Type IIx myosin heavy chain promoter activity during hindlimb immobilization

Abstract

Myosin heavy chain (MyHC) is an important skeletal muscle contractile protein that is often regulated at the transcriptional level. During atrophy, fast IIx MyHC mRNA levels are increased 35 fold in young, predominantly slow-twitch soleus muscle. The purpose of this study was to determine if the 5′-flanking region of the rat MyHC IIx gene contains an atrophy-response element. 1007 base pairs of the 5′-flanking region (−1 kb promoter) of the rat MyHC IIx gene were cloned, and deletion mutations were made. Intramuscular injections of plasmid DNA containing the firefly luciferase gene under control of the rat MyHC IIx promoter were made into the soleus of 2-mo-old rats undergoing normal cage activity or 6 days of hindlimb immobilization (HI). Aliquots of soleus muscle homogenates were used for firefly luciferase activity and plasmid DNA isolation for normalization (using real-time PCR with primers and probe designed against the firefly luciferase gene). The −1 and −0.889 kb promoter activities were 1.46-fold and 1.3-fold higher, respectively, in HI soleus compared to control. Deletion to −0.754 and −0.617 kb decreased promoter activity 70% and 30%, respectively, in HI soleus compared to control. In summary, the −1 kb MyHC IIx promoter is responsive to HI, and deletional analyses suggest that the MyHC IIx promoter potentially contains an atrophy-response element between −0.889 and −0.754 kb. Sponsored by the National Institutes of Health: 5F32AR051640-02 (RAS).