Skeletal Muscle Regrowth Research Articles

The Impact Of CD146 +/- Serum Extracellular Vesicles On Recovery Of Skeletal Muscle Following Hindlimb Immobilization

Our laboratory recently reported a significant decline in muscle-resident pericyte quantity following hindlimb immobilization, and subsequently demonstrated the capacity for pericyte transplantation to accelerate recovery of skeletal muscle mass during the rehabilitation period. PURPOSE: The purpose of this study was to determine the extent to which pericyte-derived extracellular vesicles (EVs) effectively recover skeletal muscle mass following hindlimb immobilization. METHODS: Two groups of donor mice (n=6/group) were used to isolate serum EVs before (“Pre”) and after (“Post”) an acute bout of contraction using a sciatic nerve stimulation procedure. Serum EVs were isolated using ultracentrifugation followed by magnetic bead sorting to isolate CD146+ EVs and CD146- EVs. Five groups of mice (n=4/group) underwent unilateral hindlimb immobilization for 14 days. At 14 days, the mice were intramuscularly (tibialis anterior) injected with 1) PBS, 2) CD146+Pre EVs 3) CD146+Post EVs, 4) CD146-Pre EVs, or 5) CD146-Post EVs (in 40 μL of PBS), then remobilized for 2 weeks to determine therapeutic capacity. RESULTS: A significant decrease in serum CD146+ EVs was observed following 14 days of hindlimb immobilization (p<0.05). CD146-Post EVs demonstrated significant recovery of myofiber cross-sectional area compared to PBS control (p<0.05). CD146+Pre and CD146+Post EVs significantly restored capillary density compared to PBS control (p<0.05). CD146-Pre EVs recovered capacity for collagen remodeling compared to PBS control (p<0.05). CONCLUSIONS: The results from this study suggest that CD146- serum EVs positively benefit regrowth of skeletal muscle following a period of disuse. Additionally, CD146+ serum EVs enhance skeletal muscle capillarization. Overall, a combination of both EV fractions may optimize recovery of skeletal muscle mass following disuse. Supported by NIH NIAMS R01 AR072735 (to MDB) and ACSM NASA Space Physiology Research Grant (to SD).

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.

Aging impairs mouse skeletal muscle macrophage polarization and muscle-specific abundance during recovery from disuse.

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.

Enhanced skeletal muscle regrowth and remodelling in massaged and contralateral non-massaged hindlimb.

Muscle fibre cross sectional area is enhanced with massage in the form of cyclic compressive loading during regrowth after atrophy. Massage enhances protein synthesis of the myofibrillar and cytosolic, but not the mitochondrial fraction, in muscle during regrowth. Focal adhesion kinase activation and satellite cell number are elevated in muscles undergoing massage during regrowth. Muscle fibre cross sectional area and protein synthesis of the myofibrillar fraction, but not DNA synthesis, are elevated in muscle of the contralateral non-massaged limb. Massage in the form of cyclic compressive loading is a potential anabolic intervention during muscle regrowth after atrophy. Massage, in the form of cyclic compressive loading (CCL), is associated with multiple health benefits, but its potential anabolic effect on atrophied muscle has not been investigated. We hypothesized that the mechanical activity associated with CCL induces an anabolic effect in skeletal muscle undergoing regrowth after a period of atrophy. Fischer-Brown Norway rats at 10months of age were hindlimb unloaded for a period of 2weeks. The rats were then allowed reambulation with CCL applied at a 4.5N load at 0.5Hz frequency for 30min every other day for four bouts during a regrowth period of 8days. Muscle fibre cross sectional area was enhanced by 18% with massage during regrowth compared to reloading alone, and this was accompanied by elevated myofibrillar and cytosolic protein as well as DNA synthesis. Focal adhesion kinase phosphorylation indicated that CCL increased mechanical stimulation, while a higher number of Pax7+ cells likely explains the elevated DNA synthesis. Surprisingly, the contralateral non-massaged limb exhibited a comparable 17% higher muscle fibre size compared to reloading alone, and myofibrillar protein synthesis, but not DNA synthesis, was also elevated. We conclude that massage in the form of CCL induces an anabolic response in muscles regrowing after an atrophy-inducing event. We suggest that massage can be used as an intervention to aid in the regrowth of muscle lost during immobilization.

