Transcriptional regulation of the type I myosin heavy chain gene in denervated rat soleus

Abstract

Denervation (DEN) of rat soleus is associated with a decreased expression of slow type I myosin heavy chain (MHC) and an increased expression of the faster MHC isoforms. The molecular mechanisms behind these shifts remain unclear. We first investigated endogenous transcriptional activity of the type I MHC gene in normal and denervated soleus muscles via pre-mRNA analysis. Our results suggest that the type I MHC gene is regulated via transcriptional processes in the denervated soleus. Deletion and mutational analysis of the rat type I MHC promoter was then used to identify cis elements or regions of the promoter involved in this response. DEN significantly decreased in vivo activity of the −3,500, −2,500, −914, −408, −299, and −215 bp type I MHC promoters, relative to the α-skeletal actin promoter. In contrast, normalized −171 promoter activity was unchanged. Mutation of the βe3 element (−214/−190) in the −215 promoter and deletion of this element (−171 promoter) blunted type I downregulation with DEN. In contrast, βe3 mutation in the −408 promoters was not effective in attenuating the DEN response, suggesting the existence of additional DEN-responsive sites between −408 and −215. Western blotting and gel mobility supershift assays demonstrated decreased expression and DNA binding of transcription enhancer factor 1 (TEF-1) with DEN, suggesting that this decrease may contribute to type I MHC downregulation in denervated muscle.