Insulin-like growth factor-I biocompartmentalization across blood, interstitial fluid and muscle, before and after 3 months of chronic resistance exercise.

This investigation examined the influence of 12-week ballistic resistance training programs on the IGF-I system in circulation, interstitial fluid, and skeletal muscle, at rest and in response to acute exercise. Seventeen college-aged subjects (11 women/6 men; 21.7 ± 3.7 yr) completed an acute ballistic exercise bout before and after the training program. Blood samples were collected pre-, mid-, and postexercise and analyzed for serum total IGF-I, free IGF-I, and IGF binding proteins (IGFBPs) 1-4. Dialysate and interstitial free IGF-I were analyzed in vastus lateralis (VL) interstitial fluid collected pre- and postexercise via microdialysis. Pre- and postexercise VL muscle biopsies were analyzed for IGF-I protein expression, IGF-I receptor phosphorylation (p-IGF-IR), and AKT phosphorylation (p-AKT). Following training, basal serum IGF-I, free IGF-I, IGFBP-2, and IGFBP-3 decreased whereas IGFBP-1 and IGFBP-4 increased. Training reduced basal dialysate and interstitial free IGF-I but had no effect on basal skeletal muscle IGF-I, p-IGF-IR, or p-AKT. Acute exercise elicited transient changes in IGF-I system concentrations and downstream anabolic signaling both pre- and posttraining; training did not affect this acute exercise response. Posttraining, acute exercise-induced changes in dialysate/interstitial free IGF-I were strongly correlated with the changes in intramuscular IGF-I expression, p-IGF-IR, and p-AKT. The divergent influence of resistance training on circulating/interstitial and skeletal muscle IGF-I demonstrates the importance of concurrent, multiple biocompartment analysis when examining the IGF-I system. As training elicited muscle hypertrophy, these findings indicate that IGF-I's anabolic effects on skeletal muscle are mediated by local, rather than systemic mechanisms.NEW & NOTEWORTHY In the first investigation to assess resistance training's effects on the IGF-I system in serum, interstitial fluid, and skeletal muscle, training decreased basal circulating and interstitial IGF-I but did not alter basal intramuscular IGF-I protein activity. Posttraining, acute exercise-induced interstitial IGF-I increases were strongly correlated with intramuscular IGF-I expression and signaling. These findings highlight the importance of multibiocompartment measurement when analyzing IGF-I and suggest that IGF-I's role in hypertrophic adaptations is locally mediated.

Dietary Zinc Modulates Matrix Metalloproteinases in Traumatic Brain Injury.

Animal models of mild traumatic brain injury (mTBI) provide opportunity to examine the extent to which dietary interventions can be used to improve recovery after injury. Animal studies also suggest that matrix metalloproteinases (MMPs) play a role in tissue remodeling post-TBI. Because dietary zinc (Zn) improved recovery in nonblast mTBI models, and the MMPs are Zn-requiring enzymes, we evaluated the effects of low- (LoZn) and adequate-Zn (AdZn) diets on MMP expression and behavioral responses, subsequent to exposure to a single blast. MMP messenger RNA expression in soleus muscle and frontal cortex tissues were quantified at 48 h and 14 days post-blast. In muscle, blast resulted in significant upregulation of membrane-type (MT)-MMP, MMP-2, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 at 48 h post-injury in rats consuming AdZn. At 14 days post-blast, there were no blast or dietary effects observed on MMP levels in muscle, supporting the existence of a Zn-responsive, functional repair and remodeling mechanism. In contrast, blast resulted in a significant downregulation of MT-MMP, TIMP-1, and TIMP-2 and a significant upregulation of MMP-3 levels at 48 h post-injury in cortex tissue, whereas at 14 days post-blast, MT-MMP, MMP-2, and TIMP-2 were all downregulated in response to blast, independent of diet, and TIMP-1 were significantly increased in rats fed AdZn diets despite the absence of elevated MMPs. Because the blast injuries occurred while animals were under general anesthesia, the increased immobility observed post-injury in rats consuming LoZn diets suggest that blast mTBI can, in the absence of any psychological stressor, induce post-traumatic stress disorder-related traits that are chronic, but responsive to diet. Taken together, our results support a relationship between marginally Zn-deficient status and a compromised regenerative response post-injury in muscle, likely through the MMP pathway. However, in neuronal tissue, changes in MMP/TIMP levels after blast indicate a variable response to marginally Zn-deficient diets that may help explain compromised repair mechanism(s) previously associated with the systemic hypozincemia that develops in patients with TBI.