Inducible satellite cell depletion attenuates skeletal muscle regrowth following a scald-burn injury.

Severe burns result in significant skeletal muscle cachexia that impedes recovery. Activity of satellite cells, skeletal muscle stem cells, is altered following a burn injury and likely hinders regrowth of muscle. Severe burn injury induces satellite cell proliferation and fusion into myofibres with greater activity in muscles proximal to the injury site. Conditional depletion of satellite cells attenuates recovery of myofibre area and volume following a scald burn injury in mice. Skeletal muscle regrowth following a burn injury requires satellite cell activity, underscoring the therapeutic potential of satellite cells in the prevention of prolonged frailty in burn survivors. Severe burns result in profound skeletal muscle atrophy; persistent muscle atrophy and weakness are major complications that hamper recovery from burn injury. Many factors contribute to the erosion of muscle mass following burn trauma, and we have previously shown concurrent activation and apoptosis of muscle satellite cells following a burn injury in paediatric patients. To determine the necessity of satellite cells during muscle recovery following a burn injury, we utilized a genetically modified mouse model (Pax7CreER -DTA) that allows for the conditional depletion of satellite cells in skeletal muscle. Additionally, mice were provided 5-ethynyl-2'-deoxyuridine to determine satellite cell proliferation, activation and fusion. Juvenile satellite cell-wild-type (SC-WT) and satellite cell-depleted (SC-Dep) mice (8weeks of age) were randomized to sham or burn injury consisting of a dorsal scald burn injury covering 30% of total body surface area. Both hindlimb and dorsal muscles were studied at 7, 14 and 21days post-burn. SC-Dep mice had >93% depletion of satellite cells compared to SC-WT (P<0.05). Burn injury induced robust atrophy in muscles located both proximal and distal to the injury site (∼30% decrease in fibre cross-sectional area, P<0.05). Additionally, burn injury induced skeletal muscle regeneration, satellite cell proliferation and fusion. Depletion of satellite cells impaired post-burn recovery of both muscle fibre cross-sectional area and volume (P<0.05). These findings support an integral role for satellite cells in the aetiology of lean tissue recovery following a severe burn injury.

Nitric oxide synthase inhibition impairs muscle regrowth following immobilization

Nitric oxide (NO) has been shown to increase skeletal muscle protein synthesis. However, the role of NO during skeletal muscle regrowth after immobilization remains unknown. The purpose of this study was to determine whether NO is required for muscle regrowth/recovery after a period of disuse by immobilization. Male Wistar rats were divided into 4 groups: recovered, 1-(2-trifluoromethyl-phenyl)-imidazole (TRIM; 10 mg·kg body mass−1·day−1), NG-nitro-l-arginine methyl ester (l-NAME; 90 mg·kg body mass−1·day−1), and control. The recovered, TRIM, l-NAME groups were submitted to a 7-d muscle recovery period (by remobilization), following a 10-d immobilization period (to induce plantaris [PLA] muscle atrophy). After the experimental period, the PLA muscle was collected for morphometrical (muscle fibers cross-sectional area [CSA]) and molecular (Phospho-mTORSer2448 protein expression) analysis. After 7 d of recovery, the recovered group displayed complete muscle regrowth (CSA, recovered: 2.216 ± 214 vs. control: 2.219 ± 280 cm2; P > 0.05). However, CSA of the l-NAME (1.911 ± 267 cm2) and TRIM (1.896 ± 219 cm2) groups were statistically (P < 0.05) lower than the recovered and control groups. Additionally, there was a 29% increase in Phos-mTORSer2448 protein expression levels in the recovered group compared to control group, and this increase was blocked in both TRIM and l-NAME groups. In conclusion, our results indicate that NO is crucial for skeletal muscle regrowth after an immobilization period, potentially via the mTOR signaling pathway.

The involvement of transient receptor potential canonical type 1 in skeletal muscle regrowth after unloading-induced atrophy.

