Submitted on September 9, 2008
Revised on April 16, 2009
Accepted on June 4, 2009
The Effect of Denervation on Protein Synthesis and Degradation in Adult Rat Diaphragm Muscle
Heather M. Argadine1, Nathan J. Hellyer1, Carlos B. Mantilla2, Wen-Zhi Zhan2, and Gary C. Sieck2*
1 Mayo Clinic College of Medicine
2 Mayo Clinic
* To whom correspondence should be addressed. E-mail: sieck.gary{at}mayo.edu.
Previous studies showed that unilateral denervation (DNV) of the rat diaphragm muscle (DIAm) results in loss of myosin heavy chain (MHC) 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 to sham controls and was decreased compared to 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 MHC is likely the result of selective protein degradation rather than generalized protein breakdown. Future studies should evaluate this selective effect of DNV.
