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Resveratrol and muscle recovery

Resveratrol and muscle recovery

Roller, M. Beetroot juice and athletic performance cycling exercise produced an abruptly systemic IL-6 response involving a significant muscle Rseveratrol in the Resveratrol and muscle recovery activity. About the journal Open Access Fees and Funding About Rrecovery Reports Muacle Journal policies Calls for Papers Guide to musclw Editor's Resveratrol and muscle recovery Journal highlights. Resveratrol and muscle recovery was possibly because the initial intensity of the bicycle ergometer overwhelmed the leg muscles of the participants, who expressed severe fatigue in their legs. No use, distribution or reproduction is permitted which does not comply with these terms. Resveratrol protects against physical fatigue and improves exercise performance in mice. Although the cumulative frequency curves for the type IIA fibers shifted to the right after exercise as compared to before exercise, the shift to larger fibers shift to the right was more pronounced in type IIA fibers of the RSV group Figure 3D — F ; Supplementary Figure 6. Resveratrol and muscle recovery

Resveratrol and muscle recovery -

Nevertheless, our current results are consistent with data from reloading after atrophy in rodents, where improvement in especially type II fiber size was reported in animals given RSV Along with the larger fibers Figure 4 , RSV induced an increase in the exercise-associated nuclear and satellite cell numbers Figure 4.

However, it is not necessary for RSV to enhance satellite cell proliferation, as RSV could potentially contribute to a greater total nuclear pool by reducing apoptotic signaling such as proapoptotic proteins Bax, cleaved caspase 3, and cleaved caspase 9 in exercised muscles 15 , and therefore reducing the potential for elimination of muscle nuclei.

As we did not conduct a time course evaluation, we do not know if the total satellite cell and nuclear abundance was increased early as a result of hypertrophic adaptations to exercise training, or if it were accomplished more slowly and in part by eliminating fewer nuclei through apoptotic pathways.

The association of RSV-mediated increase in fiber size and total myonuclei satellite cells and muscle nuclei is consistent with the nuclear domain hypothesis, but additional time course studies should be conducted to provide a better documentation of satellite cell and myonuclei accretion with the combined effects of exercise and RSV treatments.

A potential limitation of this study is that we used a somewhat moderate training protocol, where only small or in some cases, no changes were noted in the variables that where measured. Thus, we cannot rule out the possibility that if we had used a training protocol that had been more intensive and had resulted in greater exercise-induced improvements, that the effects of RSV might have been different than what we report in this study.

This is because our findings will be relevant for average older persons who are interested in increasing their physical activity but who are not interested in engaging in training at high intensity. However, it is exciting to note that even with this modest training approach, we saw some very important structural adaptations in muscle to exercise with RSV treatment.

We propose that exercise-initiated an overall improvement in mitochondrial function that was linked to improved anabolic signaling pathway leading to hypertrophy of type I and IIA fiber sizes, and an increase in total muscle nuclei satellite cells and myonuclei that was enhanced in muscles from RSV-exercised versus PL-exercised subjects.

The greater fiber sizes account for the RSV-induced elevation in muscle torque and power. These data support a role for RSV treatment combined with exercise as a potential means for reducing or reversing sarcopenia in elderly persons. Supplementary data is available at The Journals of Gerontology, Series A: Biological Sciences and Medical Sciences online.

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Supplementary Material. Conflict of Interest. Journal Article Editor's Choice. Resveratrol Enhances Exercise-Induced Cellular and Functional Adaptations of Skeletal Muscle in Older Men and Women.

Stephen E Alway , Stephen E Alway. Oxford Academic. Jean L McCrory. Kalen Kearcher. Austen Vickers. Benjamin Frear.

Diana L Gilleland. Daniel E Bonner. James M Thomas. David A Donley. Mathew W Lively. Junaith S Mohamed Junaith S Mohamed. Decision Editor: Rafael de Cabo, PhD. PDF Split View Views. Select Format Select format.

ris Mendeley, Papers, Zotero. enw EndNote. bibtex BibTex. txt Medlars, RefWorks Download citation. Permissions Icon Permissions. Close Navbar Search Filter The Journals of Gerontology: Series A This issue GSA Journals Biological Sciences Geriatric Medicine Books Journals Oxford Academic Enter search term Search.

