Suppression of protein kinase C theta contributes to enhanced myogenesis in vitro via IRS1 and ERK1/2 phosphorylation.

BACKGROUND: Differentiation and fusion of skeletal muscle myoblasts into multi-nucleated myotubes is required for neonatal development and regeneration in adult skeletal muscle. Herein, we report novel findings that protein kinase C theta (PKCθ) regulates myoblast differentiation via phosphorylation of insulin receptor substrate-1 and ERK1/2. RESULTS: In this study, PKCθ knockdown (PKCθshRNA) myotubes had reduced inhibitory insulin receptor substrate-1 ser1095 phosphorylation, enhanced myoblast differentiation and cell fusion, and increased rates of protein synthesis as determined by [3H] phenylalanine incorporation. Phosphorylation of insulin receptor substrate-1 ser632/635 and extracellular signal-regulated kinase1/2 (ERK1/2) was increased in PKCθshRNA cells, with no change in ERK5 phosphorylation, highlighting a PKCθ-regulated myogenic pathway. Inhibition of PI3-kinase prevented cell differentiation and fusion in control cells, which was attenuated in PKCθshRNA cells. Thus, with reduced PKCθ, differentiation and fusion occur in the absence of PI3-kinase activity. Inhibition of the ERK kinase, MEK1/2, impaired differentiation and cell fusion in control cells. Differentiation was preserved in PKCθshRNA cells treated with a MEK1/2 inhibitor, although cell fusion was blunted, indicating PKCθ regulates differentiation via IRS1 and ERK1/2, and this occurs independently of MEK1/2 activation. CONCLUSION: Cellular signaling regulating the myogenic program and protein synthesis are complex and intertwined. These studies suggest that PKCθ regulates myogenic and protein synthetic signaling via the modulation of IRS1and ERK1/2 phosphorylation. Myotubes lacking PKCθ had increased rates of protein synthesis and enhanced myotube development despite reduced activation of the canonical anabolic-signaling pathway. Further investigation of PKCθ regulated signaling may reveal important interactions regulating skeletal muscle health in an insulin resistant state.

Influence of Anticipation and Motor-Motor Task Performance on Cutting Biomechanics in Healthy Men.

CONTEXT: Biomechanical analyses of cutting tasks have demonstrated kinematic differences associated with the noncontact knee-injury risk when the movement direction is unanticipated. Motor-motor dual tasks occur within dynamic environments and change the demand for attentional resources needed to complete athletic maneuvers, which may contribute to injury risk. OBJECTIVE: To investigate the influence of anticipation and motor-motor task performance on cutting biomechanics. DESIGN: Cross-sectional study. SETTING: Laboratory. PATIENTS OR OTHER PARTICIPANTS: A total of 32 healthy, recreationally active men (age = 23.1 ± 3.6 years, height = 180.0 ± 7.0 cm, mass = 81.3 ± 17.3 kg) who self-reported regular participation in cutting sports. INTERVENTION(S): Participants performed a 45° side-step cut on the dominant limb in a random order of conditions: anticipation (anticipated, unanticipated) and task (no ball throw, ball fake, ball throw). MAIN OUTCOME MEASURE(S): Triplanar trunk, hip, and knee angles were assessed throughout the stance phase using 3-dimensional motion capture. Data were analyzed using a time series of means calculated from initial contact to toe-off (0%-100%) with 90% confidence intervals. Mean differences between conditions were identified as regions of nonoverlapping confidence intervals, and those that occurred during the region of peak vertical ground reaction force (0%-25%) are presented. RESULTS: Regardless of anticipation, attending to a ball (ball throw) resulted in more trunk extension (range = 2.9°-3.7°) and less lateral trunk flexion toward the cutting direction (range = 5.2°-5.9°). Planning to attend to a ball (ball fake) resulted in less lateral trunk flexion toward the cutting direction (4.7°). During unanticipated cutting, more trunk rotation away from the cutting direction was observed when attending to a ball (range = 5.3°-7.1°). The interaction of anticipation and task had a similar influence on sagittal- and frontal-plane trunk position. CONCLUSIONS: Motor-motor task performance and its interaction with anticipation induced an upright, neutral trunk position during side-step cutting, which has been associated with the risk for noncontact knee injury. Promoting task complexity during rehabilitation and injury-prevention programs may better prepare individuals to succeed when performing high-risk athletic maneuvers.

