| BackgroundSkeletal muscle,which comprises about 40%of the human body mass,is necessary for motion and optimal health.Decreased muscle mass can reduce quality of life as well as health.Skeletal muscle phenotypes change depending on the mechanical load placed upon them.For instance,increased mechanical load stimulates hypertrophy,whereas an absence of mechanical load(or a normal gravitational load)causes muscle atrophy and this can be seen in astronauts during adaptation to hypogravity.Muscle disuse includes reduced mechanical load or the mechanical unloading of muscle and both cause decreased volume of existing skeletal muscle myofibers,which is manifested as reduced muscle size or "disuse atrophy." Such atrophy occurs after bone or CNS injury,prolonged disease states,or neurological illnesses.Muscles that must work against gravity,such as the soleus,gastrocnemius,and vastus lateralis,are most commonly adversely affected by immobilization and their fibers undergo apoptosis during atrophy.PI3K/Akt/mTOR is a crucial signaling pathway that increases protein synthesis and muscle mass recovery after immobilization depends on generation of a sustained positive nitrogen balance due to normalization of changes in protein metabolism associated with increased protein synthesis.Akt phosphorylation is sufficient for dramatic muscle hypertrophy and inhibition of atrophy.Simultaneously,Apoptosis is generally regulated by complex proteins such as pro-apoptosis(Bax)and anti-apoptosis(Bcl-2)proteins.The balance between Bax and Bcl-2 determines whether a cell will undergo apoptosis process or to survive.Because adult myofibers are terminally differentiated,the regeneration of skeletal muscle is largely dependent on a small population of satellite cells,which are located between the sarcolemma and the basal lamina of the muscle fiber.Satellite cells are responsible for providing myonuclei for postnatal growth or repair.and after muscle injury,satellite cells become activated and divide,forming new myofibers or repairing existing fibers.Previously,satellite cells were thought to be optimal for transplant into injured or dystrophic skeletal muscles.However,many studies indicated that satellite cells were limited as donor cells.First,self-renewal potential of adult satellite cells is limited,decreases with age,and can be exhausted by a chronic regenerative process such as severe muscular dystrophy,for which most muscle tissue is eventually lost and is replaced by connective tissue.Secondly,satellite cells transplanted into damaged skeletal muscle have restricted migration and these cells undergo apoptosis readily.In vitro culture of satellite cell-derived myoblasts to expand populations causes loss of their regenerative ability.Bone marrow stromal cells(MSCs)are under consideration for regenerative medicine as they are easy to isolate and can be rapidly expanded from patients.After muscle injury,or for individuals with chronic degenerative myopathies,MSCs divide and fuse to repair or replace damaged fibers.MSCs were reported to contribute to satellite cell function in cardiotoxin-injured muscle.Research indicates that MSCs transplantation have therapeutic potential in animal experiments.Indeed,MSCs has been confirmed to contribute to myofiber formation and to functional recovery of muscle tissue.However,the effect of MSCs on muscle atrophy induced by immobilization is not clear.ObjectivesWe investigated whether MSCs could enhance the proliferation of satellite cell and suppress myonuclear apoptosis during immobilization.Materials and Methods1.Isolation and culture of MSCsMSCs were generated from bone marrow aspirates of normal male Wistar rats.Briefly,five rats were anesthetized for surgery and femur and tibial whole marrow was removed and cleaned of all connective tissue.MSCs were cultured in α-modified DMEM with low glucose supplemented with 10%FBS,100 U/ml penicillin,and 100 mg/ml streptomycin and incubated in a humidified incubator.After 48 h,nonadherent cells were removed,fresh medium was added,and medium was changed weekly.When adherent cells were 90%confluent,they were trypsinized and seeded onto fresh plates(split 1:3)All experiments were performed using cells at 3-5 passages.2.Cell-surface analysis and flow cytometryCells were seeded into 12-well culture plates and culture slides.When cultures reached 80-90%confluence,MSCs were fixed with 4%paraformaldehyde for 40 min and were washed with PBS.FITC-conjugated antibodies against rat CD34 or CD44,and phycoerythrin(PE)-conjugated antibodies against rat CD45 or CD90 were added to wells in the dark.After 60 min,MSCs seeded into 