Sebag IA, Gillis MA, Calderone A, Kasneci A, Meilleur M, Haddad R, Noiles W, Patel B, Chalifour LE. Sex hormone control of left ventricular structure/function: mechanistic insights using echocardiography, expression, and DNA methylation analyses in adult mice. Am J Physiol Heart Circ Physiol 301: H1706 -H1715, 2011. First published July 29, 2011; doi:10.1152/ajpheart.00088.2011.-Calcium flux into and out of the sarco(endo)plasmic reticulum is vitally important to cardiac function because the cycle of calcium entry and exit controls contraction and relaxation. Putative estrogen and androgen consensus binding sites near to a CpG island are present in the cardiac calsequestrin 2 (CSQ2) promoter. Cardiomyocytes express sex hormone receptors and respond to sex hormones. We hypothesized that sex hormones control CSQ2 expression in cardiomyocytes and so affect cardiac structure/function. Echocardiographic analysis of male and female C57bl6n mice identified thinner walled and lighter hearts in females and significant concentric remodeling after long-term gonadectomy. CSQ2 and sodium-calcium exchanger-1 (NCX1) expression was significantly increased in female compared with male hearts and decreased postovariectomy. NCX1, but not CSQ2, expression was increased postcastration. CSQ2 expression was reduced when H9c2 cells were cultured in hormone-deficient media; increased when estrogen receptor-␣ (ER␣), estrogen receptor-␤ (ER␤), or androgen agonists were added; and increased in hearts from ER␤-deficient mice. CSQ2 expression was reduced in mice fed a diet low in the methyl donor folic acid and in cells treated with 5-azadeoxycytidine suggesting an involvement of DNA methylation. DNA methylation in CpG in the CSQ2 CpG island was significantly different in males and females and was additionally changed postgonadectomy. Expression of DNA methyltransferases 1, 3a, and 3b was unchanged. These studies strongly link sex hormone-directed changes in CSQ2 expression to DNA methylation with changed expression correlated with altered left ventricular structure and function. protein expression; cardiac function MAINTENANCE OF THE EXPRESSION of calcium homeostasis is important for normal cardiac function (1, 13). Contraction is the consequence of the release of massive amounts of calcium into the cytosol from the sarco(endo)plasmic reticulum (SER) by ryanodine receptor 2. Relaxation is established by the combined action of the sodium-calcium exchanger-1 (NCX1) (30), which removes calcium to the cell exterior, and the sarco(endo)plasmic reticulum ATPase 2a (SERCA2a), which resequesters calcium back to the SER (19). Phospholamban (PLB) inhibits SERCA2a activity, but phosphorylation on either serine 16 (S 16 ) or threonine 17 (T 17 ) relieves this inhibition. Calsequestrin 2 (CSQ2) may be uniquely important for calcium signaling because it is the principal calcium storage protein in the SER and, as part of a protein complex with the ryanodine receptor 2, helps to control the amount of calcium released for contraction (7).The available evide...

Estrogenic compounds such as bisphenol A (BPA) leach from plastics into food and beverage containers. Increased BPA exposure has been correlated with increased cardiovascular disease. To test the hypothesis that increased BPA exposure reduces cardiovascular remodeling, we chronically exposed C57bl/6n male mice to BPA and performed a myocardial infarction (MI). We measured cardiac function, as well as myeloid and cardiac fibroblast accumulation and activity. We found increased early death as well as increased cardiac dilation and reduced cardiac function in surviving BPA-exposed mice. Matrix metalloproteinase-2 (MMP2) protein and activity were increased 1.5-fold in BPA-exposed heart. BPA-exposed mice had similar neutrophil infiltration; however, monocyte and macrophage (MΦ) infiltration into the ischemic area was 5-fold greater than VEH mice potentially due to a 2-fold increase in monocyte chemoattractant protein-1. Monocyte and MΦ exposure to BPA in vitro in primary bone marrow cultures or in isolated peritoneal MΦ increased polarization to an activated MΦ, increased MMP2 and MMP9 expression 2-fold and activity 3-fold, and increased uptake of microspheres 3-fold. Cardiac fibroblasts (CF) differentiate to α-smooth muscle actin (αSMA) expressing myofibroblasts, migrate to the ischemic area and secrete collagen to strengthen the scar. Collagen and αSMA expression were reduced 50% in BPA-exposed hearts. Chronic in vivo or continuous in vitro BPA exposure ablated transforming growth factor beta-mediated differentiation of CF, reduced αSMA expression 50% and reduced migration 40% yet increased secreted MMP2 activity 2-fold. We conclude that chronic BPA exposure reduces the ability to successfully remodel after an MI by increasing MΦ-based inflammation and reducing myofibroblast repair function.

Bisphenol A (BPA) leaches from plastics to contaminate foodstuffs. Analogs, such as bisphenol S (BPS), are now used increasingly in manufacturing. Greater BPA exposure has been correlated with exacerbation of cardiovascular disease, including myocardial infarction (MI). To test the hypothesis that bisphenol exposure impairs cardiac healing, we exposed C57bl/6n mice to water containing 25ng/ml BPA or BPS from conception and surgically induced an MI in adult male progeny. Increased early death and cardiac dilation, and reduced cardiac function were found post-MI in BPA- and BPS-exposed mice. Flow cytometry revealed increased monocyte and macrophage infiltration that correlated with increased chemokine C-C motif ligand-2 expression in the infarct. In vitro BPA and BPS addition increased matrix metalloproteinase-9 (MMP) protein and secreted activity in RAW264.7 macrophage cells suggesting that invivo increases in MMP2 and MMP9 in exposed infarcts were myeloid-derived. Bone marrow-derived monocytes isolated from exposed mice had greater expression of pro-inflammatory polarization markers when chemokine stimulated indicating an enhanced susceptibility to develop a pro-inflammatory monocyte population. Chronic BPA exposure of estrogen receptor beta (ERβ) deficient mice did not worsen early death, cardiac structure/function, or expression of myeloid markers after an MI. In contrast, BPS exposure of ERβ-deficient mice resulted in greater death and expression of myeloid markers. We conclude that lifelong exposure to BPA or BPS augmented the monocyte/macrophage inflammatory response and adverse remodeling from an MI thereby reducing the ability to survive and successfully recover, and that the adverse effect of BPA, but not BPS, is downstream of ERβ signaling.

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.