FoxO1 induces apoptosis in skeletal myotubes in a DNA-binding-dependent manner.

Recent studies indicate that FoxO transcription factors play an important role in promoting muscle atrophy. To study mechanisms mediating effects of FoxO proteins on muscle wasting, FoxO1-estrogen receptor fusion proteins that are activated by treatment with 4-hydroxytamoxifen (4-OH-T) were stably transfected in C(2)C(12) skeletal myoblasts using the pBABE retroviral system and grown into multinucleated skeletal myotubes. Activation of FoxO1 resulted in significant muscle atrophy, which was accompanied by DNA fragmentation, evidenced by terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling. Cells expressing a DNA-binding-deficient form of FoxO1 also exhibited significant atrophy on FoxO1 activation but no hallmark signs of apoptosis. FoxO1 activation resulted in a significant increase in muscle atrophy F-box (MAFbx)/atrogin-1, muscle-specific RING finger protein 1 (MuRF-1), and Bcl-2-interacting mediator of cell death (Bim) gene expression, with no significant increase in Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNip3) gene expression. Although the ability of FoxO1 to induce MuRF-1 gene expression appeared to be independent of DNA binding, expression of MAFbx/atrogin-1 and Bim was significantly blunted in cells expressing DNA-binding-deficient FoxO1. BNip3 gene expression was significantly elevated in DNA-binding-deficient mutant cells. These findings indicate that FoxO1 promotes skeletal muscle atrophy through induction of proteolytic and apoptotic machinery via DNA-binding-dependent and -independent mechanisms.

Surface electromyography of the forearm musculature during an overhead throwing rehabilitation progression program.

OBJECTIVE: The flexor carpi ulnaris (FCU) and flexor digitorum superficialis (FDS) provide dynamic stabilization to the medial elbow. It remains unclear how these muscles function during progressive throwing exercises. Our objective was to compare FCU and FDS surface electromyography (sEMG) during a throwing progression. DESIGN: Crossover. SETTING: Laboratory. PARTICIPANTS: Sixteen healthy males. MAIN OUTCOME MEASURES: Participants completed a plyometric throw (PLYO), long-toss 50% (LT50), long-toss 75% (LT75), and pitch (PITCH). sEMG was synchronized with three-dimensional kinematics to assess the acceleration phase of each exercise. Peak sEMG amplitude (%MVIC) and percentage change between progressive exercises was measured. Continuous sEMG data were assessed to determine when peak activation occurred during acceleration. RESULTS: FCU activity was greater during PITCH than LT50, and during LT75 than LT50. Percentage change was greater from LT50-to-LT75 than PLYO-to-LT50 for both muscles. PLYO and PITCH increased most during late acceleration, whereas LT50 and LT75 increased most during mid-acceleration. CONCLUSIONS: FCU activity did not increase in a stepwise manner, and FDS remained unchanged. Each muscle demonstrated a disproportionate increase in activation during the second exercise progression (LT50-to-LT75) compared to the first (PLYO-to-LT50), suggesting that additional exercises may be required to achieve a stepwise progression relative to forearm muscle activation.

Cortisol concentrations in human skeletal muscle tissue after phonophoresis with 10% hydrocortisone gel.

CONTEXT: The delivery of hydrocortisone through phonophoresis is a widely prescribed technique for the treatment of various musculoskeletal inflammatory conditions. However, limited scientific evidence exists to support the efficacy of phonophoresis in delivering hydrocortisone to skeletal muscle tissue in humans. OBJECTIVE: To determine hydrocortisone (cortisol) concentrations in human skeletal muscle tissue after a phonophoresis treatment using 10% hydrocortisone gel. DESIGN: Randomized design in which 12 subjects were randomly assigned to either an ultrasound (sham) treatment or a 10% hydrocortisone phonophoresis treatment. SETTING: Laboratory. PATIENTS OR OTHER PARTICIPANTS: Twelve healthy subjects (8 women, 4 men: age = 22.3 +/- 2.64 years, height = 168.28 +/- 8.19 cm, mass = 69.58 +/- 9.05 kg) with no history of musculoskeletal disease, preexisting inflammatory conditions, or recent orthopaedic injuries. INTERVENTION(S): Ultrasound at 1.0 MHz, 1.0 W/cm (2), at a continuous setting for 7 minutes was applied to a standardized area of the vastus lateralis muscle in both groups. The contralateral limb served as the control (no treatment) for both the sham and the phonophoresis groups. MAIN OUTCOME MEASURE(S): Vastus lateralis muscle biopsies were taken from both legs immediately after treatment, and cortisol concentrations were analyzed using an enzyme-linked immunosorbent assay. RESULTS: We observed no significant difference in muscle cortisol concentration between the contralateral control limb and the treatment limb in either the sham or the phonophoresis group ( P > .05). No significant difference was noted when the treatment limbs in the sham and phonophoresis groups were compared ( P > .05). CONCLUSIONS: Our data suggest that a 10% hydrocortisone-based phonophoresis treatment did not increase cortisol concentrations in human skeletal muscle tissue.

