Time-dependent changes in myosin heavy chain (MHC) isoform expression were investigated in rat soleus muscle unloaded by hindlimb suspension. Changes at the mRNA level were measured by RT-PCR and correlated with changes in the pattern of MHC protein isoforms. Protein analyses of whole muscle revealed that MHCI decreased after 7 days, when MHCIIa had increased, reaching a transient maximum by 15 days. Longer periods led to inductions and progressive increases of MHCIId(x) and MHCIIb. mRNA analyses of whole muscle showed that MHCIId(x) displayed the steepest increase after 4 days and continued to rise until 28 days, the longest time period investigated. MHCIIb mRNA followed a similar time course, although at lower levels. MHCIalpha mRNA, present at extremely low levels in control soleus, peaked after 4 days, stayed elevated until 15 days, and then decayed. Immunohistochemistry of 15-day unloaded muscles revealed that MHCIalpha was present in muscle spindles but at low amounts also in extrafusal fibers. The slow-to-fast transitions thus seem to proceed in the order MHCIbeta --> MHCIIa --> MHCIId(x) --> MHCIIb. Our findings indicate that MHCIalpha is transiently upregulated in some fibers as an intermediate step during the transition from MHCIbeta to MHCIIa.

O-linked N-acetylglucosaminylation (O-GlcNAc) is a regulatory post-translational modification of nucleo-cytoplasmic proteins that has a complex interplay with phosphorylation. O-GlcNAc has been described as a nutritional sensor, the level of UDP-GlcNAc that serves as a donor for the uridine diphospho-N-acetylglucosamine:polypeptide ␤-N-acetyl-glucosaminyltransferase being regulated by the cellular fate of glucose. Because muscular contraction is both dependent on glucose metabolism and is highly regulated by phosphorylation/dephosphorylation processes, we decided to investigate the identification of O-GlcNAc-modified proteins in skeletal muscle using a proteomic approach. Fourteen proteins were identified as being O-GlcNAc modified. These proteins can be classified in three main classes: i) proteins implicated in the signal transduction and in the translocation between the cytoplasm and the nucleus or structural proteins, ii) proteins of the glycolytic pathway and energetic metabolism, and iii) contractile proteins (myosin heavy chain). A decrease in the O-GlcNAc level was measured in the slow postural soleus muscle after 14-day hindlimb unloading, a model of functional atrophy characterized by a decrease in the force of contraction. These results strongly suggest that O-GlcNAc modification may serve as an important regulation system in skeletal muscle physiology.

To investigate the plasticity of slow and fast muscles undergoing slow-to-fast transition, rat soleus (SOL), gastrocnemius (GAS), and extensor digitorum longus (EDL) muscles were exposed for 14 days to 1) unweighting by hindlimb suspension (HU), or 2) treatment with the beta(2)-adrenergic agonist clenbuterol (CB), or 3) a combination of both (HU-CB). In general, HU elicited atrophy, CB induced hypertrophy, and HU-CB partially counteracted the HU-induced atrophy. Analyses of myosin heavy (MHC) and light chain (MLC) isoforms revealed HU- and CB-induced slow-to-fast transitions in SOL (increases of MHCIIa with small amounts of MHCIId and MHCIIb) and the upregulation of the slow MHCIa isoform. The HU- and CB-induced changes in GAS consisted of increases in MHCIId and MHCIIb ("fast-to-faster transitions"). Changes in the MLC composition of SOL and GAS consisted of slow-to-fast transitions and mainly encompassed an exchange of MLC1s with MLC1f. In addition, MLC3f was elevated whenever MHCIId and MHCIIb isoforms were increased. Because the EDL is predominantly composed of type IID and IIB fibers, HU, CB, and HU-CB had no significant effect on the MHC and MLC patterns.

In this work we studied changes in passive elastic properties of rat soleus muscle fibers subjected to 14 days of hindlimb unloading (HU). For this purpose, we investigated the titin isoform expression in soleus muscles, passive tension-fiber strain relationships of single fibers, and the effects of the thick filament depolymerization on passive tension development. The myosin heavy chain composition was also measured for all fibers studied. Despite a slow-to-fast transformation of the soleus muscles on the basis of their myosin heavy chain content, no modification in the titin isoform expression was detected after 14 days of HU. However, the passive tension-fiber strain relationships revealed that passive tension of both slow and fast HU soleus fibers increased less steeply with sarcomere length than that of control fibers. Gel analysis suggested that this result could be explained by a decrease in the amount of titin in soleus muscle after HU. Furthermore, the thick filament depolymerization was found to similarly decrease passive tension in control and HU soleus fibers. Taken together, these results suggested that HU did not change titin isoform expression in the soleus muscle, but rather modified muscle stiffness by decreasing the amount of titin.