Egr-1 negatively regulates calsequestrin expression and calcium dynamics in ventricular cells

被引:26
作者
Kasneci, Amanda [1 ,2 ]
Kemeny-Suss, Naomi M. [3 ]
Komarova, Svetlana V.
Chalifour, Lorraine E. [1 ,2 ,4 ]
机构
[1] Sir Mortimer B Davis Jewish Hosp, Lady Davis Inst Med Res, Montreal, PQ H3T 1E2, Canada
[2] McGill Univ, Fac Med, Dept Med, Div Expt Med, Montreal, PQ, Canada
[3] McGill Univ, Fac Dent, Montreal, PQ, Canada
[4] Sir Mortimer B Davis Jewish Hosp, Bank Montreal Res Ctr Study Heart Dis Women, Montreal, PQ H3T 1E2, Canada
关键词
Calsequestrin; Early growth response gene-1; Calcium dynamics; Cardiomyocytes; INDUCED CARDIAC-HYPERTROPHY; ACTIVATED T-CELLS; MOLECULAR RESPONSE; NUCLEAR FACTOR; GROWTH-FACTOR; TRANSCRIPTION; CA2+; COREPRESSOR; DEFICIENT; INDUCTION;
D O I
10.1093/cvr/cvn357
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
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. 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. 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. 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.
引用
收藏
页码:695 / 702
页数:8
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