Decreased mechanical loading results in skeletal muscle atrophy. The transient receptor potential canonical type 1 (TRPC1) protein is implicated in this process. Investigation of the regulation of TRPC1 in vivo has rarely been reported. In the present study, we employ the mouse hindlimb unloading and reloading model to examine the involvement of TRPC1 in the regulation of muscle atrophy and regrowth, respectively. We establish the physiological relevance of the concept that manipulation of TRPC1 could interfere with muscle regrowth processes following an atrophy-inducing event. Specifically, we show that suppressing TRPC1 expression during reloading impairs the recovery of the muscle mass and slow myosin heavy chain profile. Calcineurin appears to be part of the signalling pathway involved in the regulation of TRPC1 expression during muscle regrowth. These results provide new insights concerning the function of TRPC1. Interventions targeting TRPC1 or its downstream or upstream pathways could be useful for promoting muscle regeneration. Decreased mechanical loading, such as bed rest, results in skeletal muscle atrophy. The functional consequences of decreased mechanical loading include a loss of muscle mass and decreased muscle strength, particularly in anti-gravity muscles. The purpose of this investigation was to clarify the regulatory role of the transient receptor potential canonical type 1 (TRPC1) protein during muscle atrophy and regrowth. Mice were subjected to 14days of hindlimb unloading followed by 3, 7, 14 and 28days of reloading. Weight-bearing mice were used as controls. TRPC1 expression in the soleus muscle decreased significantly and persisted at 7days of reloading. Small interfering RNA (siRNA)-mediated downregulation of TRPC1 in weight-bearing soleus muscles resulted in a reduced muscle mass and a reduced myofibre cross-sectional area (CSA). Microinjecting siRNA into soleus muscles in vivo after 7days of reloading provided further evidence for the role of TRPC1 in regulating muscle regrowth. Myofibre CSA, as well as the percentage of slow myosin heavy chain-positive myofibres, was significantly lower in TRPC1-siRNA-expressing muscles than in control muscles after 14days of reloading. Additionally, inhibition of calcineurin (CaN) activity downregulated TRPC1 expression in both weight-bearing and reloaded muscles, suggesting a possible association between CaN and TRPC1 during skeletal muscle regrowth.

Regrowth after skeletal muscle atrophy is impaired in aged rats, despite similar responses in signaling pathways

Skeletal muscle regrowth after atrophy is impaired in the aged and in this study we hypothesized that this can be explained by a blunted response of signaling pathways and cellular processes during reloading after hind limb suspension in muscles from old rats. Male Brown Norway Fisher 344 rats at 6 (young) and 32 (old) months of age were subjected to normal ambulatory conditions (amb), hind limb suspension for 14days (HS), and HS followed by reloading through normal ambulation for 14days (RE); soleus muscles were used for analysis of intracellular signaling pathways and cellular processes. Soleus muscle regrowth was blunted in old compared to young rats which coincided with a recovery of serum IGF-1 and IGFBP-3 levels in young but not old. However, the response to reloading for p-Akt, p-p70s6k and p-GSK3β protein abundance was similar between muscles from young and old rats, even though main effects for age indicate an increase in activation of this protein synthesis pathway in the aged. Similarly, MAFbx mRNA levels in soleus muscle from old rats recovered to the same extent as in the young, while Murf-1 was unchanged. mRNA abundance of autophagy markers Atg5 and Atg7 showed an identical response in muscle from old compared to young rats, but beclin did not. Autophagic flux was not changed at either age at the measured time point. Apoptosis was elevated in soleus muscle from old rats particularly with HS, but recovered in HSRE and these changes were not associated with differences in caspase-3, -8 or -9 activity in any group. Protein abundance of apoptosis repressor with caspase-recruitment domain (ARC), cytosolic EndoG, as well as cytosolic and nuclear apoptosis inducing factor (AIF) were lower in muscle from old rats, and there was no age-related difference in the response to atrophy or regrowth. Soleus muscles from old rats had a higher number of ED2 positive macrophages in all groups and these decreased with HS, but recovered in HSRE in the old, while no changes were observed in the young. Pro-inflammatory cytokines in serum did not show a differential response with age to different loading conditions. Results indicate that at the measured time point the impaired skeletal muscle regrowth after atrophy in aged animals is not associated with a general lack of responsiveness to changes in loading conditions.

