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.
