Heather M. Argadine

The effect of denervation on protein synthesis and degradation in adult rat diaphragm muscle

Previous studies showed that unilateral denervation (DNV) of the rat diaphragm muscle (DIAm) results in loss of myosin heavy chain protein by 1 day after DNV. We hypothesize that DNV decreases net protein balance as a result of activation of the ubiquitin-proteasome pathway. In DIAm strips, protein synthesis was measured by incorporation of 3H-Tyr, and protein degradation was measured by Tyr release at 1, 3, 5, 7, and 14 days after DNV. Total protein ubiquitination, caspase-3 expression/activity, and actin fragmentation were analyzed by Western analysis. We found that, at 3 days after DNV, protein synthesis increased by 77% relative to sham controls. Protein synthesis remained elevated at 5 (85%), 7 (53%), and 14 days (123%) after DNV. At 5 days after DNV, protein degradation increased by 43% relative to sham controls and remained elevated at 7 (49%) and 14 days (74%) after DNV. Thus, by 5 days after DNV, net protein balance decreased by 43% compared with sham controls and was decreased compared with sham at 7 (49%) and 14 days (72%) after DNV. Protein ubiquitination increased at 5 days after DNV and remained elevated. DNV had no effect on caspase-3 activity or actin fragmentation, suggesting that the ubiquitin-proteasome pathway rather than caspase-3 activation is important in the DIAm response to DNV. Early loss of contractile proteins, such as myosin heavy chain, is likely the result of selective protein degradation rather than generalized protein breakdown. Future studies should evaluate this selective effect of DNV.

Intracellular signaling pathways regulating net protein balance following diaphragm muscle denervation.

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.

1 kHz low power sound stimulates ATDC5 chondrocytes

A current clinical product from Smith and Nephew uses a pulsed 1.5 MHz signal for treating fractures 20 minutes per day. This pulsed 1.5 MHz signal produces radiation force vibration at 1 kHz. It was hypothesized that the radiation force, not the ultrasound, is responsible for the biological effect of the Smith and Nephew system of stimulating chondrocytes. In vitro experiments using the following method indeed showed that treatment with 1 kHz induced chondrogenesis similar to treatment with 1.5 MHz pulsed ultrasound. This study provides the first evidence for 1 kHz activation of chondrocytes and for the potential mechanisms with which this vibration is sensed in the cell.

1I-4 Stimulation of Proteoglycan Synthesis with Low-Intensity 1 kHz Vibration

Pulsed ultrasound has become a common therapy for delayed unions and nonunions after fractures. Experiments using ATDC5 cells, a mouse clonal chondrogenic cell line, have shown that 1 kHz dynamic acoustic radiation force stimulates proteoglycan synthesis similar to 1.5 MHz pulsed ultrasound. The results of two experiments performed 8 months apart showed that chondrocytes treated with 1 kHz squarewave had a 2 to 4-fold (p > 0.01) increase in total area of nodules compared to control. When the same experiments were repeated 2 months later with the same frozen cell stock, the cells were unresponsive to mechanical stimulation, suggesting the cell line had transformed and was unable to differentiate into chondrocytes. This study demonstrates the need for primary chondrocytes to further examine the biological effects of 1 kHz vibration

1 kHz Vibration Stimulates ATDC5 Chondrocytes

Low‐intensity pulsed ultrasound is a commonly prescribed therapy for delayed unions and nonunions after fractures. Several clinical trials have shown that a 1.5 MHz ultrasound signal at a 200 μs tone burst repeating at 1 kHz shortens the time to normal bone strength by 30%. In vitro studies have shown that pulsed ultrasound increases aggrecan gene expression in chondrocytes. The pulsed 1.5 MHz signal produces radiation force vibration at 1 kHz. It was hypothesized that dynamic radiation force, not ultrasound, is responsible for the biological effect of the signal. Experiments showed that 1 kHz induced chondrogenesis similar to pulsed ultrasound treatment. These results have implications for stimulation of different types of strain‐sensitive cells.

1 kHz vibration increases proteoglycan production in ATDC5 chondrocytes

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 chondrogen...