Inflammatory cells in rat skeletal muscle are elevated after electrically stimulated contractions.

We determined the effect of muscle contractions resulting from high-frequency electrical stimulation (HFES) on inflammatory cells in rat tibialis anterior (TA), plantaris (Pln), and soleus (Sol) muscles at 6, 24, and 72 h post-HFES. A minimum of four and a maximum of seven rats were analyzed at each time point. HFES, applied to the sciatic nerve, caused the Sol and Pln to contract concentrically and the TA to contract eccentrically. Neutrophils were higher (P < 0.05) at 6 and 24 h after HFES in the Sol, Pln, and TA muscles relative to control muscles. ED1(+) macrophages in the Pln were elevated at 6 and 24 h after HFES and were also elevated in the Sol and TA after HFES relative to controls. ED2(+) macrophages in the Sol and TA were elevated at 24 and 72 h after HFES, respectively, and were also elevated in the Pln after HFES relative to controls. In contrast to the TA muscles, the Pln and Sol muscles showed no gross histological abnormalities. Collectively, these data indicate that both eccentric and concentric contractions can increase inflammatory cells in muscle, regardless of whether overt histological signs of injury are apparent.

COX-2 inhibitor reduces skeletal muscle hypertrophy in mice.

Anti-inflammatory strategies are often used to reduce muscle pain and soreness that can result from high-intensity muscular activity. However, studies indicate that components of the acute inflammatory response may be required for muscle repair and growth. The hypothesis of this study was that cyclooxygenase (COX)-2 activity is required for compensatory hypertrophy of skeletal muscle. We used the synergist ablation model of skeletal muscle hypertrophy, along with the specific COX-2 inhibitor NS-398, to investigate the role of COX-2 in overload-induced muscle growth in mice. COX-2 was expressed in plantaris muscles during compensatory hypertrophy and was localized mainly in or near muscle cell nuclei. Treatment with NS-398 blunted the increases in mass and protein content in overloaded muscles compared with vehicle-treated controls. Additionally, the COX-2 inhibitor decreased activity of the urokinase type plasminogen activator, macrophage accumulation, and cell proliferation, all of which are required for hypertrophy after synergist ablation. Expression of insulin-like growth factor-1 and phosphorylation of Akt, mammalian target of rapamycin, and p70S6K were increased following synergist ablation, but were not affected by NS-398. Additionally, expression of atrogin-1 was reduced during hypertrophy, but was also not affected by NS-398. These results demonstrate that COX-2 activity is required for skeletal muscle hypertrophy, possibly through facilitation of extracellular protease activity, macrophage accumulation, and cell proliferation.

TNF induction of atrogin-1/MAFbx mRNA depends on Foxo4 expression but not AKT-Foxo1/3 signaling.