12-well culture plates were washed with PBS and harvested with 0.25%trypsin for 3 min at 37 ℃.Samples were then centrifuged and supernatant was removed and resuspended in 500 μl HBS.Finally,cells were measured with flow cytometry and analyzed with Facs Canto Ⅱ and a Facs DiVa software program.3.Lentiviral transduction of MSCSelf-inactivating lentivirus expressing enhanced green fluorescent protein(GFP)cDNA under control of the p-actin/cytomegalovirus(CMV)/β-globin intron hybrid promoter(LV-GFP)was used.Briefly,MSC were seeded at a density of 5× 104 cells/well in six-well plates and exposed to lentivirus for 24 h at 37 ℃ with a multiplicity of infection(MOI)of 50.Virus-containing medium was removed and MSC were cultured for another 48 h in standard medium.GFP-expressing MSC were selected by DiVa cell sorting and characterized as described above.4.Animal immobilization with plaster castsRats(n = 48)were randomized into three groups:WB(n = 16)received no treatment but plaster casts;IM-PBS(n = 16)were immobilized and received vehicle PBS;and IM-MSC(n = 16)were immobilized and received MSCs.Modified plastic casts were applied as described previously[16].First,rats were anesthetized with 8%chloral hydrate(400 mg/kg,ip).Plaster casts were cut to 1.5 cm widths and 20-25 cm lengths and these were used to immobilize the right hind limb from the thigh to the foot,resulting in immobilization of the knee in the extension position,and the ankle in the plantar flexion position.Sham-immobilized animals were immobilized such that the knee and ankle could move without restraint and this ensured that the approximately equal plaster casts were appended to the right hind limb of each rat.The animals were immobilized for 14 d.4.Transplantation of BM-MSCs in RatsAfter 3-5 passages,LV-GFP-MSCs were detached with Trypsin/EDTA,washed,and re-suspended in PBS(final concentration 20,000 cells/ul)for transplantation.Plaster casts were removed after 24 hours.MSCs(1 ×106 in PBS 50ul)were injected into soleus in fixed limbs and this was the IM-MSC treatment group.The same volume of PBS without MSCs was given to controls(IM-PBS treatment group).Then all animals were immobilized with previously methods.5.Soleus histology and force measurementAfter 14 d of immobilization,rats were anesthetized with 8%chloral hydrate(400 mg/kg,ip).Soleus were surgically excised,and both ends of the dissected muscle were tied firmly with non-absorbable black surgical silk.Muscles were weighed and mounted on an experimental chamber with a vertical organ bath containing mammalian Ringer’ s solution(22-24 oC).Relationships of force-length,force-frequency,and peak tetanic force(Po)were established.The protocol has been previously described in detail.Immediately following force mechanics,muscle weights were measured.For routine histopathology,tissues were formalin-fixed,paraffin-embedded,and transverse soleus sections were cut(3-μm thickness)and stained with hematoxylin and eosin(H&E).Slides were evaluated under light microscopy,and microphotographs were taken with a digital camera attached to a microscope.6.Bromodeoxyuridine(Brdu)incorporation and measurementAnimals were given water with BrdU(0.8 mg/ml)(Sigma,Saint Louis,Mo.,USA)for 3 days after injection and then animals were sacrificed and soleus were removed 14 d after immobilization.Muscles were fixed and incubated with 2 N HCl for 20 min.Samples were washed and sections were incubated with anti-BrdU(1:200;Sigma,Saint Louis,Mo.,USA)and anti-Pax7(1:200,Abcam)at 4 ℃ overnight,followed by Alexa 594-conjugated goat anti-mouse IgG(1:500,Abcam)and Alexa 488-conjugated goat anti-rabbit IgG(1:500,Abcam).Finally,the sections were incubated with DAPI(Life Technologies Japan)for 1 min,washed in PBS,and mounted in mounting solution(Thermo Fisher Scientific).Positive cells were counted under 20 high-power fields(HPFs)of 25 sections and the relative number of Pax7/BrdU-positive cells in HPF was noted.7.Apoptosis Detection Iterminal deoxynucIeoUdyI transferase-mediated UTP nick end labeling,TUNEL)Apoptosis was assessed by terminal deoxynucleotidyl transferase(TdT)-mediated deoxy-UTP nick end labeling(TUNEL)kit(Roche Applied Science,Indianapolis,IN,USA).Six micron thick sections were cut and positioned on poly-lysine coated glass slides for 20 min and incubated with TUNEL reaction mixture(freshly prepared)for 60 min at 37 C in the dark.Nulcei were counterstained with DAPI.Sections were visualized under a fluorescence microscope.The photographs were analyzed using a graphic program(Image J).8.Western Blot AnalysisSoleus were removed under sterile conditions for Western blot.Briefly,muscles