Leukemia inhibitory factor restores the hypertrophic response to increased loading in the LIF(-/-) mouse

Cytokines and growth factors are thought to contribute to skeletal muscle hypertrophy. Leukemia inhibitory factor (LIF), a cytokine, enhances skeletal muscle regeneration; however the role of LIF in skeletal muscle hypertrophy remains uncertain. We examined the hypertrophic ability of the plantaris and soleus muscles in wild-type mice (WT) and LIF knock-out mice [LIF(-/-)] in response to increased mechanical load. Using the functional overload model to induce increases in mechanical load on the plantaris and soleus muscle, WT mice demonstrated increases in plantaris and soleus mass after 7, 21, and 42 days of loading. However, the LIF(-/-) mice had no significant increases in plantaris muscle mass at any time point, while the soleus muscle exhibited a delayed hypertrophic response. Systemic delivery of LIF to the LIF(-/-) mice returned the hypertrophic response to the same levels as the WT mice after 21 days of functional overload. These data demonstrate for the first time that LIF expression in loaded skeletal muscle is critical for the development of skeletal muscle hypertrophy in the functional overload model.     

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.     

Heat stress facilitates stretch-induced hypertrophy of cultured muscle cells

Increased mechanical stress induced by stretch is an important growth stimulus in skeletal muscle. Heat shock proteins (HSPs) are an important family of endogenous, protective proteins. HSP90 and HSP70 families show elevated levels under beat stress. Mechanical stress, such as physical exercise, is known to induce not only muscular hypertrophy but also the elevation of HSPs expression in skeletal muscle. The purpose of this study was to determine whether heat stress facilitates the stretch-induced hypertrophy of skeletal muscle cells. Cultured rat myotubes (L6) were plated on collagenized Silastic membranes and incubated at 41 degrees C for 60 and 75 minutes (heat shock). Following the incubation, the cells were subjected two-second stretching and four-second releasing for 4 days at 37 degrees C. Protein concentrations in the homogenates and pellets of the cultured skeletal muscle cells increased under heat shock and/or mechanical stretching. The protein concentration of cells following mechanical stretching following heat shock was significantly higher than that following either heat shock or mechanical stretching alone. HSP72 in supernatants and HSP90 in pellets increased under heat shock and/or mechanical stretching. HSP90 in supernatants decreased following heat shock and/or mechanical stretching. Changes in HSPs and cellular protein concentrations in stressed cells suggest that the expression of HSPs may be closely related with muscular hypertrophy.     

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 novel cell-based system for evaluating skeletal muscle cell hypertrophy-inducing agents

Skeletal muscle is a tissue that adapts to increased use by increasing contractile protein gene expression and ultimately skeletal muscle mass (hypertrophy). To identify hypertrophy-inducing agents that may be potentially useful in the treatment of age-related muscle loss (sarcopenia) and to better understand hypertrophy signal transduction pathways, we have created a skeletal muscle cell-based hypertrophy-responsive system. This system was created by permanently modifying the relatively undifferentiated C2C12 cell line so that it contains the beta-myosin heavy chain (beta-MHC) gene promoter and enhancer regions fused to a luciferase reporter gene. This cell line responds, by increasing luciferase expression, to a variety of skeletal muscle hypertrophy-inducing agents, including insulin, insulin-like growth factor I, testosterone, and the beta-adrenergic receptor agonist isoproterenol, in both the undifferentiated and differentiated states. This cell-based system should be useful for identifying novel hypertrophy-inducing agents as well as understanding hypertrophy signal transduction.     

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.     

Time course adaptations in rat skeletal muscle isomyosins during compensatory growth and regression

The purpose of this study was to ascertain the time course of change during both compensatory growth (hypertrophy) and subsequent growth regression on myosin isoform expression in rodent fast-twitch plantaris muscle in response to functional overload (induced by removal of synergists). Peak hypertrophy of the plantaris muscle (92%) occurred after 9 wk of overload. After 7 wk of overload regression (induced by a model of hindlimb unweighting), muscle weight returned to within 30% of control values. Myofibril protein content (mg/g muscle) remained relatively constant throughout the overload period but became significantly depressed relative to control values after 7 wk of regression. However, when expressed on a per muscle basis (mg/muscle) no differences existed at this time point (t = 7 wk regression). The distribution of native myosin isoforms in the myofibril protein pool of the overloaded plantaris muscle reflected a progressive increase (23% at t = 9 wk; P less than 0.001) in the relative proportion of slow myosin (Sm). This change was also accompanied by increases in intermediate myosin (Im) as well as the repression of the fast myosin one (Fm1) isoform (P less than 0.001). These shifts in Sm and Fm1 isoform expression were gradually reversed during the regression period, whereas Im remained elevated relative to control values. These adaptive changes in myosin isoform expression during both hypertrophy and regression were further supported by concomitant shifts in both myosin adenosinetriphosphatase (ATPase) activity (decreased during overload) and slow myosin light chain (SLC) expression. However, during regression the changes in myosin isoform expression and myosin ATPase were not as synchronous as they were during overload. Estimation of the mixed myosin heavy chain (MHC) half-life (t 1/2), using a linear model that assumes zero-order synthesis and first-order degradation kinetics, revealed t 1/2 values of approximately 19 and 10 days for the overload and regression periods, respectively. Collectively these data suggest that 1) skeletal muscle myosin isoforms and corresponding ATPase activity are in a dynamic state of change, although not completely synchronous, in response to altered muscle stress, and 2) the kinetics of change in the mixed MHC protein pool are slower during compensatory growth compared with regression of growth.     