Mitochondria , Sarcopenia , Fiber type , Exercise , Muscle , Strength , Fatigue. Table 1. p Values. Condition pre, post.

Group PL, RSV. Condition × Group Interaction. Age y Open in new tab. Figure 1. Open in new tab Download slide. The Heat Map provides a visualization of the fold changes in mitochondrial log2 fold gene expression between the selected groups for every gene with the layout for the Heat Map as follows:.

The legend for the color intensity is shown below each heat map. Table 2. Gene Symbol. Pre-PL vs Pre-RSV. Post-PL vs Post-RSV. Pre- vs Post-Exercise PL.

Pre- vs Post-Exercise RSV. Apoptosis BAK1 3. Table 3. Figure 2. Figure 3. Figure 4. Google Scholar Crossref. Search ADS. Published by Oxford University Press on behalf of The Gerontological Society of America.

All rights reserved. For permissions, please e-mail: journals. permissions oup. Issue Section:. Download all slides. Supplementary data. Views 6, More metrics information. Total Views 6, Email alerts Article activity alert. New issue alert. Nevertheless, the function of resveratrol on skeletal muscle stem cell function during muscle repair is less clear.

As resveratrol may function differently on skeletal muscle satellite cells in vivo in aging vs. these in vitro studies, we tested the hypothesis that resveratrol supplementation would alter satellite cell proliferation and reduce pro-apoptotic signaling to improve muscle regeneration in muscles of aged rats following muscle disuse.

This line of inquiry is important because muscles from aged animals recover very little after disuse 26 and this is confirmed in the current study. The findings of our study suggest that resveratrol may modestly improve the apoptotic environment of muscles in aged rodents during reloading following disuse, but it has a limited ability to alter satellite cell proliferation as a mechanism for recovering muscle mass that was lost during the period of disuse.

Thirty-six male Fisher × Brown Norway rats were obtained from the National Institute on Aging NIA colony that is housed at Harlan Indianapolis, IN. All of these animals were 32 months of age. The animals were housed at 20°C in barrier-controlled conditions under a hour light-dark cycle.

Proper animal care standards were followed by adhering to the recommendations for the care of laboratory animals as advocated by the American Association for Accreditation of Laboratory Animal Care and by following the policies and procedures detailed in the Guide for the Care and Use of Laboratory Animals as published by the U.

Department of Health and Human Services and proclaimed in the Animal Welfare Act PL, PL, and PL All experimental procedures carried approval by the Institutional Animal Care and Use Committee from the West Virginia University.

The hindlimb suspension lasted fourteen days as previously described [ 25 ]. This was fixed to the top of a specially designed hindlimb suspension cage which provided the rats with ° of movement around the cage. The forelimbs maintained contact with a grid pattern floor, allowing the animals to move, groom themselves, and obtain food and water freely.

The exposed tip of the tail was closely monitored to ensure that it remained pink, indicating that hindlimb suspension did not interfere with blood flow to the tail. The suspension height was monitored and adjusted to prevent contact between the hindlimbs and any supportive surface of the cage. Care was taken so that the suspension angle of the torso of the animals to the cage floor did not exceed 30°.

All animals were weighed prior to and following the suspension period to determine total body mass changes. Furthermore, the recovery animals were released from suspension after the fourteen day protocol and provided an additional fourteen days of normal ambulation, at which point they were weighed and sacrificed.

Beginning one week prior to suspension, the rats received 1ml of 0. Previous studies in our lab [ 19 , 26 ] using resveratrol This provided a bolus increase in resveratrol every 24 hours.

To ensure resveratrol was also available systemically throughout the day, the resveratrol treated animals were given food pellets that contained 0. Vehicle treated animals had an identical diet to the resveratrol treated animals, but their chow did not contain resveratrol. This provided a mechanism to provide a more consistently high level of resveratrol throughout the day.

Food and water was provided ad libitum. Upon release from suspension, a small incision was made in the upper back of anesthetized animals whereupon the BrdU pellet was inserted subcutaneously. The incision was closed with a wound clip MikRon Autoclip 9 mm; Becton Dickinson; Sparks, MD and the animals were allowed to recover from anesthesia before being returned to their cage.

Muscle data were obtained from both the resveratrol and vehicle-treated animals after hindlimb suspension, or recovery. In addition, two groups of ambulatory non-suspended cage control animals were used.