Non‐passaged muscle precursor cells from 32‐month old rat skeletal muscle have delayed proliferation and differentiation

The systemic environment and satellite cell dysfunction have been proposed as important contributors in the development of sarcopenia and impaired skeletal muscle regrowth with ageing. In the present study, we investigated effects of serum age on proliferation of muscle precursor cells (MPCs) isolated from skeletal muscles of young and old rats. We examined proliferation and subsequent differentiation of non-passaged MPCs isolated from skeletal muscles of 1-, 3- and 32-month old rats over a 72-h time course, using a serum cross-over design. We found no effect of serum age on MPC proliferation, but we did discover that MPCs isolated from skeletal muscle of 32-month old rats had delayed onset of, and exit from proliferation, compared to MPCs isolated from skeletal muscle of 1-month old rats. Delayed proliferation of MPCs from 32-month old rats was associated with delayed p38 MAPK phosphorylation, and MyoD and p21(Cip1) protein expression. We also demonstrate that MPCs from 32-month old rats exhibited lower levels of muscle creatine kinase mRNA compared to 1-month old rats, but elevated levels of myogenin mRNA, when stimulated to differentiate after 36 h proliferation. These findings suggest that delayed entry and exit of the cell cycle observed in MPCs from 32-month old rats may compromise their ability to respond to differentiation stimuli and subsequently impair myogenic potential of 32-month old skeletal muscle, in this model.

Satellite cell depletion does not inhibit adult skeletal muscle regrowth following unloading-induced atrophy

Resident muscle stem cells, known as satellite cells, are thought to be the main mediators of skeletal muscle plasticity. Satellite cells are activated, replicate, and fuse into existing muscle fibers in response to both muscle injury and mechanical load. It is generally well-accepted that satellite cells participate in postnatal growth, hypertrophy, and muscle regeneration following injury; however, their role in muscle regrowth following an atrophic stimulus remains equivocal. The current study employed a genetic mouse model (Pax7-DTA) that allowed for the effective depletion of >90% of satellite cells in adult muscle upon the administration of tamoxifen. Vehicle and tamoxifen-treated young adult female mice were either hindlimb suspended for 14 days to induce muscle atrophy or hindlimb suspended for 14 days followed by 14 days of reloading to allow regrowth, or they remained ambulatory for the duration of the experimental protocol. Additionally, 5-bromo-2'-deoxyuridine (BrdU) was added to the drinking water to track cell proliferation. Soleus muscle atrophy, as measured by whole muscle wet weight, fiber cross-sectional area, and single-fiber width, occurred in response to suspension and did not differ between satellite cell-depleted and control muscles. Furthermore, the depletion of satellite cells did not attenuate muscle mass or force recovery during the 14-day reloading period, suggesting that satellite cells are not required for muscle regrowth. Myonuclear number was not altered during either the suspension or the reloading period in soleus muscle fibers from vehicle-treated or satellite cell-depleted animals. Thus, myonuclear domain size was reduced following suspension due to decreased cytoplasmic volume and was completely restored following reloading, independent of the presence of satellite cells. These results provide convincing evidence that satellite cells are not required for muscle regrowth following atrophy and that, instead, the myonuclear domain size changes as myofibers adapt.