RNA transcript expression of IGF-I/PI3K pathway components in regenerating skeletal muscle is sensitive to initial injury intensity.

OBJECTIVE: Skeletal muscle regeneration is a complex process involving the coordinated input from multiple stimuli. Of these processes, actions of the insulin-like growth factor-I (IGF-I) and phosphoinositide 3-kinase (PI3K) pathways are vital; however, whether IGF-I or PI3K expression is modified during regeneration relative to initial damage intensity is unknown. The objective of this study was to determine whether mRNA expression of IGF-I/PI3K pathway components was differentially regulated during muscle regeneration in mice in response to traumatic injury induced by freezing of two different durations. DESIGN: Traumatic injury was imposed by applying a 6-mm diameter cylindrical steel probe, cooled to the temperature of dry ice (-79°C), to the belly of the left tibialis anterior muscle of 12-week-old C57BL/6J mice for either 5s (5s) or 10s (10s). The right leg served as the uninjured control. RNA was obtained from injured and control muscles following 3, 7, and 21days recovery and examined by real-time PCR. Expression of transcripts within the IGF, PI3K, and Akt families, as well as for myogenic regulatory factors and micro-RNAs were studied. RESULTS: Three days following injury, there was significantly increased expression of Igf1, Igf2, Igf1r, Igf2r, Pik3cb, Pik3cd, Pik3cg, Pik3r1, Pik3r5, Akt1, and Akt3 in response to either 5s or 10s injury compared to uninjured control muscle. There was a significantly greater expression of Pik3cb, Pik3cd, Pik3cg, Pik3r5, Akt1, and Akt3 in 10s injured muscle compared to 5s injured muscle. Seven days following injury, we observed significantly increased expression of Igf1, Igf2, Pik3cd, and Pik3cg in injured muscle compared to control muscle in response to 10s freeze injury. We also observed significantly reduced expression of Igf1r and miR-133a in response to 5s freeze injury compared to control muscle, and significantly reduced expression of Ckm, miR-1 and miR-133a in response to 10s freeze injury as compared to control. Twenty-one days following injury, 5s freeze-injured muscle exhibited significantly increased expression of Igf2, Igf2r, Pik3cg, Akt3, Myod1, Myog, Myf5, and miR-206 compared to control muscle, while 10s freeze-injured muscles showed significantly increased expression of Igf2, Igf2r, Pik3cb, Pik3cd, Pik3r5, Akt1, Akt3, and Myog compared to control. Expression of miR-1 was significantly reduced in 10s freeze-injured muscle compared to control muscle at this time. There were no significant differences in RNA expression between 5s and 10s injury at either 7d or 21d recovery in any transcript examined. CONCLUSIONS: During early skeletal muscle regeneration in mice, transcript expressions for some components of the IGF-I/PI3K pathway are sensitive to initial injury intensity induced by freeze damage.

Transcriptional and Chromatin Dynamics of Muscle Regeneration after Severe Trauma.