Six cage control animals were examined 21 days after the initiation of the study. These animals acted as controls for the hindlimb suspension groups.

A second group of six cage control animals were examined 35 days after the initiation of the study and were used as controls for the recovery groups.

Among the suspended animals, six per diet group i. The six hindlimb suspension cage control and six recovery cage control animals were also sacrificed at these respective time points.

The soleus, gastrocnemius, and plantaris muscles were excised from both hind limbs, blotted to remove excess fluid, and individually weighed.

A block obtained from the mid-belly of the muscle was embedded in optimal cutting temperature OCT compound, flash frozen in liquid nitrogen cooled isopentane, and stored at °C until later analyses.

Blood samples were obtained from the left ventricle of each animal via a stainless steel needle 19G1½; Becton Dickinson; Rutherford, NJ. The concentration of resveratrol was determined in the serum samples by high-performance liquid chromatography against known standards Protea Biosciences, Inc.

Following blood collection, the animals were immediately euthanized by removing the heart with surgical scissors. Plasma from resveratrol and control animals was analyzed for resveratrol and its metabolites: Trans-ResveratroiSulfate, Trans-ResveratroiGiucuronide, and Trans-ResveratroiGiucuronide.

Plasma was analyzed by Protea Biosciences Morgantown, WV using high performance liquid chromatography HPLC. Quantification of the data was made against standard curves generated in duplicate for each metabolite. Sirt1 activity was measured in total plantaris muscle homogenates using a fluorescent deacetylase substrate using a commercially available kit BML-AK; Enzo Life Sciences Inc.

Briefly, muscles were homogenized in ice-cold distilled water and the protein concentrations were determined using a DC protein concentration assay BioRad. The tissue samples were diluted to 2. Once complete, 2mM nicotinamide a Sirt1 inhibitor and the provided fluorescent developer were added to each well to halt the reaction and produce a fluorophore which is linearly related to Sirt1 activity.

The intensity of the fluorescent signal was detected with an excitation wavelength of nm and an emission wavelength of nm. Data are presented as fluorescent units normalized to the respective milligrams of protein used in each homogenate.

Isometric muscle contractile properties were examined in the plantaris muscles from the left leg of all experimental animals. The distal end of the muscle was attached to a stationary plexiglass plate, and the proximal end fixed to the lever arm of a C dynamometer Aurora Scientific, Aurora Ontario, Canada.

The muscles were stimulated by passing a constant current through platinum plates positioned on each side of the muscle. Muscles were adjusted to the optimal muscle length L o by a micromanipulator that controls the base position of the electrode clamp.

L o was established as the muscle length that produced maximal isometric twitch tension and was periodically checked by the same procedure throughout each experiment to maintain this length.

Force-frequency isometric force records were obtained by stimulating the muscle at 10, 20, 40, 50, 75 and Hz, with 3 minutes of rest between each contraction.

Physiological contractile measures included peak isometric twitch force PT , time to peak twitch contraction tension CT , and the ½ relaxation time of twitch contraction ½ RT , as previously described [ 27 ].

Muscle fatigue was assessed by stimulating the muscle at 40Hz for 3 minutes utilizing a duty cycle of ms of stimulation followed by ms of rest [ 28 ]. The fatigue index was calculated as the force from the last contraction expressed as a percentage of the force obtained on the first contraction.

The contractile and fatigue measurements were analyzed off line using commercial software DMI, Aurora Scientific. Muscle fiber type and cross-sectional area CSA analyses were performed in the plantaris muscles.

Frozen tissue cross sections measuring μm thick were cut from the mid-belly of the muscles, mounted on charged microscope slides Fisher Scientific; Pittsburgh, PA , and stored at °C. The tissue sections were incubated overnight at 4°C with antibodies directed against laminin MAB; Millipore; Temecula, CA to visualize the basal lamina of each muscle fiber, as well as one of the following individual myosin heavy chain MyHC antibodies: BA-D5 for MyHC I; Developmental Studies Hybridoma Bank; Iowa City, IA , SC for MyHC IIA; DSHB , and BF-F3 for MyHC IIB; DSHB in order to determine both the whole muscle composition and mean cross-sectional area CSA for each respective fiber type.