Satellite Cells are not Prerequisite for Skeletal Muscle Regrowth Following Unloading‐Induced Atrophy

Resident muscle stem cells, known as satellite cells, are thought to be hallmarks of skeletal muscle plasticity. It is generally well accepted that satellite cells are essential for muscle regeneration following injury, however, their role in muscle regrowth following an atrophic stimuli remains unclear. The current study employed a genetically modified mouse model (Pax7‐DTA) that allowed for the effective ablation of &gt;90% of satellite cells in adulthood. Control and satellite cell ablated young female mice were either hindlimb suspended (HS) for 14 days, or HS for 14 days followed by 14 days of reloading to follow regrowth. Muscle atrophy as measured by whole muscle wet weight, fiber cross‐sectional area and single‐fiber width occurred in response to HS and did not differ between satellite cell ablated muscles and control muscles. Furthermore, the absence of satellite cells did not attenuate muscle mass recovery during the reloading period, suggesting that satellite cells are not mandatory for muscle regrowth. Interestingly, the myonuclei from satellite cell ablated muscles were longer than myonuclei from non‐ablated muscles, alluding to a potential mechanism of regulation between satellite cells and myonuclear size. The data from the present study suggest that satellite cells are not a requirement for muscle regrowth following atrophy; however, their absence may alter myonuclear size. NIAMS: R01AR060701

Absence of heat shock transcription factor 1 retards the regrowth of atrophied soleus muscle in mice

Effects of heat shock transcription factor 1 (HSF1) gene on the regrowth of atrophied mouse soleus muscles were studied. Both HSF1-null and wild-type mice were subjected to continuous hindlimb suspension for 2 wk followed by 4 wk of ambulation recovery. There was no difference in the magnitude of suspension-related decrease of muscle weight, protein content, and the cross-sectional area of muscle fibers between both types of mice. However, the regrowth of atrophied soleus muscle in HSF1-null mice was slower compared with that in wild-type mice. Lower baseline expression level of HSP25, HSC70, and HSP72 were noted in soleus muscle of HSF1-null mice. Unloading-associated downregulation and reloading-associated upregulation of HSP25 and HSP72 mRNA were observed not only in wild-type mice but also in HSF1-null mice. Reloading-associated upregulation of HSP72 and HSP25 during the regrowth of atrophied muscle was observed in wild-type mice. Minor and delayed upregulation of HSP72 at mRNA and protein levels was also seen in HSF1-null mice. Significant upregulations of HSF2 and HSF4 were observed immediately after the suspension in HSF1-null mice, but not in wild-type mice. Therefore, HSP72 expression in soleus muscle might be regulated by the posttranscriptional level, but not by the stress response. Evidence from this study suggested that the upregulation of HSPs induced by HSF1-associated stress response might play, in part, important roles in the mechanical loading (stress)-associated regrowth of skeletal muscle.

The Radiosensitivity of Satellite Cells: Cell Cycle Regulation, Apoptosis and Oxidative Stress

Skeletal muscles are the organ of movement, and their growth, regeneration and maintenance are dependent in large part on a population of myogenic stem cells known as satellite cells. Skeletal muscles and these resident myogenic stem cells (i.e., satellite cells) are commonly exposed to significant doses of radiation during diagnostic procedures and/or during the radiotherapeutic management of cancer. The main objective of this study was to examine the effects of clinically relevant doses of γ radiation on satellite cell survival and proliferation, cell cycle regulation, apoptosis, DNA double-strand break repair, oxidative stress and NO production. Overall, our findings demonstrate that doses of γ radiation ≥5 Gy reduced satellite cell numbers by at least 70% due in part to elevated apoptosis and the inhibition of cell cycle progression. Radiation was also found to cause a significant and persistent increase in the level of reactive oxygen and nitrogen species. Interestingly, and within this backdrop of elevated oxidative stress, similar doses were found to produce substantial reductions in the levels of nitric oxide (NO). Proliferation of satellite cells has been shown to depend in part on the production of NO, and our findings give rise to the possibility that radiation-induced reductions in NO levels may provide a mechanism for the inhibition of satellite cell proliferation in vitro and possibly the regrowth of skeletal muscle exposed during clinical irradiation procedures.

