Cardiovascular Research Advance Access [Accepted Manuscript] published online on December 22, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn357
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Egr-1 negatively regulates calsequestrin expression and calcium dynamics in ventricular cells
1 Lady Davis Institute for Medical Research, Sir Mortimer B. Davis - Jewish General Hospital, 3755 chemin Côte Ste Catherine, Montréal, Québec, Canada, H3T 1E2.
2 Division of Experimental Medicine, Department of Medicine, Faculty of Medicine, McGill University.
3 Faculty of Dentistry, McGill University.
4 The Bank of Montréal Research Center for the Study of Heart Disease in Women, Sir Mortimer B. Davis - Jewish General Hospital.
* corresponding author Mailing address: Lorraine E. Chalifour, PhD. Lady Davis Institute for Medical Research, Sir Mortimer B. Davis - Jewish General Hospital, 3755 chemin Côte Ste Catherine, Montréal, Québec, Canada, H3T 1E2 Telephone: (514) 340-8222 ext. 4295 Telefax: (514) 340-7502 E-mail: lorraine.chalifour{at}mcgill.ca
Aim: The transcription factor early growth response-1 (Egr-1) is increased in models of cardiac pathology; however, it is unclear how Egr-1 impacts the heart. We sought to identify how Egr-1 regulates expression of proteins involved in cardiomyocyte calcium homeostasis.
Methods: Protein expression was measured by immunoblotting in control cardiac differentiated H9c2 cells or in H9c2 cells overexpressing wild-type Egr-1 (Egr-1) or an Egr-1 (I293F) mutant. Microspectrofluorimetry of fura-2-loaded cells was used to study calcium dynamics. Chromatin immunoprecipitation with anti-Egr-1 antibody was used to identify Egr-1-associated DNA.
Results: Calsequestrin (CSQ) expression was reduced in Egr-1- and profoundly reduced in I293F-expressing cells. Calreticulin, triadin, sarcoendoplasmic reticulum ATPase 2a, phospholamban and phosphoserine16-phospholamban expression was unaffected. Calcium release from CSQ-dependent ryanodine-sensitive stores was reduced in Egr-1 and absent in I293F-expressing cells. In contrast, calcium release from calreticulin-dependent inositol 1,4,5-trisphosphate stores was unaffected. In vivo and in vitro chromatin immunoprecipitation demonstrated Egr-1 binding to the CSQ2 promoter. The Egr-1 binding region contains overlapping Egr-1, SP1 and nuclear factor of activated T-cells (NFAT) sites and a CpG island. Reciprocal immunoprecipitation coupled to immunoblots indicated Egr-1:NFAT3 binding was present in all cells lines. Treatment with cyclosporin A, inhibition of DNA methylation using 5-azadeoxycytidine or inhibition of protein acetylation using sodium butyrate reduced CSQ expression.
Conclusions: Our data suggest that Egr-1:DNA binding at the promoter, DNA methylation and protein acetylation are important in CSQ repression. Moreover, we demonstrate that a reduction in CSQ protein is associated with abnormal calcium dynamics. We conclude that Egr-1 acts as a transcriptional repressor at the CSQ promoter, resulting in downregulation of CSQ, the major calcium storage protein that links excitation-contraction coupling in the cardiac sarcoendoplasmic reticulum.
Time for primary review: 39 days