Time course of the MAPK and PI3-kinase response within 24 h of skeletal muscle overload.

Knowledge of the molecular mechanisms by which skeletal muscle hypertrophies in response to increased mechanical loading may lead to the discovery of novel treatment strategies for muscle wasting and frailty. To gain insight into potential early signaling mechanisms associated with skeletal muscle hypertrophy, the temporal pattern of mitogen-activated protein kinase (MAPK) phosphorylation and phosphatidylinositol 3-kinase (PI3-kinase) activity during the first 24 h of muscle overload was determined in the rat slow-twitch soleus and fast-twitch plantaris muscles after ablation of the gastrocnemius muscle. p38alpha MAPK phosphorylation was elevated for the entire 24-h overload period in both muscles. In contrast, Erk 2 and p54 JNK phosphorylation were transiently increased by overload, returning to the levels of sham-operated controls by 24 h. PI3-kinase activity was increased by muscle overload only at 12 h of overload and only in the plantaris muscle. In summary, sustained elevation of p38alpha MAPK phosphorylation occurred early in response to muscle overload, identifying this pathway as a potential candidate for mediating early hypertrophic signals in response to skeletal muscle overload.     

Angiotensin-converting enzyme inhibition attenuates myonuclear addition in overloaded slow-twitch skeletal muscle

Because optimal overload-induced skeletal muscle hypertrophy requires ANG II, we aimed to determine the effects of blocking ANG II production [via angiotensin-converting enzyme (ACE) inhibition] on potential mediators of hypertrophy in overloaded skeletal muscle, namely, myonuclear addition and fibroblast content. In a 2 x 2 design, adult (200-225 g) female Sprague-Dawley rats were placed into one of four groups (n = 8/group): 7-day skeletal muscle overload, sham operation, 7-day skeletal muscle overload with ACE inhibition, or sham operation with ACE inhibition. Functional overloads of the plantaris and soleus muscles were produced via bilateral surgical ablation of the synergistic gastrocnemius muscle, and ACE inhibition was accomplished by the addition of the ACE inhibitor enalapril maleate to the animals' daily drinking water (0.3 mg/ml). Myonuclear addition and extrasarcolemmal nuclear proliferation, as measured by in vivo 5-bromo-2'-deoxyuridine labeling, were significantly (P < or = 0.05) increased by overload in both the slow-twitch soleus and fast-twitch plantaris muscles. Furthermore, ACE inhibition attenuated these overload-induced increases in the soleus muscle but not in the plantaris muscle. However, the effect of ACE inhibition on soleus extrasarcolemmal nuclei was not likely due to differences in fibroblast content because overload elicited significant increases in vimentin-positive areas in soleus and plantaris muscles, and these areas were unaffected by ACE inhibition in either muscle. There was no effect of ACE inhibition on any measure in sham-operated muscles. Collectively, these data indicate that ANG II may mediate the satellite cell response to overload in slow-twitch soleus but not in fast-twitch plantaris muscles and that this effect may occur independently of changes in fibroblast content.     

Diminished overload-induced hypertrophy in aged fast-twitch skeletal muscle is associated with AMPK hyperphosphorylation.

Skeletal muscle mass declines with age, as does the potential for overload-induced fast-twitch skeletal muscle hypertrophy. Because 5'-AMP-activated protein kinase (AMPK) activity is thought to inhibit skeletal muscle protein synthesis and may therefore modulate muscle mass and hypertrophy, the purpose of this investigation was to examine AMPK phosphorylation status (a marker of AMPK activity) and its potential association with the attenuated overload-induced hypertrophy observed in aged skeletal muscle. One-week overload of fast-twitch plantaris and slow-twitch soleus muscles was achieved in young adult (8 mo; n = 7) and old (30 mo; n = 7) Fischer344 x Brown Norway male rats via unilateral gastrocnemius ablation. Significant (P < or = 0.05) age-related atrophy (as measured by total protein content) was noted in plantaris and soleus control (sham-operated) muscles. In fast-twitch plantaris muscles, percent hypertrophy with overload was significantly attenuated with age, whereas AMPK phosphorylation status as determined by Western blotting [phospho-AMPK (Thr172)/total AMPK] was significantly elevated with age (regardless of loading status). There was also a main effect of loading on AMPK phosphorylation status in plantaris muscles (overload > control). Moreover, a strong and significant negative correlation (r = -0.82) was observed between AMPK phosphorylation status and percent hypertrophy in the overloaded plantaris muscles of all animals. In contrast to the plantaris, overload-induced hypertrophy of the slow-twitch soleus muscle was similar between ages, and AMPK phosphorylation in this muscle was also unaffected by age or overload. These data support the possibility that an age-related elevation in AMPK phosphorylation may partly contribute to the attenuated hypertrophic response observed with age in overloaded fast-twitch plantaris muscle.     