Examination of FGF2‐dependent proliferation in muscle precursor cells isolated from 3‐mo‐old and 32‐mo‐old rats: Implication for regrowth, repair, and regeneration of skeletal muscle

Examination of FGF2‐dependent proliferation in muscle precursor cells isolated from 3‐mo‐old and 32‐mo‐old rats: Implication for regrowth, repair, and regeneration of skeletal muscle

Heat stress facilitates the regeneration of injured skeletal muscle in rats

BackgroundSkeletal muscle stem cells, so-called muscle satellite cells, are responsible for the repair and the regeneration of adult skeletal muscle tissues. Heat stress can facilitate the proliferation and the differentiation of myoblasts in vitro and can enhance their proliferative potential, which may stimulate the regrowth of atrophied skeletal muscle. The purpose of this study was to investigate the effect of heat stress on the regeneration of skeletal muscle injury induced by cardiotoxin. MethodsMale Wistar rats, aged 7 weeks, were randomly divided into six groups: a nonheated control group that received a physiological saline injection, a group heat stressed before physiological saline injection, a group heat stressed after physiological saline injection, a group injected with cardiotoxin without heat stress, a group heat stressed before cardiotoxin injection, and a group heat stressed after cardiotoxin injection (25 in each group). To initiate muscle injury and regeneration, 0.5ml of 10μM cardiotoxin was injected into the left tibialis anterior muscle. Conscious rats in some groups were exposed to environmental heat stress (41°C for 60 min) in a heat chamber 24h before or immediately after cardiotoxin or physiological saline injection. The heating protocol in the present study causes an increase in the colonic temperature to 41°C. The left tibialis anterior muscles were dissected 1, 3, 7, 14, and 28 days after injection of cardiotoxin or physiological saline. ResultsThe wet weight and water content of muscles increased 1 day after cardiotoxin injection regardless of the application of heat stress, but normalized after 7–14 days. The muscle protein content in control rats had increased 7 days after heat stress. Although the muscle protein content decreased on cardiotoxin injection, heat stress caused a significant recovery in protein level. Expression of heat shock protein 72 (HSP72) and the number of Pax7-positive nuclei decreased after cardiotoxin injection but increased on the application of heat stress in both normal control and cardiotox-in-injected groups. ConclusionsHeat stress stimulated not only the proliferation of satellite cells but also protein synthesis during the regeneration of injured skeletal muscle. It is thus strongly suggested that the heating of injured skeletal muscle may facilitate recovery. There was no direct relationship between the level of HSP72 expression and muscle protein content, suggesting that HSP72 expression may not be the key signal for protein synthesis in the necrosis–regeneration process.

Sirt1 increases satellite cell cycle progression

The repair, regrowth, and hypertrophy of skeletal muscle is largely dependent on the proliferation of satellite cells, muscle precursor cells located between the basal lamina and plasmalemma of mature muscle fibers. The objective of the present study was to determine the effect of the class III, NAD+-dependent histone deacetylase, Sirt1, on satellite cell cycle progression. Satellite cells were isolated from Fischer 344 × Brown Norway F1 hybrid male rats (3–4 months old), cultured for 5–6 days, passagedonce, and seeded onto Matrigel-coated tissue culture plates. 18 hours later, cells were infected with a recombinant adenovirus engineered to overexpress Sirt1 (Ad-Sirt1) or β-galactosidase (Ad-LacZ) to determine the effects of increasing Sirt1 on satellite cell cycle progression. Ad-Sirt1 infection increased MPC cell cycle progression and markers of the proliferative state as evidenced by an increase in cell number, PCNA expression, and the phosphorylation of retinoblastoma protein. Ad-Sirt1 infection decreased and increased the expression of the cyclin dependent kinase inhibitors p21Waf/Cip1and p27Kip1, respectively. These studies identify Sirt1 as a positive regulator of MPC cycle progression, potentially through its ability to decrease the levels of the cdk inhibitor p21Waf/Cip1. Supported by NIH AG18780 and NIH AR048523

Myogenic differentiation during regrowth of atrophied skeletal muscle is associated with inactivation of GSK-3β