were homogenized in lysis buffer and protease inhibitors.Protein was separated with 12%SDS-PAGE and transferred onto PVDF membranes After overnight blocking with 5%non-fat milk in PBS at 4℃,membranes were incubated with primary antibodies for Phospho-AktSer473(1:1,000,#9271,Cell Signaling Technology,Danvers,MA,USA),Akt(1:1,000,#9272 Cell Signaling Technology),Bcl-2(1:1,000,G9545,Sigma-Aldrich),and Bax(1:1,000,B3428,Sigma-Aldrich)overnight at 4℃.Subsequently,membranes were incubated in appropriate dilutions of goat anti-rabbit IgG H&L(HRP)secondary antibodies.Immunoreactive bands were detected using the BioSpectrum Imaging 810 System and quantified with ImageJ software.9.Statistical analysis.Statistical analysis was conducted by GraphPad Prism software(GraphPad Software Inc.,USA).Statistical comparisons were made using t-test and one-way analysis of variance(ANOVA).Differences were considered statistically significant at a value of P<0.05.All results were expressed as means 土 SEM of the indicated number of experiments and represent at least 3 independent experiments.Results1.Morphological characteristics of MSCs and flow cytometry analysisIn primary culture,MSCs were mixed with non-adherent cells.However,following two to three passages,the cells eliminated from the population.MSCs contained two morphologically distinct cell types:spindle-shaped cells and large,flat cells.The specific surface antigen markers of MSCs were detected via immunofluorescence.The results of immunofluorescence staining demonstrated that the MSCs expressed CD44,CD90.However,the expression of CD34,CD45 and was negative.The cells were analyzed for expression of MSCs markers using flow cytometry.The percentage of cells expressing the MSCs markers CD44,CD90,CD34 and CD45 are shown in Fig.2B.The percentages of CD44+,CD90+,CD34+ and CD45+ cells were calculated to be 93.83,96.88,6.32,and 8.32%,respectively.2.MSCs transplatantion has no effect on soleus muscle mass,peak tetanic force(Po)and cross-sectional area.Soleus mass were normalized to body weight for comparisons.Soleus wet masses in the IM-PBS group were reduced compared with wet masses in the WB group(p<0.001).MSCs improved muscle mass compared to WB controls but compared with PBS-treated animals,MSCs(IM-MSC group)did not stop muscle mass diminishment.Mean muscle fiber cross-sectional solei areas in the IM-PBS group were reduced significantly compared with the WB group but differences between IM-PBS and IM-MSC groups were significant.After normalizing Po to the cross-sectional area,IM-PBS Po was reduced compared with WB group(p<0.05)but no significant difference was noted between IM-PBS and IM-MSC groups.2.Satellite cell proliferationTo detect changes in total satellite pools and proliferation after hind limb immobilization,we measured Pax7-immunoreactive nuclei and co-labeled them with BrdU.Total satellite cells as indicated by Pax7 nuclei decreased by about half after 14 d of hind limb immobilization.Satellite cell proliferation was also reduced after hind limb immobilization.Measurements of BrdU+/Pax7+ double-positive nuclei among Pax7-immunoreactive cells indicated that MSCs not only prevented satellite cell decline but also significantly induced satellite cell proliferation(p<0.05).Compared with the IM-PBS group,the ratio of BrdU+/Pax7+ nuclei within the total Pax7+ population in the IM-MSC group was increased and was greater than that of WB group(p<0.05).3.MSCs transplantation suppresses apoptosisTUNEL was used to visualize DNA fragmentation to identify apoptotic myonuclei,and apoptotic indices,represented by total apoptotic myonuclei per 103 myofibers in solei,were significantly increased(p<0.05)in the IM-PBS group compared with WB controls.MSCs significantly reduced apoptotic indices in the IM-MSC group compared with the IM-PBS group but the index was greater than in WB controls(p<0.05).4.MSCs activate the Akt pathway and regulate the expression of Bcl-2 and BaxWe assessed the expression of these proteins in soleus after immobilization and investigated whether MSCs could change expression of these proteins.Western blot indicated that in the IM-PBS group,Bax expression was higher(p<0.05)and after MSC treatment Bax was reduced in the IM-MSC group compared with the IM-PBS group(p<0.05).Bcl-2 and p-Akt expression was greater in the IM-PBS group and Bcl-2 expression was the greatest in the WB group and this group had the least Bax expression(p<0.05).ConclusionMSCs injected during hind limb immobilization can maintain satellite cell activity by suppressing myonuclear apoptosis. |
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