Identification and Characterization of Calcium Binding and Coiled Coil Domain 1 (Calcoco1) in Skeletal Muscle

Abstract

Skeletal muscle atrophy results in reduced muscle size and strength and is caused by a range of physiological conditions, including aging, cancer, corticosteroid use, and denervation. In order to better characterize the molecular genetic events of atrophy, skeletal muscle was isolated from mice following 3 days and 14 days of denervation. The gene expression profile of the denervated muscle tissue was analyzed by microarray and compared to control muscle in order to identify novel atrophy‐induced genes. The microarray revealed that Calcium Binding and Coiled Coil Domain 1 (Calcoco1) is expressed in skeletal muscle and is induced in response to denervation. To confirm that Calcoco1 is expressed in muscle, we successfully cloned the cDNA of Calcoco1 from cultured muscle cells, demonstrating that this gene is transcriptionally active. In addition, quantitative PCR (qPCR) was used to assess Calcoco1 expression in both proliferating and differentiated muscle cells and the results demonstrated that Calcoco1 expression levels are moderate in proliferating myoblasts, but show significant elevation in differentiated myotubes. Furthermore, Western Blot analysis of protein homogenates isolated from muscle cells confirmed that Calcoco1 is expressed in both proliferating myoblasts and differentiated myotubes. In order to better characterize the transcriptional regulation of Calcoco1, fragments of the proximal promoter located immediately upstream of the start of transcription were cloned and fused to 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. Furthermore, bioinformatic analysis of the promoter region of Calcoco1 revealed a conserved canonical E‐box element, which is a known myogenic regulatory factor (MRF) binding site, further suggesting that Calcoco1 may be regulated by muscle specific transcription factors. Finally, the Calcoco1 protein is predicted to have both a calcium binding domain and a coiled‐coil domain, suggesting that it may function in protein‐protein interactions and/or as a putative transcription factor. Therefore, in order to determine the sub‐cellular localization of Calcoco1 in muscle cells, the Calcoco1 cDNA was fused with the green fluorescent protein (GFP), expressed in muscle cells, and visualized by confocal microscopy revealing that Calcoco1 is localized exclusively to the cytoplasm in unchallenged myoblast cells. The discovery that Calcoco1 is expressed in skeletal muscle combined with the observation that this gene is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of muscle atrophy and may eventually lead to the identification of new therapeutic targets for the treatment and prevention of muscle wasting.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants to D.W.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.