Identification and Characterization TSSK6 Activating Co‐chaperone (TSACC) in Skeletal Muscle

Abstract

Skeletal muscle atrophy is a physiological condition that is caused by a range of conditions, including immobilization, denervation, spinal cord injury, and aging. To better characterize the molecular genetic events of neurogenic atrophy, a previous study isolated the gastrocnemius muscle from mice following 3 and 14 days of sciatic nerve transection. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that TSSK6 Activating Co‐chaperone (TSACC) is expressed in skeletal muscle and is significantly induced in response to denervation. To confirm that TSACC is expressed in muscle, the predicted TSACC cDNA was cloned from cultured muscle cells, as was a novel TSACC splice variant that contains an additional exon. Quantitative PCR (qPCR) was performed to assess TSACC expression levels of the full‐length and novel splice variants in proliferating and differentiated muscle cells. The results demonstrate that TSACC expression levels are relatively low in proliferating myoblasts but show significantly elevated expression in differentiated myotubes. In addition, characterization of the transcriptional regulation of TSACC was assessed by fusing fragments of the proximal promoter located immediately upstream of the start of transcription with a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells in combination with myogenic regulatory factor (MRF) expression plasmids, which resulted in significant activation of reporter gene activity. Interestingly, there are several predicted E‐box elements in the proximal promoter region of the TSACC gene further suggesting that TSACC may be regulated by muscle‐specific transcription factors. To identify a possible role for TSACC in skeletal muscle, sub‐cellular localization was assessed by fusing TSACC with GFP and expressing the fusion protein in cultured muscle cells, revealing that TSACC is localized predominantly to the cytoplasm. The discovery that TSACC is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of muscle wasting and may eventually lead to the identification of new therapeutic targets for the treatment of muscle atrophy.