Following injury, adult skeletal muscle undergoes a well-coordinated sequence of molecular and physiological events to promote repair and regeneration. However, a thorough understanding of the in vivo epigenomic and transcriptional mechanisms that control these reparative events is lacking. To address this, we monitored the in vivo dynamics of three histone modifications and coding and noncoding RNA expression throughout the regenerative process in a mouse model of traumatic muscle injury. We first illustrate how both coding and noncoding RNAs in tissues and sorted satellite cells are modified and regulated during various stages after trauma. Next, we use chromatin immunoprecipitation followed by sequencing to evaluate the chromatin state of cis-regulatory elements (promoters and enhancers) and view how these elements evolve and influence various muscle repair and regeneration transcriptional programs. These results provide a comprehensive view of the central factors that regulate muscle regeneration and underscore the multiple levels through which both transcriptional and epigenetic patterns are regulated to enact appropriate repair and regeneration.

Does high muscle temperature accentuate skeletal muscle injury from eccentric exercise?

Hyperthermia is suspected of accentuating skeletal muscle injury from novel exercise, but this has not been well studied. This study examined if high muscle temperatures alters skeletal muscle injury induced by eccentric exercise (ECC). Eight volunteers (age, 22.5 ± 4.1 year; height, 169.5 ± 10.8 cm; body mass, 76.2 ± 12.6 kg), serving as their own control, and who were not heat acclimatized, completed two elbow flexor ECC trials; in one trial the biceps were heated >40°C (HEAT) and in the other trial there was no heating (NON). HEAT was applied with shortwave diathermy (100 W) for 15 min immediately before the first ECC bout and for 2 min in between each bout. Individuals were followed for 10 days after each ECC session, with a 6-week washout period between arms. The maximal voluntary isometric contraction decreased by 41 ± 17% and 46 ± 20% in the NON and HEAT trials, respectively. Bicep circumference increased by 0.07 ± 0.08 mm (4%, P = 0.04) and relaxed range of motion decreased by 11.5 ± 8.2° (30%, P < 0.001) in both trials. Serum creatine kinase peaked 72-h following ECC (NON: 6289 ± 10407; HEAT: 5486 ± 6229 IU L(-1), 38-fold increase, P < 0.01) as did serum myoglobin (NON: 362 ± 483; HEAT: 355 ± 373 µg L(-1), 13-fold increase, P < 0.03). Plasma HSP 70 was higher (P < 0.02) in HEAT after 120-h of recovery. There were no differences between treatments for plasma HSP27 and interleukins 1ß, 6, and 10. The results indicate that >40°C muscle temperature does not alter skeletal muscle injury or functional impairments induced by novel ECC.

Combined use of the Ilizarov method, concentrated bone marrow aspirate (cBMA), and platelet-rich plasma (PRP) to expedite healing of bimalleolar fractures.

Distal tibial and fibular fractures, particularly in patients with comorbidities, heal slowly and have a high incidence of postoperative nonunion and infection. Autologous concentrated bone marrow aspirate (cBMA) and platelet-rich plasma (PRP) increase osteogenic potential of demineralized bone matrix (DBM). The purpose of this case series was to evaluate the efficacy of cBMA, PRP, DBM in conjunction with the Ilizarov fixator as compared to DBM and the Ilizarov fixator alone in expediting fracture healing. Ten patients (mean age 52.9 years) were in the cBMA Group, and 10 patients (mean age 54 years) were in the Control Group. Comorbidities included diabetes, obesity, smoking, and renal disease. Radiographs showed a significant difference in the rate of complete healing in the cBMA Group at 16 ± 1.6 weeks post-surgery as compared to 24 ± 1.3 weeks in the Control Group (P < 0.001). No differences were observed between groups in infection rate or nonunions. We conclude that the Ilizarov fixator combined with DBM, cBMA, and PRP expedites fracture healing of the distal tibia and fibula in patients with significant comorbidities.

In vivo Monitoring of Transcriptional Dynamics After Lower-Limb Muscle Injury Enables Quantitative Classification of Healing.