Effective fiber hypertrophy in satellite cell-depleted skeletal muscle

An important unresolved question in skeletal muscle plasticity is whether satellite cells are necessary for muscle fiber hypertrophy. To address this issue, a novel mouse strain (Pax7-DTA) was created which enabled the conditional ablation of >90% of satellite cells in mature skeletal muscle following tamoxifen administration. To test the hypothesis that satellite cells are necessary for skeletal muscle hypertrophy, the plantaris muscle of adult Pax7-DTA mice was subjected to mechanical overload by surgical removal of the synergist muscle. Following two weeks of overload, satellite cell-depleted muscle showed the same increases in muscle mass (approximately twofold) and fiber cross-sectional area with hypertrophy as observed in the vehicle-treated group. The typical increase in myonuclei with hypertrophy was absent in satellite cell-depleted fibers, resulting in expansion of the myonuclear domain. Consistent with lack of nuclear addition to enlarged fibers, long-term BrdU labeling showed a significant reduction in the number of BrdU-positive myonuclei in satellite cell-depleted muscle compared with vehicle-treated muscle. Single fiber functional analyses showed no difference in specific force, Ca(2+) sensitivity, rate of cross-bridge cycling and cooperativity between hypertrophied fibers from vehicle and tamoxifen-treated groups. Although a small component of the hypertrophic response, both fiber hyperplasia and regeneration were significantly blunted following satellite cell depletion, indicating a distinct requirement for satellite cells during these processes. These results provide convincing evidence that skeletal muscle fibers are capable of mounting a robust hypertrophic response to mechanical overload that is not dependent on satellite cells.     

ANG II is required for optimal overload-induced skeletal muscle hypertrophy

ANG II mediates the hypertrophic response of overloaded cardiac muscle, likely via the ANG II type 1 (AT(1)) receptor. To examine the potential role of ANG II in overload-induced skeletal muscle hypertrophy, plantaris and/or soleus muscle overload was produced in female Sprague-Dawley rats (225-250 g) by the bilateral surgical ablation of either the synergistic gastrocnemius muscle (experiment 1) or both the gastrocnemius and plantaris muscles (experiment 2). In experiment 1 (n = 10/group), inhibiting endogenous ANG II production by oral administration of an angiotensin-converting enzyme (ACE) inhibitor during a 28-day overloading protocol attenuated plantaris and soleus muscle hypertrophy by 57 and 96%, respectively (as measured by total muscle protein content). ACE inhibition had no effect on nonoverloaded (sham-operated) muscles. With the use of new animals (experiment 2; n = 8/group), locally perfusing overloaded soleus muscles with exogenous ANG II (via osmotic pump) rescued the lost hypertrophic response in ACE-inhibited animals by 71%. Furthermore, orally administering an AT(1) receptor antagonist instead of an ACE inhibitor produced a 48% attenuation of overload-induced hypertrophy that could not be rescued by ANG II perfusion. Thus ANG II may be necessary for optimal overload-induced skeletal muscle hypertrophy, acting at least in part via an AT(1) receptor-dependent pathway.     

