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

Abstract

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.

Fig. 1.

Unilateral denervation (DNV)-induced increase in rat diaphragm muscle (DIAm) protein synthesis, as determined by incorporation of ³H-Tyr. Mean and SE of the percent increase relative to sham controls are plotted over time (in days, D) after DNV. *Significantly different from the sham control group at the same DNV time point; †significantly different from 1 day after DNV; ±significantly different from 7 days after DNV (P < 0.05 for all comparisons).

Fig. 2.

DNV-induced increase in rat DIAm protein degradation, in the presence of cycloheximide, as determined by Tyr release. Mean and SE of the percent increase relative to sham controls are plotted over time after DNV. *Significantly different from the sham control group at the same DNV time point; †significantly different from 1 and 3 days after DNV; #significantly different from 5 days after DNV (P < 0.05 for all comparisons).

Fig. 3.

DNV-induced change in rat DIAm net protein balance as determined by performing parallel, but separate, incubations of strips from the same diaphragm for protein synthesis and protein degradation measurements. Mean and SE of the percent change relative to sham controls are plotted over time after DNV. *Significantly different from the sham control group at the same DNV time point; †significantly different from 1 and 3 days after DNV; #significantly different from 5 days after DNV (P < 0.05 for all comparisons).

Fig. 4.

DNV-induced increase in total protein ubiquitination, as detected by Western analyses. Top: representative immunoblot of ubiquitin and β-tubulin for each DNV time point (S, sham control; D, DNV). Bottom: mean intensity profile of protein ubiquitination across molecular weight, separated by groups determined by measuring area under the intensity profile curve. *Significantly different (P < 0.05) from the sham controls and from 1 and 3 days after DNV.

Fig. 5.

DNV-induced change in procaspase-3 (pro) and cleaved capase-3 protein expression in rat DIAm, as detected by Western analyses. Representative immunoblots are shown of procaspase-3 (35 kDa), cleaved caspase-3 (17 kDa), and actin (43 kDa) for each DNV time point. Cytochrome c-treated Jurkat cell extract was used as positive control.

Fig. 6.

DNV-induced change in actin fragmentation as detected by Western analyses. Top: ratio of actin fragment to total actin for 1 and 3 days after DNV. Bottom: representative immunoblot of intact actin and actin fragment for 1 and 3 days after DNV.