Characterization and Expression Analysis of Zinc Finger Protein 593 (Zfp593) in Skeletal Muscle

Skeletal muscle atrophy is a serious medical condition resulting from an array of physiological conditions such as aging, corticosteroid use, cancer and loss of innervation. In order to better characterize the molecular genetic events of neurogenic atrophy, the gastrocnemius muscle was isolated from mice following 3 days and 14 days of sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue in order to identify novel genes showing differential expression in response to neurogenic atrophy. The microarray data revealed for the first time that zinc finger protein 593 (Zfp593) is expressed in skeletal muscle and is significantly induced in response to denervation. Furthermore, Western blot analysis confirmed that Zfp593 is expressed at the protein level in C 2 C 12 mouse muscle cells. In order to characterize the transcriptional regulation of Zfp593, several fragments of the proximal regulatory region were cloned and fused to a reporter gene. The reporter plasmids were then transfected into C 2 C 12 mouse muscle cells alone or in combination with a myogenin expression plasmid, resulting in myogenin dependent induction of the Zfp593 reporter genes. Bioinformatic analysis of the promoter region of Zfp593 revealed two conserved E‐box elements, which are known myogenic regulatory factor (MRF) binding sites. Myogenin is a member of the myogenic regulatory factor family of transcription factors, which are important in muscle cell differentiation and are also significantly induced in response to neurogenic atrophy. Interestingly, Zfp593 is predicted to possess a zinc finger domain and is believed to function as a transcription factor. Therefore, in order to determine if Zfp593 is localized to the nucleus of muscle cells, the Zfp593 cDNA was amplified from C 2 C 12 cells and sub‐cloned into an expression plasmid that produces a GFP‐Zfp593 fusion protein. The GFP‐Zfp593 expression plasmid was then transfected into proliferating C 2 C 12 cells followed by visualization using confocal microscopy. Cells transfected with the GFP‐Zfp593 expression plasmid showed fluorescence that is exclusively localized to the nucleus of proliferating myoblasts. The discovery that Zfp593 is expressed in skeletal muscle combined with the observation that this gene, a potential modulator of gene expression itself, is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of skeletal muscle wasting and may eventually lead to the identification of new therapeutic targets for the treatment of muscle atrophy. Support or Funding Information University of North Florida Transformational Learning Opportunity Grant