Traumatic lower-limb musculoskeletal injuries are pervasive amongst athletes and the military and typically an individual returns to activity prior to fully healing, increasing a predisposition for additional injuries and chronic pain. Monitoring healing progression after a musculoskeletal injury typically involves different types of imaging but these approaches suffer from several disadvantages. Isolating and profiling transcripts from the injured site would abrogate these shortcomings and provide enumerative insights into the regenerative potential of an individual's muscle after injury. In this study, a traumatic injury was administered to a mouse model and healing progression was examined from 3 hours to 1 month using high-throughput RNA-Sequencing (RNA-Seq). Comprehensive dissection of the genome-wide datasets revealed the injured site to be a dynamic, heterogeneous environment composed of multiple cell types and thousands of genes undergoing significant expression changes in highly regulated networks. Four independent approaches were used to determine the set of genes, isoforms, and genetic pathways most characteristic of different time points post-injury and two novel approaches were developed to classify injured tissues at different time points. These results highlight the possibility to quantitatively track healing progression in situ via transcript profiling using high- throughput sequencing.

Anti-inflammatory interventions and skeletal muscle injury: benefit or detriment?

Exercise, eccentric contractions, acute trauma, and disease are all causal mechanisms of skeletal muscle injury. After skeletal muscle is injured, it undergoes sequential phases of degeneration, inflammation, regeneration, and fibrosis. Events that occur in response to inflammation trigger regenerative processes. However, since inflammation causes pain, decreases skeletal muscle function, has a negative effect on performance, and contributes to fibrosis, which is one of the leading causes of delayed regeneration, the general practice has been to reduce inflammation. The problem with this approach is that preventing inflammation may hinder recovery. Current treatment options for inflammation are not necessarily effective and, in some cases, they may be unsafe. This review focuses on the question of whether the most beneficial course of treatment should be to block inflammation or if it is sensible to allow inflammatory processes to progress naturally. If blocking inflammation is perceived as a beneficial approach, it is not yet known at what time point during the inflammatory response it is most sensible to interfere. To address these issues, this review evaluates the effects of various anti-inflammatory agents on recovery processes in response to exercise-induced, traumatic, and disease-associated models of skeletal muscle injury. A collective analysis such as this should lay the foundation for future work that systematically manipulates the inflammatory response to most effectively promote regeneration and functional recovery in injured skeletal muscle, while reducing the negative effects of inflammatory processes such as pain and fibrosis.

Postmenopausal effects of resistance training on muscle damage and mitochondria.

The purpose of this study was to measure the effects of a 12-month progressive resistance training intervention on muscle morphology and strength gains in postmenopausal women. Skeletal muscle biopsies were obtained from the vastus lateralis of 5 independent community-dwelling women (mean age: 75.6 ± 4.28 years; mean height: 163 ± 5.34 cm; mean weight: 72 ± 17.5 kg) before 6 months and 12 months after progressive resistance training. Muscle strength (1 repetition maximum) was measured at the same time points. After 6 months of training, morphological analysis revealed evidence of increased proteolysis and tissue repair, and rudimentary fiber development. The percent of Z-bands with mild Z-band disruption increased from 43.9% at baseline to 66.7% after 6 months of training (p < 0.01). Mitochondrial volume also increased (percent of mitochondria = 0.86% at baseline, 1.19% at 6 months, and 1.04% at 12 months, p < 0.05), and there was a shift to larger sized mitochondria. The training did not result in statistically significant increases in muscle leg strength (p < 0.18). It appears that mild Z-band disruption acts as a precursor for increased protein synthesis and stimulates an increase in mitochondrial mass. Therefore, although a progressive resistance training program in this population did not increase muscle strength, it did demonstrate clinical applications that lend support to the importance of resistance training in older adults.

IGF-I measurement across blood, interstitial fluid, and muscle biocompartments following explosive, high-power exercise.