Murine models of starvation-induced muscle atrophy demonstrate that reduced protein kinase B (AKT) function upregulates the atrophy-related gene atrogin-1/MAFbx (atrogin). The mechanism involves release of inhibition of Forkhead transcription factors, namely Foxo1 and Foxo3. Elevated atrogin mRNA also corresponds with elevated TNF in inflammatory catabolic states, including cancer and chronic heart failure. Exogenous tumor necrosis factor (TNF) increases atrogin mRNA in vivo and in vitro. We used TNF-treated C2C12 myotubes to test the hypothesis that AKT-Foxo1/3 signaling mediates TNF regulation of atrogin mRNA. Here we confirm that exposure to TNF increases atrogin mRNA (+125%). We also confirm that canonical AKT-mediated regulation of atrogin is active in C2C12 myotubes. Inhibition of phosphoinositol-3 kinase (PI3K)/AKT signaling with wortmannin reduces AKT phosphorylation (-87%) and increases atrogin mRNA (+340%). Activation with insulin-like growth factor (IGF) increases AKT phosphorylation (+126%) and reduces atrogin mRNA (-15%). Although AKT regulation is intact, our data suggest it does not mediate TNF effects on atrogin. TNF increases AKT phosphorylation (+50%) and stimulation of AKT with IGF does not prevent TNF induction of atrogin mRNA. Nor does TNF appear to signal through Foxo1/3 proteins. TNF has no effect on Foxo1/3 mRNA or Foxo1/3 nuclear localization. Instead, TNF increases nuclear Foxo4 protein (+55%). Small interfering RNA oligos targeted to two distinct regions of Foxo4 mRNA reduce the TNF-induced increase in atrogin mRNA (-34% and -32%). We conclude that TNF increases atrogin mRNA independent of AKT via Foxo4. These results suggest a mechanism by which inflammatory catabolic states may persist in the presence of adequate growth factors and nutrition.

Beta2-integrins contribute to skeletal muscle hypertrophy in mice.

We tested the contribution of beta(2)-integrins, which are important for normal function of neutrophils and macrophages, to skeletal muscle hypertrophy after mechanical loading. Using the synergist ablation model of hypertrophy and mice deficient in the common beta-subunit of beta(2)-integrins (CD18(-/-)), we found that overloaded muscles of wild-type mice had greater myofiber size, dry muscle mass, and total protein content compared with CD18(-/-) mice. The hypertrophy in wild-type mice was preceded by elevations in neutrophils, macrophages, satellite cell/myoblast proliferation (5'-bromo-2'-deoxyuridine- and desmin-positive cells), markers of muscle differentiation (MyoD1 and myogenin gene expression and formation and size of regenerating myofibers), signaling for protein synthesis [phosphorylation of Akt and 70-kDa ribosomal protein S6 kinase (p70S6k)], and reduced signaling for protein degradation (decreased gene expression of muscle atrophy F box/atrogin-1). The deficiency in beta(2)-integrins, however, altered the accumulation profile of neutrophils and macrophages, disrupted the temporal profile of satellite cell/myoblast proliferation, reduced the markers of muscle differentiation, and impaired the p70S6k signaling, all of which could serve as mechanisms for the impaired hypertrophy in overloaded CD18(-/-) mice. In conclusion, our findings indicate that beta(2)-integrins contribute to the hypertrophic response to muscle overload by temporally regulating satellite cells/myoblast proliferation and by enhancing muscle differentiation and p70S6k signaling.

FOXO1 regulates the expression of 4E-BP1 and inhibits mTOR signaling in mammalian skeletal muscle.

The mammalian target of rapamycin (mTOR) is regulated by growth factors to promote protein synthesis. In mammalian skeletal muscle, the Forkhead-O1 transcription factor (FOXO1) promotes catabolism by activating ubiquitin-protein ligases. Using C2C12 mouse myoblasts that stably express inducible FOXO1-ER fusion proteins and transgenic mice that specifically overexpress constitutively active FOXO1 in skeletal muscle (FOXO(++/+)), we show that FOXO1 inhibits mTOR signaling and protein synthesis. Activation of constitutively active FOXO1 induced the expression of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) mRNA via binding to the promoter. This resulted in an increased total 4E-BP1 abundance and a reduced 4E-BP1 (Thr-37/46) phosphorylation. The reduction in 4E-BP1 phosphorylation was associated with a reduction in the abundance of Raptor and mTOR proteins, Raptor-associated mTOR, reduced phosphorylation of the downstream protein p70S6 kinase, and attenuated incorporation of [(14)C]phenylalanine into protein. The FOXO(++/+) mice, characterized by severe skeletal muscle atrophy, displayed similar patterns of mRNA expression and protein abundance to those observed in the constitutively active FOXO1 C2C12 myotubes. These data suggest that FOXO1 may be an important therapeutic target for human diseases where anabolism is impaired.

mTOR function in skeletal muscle hypertrophy: increased ribosomal RNA via cell cycle regulators.

