Carbonic anhydrase III and four-and-a-half LIM protein 1 are preferentially oxidized with muscle unloading

Abstract

The identities of proteins that show disuse-related changes in the content of oxidative modification are unknown. Furthermore, it is unknown whether the global accumulation of oxidized proteins is greater in aged animals with muscle disuse. The purposes of this study are 1) to identify the exact proteins that show disuse-related changes in oxidation levels and 2) to test the hypothesis that the global accumulation of oxidized proteins with muscle disuse would be greater in aged animals. Adult and old rats were randomized into four groups: weight bearing and 3, 7, or 14 days of hindlimb unloading. Soleus muscles were harvested to investigate the protein oxidation with unloading. Slot blot, SDS-PAGE, and Western blot analyses were used to detect the accumulation of 4-hydroxy-2-nonenol (HNE)- and nitrotyrosine (NT)-modified proteins. Matrix-assisted laser desorption ionization-time of flight and tandem mass spectroscopy were used to identify modified proteins. We found that global HNE- and NT-modified proteins accumulated significantly with aging but not with muscle unloading. Two HNE and NT target proteins, four-and-a-half LIM protein 1 (FHL1) and carbonic anhydrase III (CAIII), showed changes in the oxidation levels with muscle unloading. The changes in the oxidation levels happened to adult rats but not old rats. However, old rats had higher baseline levels of HNE-modified FHL1. In summary, the data suggest that the muscle unloading-related changes of protein oxidation are more significant in specific proteins and that the changes are age related.

Fig. 1.

Content of 4-hydroxy-2-nonenol (HNE)- and nitrotyrosine (NT)-modified proteins in soleus muscles of adult and old rats. Densitometric analysis is shown of immune reactions (slot blots) from muscles of rats with weight bearing (C) and 3, 7, and 14 days (d) of hindlimb unloading (HU). A: content of HNE-modified proteins in the soluble fraction. B: content of NT-modified proteins in the soluble fraction. C: content of HNE-modified proteins in the myofibrillar fraction. D: content of NT-modified proteins in the myofibrillar fraction. Data are means ± SE; n = 5–8/group.

Fig. 2.

Four-and-a-half LIM protein 1 (FHL1) identification and confirmation. A: peptide mass fingerprints of FHL1 determined using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The spectrum shows the 8 peaks that matched the theoretical mass-to-charge ratio (m/z) values for peptides from FHL1. B: the tandem mass spectrometry (MS/MS) ion spectrum of peptide 1,180 m/z corresponds with QVIGTGSFFPK, a peptide sequence that matches FHL1. The sequence is displayed above the spectrum. The y and b ions found experimentally are noted above the corresponding peak in the spectrum. amu, Atomic mass units. C: the first and second panels show the immune reaction of FHL1 to HNE and NT antibodies, respectively. The third panel shows two corresponding protein bands (arrows) on the silver-stained (SS) gel that were both identified as FHL1 from mass spectrometric analysis. The fourth panel shows the immune reaction of protein to the FHL1 antibody. The position of the molecular mass marker at 31 kDa is shown at left.

Fig. 3.

HU effect on the HNE-modified carbonic anhydrase III (CAIII). Representative immunoblots (top) and densitometric analysis (bottom) are shown of immune reactions from the soluble fraction of rats with weight bearing and 3, 7, and 14 days of HU. A: HNE-modified CAIII in soleus muscles of adult rats. B: HNE-modified CAIII in soleus muscles of old rats. Values are normalized to the weight-bearing control in each age group. Data are means ± SE; n = 5–8/group. WB, Western blot.

Fig. 4.

HU effect on the HNE- and NT-modified FHL1. Representative immunoblots (top) and densitometric analysis (bottom) are shown of immune reactions (including both bands) from the myofibrillar fraction of rats with weight bearing and 3, 7, and 14 days of HU. A: HNE-modified FHL1 in soleus muscles of adult rats. B: NT-modified FHL1 in soleus muscles of adult rats. C: HNE-modified FHL1 in soleus muscles of old rats. D: NT-modified FHL1 in soleus muscles of old rats. Values are normalized to the weight-bearing control in each age group. Data are means ± SE; n = 5–8/group.

Fig. 5.

Age effect on the levels of HNE-modified FHL1. Representative immunoblots (top) and densitometric analysis of immune reactions (bottom) are shown for HNE-modified FHL1 in muscles of adult and old rats. Values are normalized to the adult value. Data are means ± SE; n = 6 per group. *Significantly different from muscles of adult rats P < 0.05.