Insulin-like growth factor-I (IGF-I) resides across different biocompartments [blood, interstitial fluid (ISF), and muscle]. Whether circulating IGF-I responses to exercise reflect local events remains uncertain. We measured the IGF-I response to plyometric exercise across blood, ISF, and muscle biopsy from the vastus lateralis. Twenty volunteers (8 men, 12 women, 22 ± 1 yr) performed 10 sets of 10 plyometric jump repetitions at a 40% 1-repetition maximum. Blood, ISF, and muscle samples were taken pre- and postexercise. Circulating IGF-I increased postexercise: total IGF-I (preexercise = 546 ± 42, midexercise = 585 ± 43, postexercise = 597 ± 45, +30 = 557 ± 42, +60 = 536 ± 40, +120 = 567 ± 42 ng/ml; midexercise, postexercise, and +120 greater than preexercise, P < 0.05); Free IGF-I (preexercise = 0.83 ± 0.09, midexercise = 0.78 ± 0.10, postexercise = 0.79 ± 0.11, +30 = 0.93 ± 0.10, +60 = 0.88 ± 0.10, + 120 = 0.91 ± 0.11 ng/ml; +30 greater than all other preceding time points, P < 0.05). No exercise-induced changes were observed for ISF IGF-I (preexercise = 2.35 ± 0.29, postexercise = 2.46 ± 0.35 ng/ml). No changes were observed for skeletal muscle IGF-I protein, although IGF-I mRNA content increased ∼40% postexercise. The increase in circulating total and free IGF-I was not correlated with increases in ISF IGF-I or muscle IGF-I protein content. Our data indicate that exercise-induced increases in circulating IGF-I are not reflective of local IGF-I signaling.

Time-course analysis of injured skeletal muscle suggests a critical involvement of ERK1/2 signaling in the acute inflammatory response.

INTRODUCTION: The coupling and timing of pro- and anti-inflammatory processes in skeletal muscle injury is poorly understood. We investigated the temporal response and regulated processes of extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38, and IkappaB kinase (IKK) α/ß signaling pathways after traumatic injury. METHODS: Traumatic freeze injury was delivered to the tibialis anterior (TA) muscle in C57BL/6J mice, and injured and uninjured TA muscles were analyzed 3-72 h into the recovery period. RESULTS: Significant increases in pro-inflammatory cytokine transcription accompanied IKKß phosphorylation, robust ERK pathway activation, and reduced heat shock protein (Hsp) protein expression at 3-24 h. At 24 h, ERK activation was abolished concomitantly with a significant increase in mitogen-activated protein kinase phosphatase-1 (MKP-1). After 24 h, cytokine transcription along with ERK1/2 and IKKß phosphorylation remained suppressed, whereas Hsp protein expression rose to significant levels by 72 h and associated with IKKß. CONCLUSIONS: Results indicate a bimodal regulation of ERK1/2 in acute inflammation in which it is supportive from 3 to 24 h, and suppressive from 24 to 72 h.

Adenosine A3 receptor stimulation reduces muscle injury following physical trauma and is associated with alterations in the MMP/TIMP response.

We have previously demonstrated that in response to traumatic injury in skeletal muscle, there is a dysregulation of the matrix metalloproteases (MMPs) and their inhibitors (TIMPs), a response hypothesized to interfere with proper skeletal muscle regeneration. Moreover, we have shown that pharmacological activation of the adenosine A(3) receptor by Cl-IBMECA in skeletal muscle can protect against ischemia-reperfusion and eccentric exercise injury. However, the mechanism by which Cl-IBMECA protects muscle tissue is poorly defined. This study evaluated the effects of Cl-IBMECA on MMP/TIMP expression in skeletal muscle and tested the hypothesis that adenosine A(3) receptor-stimulated protection of skeletal muscle following traumatic injury is associated with a blunting of MMPs involved in inflammatory processes and collagen degradation, and an increase in MMPs associated with extracellular matrix remodeling. Sixty C57BL/6J male mice were injected with Cl-IBMECA (n = 30) or a vehicle (n = 30), and Evans blue dye. Injury was induced by applying a cold steel probe (-79°C) to the tibialis anterior (TA) muscle for 10 s. TA muscles from uninjured and injured legs were collected 3, 10, and 24 h postinjury for analysis of muscle injury and MMP/TIMP mRNA and protein levels. Twenty-four hours postinjury, 56.8% of the fibers were damaged in vehicle-treated mice vs. 35.4% in Cl-IBMECA-treated mice (P = 0.02). Cl-IBMECA treatment reduced membrane type 1 (MT1)-MMP, MMP-3, MMP-9, and TIMP-1 mRNA expression 2- to 20-fold compared with vehicle-treated mice (P < 0.05). Cl-IBMECA decreased protein levels of latent/shed MT1-MMP 23-2,000%, respectively, 3-10 h postinjury. In Cl-IBMECA-treated mice, latent MMP-2 was decreased 20% 3 h postinjury, active MMP-3 was decreased 64% 3 h postinjury, and latent/active MMP-9 was decreased 417,631% 3 h postinjury and 20% 10 h postinjury. Protein levels of active MMP-2 and latent MMP-3 were increased 25% and 74% 3 h postinjury, respectively. The present study elucidates a new protective role of adenosine A(3) receptor stimulation in posttraumatic skeletal muscle injury.