Effects of mechanical over-loading on the properties of soleus muscle fibers, with or without damage, in wild type and mdx mice

Effects of mechanical over-loading on the characteristics of regenerating or normal soleus muscle fibers were studied in dystrophin-deficient (mdx) and wild type (WT) mice. Damage was also induced in WT mice by injection of cardiotoxin (CTX) into soleus muscle. Over-loading was applied for 14 days to the left soleus muscle in mdx and intact and CTX-injected WT mouse muscles by ablation of the distal tendons of plantaris and gastrocnemius muscles. All of the myonuclei in normal muscle of WT mice were distributed at the peripheral region. But, central myonuclei were noted in all fibers of WT mice regenerating from CTX-injection-related injury. Further, many fibers of mdx mice possessed central myonuclei and the distribution of such fibers was increased in response to over-loading, suggesting a shift of myonuclei from peripheral to central region. Approximately 1.4% branched fibers were seen in the intact muscle of mdx mice, although these fibers were not detected in WT mice. The percentage of these fibers in mdx, not in WT, mice was increased by over-loading (～51.2%). The fiber CSA in normal WT mice was increased by over-loading (p<0.05), but not in mdx and CTX-injected WT mice. It was suggested that compensatory hypertrophy is induced in normal muscle fibers of WT mice following functional over-loading. But, it was also indicated that muscle fibers in mdx mice are susceptible to mechanical over-loading and fiber splitting and shift of myonuclei from peripheral to central region are induced.     

Age-related loss of skeletal muscle function and the inability to express the autocrine form of insulin-like growth factor-1 (MGF) in response to mechanical overload

The response of insulin-like growth factor-1 (IGF-1) signalling and the capacity of skeletal muscle to adapt to mechanical overload was studied using synergistic muscle ablation. Overload of the plantaris and soleus resulted in marked hypertrophy and activation of satellite cells (as indicated by MyoD expression), particularly in young rats. Two muscle IGF-1 splice variants were measured and found to be differentially regulated at the RNA level. The significant changes associated with the inability of the older muscles to respond to mechanical overload included the considerably lower expression of the local splice variant mechano growth factor, and the failure to up-regulate IGF-1 receptor and MyoD mRNA.     

A histochemical study of postnatal differentiation of skeletal muscle with reference to functional overload.

Postnatal differentiation and growth of the fibers comprising the tonic soleus and phasic plantaris muscles, were investigated histochemically in kittens. Compensatory hypertrophy was induced by ablation of the synergistic gastrocnemius muscle.At birth both muscles consist of relatively homogeneous fiber populations as demonstrated by myosin ATPase and succinic dehydrogenase activities and glycogen content (PAS method). Differential myosin ATPase activities become evident during the first week (soleus and plantaris), while diversification of fiber types according to SDH and glycogen develop gradually and independently during the first 2 mo of life (plantaris).Compensatory hypertrophy is associated with a substantial enlargement of both dark and light fibers (incubated for myosin ATPase) and, with increases in SDH activity which are most notable in fibers that normally are low in this enzyme. The normal growth associated reduction in the percentage of fibers with high myosin ATPase activity is significantly accelerated in the hypertrophic soleus, while the hypertrophic plantaris, which normally undergoes only a slight reduction in the percentage of such fibers, is unaffected. These results underline the paramount role of the nerve fiber in the process of differentiation but also indicate that functional overload exerts a modifying influence on this process.     

Perlecan deficiency causes muscle hypertrophy,a decrease in myostatin expression,and changes in muscle fiber composition

Perlecan is a component of the basement membrane that surrounds skeletal muscle. The aim of the present study is to identify the role of perlecan in skeletal muscle hypertrophy and myostatin signaling, with and without mechanical stress, using a mouse model (Hspg2^?/?-Tg) deficient in skeletal muscle perlecan. We found that myosin heavy chain (MHC) type IIb fibers in the tibialis anterior (TA) muscle of Hspg2^?/?-Tg mice had a significantly increased fiber cross-sectional area (CSA) compared to control (WT-Tg) mice. Hspg2^?/?-Tg mice also had an increased number of type IIx fibers in the TA muscle. Myostatin and its type I receptor (ALK4) expression was substantially decreased in the Hspg2^?/?-Tg TA muscle. Myostatin-induced Smad activation was also reduced in a culture of myotubes from the Hspg2^?/?-Tg muscle, suggesting that myostatin expression and its signaling were decreased in the Hspg2^?/?-Tg muscle. To examine the effects of mechanical overload or unload on fast and slow muscles in Hspg2^?/?-Tg mice, we performed tenotomy of the plantaris (fast) muscle and the soleus (slow) muscle. Mechanical overload on the plantaris muscle of Hspg2^?/?-Tg mice significantly increased wet weights compared to those of control mice, and unloaded plantaris muscles of Hspg2^?/?-Tg mice caused less decrease in wet weights compared to those of control mice. The decrease in myostatin expression was significantly profound in the overloaded plantaris muscle of Hspg2^?/?-Tg mice, compared with that of control mice. In contrast, overloading the soleus muscle caused no changes in either type of muscle. These results suggest that perlecan is critical for maintaining fast muscle mass and fiber composition, and for regulating myostatin signaling.     