The next day, the slides were washed in PBS and incubated 1 hour at 37°C with secondary antibodies of donkey anti-rat rhodamine conjugate ; Jackson ImmunoResearch Laboratories; West Grove, PA to visualize the basal lamina, and Alexa Fluor A; Invitrogen; Eugene, OR to visualize the MyHC expression in the muscle fibers.

The sections were mounted with 4', 6-diamidinophenylindole DAPI Vectashield Mounting Medium; Vector Laboratories; Burlingame, CA to visualize the nuclei. All images were taken with a Zeiss LSM Meta confocal microscope Carl Zeiss Microimaging Inc. Mean fiber CSA of respective fiber types was determined by planimetry and calculated by the ImageJ software NIH; Frederick, MD.

Fiber types are expressed as a proportion of MyHC-positive fibers to total fibers for each individual MyHC examined. The percentage of type IIX MyHC fibers was calculated from fibers that were negative to type I, IIA, and IIB MyHC antibodies.

Western blots were used to determine the relative amount of proteins in apoptotic signaling pathways following hindlimb suspension and recovery. The muscle homogenates were centrifuged at 1, g for 5 minutes at 4°C, and the resulting supernatant was collected. The protein content of the samples was measured using the DC protein assay kit BioRad; Hercules, CA.

The proteins were then transferred to a nitrocellulose membrane for 2 hours at 35V. Membranes were incubated overnight at 4°C on a rocking table with primary antibodies , directed against Bcl-xL ; Cell Signaling Technology; Boston, MA , Bax ; Cell Signaling Bcl-2 ; Cell Signaling , cleaved caspase 3, ; Cell Signaling , cleaved caspase 9 ; Cell Signaling , AMPK ; Cell Signaling , pAMPK ; Cell Signaling , Sirt1 ; Cell Signaling and PGC1 sc; Santa Cruz Biotech; Dallas, TX.

Furthermore, a primary antibody , was directed against GAPDH ab; Abcam; Cambridge, MA for use as a loading control. The signals were developed using a chemiluminescent substrate Lumigen; Southfield, MI and visualized by exposing the membranes to x-ray film BioMax MS-1; Eastman Kodak, Rochester, NY.

Digital records of the bands were captured using a Kodak camera and quantified using one-dimensional analysis software Eastman Kodak as optical density × band area, expressed in arbitrary units relative to appropriate loading controls.

Frozen tissue μm thick cross sections were obtained from the plantaris muscles and mounted on charged microscope slides Fisher Scientific; Pittsburgh, PA. Fluorescent labeling of terminal dUTP nick-end labeling TUNEL with lamina allowed the detection of apoptotic nuclei in the muscle sections.

This was accomplished using a slight modification to the method previously reported for our lab [ 29 ]. The tissues were incubated overnight at 4°C in a rat anti-lamina monoclonal antibody Millipore.

Sections were incubated the following day with donkey anti-rat rhodamine conjugated secondary antibody Jackson ImmunoResearch , along with the TUNEL reaction mixture ; Roche Diagnostics; Indianapolis, IN in a humidified chamber at 37°C for 1 hour in the dark.

The exclusion of the TdT enzyme in the TUNEL reaction mixture on one of the tissue sections on each slide was included as a negative control. The sections were mounted with DAPI Vectashield in order to visualize nuclei and viewed under a Zeiss LSM Meta confocal microscope Carl Zeiss Microimaging Inc.

The number of TUNEL and DAPI-positive nuclei that were immediately adjacent to, or beneath the basal lamina were counted. The data are expressed as an apoptotic index, calculated as the percentage of TUNEL-positive nuclei out of the total myonuclei i.

The apoptotic index was determined from four non-overlapping regions of each tissue cross section visualized with a 20X objective. Frozen tissue cross sections measuring μm thick from the plantaris muscle were mounted on charged microscope slides Fisher Scientific; Pittsburgh, PA.

Briefly, tissue sections were air dried, washed in PBS, fixed in methanol: acetone at °C for 5 minutes, and permeabilized with 0. Sections were then denatured, blocked with serum and incubated overnight in a biotinylated mouse anti-BrdU primary antibody HCS30, EMD Biosciences, Inc.

On the following day, the sections were incubated with a secondary antibody of Fluorescein Avidin DCS A; Vector Laboratories, Inc.

