IIx myosin heavy chain promoter regulation cannot be characterized in vivo by direct gene transfer.

In skeletal muscle of the adult mammal IIx is a pivotal myosin heavy chain (MHC) isoform that can be either up- or downregulated depending on both the fiber type of the target muscle and the type of external stimulus imposed. Since little is known about promoter elements of the IIx MHC gene that are important for its transcriptional regulation in vivo,the main goal of this study was to characterize IIx MHC promoter activity and identify potential regulatory elements on the IIx MHC promoter. A direct gene transfer approach was used, and this approach involved transfection of promoter-reporter constructs into intact rat soleus and plantaris muscle under control and denervated conditions, as well as hindlimb suspension (i.e., models to upregulate IIx MHC transcription). Fast-twitch (plantaris) muscle fibers were confirmed to have significantly greater IIx MHC transcriptional products (pre-mRNA and mRNA) than slow-twitch (soleus) muscle fibers. However, promoter sequences corresponding to -2671 to +1720, -1000 to +392, and -605/+392 relative to the IIx MHC transcription start site, plus an additional construct ligated to a putative embryonic MHC enhancer, failed to produce a fiber type-specific response that is characteristic of the endogenous IIx MHC promoter. Furthermore, the activity of these promoter constructs did not demonstrate the expected response to denervation or hindlimb suspension (i.e., marked upregulation), despite normal uptake and activity of a coinjected alpha-actin reference promoter. On the basis of these findings with IIx MHC promoter-reporters we conclude that the loss of the native chromatin environment as well as other necessary cis elements may preclude use of the gene transfer approach, thereby suggesting that there are hidden layers of regulation for the IIx MHC gene.