Expression profiling identifies dysregulation of myosin heavy chains IIb and IIx during limb immobilization in the soleus muscles of old rats

Abstract

Aged individuals suffer from multiple dysfunctions during skeletal muscle atrophy. The purpose of this study was to determine differential changes in gene expression in atrophied soleus muscle induced by hindlimb immobilization in young (3-4 months) and old (30-31 months) rats. The hypothesis was that differentially expressed mRNAs with age-atrophy interactions would reveal candidates that induce loss of function responses in aged animals. Each muscle was applied to an independent set of Affymetrix micoarrays, with 385 differentially expressed mRNAs with atrophy and 354 age-atrophy interactions detected by two-factor ANOVA (alpha of 0.05 with a Bonferroni adjustment). Functional trends were observed for 23 and 15 probe sets involved in electron transport and the extracellular matrix, respectively, decreasing more in the young than in the old. Other functional categories with atrophy in both ages included chaperones, glutathione-S-transferases, the tricarboxylic acid cycle, reductions in Z-line-associated proteins and increases in probe sets for protein degradation. Surprisingly, myosin heavy chain IIb and IIx mRNAs were suppressed in the atrophied soleus muscle of old rats as opposed to the large increases in the young animals (16- and 25-fold, respectively, with microarrays, and 61- and 68-fold, respectively, with real-time PCR). No significant changes were observed in myosin heavy chain IIb and IIx mRNA with micoarrays in the atrophied soleus muscles of old rats, but they were found to increase six- and fivefold, respectively, with real-time PCR. Therefore, deficiencies in pre-translational signals that normally upregulate myosin heavy chain IIb and IIx mRNAs during atrophy may exist in the soleus muscle of old animals.

Figure 1. Failure of MHC IIb and IIx mRNAs to increase in the soleus muscle of old rats after 10 days of hindlimb immobilization

Failure of MHC IIb (A) and IIx (B) mRNAs to increase in the soleus muscle of old rats after 10 days of hindlimb immobilization (OI) when analysed by microarray analysis. Fold changes are shown above immobilized groups as the difference from the respective age-matched control. Values are the means ± s.e.m. of n = 5 per group. OI, old immobilized; OC, old control; YI, young immobilized; YC, young control. *Significant at P < 4.0 × 10⁻⁶.

Figure 2. An example of homer2 mRNA expression

This example was selected from the five different probe sets for homer2 as detected from microarray analysis because this particular set was used for the design of primers and probes utilized in real-time PCR. The fold change is shown above the immobilized groups as the difference from respective age-matched controls. Values are the means ± s.e.m. of n = 5 per group. *Significant at P < 4.0 × 10⁻⁶.

Figure 3. Hierarchical clustering and probe-set expression patterns

A, a cluster is shown that is composed predominantly of extracellular matrix genes obtained with hierarchical clustering analysis of expression profiles for age-atrophy interactions. B, representative examples of probe-set expression patterns from the shown hierarchical cluster of age-atrophy interactions.