They were then incubated overnight in a rat anti-lamina monoclonal Millipore primary antibody. The tissue sections were incubated the following day with a donkey anti-rat rhodamine conjugated Jackson ImmunoResearch secondary antibody in a humidified chamber at 37°C for 1 hour in the dark. Finally, the sections were mounted with 4',6-diamidinophenylindole DAPI -containing mounting medium Vectashield in order to visualize nuclei.

Immunofluorescence was visualized with a Zeiss LSM Meta confocal microscope Carl Zeiss. Images were taken from four non-overlapping regions of each tissue section with a 20X objective.

All results are reported as means ± SD. Differences in means between groups were determined by multiple analysis of variance MANOVA Hotelling's T-Square test. Bonferroni post hoc analyses were subsequently performed between significant means. We measured the bodyweight of all animals at the beginning of the one-week pretreatment period Start , immediately prior to suspension Day 0 , following hindlimb suspension, and following the recovery period.

There were no differences in bodyweight between any of the groups at the beginning of the experimental protocol. Furthermore, no significant changes in bodyweight were observed in the cage control animals at any of the time points.

A significant loss of bodyweight occurred in all of the suspended animals as compared to the first day Start of the experiment, with decreases of These significant losses continued into the recovery period as well, with vehicle and resveratrol treated animals losing an additional 3.

However, resveratrol was unable to attenuate any of these bodyweight reductions, as there were no significant differences observed between the vehicle and resveratrol treated animals at any of the individual time points of the study Figure 1A. Plantaris muscle wet weights were obtained following HS or R.

cage control. Upon sacrifice, the plantaris muscle wet weights were examined to assess changes in muscle mass following the suspension and recovery periods. However, there were no differences between the vehicle and the resveratrol treated animals.

Plantaris muscles were attached to a force transducer in oxygenated Ringers solution, to determine various muscle contractile properties following hindlimb suspension and recovery.

However, resveratrol was unable to attenuate this increase Figure 2A. In the recovery group, the CT of both the vehicle There were no differences observed when comparing the CT of the vehicle and resveratrol treated animals.

Muscle twitch contractile properties including CT and ½ RT were analyzed in the plantaris muscles of both the hindlimb suspension A and recovery B groups. Each data point represents the average of three measures.

Next, we examined the ½ RT during a twitch contraction. In the hindlimb suspension group, the ½ RT was significantly shorter in the vehicle treated Moreover, there were no differences between the resveratrol and cage control groups at this time point.

This same trend also continued into the recovery period Figure 2B. While there were no differences in the ½ RT between the resveratrol Lastly, we plotted the force-frequency curve for the hindlimb suspension and recovery groups.

In the hindlimb suspension animals, a frequency of 20 Hz produced a significant rightward shift in both the vehicle and resveratrol treated groups Figure 3A.

At 50 Hz, there remained a significant rightward shift in the resveratrol animals curve compared to both the cage control and vehicle treated animals. In contrast, there were no differences observed between the vehicle treated and cage control groups. At 75 Hz, both the vehicle and resveratrol treated groups reached their peak relative forces.

A force-frequency curve was plotted for both the hindlimb suspension A and recovery B groups. Each force-frequency measurement was made three times with a minimum of 5 minutes rest between each contraction, and the average of the three trials was recorded for each animal.

In the recovery animals, the force frequency curves from both the vehicle and resveratrol treated groups were significantly shifted to the right of the cage control curve at 10, 20, and 50 Hz Figure 3B. However, the force-frequency curves of the vehicle and the resveratrol groups did not differ.

The force record at 75 Hz in the resveratrol treated animals had a significant rightward shift as compared to the cage controls. The fatigue index was examined in order to determine if resveratrol had improved the muscles fatigue tolerance during a series of contractions.

Both the vehicle However, there were no differences observed between the resveratrol and the vehicle treated animals. In the recovery animals, the vehicle treated animals had a similar fatigue index Plantaris muscle fatigability was expressed as a fatigue index, and calculated as the percent of the initial force average of the first three contractions, divided by the force at the end of the fatigue protocol an average of the force generated in the final three contractions in a series of consecutive contractions.

The plasma level of resveratrol, in resveratrol-treated animals averaged There were no differences between the animals in the hindlimb suspension or control groups. Metabolites of resveratrol were similarly very high in the resveratrol treated animals Table S1.

