Use of DNA injection for identification of slow nerve-dependent regions of the MLC2s gene.

It has been well established that expression of slow contractile protein genes in skeletal muscle is regulated, in part, by activity from slow motoneurons. However, very little is understood about the mechanism by which neural activity regulates transcription of slow isoform genes. The purpose of this investigation was first to more fully define the in vivo DNA injection technique for use in both fast-twitch and slow-twitch muscles and second to use the injection technique for the identification of slow nerve-dependent regions of the myosin light chain 2 slow (MLC2s) gene. Initial experiments determined that the same amount of plasmid DNA was taken up by both the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles and that injection of from 0.5 to 10 micrograms DNA/muscle is ideal for analysis of promoter activity during regeneration. This technique was subsequently used to identify that the region from -800 to +12 base pairs of MLC2s gene directed approximately 100 times higher activity in the innervated soleus than in innervated EDL, denervated soleus, or denervated EDL muscles. Placing the introns upstream of either the MLC2s or SV40 promoter increased expression 5- and 2.7-fold, respectively, in innervated soleus but not in innervated EDL, denervated soleus, or denervated EDL muscles. These results demonstrate that 1) in vivo DNA injection is a sensitive assay for promoter analysis in both fast-twitch and slow-twitch skeletal muscles and 2) both 5' flanking and intronic regions of the MLC2s gene can independently and synergistically direct slow nerve-dependent transcription in vivo.