Relaxin regulates MMP expression and promotes satellite cell mobilization during muscle healing in both young and aged mice.

The polypeptide hormone relaxin has been proven to be effective in promoting both the remodeling and regeneration of various tissues, including cardiac muscle. In addition, our previous study demonstrated that relaxin is beneficial to skeletal muscle healing by both promoting muscle regeneration and preventing fibrosis formation. However, the molecular and cellular mechanisms of relaxin in regulating both myogenic cell differentiation and muscle healing process are still unclear. In this study, C2C12 mouse myoblasts and primary human myoblasts were treated with relaxin to investigate its potential effect in vitro; relaxin was also injected intramuscularly into the injured site of the mouse on the second day after injury to observe its function in vivo, especially in the aged muscle. Results showed that relaxin promoted myogenic differentiation, migration, and activation of matrix metalloproteinases (MMPs) of cultured myoblasts in vitro. In the injured muscle, relaxin administration promoted the activation of Pax7-positive skeletal muscle satellite cells and increased its local population compared with nontreated control muscles. Meanwhile, both angiogenesis and revascularization were increased, while the extended inflammatory reaction was repressed in the relaxin-treated injured muscle. Moreover, relaxin similarly promoted muscle healing in mice with aged muscle. These results revealed the multiple effects of relaxin in systematically improving muscle healing as well as its potential for clinical applications in patients with skeletal muscle injuries and diseases.

Molecular responses to moderate endurance exercise in skeletal muscle.

This study examined alterations in skeletal-muscle growth and atrophy-related molecular events after a single bout of moderate-intensity endurance exercise. Muscle biopsies were obtained from 10 men (23 ± 1 yr, body mass 80 ± 2 kg, and VO(2peak) 45 ± 1 ml x kg⁻¹ x min⁻¹) immediately (0 hr) and 3 hr after a 60-min bout of cycle exercise (60% +/- 5% VO(2peak)). Corresponding muscle biopsies were also obtained under resting conditions. The phosphorylation status of insulin/IGF-PI3K molecular-signaling proteins, ubiquitin-proteasome-related gene expression, FOXO transcription factors, and myogenic regulatory factors in muscle samples was analyzed using multiplex analysis, Western blotting, and quantitative real-time polymerase chain reaction (qRT-PCR). A condition-time interaction was observed for Akt phosphorylation (p < .05) with multiplexing. Regardless of endurance exercise, Akt phosphorylation decreased and ERK phosphorylation increased at 3 hr compared with 0 hr (p < .05). Levels of p70(S6K) phosphorylation were 110% greater (p < .05) at 3 hr than at 0 hr using Western blots. MuRF mRNA expression postexercise increased; levels were 4.7- and 5.7-fold greater (p < .05) at 0 hr and 3 hr, respectively, than at rest with qRT-PCR. Atrogin mRNA expression was up-regulated 3.2-fold 3 hr postexercise compared with rest. These findings demonstrate modest changes in the molecular responses to moderate endurance exercise in the absence of nutrition. This study provides the groundwork for future investigations designed to optimize the metabolic conditions necessary to positively influence the cellular mechanisms specific to skeletal-muscle protein turnover during recovery from endurance exercise.