While there were no traces of resveratrol in the plasma of any of the vehicle treated animals, there was a detectible about of several resveratrol metabolites in the plasma of vehicle treated animals Table S1 suggesting that perhaps the control diet contained small but detectable levels of resveratrol which had been metabolized by the vehicle-treated animals.

The relative activity of Sirt1 in the plantaris muscles of all of the animals was assessed using a fluorometric assay.

In the hindlimb suspension animals, there were no significant differences in Sirt1 activity between any of the groups, although there was a trend towards higher levels in the resveratrol treated animals Figure 5.

This trend continued with the recovery groups as well, although the differences between the groups were less than it was in the hindlimb suspension animals.

This was due to a slight elevation in Sirt1 activity in the muscles of both the recovery control and the vehicle treated animals as compared to their respective hindlimb suspension groups. Western blots were conducted for plantaris muscles after hindlimb suspension or following recovery from hindlimb suspension.

The protein content was measured in total plantaris muscle homogenates via immunoblotting. The proteins were electroblotted to nitrocellulose membranes and the signals were developed by chemiluminescence.

The proteins included phosphorylated activated AMPK, total AMPK, PGC1α, and Sirt1. GAPDH was used as an internal loading control. Con, control; Veh, vehicle-treated; Res, resveratrol treated.

The digital images of the western blots were quantified as optical density x band area using ImageJ software and normalized to GAPDH, which was used as the loading control for each lane. The data include: B , total AMPK; C.

phosphorylated AMPK pAMPK ; D. PGC1α; and E. The data are expressed as the protein signal to the GAPDH signal and are reported as mean ± SEM in arbitrary units. A minimum of three western blots were completed for each protein, and the data were averaged for each animal.

Sirt1 enzyme activity was determined fluorometrically in plantaris muscle homogenates. Resveratrol treatment elevated AMPK and pAMPK abundance in the plantaris of resveratrol-fed animals in the recovery group as compared to the vehicle treated or control animals Figure 5.

Sirt1 protein abundance was not improved by resveratrol treatment Figure 5. However, resveratrol, appeared to increase the protein abundance of PGC1α, a downstream target of Sirt1, in the plantaris muscles of animals in the recovery group fed resveratrol, as compared to vehicle treated animals.

We did not determine if mitochondrial biogenesis had been altered by resveratrol treatment in the muscles of these animals, however, this seems unlikely since the fatigue characteristics of the muscles was not improved by resveratrol treatment.

The myosin heavy chain expression was identified in the plantaris muscles fibers by immunocytochemistry Figure 6. This provided both an index of fiber type and also fiber size.

Typically, hindlimb suspension results in a shift from type I to type II fibers. To determine if resveratrol reduced this fiber type shift following hindlimb suspension or recovery, we examined the myosin heavy chain MyHC profile of the plantaris muscles.

Following hindlimb suspension, the relative percentage of type I MyHC fibers was significantly reduced in both of the groups subjected to suspension. However, there were no differences between MyHC composition in the vehicle and the resveratrol treated animals, although the mean percentage was slightly higher in the resveratrol group Figure 7A.

The resveratrol treated animals in the hindlimb suspension group, had a greater percentage of type IIA MyHC fibers, than either the vehicle treated suspended animals, or cage control animals.

Suspension significantly increased the percentage of type IIB fibers, although resveratrol had no effect on changes in this fiber type compared to the vehicle treatment. In contrast, the percentage of type IIX MyHC fibers was significantly reduced after suspension in the resveratrol treated animals from that of the control muscles.

However, there were no significant differences between the two suspended groups. Frozen tissue cross sections were incubated overnight at 4°C with antibodies directed against the basal lamina red and myosin heavy chain I, IIA, or IIB green.

The sections were counterstained with DAPI blue to identify the nuclei. Digital images were taken with a confocal microscope. This figure provides a representative example of a section stained for type I myosin heavy chain that was used to measure fiber type composition and CSA analyses for this fiber type in both the hindlimb suspension and recovery groups.

Green fibers are positive for type I myosin heavy chains. Scale bars represent 50 µm. Immunocytochemical stained plantaris muscle fibers for myosin heavy chains, from control, vehicle-treated and resveratrol-treated rats were analyzed for their myosin heavy chain fiber composition in both the hindlimb suspension A and recovery B groups.

