The molecular mechanisms controlling ␤-adrenergic receptor agonist (BA)-induced skeletal muscle hypertrophy are not well known. We presently report that BA exerts a distinct muscle-and muscle fiber type-specific hypertrophy. Moreover, we have shown that pharmacologically or genetically attenuating extracellular signal-regulated kinase (ERK) signaling in muscle fibers resulted in decreases (P Ͻ 0.05) in fast but not slow fiber type-specific reporter gene expressions in response to BA exposure in vitro and in vivo. Consistent with these data, forced expression of MAPK phosphatase 1, a nuclear protein that dephosphorylates ERK1/2, in fast-twitch skeletal muscle ablated (P Ͻ 0.05) the hypertrophic effects of BA feeding (clenbuterol, 20 parts per million in water) in vivo. Further analysis has shown that BA-induced phosphorylation and activation of ERK occurred to a greater (P Ͻ 0.05) extent in fast myofibers than in slow myofibers. Analysis of the basal level of ERK activity in slow and fast muscles revealed that ERK1/2 is activated to a greater extent in fast-than in slow-twitch muscles. These data indicate that ERK signaling is differentially involved in BA-induced hypertrophy in slow and fast skeletal muscles, suggesting that the increased abundance of phospho-ERK1/2 and ERK activity found in fast-twitch myofibers, compared with their slow-twitch counterparts, may account, at least in part, for the fiber type-specific hypertrophy induced by BA stimulation. These data suggest that fast myofibers are pivotal in the adaptation of muscle to environmental cues and that the mechanism underlying this change is partially mediated by the MAPK signaling cascade. muscle fiber type; mitogen-activated protein kinase signaling pathways; mechanism SKELETAL MUSCLE ADAPTATION is triggered by a variety of cues that depend largely on a delicate balance between hypertrophy and atrophy signaling processes converging on the nucleus (34,37,39). This delicate balance suggests that hypertrophy may provide a means to antagonize skeletal muscle atrophy induced by some physiological and pathological challenges (16). ␤-Adrenergic agonists (BA) are a family of compounds that induce skeletal muscle hypertrophy in rats (7, 13, 46), mice (21), pigs (12), cattle, and sheep (27). Consistent with the aforementioned thesis, BA antagonize skeletal muscle atrophy in experimentally induced denervation or limb immobilization models (11, 21). Mechanistically, BA bind to the ␤-adrenergic receptor, a G protein-coupled receptor, and activate the G s protein and PKA signaling pathway. PKA then phosphorylates the ␤-receptor and switches its coupling from G s to G i protein. The ␤␥-subunit of G i protein stimulates the ERK-MAPK through a pathway involving c-Src and Ras (10). The activated receptor is then phosphorylated and bound by ␤-arrestin for degradation, a process called desensitization (29,38). Modulation of receptor desensitization is thought to account for the activation of MAPK in isoproterenol-stimulated cells (24). In addition, the ␤ 2 form of the ...

Skeletal muscle is composed of diverse fiber types, yet the underlying molecular mechanisms responsible for this diversification remain unclear. Herein, we report that the extracellular signal-regulated kinase (ERK) 1/2 pathway, but not p38 or c-Jun NH(2)-terminal kinase (JNK), is preferentially activated in fast-twitch muscles. Pharmacological blocking of ERK1/2 pathway increased slow-twitch fiber type-specific reporter activity and repressed those associated with the fast-twitch fiber phenotype in vitro. Overexpression of a constitutively active ERK2 had an opposite effect. Inhibition of ERK signaling in cultured myotubes increased slow-twitch fiber-specific protein accumulation while repressing those characteristic of fast-twitch fibers. Overexpression of MAP kinase phosphatase-1 (MKP1) in mouse and rat muscle fibers containing almost exclusively type IIb or IIx fast myosin heavy chain (MyHC) isoforms induced de novo synthesis of the slower, more oxidative type IIa and I MyHCs in a time-dependent manner. Conversion to the slower phenotype was confirmed by up-regulation of slow reporter gene activity and down-regulation of fast reporter activities in response to forced MKP1 expression in vivo. In addition, activation of ERK2 signaling induced up-regulation of fast-twitch fiber program in soleus. These data suggest that the MAPK signaling, most likely the ERK1/2 pathway, is necessary to preserve the fast-twitch fiber phenotype with a concomitant repression of slow-twitch fiber program.

