Cardiovascular Research Advance Access first published online on December 22, 2008
This version [Corrected Proof] published online on January 12, 2009
Cardiovascular Research, doi:10.1093/cvr/cvn357
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, Montréal, Québec, Canada
3 Faculty of Dentistry, McGill University, Montréal, Québec, Canada
4 The Bank of Montréal Research Center for the Study of Heart Disease in Women, Sir Mortimer B. Davis—Jewish General Hospital, Montréal, Québec, Canada
* Corresponding author. Tel: +1 514 340 8222 ext. 4295; fax: +1 514 340 7502. E-mail address: lorraine.chalifour{at}mcgill.ca
Aims: 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 phosphoserine 16-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.
Conclusion: 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.
KEYWORDS Calsequestrin; Early growth response gene-1; Calcium dynamics; Cardiomyocytes
Time for primary review: 39 days
These authors contributed equally to this work.