The percentage of type I fibers remained significantly less in the animals in the recovery group that had previously been suspended.

However, there was an increase in the percentage of type I fibers in the vehicle treated animals to where the resveratrol treated animals no longer had a greater proportion of this fiber type Figure 7B.

While the percentage of type IIB fibers remained significantly elevated in both the vehicle and the resveratrol treated animals in the recovery group as compared to the control group, there remained no differences between these treatment groups.

There was no difference between the percentage of type IIX MyHC fibers in the recovery group of resveratrol and the vehicle treated animals.

Muscle fiber CSA was determined by planimetry in type I, IIA, and IIB MyHC fibers Figure 6. As expected, hindlimb suspension induced significant muscle atrophy in all of the fiber types examined.

There were no differences in mean type I fiber CSA between the vehicle and resveratrol treated animals after hindlimb suspension, although both hindlimb suspension treatment groups had significantly smaller fibers than the cage control animals. The mean fiber CSA from the resveratrol treated animals in the hindlimb suspension group was not different than the cage control animals, indicating that resveratrol partially preserved muscle fiber area during hindlimb suspension in this particular fiber type.

Type IIB MyHC fibers CSA was significantly smaller in both the vehicle and the resveratrol treated groups as compared with the fiber CSA from cage control animals. However, no significant differences were noted between type IIB fiber CSA in the vehicle and resveratrol treated groups following hindlimb suspension.

Mean fiber cross sectional area CSA measurements were obtained from a minimum of fibers in each muscle for plantaris muscle fibers that expressed type I, IIA, or IIB myosin heavy chains in hindlimb suspension A and recovery B groups.

Four images from non-overlapping regions of each tissue cross-section stained for individual MyHC fibers were used for muscle fiber CSA measures. All images were taken with a Zeiss LSM Meta confocal microscope at a magnification of 20X. Mean fiber CSA of respective fiber types was determined by planimetry.

Animals in the recovery group had a similar fiber type pattern as was found in muscles from the hindlimb suspension animals Figure 8B. This suggests that there was no substantial return to control levels in fiber type distribution during the recovery period. Specifically, type I fibers CSA remained significantly smaller in both the vehicle and resveratrol treated animals as compared to the cage controls.

Mean type IIA fiber CSA was significantly reduced in the vehicle treated animals as compared to the cage control animals. Moreover, although the mean type IIB fiber CSA remained significantly reduced in the vehicle treated animals, the mean CSA of the resveratrol treated animals somewhat recovered and so that fiber CSA was no longer statistically smaller than the control fibers.

A BrdU pellet was implanted subcutaneously at point that hindlimb suspension was removed, in each of the recovery animals to identify satellite cells that had proliferated during the regenerative period following hindlimb suspension. BrdU-positive nuclei were expressed per myonuclei. BrdU incorporation was very low in muscles of the cage control animals 0.

Presumably, there was minimal stimulus to activate any available satellite cells from quiescence under these homeostatic conditions Figure 9. It was not surprising that there was a significant increase in BrdU incorporation in both the vehicle 7.

While there was a trend for slightly more BrdU positive nuclei in resveratrol treated muscles, this was not significantly different from the vehicle group. In order to analyze satellite cell proliferation during the reloading period, a time-released BrdU pellet was inserted into the recovery animals at the point that they were released from suspension.

BrdU is a thymidine analogue and it was incorporated into muscle nuclei satellite cells that divided. A representative section from the plantaris of a resveratrol-treated animal that was reloaded for 14 days following 14 days of hindlimb suspension.

The tissue was stained with DAPI to identify all of the nuclei, an anti-BrdU antibody to identify nuclei that had proliferated, and an anti-laminin antibody to identify the basal lamina.

The overlay shows the three fluorescent images superimposed. The expanded insert shows examples of BrdU positive nuclei that are co-localized to the DAPI stained myonuclei over the basal lamina white arrows.

Other examples of non-specific green staining not on nuclei are evident, but they were not included in the quantification of BrdU positive nuclei. BrdU incorporation in muscle nuclei i. We have previously characterized apoptotic protein changes during hindlimb suspension and reloading [ 13 , 30 ].

In the current study, western blots were used to determine the relative content of anti-apoptotic Bcl-xL and Bcl-2 and pro-apoptotic Bax, cleaved caspase 3 and cleaved caspase 9 proteins, as representative of changes in the apoptotic signaling pathway.