The signal transduction cascades that maintain muscle mass remain to be fully defined. Herein, we report that inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in vitro decreases myotube size and protein content after 3-day treatment with a MEK inhibitor. Neither p38 nor JNK inhibitors had any effect on myotube size or morphology. ERK1/2 inhibition also upregulated gene transcription of atrogin-1 and muscle-specific RING finger protein 1 and downregulated the phosphorylation of Akt and its downstream kinases. Forced expression of enhanced green fluorescent protein-tagged MAPK phosphatase 1 (MKP-1) in soleus and gastrocnemius muscles decreased both fiber size and reporter activity. This atrophic effect of MKP-1 was time dependent. Analysis of the reporter activity in vivo revealed that the activities of nuclear factor-κB and 26S proteasome were differentially activated in slow and fast muscles, suggesting muscle type-specific mechanisms may be utilized. Together, these findings suggest that MAPK signaling is necessary for the maintenance of skeletal muscle mass because inhibition of these signaling cascades elicits muscle atrophy in vitro and in vivo.

No abstract

The effect of hepatocyte growth factor (HGF) on turkey satellite cell proliferation and differentiation was examined in cell culture. Satellite cell clones were established from one muscle of an individual turkey. The results showed that HGF is a potent activator and mitogen of turkey satellite cells and embryonic myoblasts with maximal stimulation at 1 ng/mL. HGF is also an inhibitor of differentiation of turkey satellite cells. Heterogeneity in the responsiveness to HGF in the turkey satellite cell population was observed between clones selected for fast (Early) or slow (Late) rates of proliferation. However, two other Early clones exhibited responses similar to those of two other Late clones. When combined with insulin-like growth factor (IGF) and fibroblast growth factor (FGF), singularly or in combination, HGF did not exert any additive or synergistic effects on Early or Late clone proliferation. Whereas when combined with IGF, FGF, and platelet-derived growth factor (PDGF), HGF significantly stimulated proliferation of the Late clone but not the Early clone. Addition of anti-HGF antibody to culture media diminished proliferation and provided evidence of autocrine production of HGF by turkey satellite cell cultures. Heterogeneity also exists in the turkey satellite cell population with respect to autocrine production of HGF.

Postnatal muscle growth is accompanied by increases in fast fiber type compositions and hypertrophy, raising the possibility that a slow to fast transition may be partially requisite for increases in muscle mass. To test this hypothesis, we ablated the Myh4 gene, and thus myosin heavy chain IIB protein and corresponding fibers in mice, and examined its consequences on postnatal muscle growth. Wild-type and Myh4–/– mice had the same number of muscle fibers at 2 weeks postnatal. However, the gastrocnemius muscle lost up to 50% of its fibers between 2 and 4 weeks of age, though stabilizing thereafter. To compensate for the lack of functional IIB fibers, type I, IIA, and IIX(D) fibers increased in prevalence and size. To address whether slowing the slow-to-fast fiber transition process would rescue fiber loss in Myh4–/– mice, we stimulated the oxidative program in muscle of Myh4–/– mice either by overexpression of PGC-1α, a well-established model for fast-to-slow fiber transition, or by feeding mice AICAR, a potent AMP kinase agonist. Forcing an oxidative metabolism in muscle only partially protected the gastrocnemius muscle from loss of fibers in Myh4–/– mice. To explore whether traditional means of stimulating muscle hypertrophy could overcome the muscling deficits in postnatal Myh4–/– mice, myostatin null mice were bred with Myh4–/– mice, or Myh4–/– mice were fed the growth promotant clenbuterol. Interestingly, both genetic and pharmacological stimulations had little impact on mice lacking a functional Myh4 gene suggesting that the existing muscle fibers have maximized its capacity to enlarge to compensate for the lack of its neighboring IIB fibers. Curiously, however, cell signaling events responsible for IIB fiber formation remained intact in the tissue. These findings further show disrupting the slow-to-fast transition of muscle fibers compromises muscle growth postnatally and suggest that type IIB myosin heavy chain expression and its corresponding fiber type may be necessary for fiber maintenance, transition and hypertrophy in mice. The fact that forcing muscle metabolism toward a more oxidative phenotype can partially compensates for the lack of an intact Myh4 gene provides new avenues for attenuating the loss of fast-twitch fibers in aged or diseased muscles.