Beta2-integrins contribute to skeletal muscle hypertrophy in mice

We tested the contribution of β₂-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 β-subunit of β₂-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 β₂-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 β₂-integrins contribute to the hypertrophic response to muscle overload by temporally regulating satellite cells/myoblast proliferation and by enhancing muscle differentiation and p70S6k signaling. skeletal muscle growth; neutrophils; macrophages; compensatory hypertrophy     

3-D in vitro model of early skeletal muscle development

An understanding of the mechanical and mechano-molecular responses that occur during the differentiation of mouse C2C12 [corrected] myoblasts in 3-D culture is critical for understanding growth, which is important for progress towards producing a tissue-engineered muscle construct. We have established the main differences in force generation between skeletal myoblasts, dermal fibroblasts, and smooth muscle cells in a 3-D culture model in which cells contract a collagen gel construct. This model was developed to provide a reproducible 3-D muscle organoid in which differences in force generation could be measured, as the skeletal myoblasts fused to form myotubes within a collagen gel. Maintenance of the 3-D culture under sustained uni-axial tension, was found to promote fusion of myoblasts to form aligned multi-nucleate myotubes. Gene expression of both Insulin Like Growth Factor (IGF-1 Ea) and an isoform of IGF-1 Ea, Mechano-growth factor (IGF-1 Eb, also termed MGF), was monitored in this differentiating collagen construct over the time course of fusion and maturation (0-7 days). This identified a transient surge in both IGF-1 and MGF expression on day 3 of the developing construct. This peak of IGF-1 and MGF expression, just prior to differentiation, was consistent with the idea that IGF-1 stimulates differentiation through a Myogenin pathway [Florini et al., 1991: Mol. Endocrinol. 5:718-724]. MGF gene expression was increased 77-fold on day 3, compared to a 36-fold increase with IGF-1 on day 3. This indicates an important role for MGF in either differentiation or, more likely, a response to mechanical or tensional cues.     

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.     

Identification of Serhl, a new member of the serine hydrolase family induced by passive stretch of skeletal muscle in vivo

In response to extended periods of stretch, skeletal muscle typically exhibits cell hypertrophy associated with sustained increases in mRNA and protein synthesis. Several soluble hypertrophic agonists have been identified, yet relatively little is known as to how mechanical load is converted into intracellular signals regulating gene expression or how increased cell size is maintained. In skeletal muscle, hypertrophy is generally regarded as a beneficial adaptive response to increased workload. In some cases, however, hypertrophy can be detrimental as seen in long-term cardiac hypertrophy. Skeletal muscle wasting (atrophy) is a feature of both inherited and acquired muscle disease and normal aging. Elucidating the molecular regulation of cell size is a fundamental step toward comprehending the complex molecular systems underlying muscle hypertrophy and atrophy. Subtractive hybridization between passively stretched and control murine skeletal muscle tissue identified an mRNA that undergoes increased expression in response to passive stretch. Encoded within the mRNA is an open reading frame of 311 amino acids containing a highly conserved type 1 peroxisomal targeting signal and a serine lipase active center. The sequence shows identity to a family of serine hydrolases and thus is named serine hydrolase-like (Serhl). The predicted three-dimensional structure displays a core alpha/beta-hydrolase fold and catalytic triad characteristic of several hydrolytic enzymes. Endogenous Serhl protein immunolocalizes to perinuclear vesicles as does Serhl-FLAG fusion protein transiently expressed in muscle cells in vitro. Overexpression of Serhl-FLAG has no effect on muscle cell phenotype in vitro. Serhl's expression patterns and its response to passive stretch suggest that it may play a role in normal peroxisome function and skeletal muscle growth in response to mechanical stimuli.     

Integrin signaling's potential for mediating gene expression in hypertrophying skeletal muscle.