Protein levels of the respective proteins were normalized to GAPDH protein levels and were expressed in arbitrary units.

Although resveratrol did not reduce the hindlimb suspension-induced elevation in Bax, in the recovery group, the pro-apoptotic Bax protein abundance was lower in resveratrol than vehicle treated plantaris muscles Figure In a similar pattern, pro-apoptotic proteins cleaved caspase 3 and cleaved caspase 9 were elevated by hindlimb suspension, but the increases in cleaved caspase 9, a mitochondrial-associated pro-apoptotic protein was suppressed by resveratrol.

Resveratrol suppressed both cleaved caspase 3 and cleaved caspase 9 in the plantaris muscles of the recovery group as compared to the vehicle treated group Figure Bcl-2 was elevated in a similar fashion in vehicle treated and resveratrol treated plantaris muscles during hindlimb suspension Figure During recovery, the Bcl-2 protein abundance returned to control levels in the vehicle-treated plantaris muscle, but it remained elevated in plantaris muscles that were treated with resveratrol.

The protein abundance of the anti-apoptotic Bcl-xL was significantly increased in the resveratrol group following hindlimb suspension, while no changes were observed in the vehicle 0. During recovery, the abundance of Bcl-xL was elevated in both the vehicle 0. Representative western blots of apoptotic proteins.

Bax, Bcl-xL, Bcl-2, cleaved caspase 3 CC-3 , cleaved caspase 9 CC-9 and GAPDH. The digital images were quantified as optical density x band area using ImageJ software and normalized to GAPDH, which was used as the loading control for each lane.

These data include: Bax B Bcl-2 C , Bcl-xL D , cleaved caspase 3 E , and cleaved caspase 9 F. To complement the western blot analysis, we used a TUNEL assay to confirm the relative amount of myonuclei undergoing apoptosis immediately following hindlimb suspension Figure 11A and recovery Figure 11B.

Consistent with our previous observations [ 13 , 30 ], there was a significant increase in apoptosis following hindlimb suspension Figure 11C. In the recovery group, the TUNEL index remained significantly elevated in both the vehicle 1.

DNA fragmentation was assessed with TUNEL staining green as an indication of nuclei committed to apoptosis in the plantaris muscles from animals. The basal lamina of the muscle fibers was incubated in an anti-lamina antibody red to identify nuclei adjacent to or inside the basal lamina of the fibers.

Representative tissue cross sections are shown for animals in the hindlimb suspension A and recovery B groups. Quantification of the TUNEL positive nuclei that were located at or below the basal lamina of the muscle fibers in the recovery or cage control animals.

Recovery of skeletal muscle mass after immobilization-induced atrophy is difficult or impossible in aged animals or humans [ 8 — 10 ], but it is more complete in younger hosts. Therefore, the focus of our study was to examine the efficacy of resveratrol to improve muscle recovery following disuse in aging, and therefore we were not interested in identifying differences in response between young and old animals.

International Resveratrol and muscle recovery of Biological Sciences. Global reach, higher impact. Int Resveratrool Med Musclee ; 18 10 Jung-Piao Tsao 1Chia-Chen Liu 1Hsueh-Fang Wang 2Jeffrey R. Bernard 3Chun-Ching Huang 4I-Shiung Cheng 1. Plyometric exercise PE is an effective training method to increase muscle mass and strength. However, excessive or inappropriate conditions might Blood sugar crash and hormonal imbalances exercise-induced muscle recoery EIMD. Resveratrol RES Reckvery a natural polyphenol plant recoovery, which improves mmuscle performance, Ans exhibits Resveratrol and muscle recovery, anti-inflammatory, and anti-cancer effects. Therefore, this study investigated the effect of RES supplementation on the recovery of muscle damage, inflammation, soreness, muscle power, and anaerobic performance following plyometric-exercise-induced muscle damage PEIMD. The present study was a double-blind, placebo-controlled research trial. At baseline, to pre-PEIMD, supplements were pre-loaded 7 days before they conducted PEIMD, and the exercise performance, delayed-onset muscle soreness DOMS and muscle damage biomarkers were measured over the experimental period at baseline, pre-PEIMD, and post-PEIMD at 2, 24, 48, and 72 h.

Author: Mazumuro

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