Skeletal Muscle Cells Express ICAM-1 after Muscle Overload and ICAM-1 Contributes to the Ensuing Hypertrophic Response

We previously reported that leukocyte specific β2 integrins contribute to hypertrophy after muscle overload in mice. Because intercellular adhesion molecule-1 (ICAM-1) is an important ligand for β2 integrins, we examined ICAM-1 expression by murine skeletal muscle cells after muscle overload and its contribution to the ensuing hypertrophic response. Myofibers in control muscles of wild type mice and cultures of skeletal muscle cells (primary and C2C12) did not express ICAM-1. Overload of wild type plantaris muscles caused myofibers and satellite cells/myoblasts to express ICAM-1. Increased expression of ICAM-1 after muscle overload occurred via a β2 integrin independent mechanism as indicated by similar gene and protein expression of ICAM-1 between wild type and β2 integrin deficient (CD18-/-) mice. ICAM-1 contributed to muscle hypertrophy as demonstrated by greater (p<0.05) overload-induced elevations in muscle protein synthesis, mass, total protein, and myofiber size in wild type compared to ICAM-1-/- mice. Furthermore, expression of ICAM-1 altered (p<0.05) the temporal pattern of Pax7 expression, a marker of satellite cells/myoblasts, and regenerating myofiber formation in overloaded muscles. In conclusion, ICAM-1 expression by myofibers and satellite cells/myoblasts after muscle overload could serve as a mechanism by which ICAM-1 promotes hypertrophy by providing a means for cell-to-cell communication with β2 integrin expressing myeloid cells.     

Interleukin-15 responses to aging and unloading-induced skeletal muscle atrophy

Interleukin-15 (IL-15) mRNA is constitutively expressed in skeletal muscle. Although IL-15 has proposed hypertrophic and anti-apoptotic roles in vitro, its role in skeletal muscle cells in vivo is less clear. The purpose of this study was to determine if skeletal muscle aging and unloading, two conditions known to promote muscle atrophy, would alter basal IL-15 expression in skeletal muscle. We hypothesized that IL-15 mRNA expression would increase as a result of both aging and muscle unloading and that muscle would express the mRNA for a functional trimeric IL-15 receptor (IL-15R). Two models of unloading were used in this study: hindlimb suspension (HS) in rats and wing unloading in quail. The absolute muscle wet weight of plantaris and soleus muscles from aged rats was significantly less when compared with muscles from young adult rats. Although 14 days of HS resulted in reduced muscle mass of plantaris and soleus muscles from young adult animals, this effect was not observed in muscles from aged animals. A significant aging times unloading interaction was observed for IL-15 mRNA in both rat soleus and plantaris muscles. Patagialis (PAT) muscles from aged quail retained a significant 12 and 6% of stretch-induced hypertrophy after 7 and 14 days of unloading, respectively. PAT muscles from young quail retained 15% hypertrophy at 7 days of unloading but regressed to control levels following 14 days of unloading. A main effect of age was observed on IL-15 mRNA expression in PAT muscles at 14 days of overload, 7 days of unloading, and 14 days of unloading. Skeletal muscle also expressed the mRNAs for a functional IL-15R composed of IL-15R, IL-2/15R-, and -c. Based on these data, we speculate that increases in IL-15 mRNA in response to atrophic stimuli may be an attempt to counteract muscle mass loss in skeletal muscles of old animals. Additional research is warranted to determine the importance of the IL-15/IL-15R system to counter muscle wasting. atrophy; interleukins; sarcopenia; gene signaling     

Nifedipine does not impede clenbuterol-stimulated muscle hypertrophy.