Overloaded skeletal muscle undergoes dramatic shifts in gene expression, which alter both the phenotype and mass. Molecular biology techniques employing both in vivo and in vitro hypertrophy models have demonstrated that mechanical forces can alter skeletal muscle gene regulation. This review's purpose is to support integrin-mediated signaling as a candidate for mechanical load-induced hypertrophy. Research quantifying components of the integrin-signaling pathway in overloaded skeletal muscle have been integrated with knowledge regarding integrins role during development and cardiac hypertrophy, with the hope of demonstrating the pathway's importance. The role of integrin signaling as an integrator of mechanical forces and growth factor signaling during hypertrophy is discussed. Specific components of integrin signaling, including focal adhesion kinase and low-molecular-weight GTPase Rho are mentioned as downstream targets of this signaling pathway. There is a need for additional mechanistic studies capable of providing a stronger linkage between integrin-mediated signaling and skeletal muscle hypertrophy; however, there appears to be abundant justification for this type of research.     

Contractile skeletal muscle tissue-engineered on an acellular scaffold

For the reconstructive surgeon, tissue-engineered skeletal muscle may offer reduced donor-site morbidity and an unlimited supply of tissue. Using an acellularized mouse extensor digitorum longus muscle as a scaffold, the authors produced engineered skeletal muscle capable of generating longitudinal force. Eight extensor digitorum longus muscles from adult mice were made acellular using a protocol developed in the authors' laboratory. The acellular muscles were then placed in a bath of 20% fetal bovine serum in Dulbecco's modified Eagle's medium and 100 U/ml penicillin for 1 week at room temperature. C2C12 myoblasts were injected into the acellular muscle matrix using a 26-gauge needle and a 100-microl syringe. The resulting constructs were placed in growth medium for 1 week at 37 degrees C under 5% carbon dioxide, with media changes every 48 hours. The constructs were then placed in differentiation medium for 1 week, with media changes every 48 hours. Isometric contractile force testing of the constructs demonstrated production of longitudinal contractile force on electrical stimulation. A length-tension, or Starling, relationship was observed. Light and electron microscopy studies demonstrated recapitulation of some of the normal histologic features of developing skeletal muscle.     

Regulation of androgen receptor expression at the onset of functional overload in rat plantaris muscle

Skeletal muscle androgen receptor (AR) expression at the onset of functional overload (OV) has not been well described. It is also not known if overload and/or anabolic steroid differentially regulate AR expression. The purpose of this study was to examine AR gene expression at the onset of functional OV in rat plantaris muscle with and without nandrolone decanoate (ND) administration. The functional significance of AR protein induction was examined using skeletal alpha-actin promoter activity in transiently transfected CV-1 fibroblast cells. Male Sprague-Dawley rats ( approximately 125 g) were functionally overloaded for 1, 3, 7, or 21 days. A subset of animals was given an ND (6 mg/kg) injection at day 0 and then overloaded for 3 days. Control animals underwent sham surgeries. AR protein concentration increased 106 and 279% after 7 and 21 days of OV, respectively. AR mRNA increased 430% after 7 days of OV. AR protein expression in C2C12 murine myotubes subjected to 1% chronic radial stretch for 18 h was elevated 101% compared with control. ND treatment increased AR protein concentration 1,300% compared with controls, and there was no additional effect when ND and OV were combined. ND with 3 days of OV treatment increased AR mRNA expression 50% compared with control. AR overexpression in transiently transfected CV-1 fibroblast cells increased -424 bp skeletal alpha-actin promoter activity 80 to 1,800% in a dose-dependent fashion. Co-overexpression of either serum response factor (SRF) or active RhoA with AR overexpression induced a synergistic 36- and 28-fold induction of skeletal alpha-actin promoter. Cotransfection of AR, SRF, and active RhoA induced 180-fold increase in skeletal alpha-actin promoter activity. In conclusion, AR protein expression is increased after 7 days of functional OV, and this induction is regulated pretranslationally. AR induction in conjunction with SRF and RhoA signaling may be an important regulator of gene expression during overload-induced muscle growth.     

Mechanotransduction pathways in skeletal muscle hypertrophy

In the last decade, molecular biology has contributed to define some of the cellular events that trigger skeletal muscle hypertrophy. Recent evidence shows that insulin like growth factor 1/phosphatidyl inositol 3-kinase/protein kinase B (IGF-1/PI3K/Akt) signaling is not the main pathway towards load-induced skeletal muscle hypertrophy. During load-induced skeletal muscle hypertrophy process, activation of mTORC1 does not require classical growth factor signaling. One potential mechanism that would activate mTORC1 is increased synthesis of phosphatidic acid (PA). Despite the huge progress in this field, it is still early to affirm which molecular event induces hypertrophy in response to mechanical overload. Until now, it seems that mTORC1 is the key regulator of load-induced skeletal muscle hypertrophy. On the other hand, how mTORC1 is activated by PA is unclear, and therefore these mechanisms have to be determined in the following years. The understanding of these molecular events may result in promising therapies for the treatment of muscle-wasting diseases. For now, the best approach is a good regime of resistance exercise training. The objective of this point-of-view paper is to highlight mechanotransduction events, with focus on the mechanisms of mTORC1 and PA activation, and the role of IGF-1 on hypertrophy process.