The purpose of this study was to identify the potential downstream functions associated with mammalian target of rapamycin (mTOR) signaling during myotube hypertrophy. Terminally differentiated myotubes were serum stimulated for 3, 6, 12, 24, and 48 h. This treatment resulted in significant myotube hypertrophy (protein/DNA) and increased RNA content (RNA/DNA) with no changes in DNA content or indices of cell proliferation. During myotube hypertrophy, the increase in RNA content was accompanied by an increase in tumor suppressor protein retinoblastoma (Rb) phosphorylation and a corresponding increase in the availability of the ribosomal DNA transcription factor upstream binding factor (UBF). Serum stimulation also induced an increase in cyclin D1 protein expression in the differentiated myotubes with a concomitant increase in cyclin D1-dependent cyclin-dependent kinase (CDK)-4 activity toward Rb. The increases in myotube hypertrophy and RNA content were blocked by rapamycin treatment, which also prevented the increase in cyclin D1 protein expression, CDK-4 activity, Rb phosphorylation, and the increase in UBF availability. Our findings demonstrate that activation of mTOR is necessary for myotube hypertrophy and suggest that the role of mTOR is in part to modulate cyclin D1-dependent CDK-4 activity in the regulation of Rb and ribosomal RNA synthesis. On the basis of these results, we propose that common molecular mechanisms contribute to the regulation of myotube hypertrophy and growth during the G1 phase of the cell cycle.

Downhill running in rats: influence on neutrophils, macrophages, and MyoD+ cells in skeletal muscle.

The accumulation of neutrophils and macrophages, as well as the activation of satellite cells, are early events following skeletal muscle injury. We examined the temporal relationship between changes in neutrophils, macrophages, and MyoD protein, a marker of satellite cell activation, after injurious exercise. Male rats ( n=47) performed an intermittent downhill (-16% grade) running (17 m/min) protocol and the solei were obtained at 0, 2, 6, 24, 48, or 72 h post-exercise. Neutrophils, macrophages (ED1 and ED2), and MyoD+ cells were determined in muscle cross sections using immunohistochemistry. Downhill running increased ( P

Deferoxamine reduces and nitric oxide synthase inhibition increases neutrophil-mediated myotube injury.

We tested the contribution of reactive oxygen species (ROS), reactive nitrogen species (RNS) and the beta 2 integrin CD18 to neutrophil-mediated myotube injury. Human myotubes were cultured with human neutrophils in the presence or absence of inhibitors directed against ROS, RNS, and CD18. Muscle injury was assessed by a (51)Cr release assay. The inclusion of superoxide dismutase (50-500 U/ml) in the culture medium did not affect myotube injury. A significant protective effect was provided by including catalase (600-2400 U/ml), deferoxamine (1-2 mM), or anti-CD18 antibody (10 microg/ml) in the culture medium. S-Ethylisothiourea (500-1000 microM), an inhibitor of nitric oxide synthase (NOS), significantly increased myotube injury and reduced nitric oxide (NO) in cultures consisting of only myotubes. In conclusion, neutrophil-mediated skeletal muscle injury appears to be largely dependent on CD18-mediated neutrophil adhesion and iron-dependent hydroxyl radical production. In addition, skeletal muscle NOS activity may protect skeletal muscle against the injury caused by neutrophils.

Selenoprotein-deficient transgenic mice exhibit enhanced exercise-induced muscle growth.

Dietary intake of selenium has been implicated in a wide range of health issues, including aging, heart disease and cancer. Selenium deficiency, which can reduce selenoprotein levels, has been associated with several striated muscle pathologies. To investigate the role of selenoproteins in skeletal muscle biology, we used a transgenic mouse (referred to as i6A-) that has reduced levels of selenoproteins due to the introduction and expression of a dominantly acting mutant form of selenocysteine transfer RNA (tRNA[Ser]Sec). As a consequence, each organ contains reduced levels of most selenoproteins, yet these mice are normal with regard to fertility, overall health, behavior and blood chemistries. In the present study, although skeletal muscles from i6A- mice were phenotypically indistinguishable from those of wild-type mice, plantaris muscles were approximately 50% heavier after synergist ablation, a model of exercise overload. Like muscle in wild-type mice, the enhanced growth in the i6A- mice was completely blocked by inhibition of the mammalian target of rapamycin (mTOR) pathway. Muscles of transgenic mice exhibited increased site-specific phosphorylation on both Akt and p70 ribosomal S6 kinase (p70S6k) (P < 0.05) before ablation, perhaps accounting for the enhanced response to synergist ablation. Thus, a single genetic alteration resulted in enhanced skeletal muscle adaptation after exercise, and this is likely through subtle changes in the resting phosphorylation state of growth-related kinases.