Argadine, Heather M.

Neuregulin (NRG) is a member of the epidermal growth factor family, with 3 types varying in their structure and cellular expression but not in their receptor‐binding domain. NRG‐2 and NRG‐3 are primarily membrane bound, while NRG‐1 can be secreted. NRG‐1 is present in phrenic motor neurons. We previously reported that exogenous NRG containing only the receptor‐binding domain, increases protein synthesis in rat diaphragm. This effect was significantly inhibited by LY294002 indicating a PI 3‐kinase dependent mechanism. We also reported that NRG increases Akt phosphorylation. Interestingly, Akt phosphorylation is positively correlated with skeletal muscle growth and inversely correlated with atrophy. In this study, we hypothesized that NRG reduces protein degradation in rat diaphragm. Diaphragms were harvested from 4‐week old rats and incubated in the presence or absence of exogenous NRG. Protein degradation was measured by quantifying muscle tyrosine release via a fluorimetric assay. We found that NRG treatment resulted in a 50% reduction in tyrosine release from diaphragm preparations, indicating a reduction in basal protein degradation rates. These results suggest that phrenic motoneuron‐derived NRG‐1 regulates muscle protein synthesis and degradation rates, thereby playing a role in matching muscle fiber and motoneuron properties within motor units.

Neuregulin (NRG) is part of the epidermal growth factor family of proteins. Previous studies have shown that NRG activates PI3K/Akt pathways, leading to regulation of protein synthesis via downstream regulation of translation initiators. The principal translation initiator involved in protein synthesis is probably eIF4E, and the principal transcription factor regulating protein degradation is FOXO. We hypothesized that NRG signaling involves activation of the PI3K/Akt pathway, thus leading to an increase in expression of eIF4E and a decrease in expression of FOXO. An increase in eIF4E expression increases protein synthesis, and a decrease in FOXO expression reduces protein degradation. In this study we used dexamethasone, a synthetic glucocorticoid, to induce atrophy and cause an imbalance between protein synthesis and degradation in L6 myotubes. As expected, we found that after 48 hours of treatment with 10–100nM dexamethasone, eIF4E expression decreased and FOXO expression increased in a dose‐dependent manner. We found that treatment with 200 ng/ml NRG increased eIF4E expression. When L6 cells were treated with both NRG and dexamethasone for 48 hours, NRG blunted the catabolic effects of dexamethasone, thus increasing eIF4E expression and decreasing FOXO expression. These results indicate that NRG promotes protein synthesis and prevents protein degradation. Supported by NIH grant AR51173.

Unilateral denervation (DNV) of rat diaphragm muscle (DIAm) increases protein synthesis and degradation, with net protein breakdown by 14 days after DNV. We hypothesized that neural influences such as the nerve‐derived trophic factor neuregulin (NRG‐1) are essential in maintaining DIAm protein balance. NRG activates receptor tyrosine kinases of the ErbB family, with the ErbB2‐ErbB3 co‐receptor showing highest affinity for NRG‐1 and strong phosphoinositol‐3‐kinase (PI3K) activation. Both protein synthesis (via Akt phosphorylation) and degradation (via the transcription factor FoxO3A) are regulated by PI3K activity. We used Western blot analyses to assess NRG‐1, ErbB2, ErbB3 and FoxO3A protein expression at 1, 3, 7, and 14 days after DNV. By 1 day after DNV, NRG‐1 expression decreased, whereas ErbB2 and ErbB3 expression increased. Consistent with reduced NRG/ErbB signaling, association of the PI3K regulatory subunit p85 with ErbB3 decreased at 14 days after DNV. FoxO3A expression increased at 7 and 14 days after DNV, in agreement with delayed net protein breakdown. Importantly, DIAm inactivity induced by spinal hemisection had no effect on ErbB2, ErbB3, or FoxO3A expression. Taken together, these results indicate that DNV‐induced skeletal muscle adaptations result from removal of a trophic influence such as NRG‐1 rather than inactivity itself. Supported by NIH grant AR51173.