The mechanism(s) responsible for beta2-adrenergic receptor-mediated skeletal muscle and cardiac hypertrophy remains undefined. This study examined whether calcium influx through L-type calcium channels contributed to the development of cardiac and skeletal muscle (plantaris; gastrocnemius; soleus) hypertrophy during an 8-day treatment with the beta2-adrenergic receptor agonist clenbuterol. Concurrent blockade of L-type calcium channels with nifedipine did not reverse the hypertrophic action of clenbuterol. Moreover, nifedipine treatment alone resulted in both cardiac and soleus muscle hypertrophy (6% and 7%, respectively), and this effect was additive to the clenbuterol-mediated hypertrophy in the heart and soleus muscles. The hypertrophic effects of nifedipine were not associated with increases in total beta-adrenergic receptor density, nor did nifedipine reverse clenbuterol-mediated beta-adrenergic receptor downregulation in either the left ventricle or soleus muscle. Both nifedipine and clenbuterol-induced hypertrophy increased total protein content of the soleus and left ventricle, with no change in protein concentration. In conclusion, our results support the hypothesis that beta2-adrenergic receptor agonist-induced muscle hypertrophy is mediated by mechanisms other than calcium influx through L-type calcium channels.     

Age effects on myosin subunit and biochemical alterations with skeletal muscle hypertrophy.

The purpose of this study was to determine whether skeletal muscle mass, myofibrillar adenosinetriphosphatase activity, and the expression of myosin heavy (MHC) and light chain subunits are differentially affected in juvenile (4 wk) and young adult (12 wk) rats by a hypertrophic growth stimulus. Hypertrophy of the plantaris or soleus was studied 4 wk after ablation of either two [gastrocnemius (GTN) and soleus or plantaris] or one (GTN) synergistic muscle(s). There was no difference in the relative magnitude of hypertrophy because of age. Plantaris myofibrillar adenosinetriphosphatase activity was decreased 21 and 12% in juvenile and adult rats, respectively, as a result of ablation of both the GTN and soleus. Slow myosin light chain isoforms (1s and 2s) were expressed to a greater extent in hypertrophied plantaris muscles of both ages, but the increase in 1s was greater in juvenile rats. The relative expression of slow beta-MHC in hypertrophied plantaris muscles increased by 470 and 350%, whereas MHC IIb decreased by 70 and 33% in juvenile and adult rats, respectively. The relative expression of MHC IIa increased (56%) in the plantaris after ablation in juvenile rats only. These shifts in myosin subunit expression and the increases in mass were generally about one-half the magnitude when only the GTN was removed. There were no detectable myosin shifts in hypertrophied soleus muscles. Although the extent of muscle hypertrophy is similar, the shifts in myosin subunits were greater in juvenile than in young adult rats.     

Are region-specific changes in fibre types attributable to nonuniform muscle hypertrophy by overloading?

Muscle fibre composition was compared among the proximal (25%), middle (50%) and distal (75%) regions of the muscle length to investigate whether compensatory overload by removal of synergists induces region-specific changes of fibre types in rat soleus and plantaris muscles. In addition, we evaluated fibre cross-sectional area in each region to examine whether fibre recruitment pattern against functional overload is nonuniform in different regions. Increases in muscle mass and fibre area confirmed a significant hypertrophic response in the overloaded soleus and plantaris muscles. Overloading increased the percentage of type I fibres in both muscles and that of type IIA fibres in the plantaris muscle, with the greater changes being found in the middle and distal regions. The percentage of type I fibres in the proximal region was higher than that of the other regions in the control soleus muscle. In the control plantaris muscle, the percentage of type I and IIA fibres in the middle region were higher than that of the proximal and distal regions. With regard to fibre size, type IIB fibre area of the middle and distal regions in the plantaris increased by 51% and 57%, respectively, with the greater changes than that of the proximal region (37%) after overloading. These findings suggest that compensatory overload promoted transformation of type II fibres into type I fibres in rat soleus and plantaris muscles, with the greater changes being found in the middle and distal regions of the plantaris muscle.     

Regulation of HSP25 expression and phosphorylation in functionally overloaded rat plantaris and soleus muscles.