Adenosine A(3) receptor stimulation induces protection of skeletal muscle from eccentric exercise-mediated injury.

Effective therapy to reduce skeletal muscle injury associated with severe or eccentric exercise is needed. The purpose of this study was to determine whether adenosine receptor stimulation can mediate protection from eccentric exercise-induced muscle injury. Downhill treadmill exercise (-15 degrees ) was used to induce eccentric exercise-mediated skeletal muscle injury. Experiments were conducted in both normal wild-type (WT) mice and also in beta-sarcoglycan knockout dystrophic mice, animals that show an exaggerated muscle damage with the stress of exercise. In the vehicle-treated WT animals, eccentric exercise increased serum creatine kinase (CK) greater than 3-fold to 358.9 +/- 62.7 U/l (SE). This increase was totally abolished by stimulation of the A(3) receptor. In the dystrophic beta-sarcoglycan-null mice, eccentric exercise caused CK levels to reach 55,124 +/- 5,558 U/l. A(3) receptor stimulation in these animals reduced the CK response by nearly 50%. In the dystrophic mice at rest, 10% of the fibers were found to be damaged, as indicated by Evans blue dye staining. While this percentage was doubled after exercise, A(3) receptor stimulation eliminated this increase. Neither the A(1) receptor agonist 2-chloro-N(6)-cyclopentyladenosine (0.05 mg/kg) nor the A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (0.07 mg/kg) protected skeletal muscle from eccentric exercise injury in WT or dystrophic mice. The protective effect of adenosine A(3) receptor stimulation was absent in mice, in which genes for phospholipase C beta2/beta3 (PLCbeta2/beta3) and beta-sarcoglycan were deleted. The present study elucidates a new protective role of the A(3) receptor and PLCbeta2/beta3 and points to a potentially effective therapeutic strategy for eccentric exercise-induced skeletal muscle injury.

Matrix metalloprotease-3 and tissue inhibitor of metalloprotease-1 mRNA and protein levels are altered in response to traumatic skeletal muscle injury.

The purpose of this study was to characterize the time course of matrix metalloprotease-3 (MMP-3) and tissue inhibitor of metalloprotease-1 (TIMP-1) expression in mouse tibialis anterior (TA) muscle post-injury. Mice were anesthetized, the TA muscle exposed, and injury induced by applying a cold steel probe (-79 degrees C) to the muscle for 10 s. Muscle was collected from uninjured and injured legs at 3, 10, 24, 48, and 72 h post-injury. qRT-PCR, immunoblotting, and immunohistochemistry were used to quantify/localize MMP-3 and TIMP-1. MMP-3 transcripts increased 19- and 12-fold, 10 and 24 h post-injury (p < 0.01), respectively. TIMP-1 transcript levels increased 9-, 34-, and 60-fold, 10, 24, and 48 h post-injury (p = 0.01), respectively, with a subsequent decrease 72 h post-injury (p < 0.01). Protein levels of the pro-form of MMP-3 increased within 3 h post-injury and remained elevated (p < 0.05). Active MMP-3 decreased over time, reaching a 72% decrease 72 h post-injury (p < 0.05). TIMP-1 protein decreased 75% within 3 h post-injury, returning to baseline by 72 h post-injury. In response to injury, injured skeletal muscle preferentially produces increased levels of the latent form of the MMP-3 protein with a concomitant decrease in the active form, and a significant decrease in TIMP-1 expression. The altered pattern of MMP-3/TIMP-1 expression may be due to alterations in post-transcriptional mechanisms that are responsible for specific regulation of the MMP-3/TIMP-1 system. These data suggest that there is a disproportionate regulation of the MMP-3/TIMP-1 system following traumatic injury and this response may contribute to impaired extracellular matrix remodeling.