Muscle atrophy contributes to morbidity and mortality in aging and chronic disease, emphasizing the need to gain understanding of the mechanisms involved in muscle atrophy and (re)growth. We hypothesized that the magnitude of muscle regrowth during recovery from atrophy determines whether myonuclear accretion and myogenic differentiation are required and that insulin-like growth factor (IGF)-I/Akt/glycogen synthase kinase (GSK)-3beta signaling differs between regrowth responses. To address this hypothesis we subjected mice to hindlimb suspension (HS) to induce atrophy of soleus (-40%) and plantaris (-27%) muscle. Reloading-induced muscle regrowth was complete after 14 days and involved an increase in IGF-IEa mRNA expression that coincided with Akt phosphorylation in both muscles. In contrast, phosphorylation and inactivation of GSK-3beta were observed during soleus regrowth only. Furthermore, soleus but not plantaris regrowth involved muscle regeneration based on a transient increase in expression of histone 3.2 and myosin heavy chain-perinatal, which are markers of myoblast proliferation and differentiation, and a strong induction of muscle regulatory factor (MRF) expression. Experiments in cultured muscle cells showed that IGF-I-induced MRF expression is facilitated by inactivation of GSK-3beta and selectively occurs in the myoblast population. This study suggests that induction of IGF-I expression and Akt phosphorylation during recovery from muscle atrophy is independent of the magnitude of muscle regrowth. Moreover, our data demonstrate for the first time that the regenerative response characterized by myoblast proliferation, differentiation, and increased MRF expression in recovering muscle is associated with the magnitude of regrowth and may be regulated by inactivation of GSK-3beta.

Intermittent hyperthermia enhances skeletal muscle regrowth and attenuates oxidative damage following reloading

Skeletal muscle reloading following disuse is characterized by profound oxidative damage. This study tested the hypothesis that intermittent hyperthermia during reloading attenuates oxidative damage and augments skeletal muscle regrowth following immobilization. Forty animals were randomly divided into four groups: control (Con), immobilized (Im), reloaded (RC), and reloaded and heated (RH). All groups but Con were immobilized for 7 days. Animals in the RC and RH groups were then reloaded for 7 days with (RH) or without (RC) hyperthermia (41-41.5 degrees C for 30 min on alternating days) during reloading. Heating resulted in approximately 25% elevation in heat shock protein expression (P < 0.05) and an approximately 30% greater soleus regrowth (P < 0.05) in RH compared with RC. Furthermore, oxidant damage was lower in the RH group compared with RC because nitrotyrosine and 4-hydroxy-2-nonenol were returned to near baseline when heating was combined with reloading. Reduced oxidant damage was independent of antioxidant enzymes (manganese superoxide dismutase, copper-zinc superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase). In summary, these data suggest that intermittent hyperthermia during reloading attenuates oxidative stress and improves the rate of skeletal muscle regrowth during reloading after immobilization.

Heating enhances skeletal muscle regrowth rate and may increase IGF‐1 pathway activation

Heating enhances skeletal muscle regrowth rate and may increase IGF‐1 pathway activation

Heating enhances muscle regrowth rate and reduces oxidant stress

Muscle regrowth after disuse is characterized by a period of increased oxidant stress. We have previously shown that heating can act as an antioxidant therapy. We hypothesized that heating during reloading would induce heat shock proteins resulting in an increased rate of skeletal muscle regrowth and reduced oxidant stress when compared to reloading alone. Rats were divided into four groups: control (Con; n=10), immobilized (Im; n=10), reloaded control (R7C; n=10), and reloaded heated (R7H; n=10). One week of immobilization resulted in a 50% and 90% reduction in HSPs 25 and 72, respectively, and an ~50% induction of HSP 32 while reloading returned HSPs to Con levels. HSP content was increased ~30% in the R7H group when compared to the R7C group. Immobilization resulted in a 45% reduction in soleus muscle mass. Reloading did not significantly increase soleus muscle mass but the R7H group recovered 50% of the loss caused by atrophy. Oxidant damage to lipids was increased 20% and 25% while protein oxidation was increased 60% and 80% in the Im and R7C groups compared to Con, but was reduced to Con levels in the R7H group. CuZn SOD activity was increased ~30% and Cat activity was increased ~20% in Im and R7C compared to Con, while the R7H was similar to Con. We conclude that heating during reloading increases the rate of skeletal muscle regrowth through a reduction in oxidant stress independent of predominant antioxidant enzymes. (Supported by the NFLPA)