The balance between protein synthesis and degradation determines if there is a net loss (atrophy) or net gain (hypertrophy) in muscle mass. Recent studies suggest that PI3K‐dependent activation of Akt is an important regulator of both protein synthesis (via Erk and mTOR) and degradation (via the transcription factor FoxO1). Unilateral denervation (DNV) of the diaphragm muscle (DIAm) increases protein synthesis at 3d and degradation by 5d post‐DNV such that protein breakdown becomes evident by 5d after DNV. We hypothesized that DIAm DNV results in time‐dependent changes in Akt activation, leading to initial mTOR and subsequent FoxO1 activation. Akt, Erk, and mTOR phosphorylation and FoxO1 nuclear translocation were measured by Western blot analyses. At 1h and 6h post‐DNV, pAkt/Akt and pErk/Erk decreased. pErk/Erk increased at 1d and 3d, and pAkt/Akt increased at 5d post‐DNV. Although mTOR phosphorylation and expression increased 5d post‐DNV, the ratio of phospho‐mTOR/mTOR did not change at any time point post‐DNV. Nuclear translocation of FoxO1 increased 1d post‐DNV. Taken together, these results indicate that concomitant changes in PI3K/Akt, Erk, mTOR, and FoxO1 activity all contribute to, but do not completely account for, the time‐dependent changes in protein balance observed after DNV. Regulation of other signaling pathways, e.g., AMPK, and/or trophic influences, e.g., neuregulin, must be considered.

In vitro studies have shown that treatment with 1.5 MHz ultrasound signal (160 mW/cm2) at a 200 μs tone burst repeating at 1 kHz increases proteoglycan synthesis in chondrocytes [J. Parvisi et al., J. Orthop. Res. 17, 488–494 (1999)]. It was hypothesized that a continuous 1 kHz signal would be similar to the pulsed 1.5 MHz signal in stimulating chondrocytes to produce proteoglycan, which may cause accelerated fracture healing. In vitro experiments were performed with ATDC5 cells, a chondrogenic clonal cell line, plated in 6-well plates for 3 to 7 days before receiving ultrasound treatments. Cells were treated with either 1.5 MHz pulsed signal or 1 kHz signal for 20 minutes per day for 9 to 11 days. The signals were calibrated so that the bottom of the 6-well plate moved 10 nm for each condition. After the final treatment, cell layers were stained with Alcian blue, which stains cartilage nodules providing a measure of chondrogenesis. Both 1.5 MHz and 1 kHz led to a highly significant increase in chondrogenesis compared to control. Quantitative image analysis of stained wells showed that treatments with either signal increased number of nodules 2.3-fold (p<0.02) and total area of nodules 3-fold (p<0.02) compared to controls.

Unilateral denervation (DNV) of rat diaphragm muscle increases protein synthesis at 3 days after DNV (DNV-3D) and degradation at DNV-5D, such that net protein breakdown is evident by DNV-5D. On the basis of existing models of protein balance, we examined DNV-induced changes in Akt, AMP-activated protein kinase (AMPK), and ERK½ activation, which can lead to increased protein synthesis via mammalian target of rapamycin (mTOR)/p70S6 kinase (p70S6K), glycogen synthase kinase-3β (GSK3β), or eukaryotic initiation factor 4E (eIF4E), and increased protein degradation via forkhead box protein O (FoxO). Protein phosphorylation was measured using Western analyses through DNV-5D. Akt phosphorylation decreased at 1 h and 6 h after DNV compared with sham despite decreased AMPK phosphorylation. Both Akt and AMPK phosphorylation returned to sham levels by DNV-1D. Phosphorylation of their downstream effector mTOR (Ser2481) did not change at any time point after DNV, and phosphorylated p70S6K and eIF4E-binding protein 1 (4EBP1) increased only by DNV-5D. In contrast, ERK½ phosphorylation and its downstream effector eIF4E increased 1.7-fold at DNV-1D and phosphorylated GSK3β increased 1.5-fold at DNV-3D ( P < 0.05 for both comparisons). Thus, following DNV there are differential effects on protein synthetic pathways with preferential activation of GSK3β and eIF4E over p70S6K. FoxO1 nuclear translocation occurred by DNV-1D, consistent with its role in increasing expression of atrogenes necessary for subsequent ubiquitin-proteasome activation evident by DNV-5D. On the basis of our results, increased protein synthesis following DNV is associated with changes in ERK½-dependent pathways, but protein degradation results from downregulation of Akt and nuclear translocation of FoxO1. No single trigger is responsible for protein balance following DNV. Protein balance in skeletal muscle depends on multiple synthetic/degradation pathways that should be studied in concert.