than the soleus (Type I, fibre-rich). Myofibrillar and sarcoplasmic proteins were equally sensitive in vivo. However, protein synthesis in mixed protein and in sarcoplasmic and myofibrillar fractions of the heart was unresponsive to glucagon in vivo. In isolated perfused muscle preparations from fed animals, the addition of glucagon also decreased the synthesis of mixed muscle proteins in gastrocnemius (Type I and II fibres) and plantaris, but not in the soleus. The sarcoplasmic and myofibrillar fractions of the plantaris were also equally affected in vitro. Similar results were observed in vitro with 1-day-starved rats, but the changes were less marked.
Project description:Microgravity exposure as well as chronic disuse are two main causes of skeletal muscle atrophy in animals and humans. The antigravity calf soleus is a reference postural muscle to investigate the mechanism of disuse-induced maladaptation and plasticity of human and rodent (rats or mice) skeletal musculature. Here, we report microgravity-induced global gene expression changes in space-flown mouse skeletal muscle and the identification of yet unknown disuse susceptible transcripts found in soleus (a mainly slow phenotype) but not in extensor digitorum longus (a mainly fast phenotype dorsiflexor as functional counterpart to soleus). Adult C57Bl/N6 male mice (n = 5) flew aboard a biosatellite for 30 days on orbit (BION-M1 mission, 2013), a sex and age-matched cohort were housed in standard vivarium cages (n = 5), or in a replicate flight habitat as ground control (n = 5). Next to disuse atrophy signs (reduced size and myofiber phenotype I to II type shift) as much as 680 differentially expressed genes were found in the space-flown soleus, and only 72 in extensor digitorum longus (only 24 genes in common) compared to ground controls. Altered expression of gene transcripts matched key biological processes (contractile machinery, calcium homeostasis, muscle development, cell metabolism, inflammatory and oxidative stress response). Some transcripts (Fzd9, Casq2, Kcnma1, Ppara, Myf6) were further validated by quantitative real-time PCR (qRT-PCR). Besides previous reports on other leg muscle types we put forth for the first time a complete set of microgravity susceptible gene transcripts in soleus of mice as promising new biomarkers or targets for optimization of physical countermeasures and rehabilitation protocols to overcome disuse atrophy conditions in different clinical settings, rehabilitation and spaceflight.