Functional overload (FO) is a powerful inducer of muscle hypertrophy and both oxidative and mechanical stress in muscle fibers. Heat shock protein 25 (HSP25) may protect against both of these stressors, and its expression can be regulated by changes in muscle loading and activation. The primary purpose of the present study was to test the hypothesis that chronic FO increases HSP25 expression and phosphorylation (pHSP25) in hypertrophying rat hindlimb muscle. HSP25 and pHSP25 levels were quantified in soluble and insoluble fractions of the soleus and plantaris to determine whether 3 or 7 days of FO increase translocation of HSP25 and/or pHSP25 to the insoluble fraction. p38 protein and phosphorylation (p-p38) was measured to determine its association with changes in pHSP25. HSP25 mRNA showed time-dependent increases in both the soleus and plantaris with FO. Three or seven days of FO increased HSP25 and pHSP25 in the soluble fraction in both muscles, with a greater response in the plantaris. In the insoluble fraction, HSP25 was increased after 3 or 7 days in both muscles, whereas pHSP25 was only increased in the 7-day plantaris. p38 and p-p38 increased in the plantaris at both time points. In the soleus, p-p38 only increased after 7 days. These results show that FO is associated with changes in HSP25 expression and phosphorylation and suggest its role in the remodeling that occurs during muscle hypertrophy. Increases in HSP25 in the insoluble fraction suggest that it may help to stabilize actin and/or other cytoskeletal proteins during the stress of muscle remodeling.     

Modulation of HSP25 and TNF-alpha during the early stages of functional overload of a rat slow and fast muscle.

Early events in response to abrupt increases in activation and loading with muscle functional overload (FO) are associated with increased damage and inflammation. Heat shock protein 25 (HSP25) may protect against these stressors, and its expression can be regulated by muscle loading and activation. The purpose of this study was to investigate the responses of HSP25, phosphorylated HSP25 (pHSP25), and tumor necrosis factor-alpha (TNF-alpha) during FO of the slow soleus and fast plantaris. We compared the HSP25 mRNA, HSP25 protein, pHSP25, and TNF-alpha responses in the soleus and plantaris after 0.5, 1, 2, 3, and 7 days of FO. HSP25 and pHSP25 were quantified in soluble and insoluble fractions. HSP25 mRNA increased immediately in both muscles and decreased with continued FO. However, HSP25 mRNA levels were consistently higher in the muscles of FO than control rats. In the soluble fraction, HSP25 increased in the plantaris after 2-7 days of FO with the greatest response at 3 and 7 days. The pHSP25 response to FO was greater in the plantaris than soleus at all points in the soluble fraction and at 0.5 days in the insoluble fraction. TNF-alpha levels in the plantaris, but not soleus, were higher than control at 0.5-2 days of FO. This may have contributed to the greater FO response in pHSP25 in the plantaris than soleus as TNF-alpha increased pHSP25 in C2C12 myotubes. These results suggest that the initial responses of pHSP25 and TNF-alpha to mechanical stress and inflammation associated with FO are greater in a fast than slow extensor muscle.     

Mechanical loading stimulates hypertrophy in tissue-engineered skeletal muscle: Molecular and phenotypic responses

Mechanical loading of skeletal muscle results in molecular and phenotypic adaptations typified by enhanced muscle size. Studies on humans are limited by the need for repeated sampling, and studies on animals have methodological and ethical limitations. In this investigation, three-dimensional skeletal muscle was tissue-engineered utilizing the murine cell line C2C12, which bears resemblance to native tissue and benefits from the advantages of conventional in vitro experiments. The work aimed to determine if mechanical loading induced an anabolic hypertrophic response, akin to that described in vivo after mechanical loading in the form of resistance exercise. Specifically, we temporally investigated candidate gene expression and Akt-mechanistic target of rapamycin 1 signalling along with myotube growth and tissue function. Mechanical loading (construct length increase of 15%) significantly increased insulin-like growth factor-1 and MMP-2 messenger RNA expression 21 hr after overload, and the levels of the atrophic gene MAFbx were significantly downregulated 45 hr after mechanical overload. In addition, p70S6 kinase and 4EBP-1 phosphorylation were upregulated immediately after mechanical overload. Maximal contractile force was augmented 45 hr after load with a 265% increase in force, alongside significant hypertrophy of the myotubes within the engineered muscle. Overall, mechanical loading of tissue-engineered skeletal muscle induced hypertrophy and improved force production.