Estrogen inhibits mast cell chymase release to prevent pressure overload-induced adverse cardiac remodeling

Abstract

Estrogen regulation of myocardial chymase and chymase effects on cardiac remodeling are unknown. To test the hypothesis that estrogen prevents pressure overload-induced adverse cardiac remodeling by inhibiting mast cell (MC) chymase release, transverse aortic constriction or sham surgery was performed in 7-week-old intact and ovariectomized (OVX) rats. Three days before creating the constriction, additional groups of OVX rats began receiving 17β-estradiol, a chymase inhibitor, or a MC stabilizer. Left ventricular function, cardiomyocyte size, collagen volume fraction, MC density and degranulation, and myocardial and plasma chymase levels were assessed 18 days postsurgery. Aortic constriction resulted in ventricular hypertrophy in intact and OVX groups, whereas collagen volume fraction was increased only in OVX rats. Chymase protein content was increased by aortic constriction in the intact and OVX groups, with the magnitude of the increase being greater in OVX rats. MC density and degranulation, plasma chymase levels, and myocardial active transforming growth factor-β1 levels were increased by aortic constriction only in OVX rats. Estrogen replacement markedly attenuated the constriction-increased myocardial chymase, MC density and degranulation, plasma chymase, and myocardial active transforming growth factor-β1, as well as prevented ventricular hypertrophy and increased collagen volume fraction. Chymostatin attenuated the aortic constriction-induced ventricular hypertrophy and collagen volume fraction in the OVX rats similar to that achieved by estrogen replacement. Nedocromil yielded similar effects, except for the reduction of chymase content. We conclude that the estrogen-inhibited release of MC chymase is responsible for the cardioprotection against transverse aortic constriction-induced adverse cardiac remodeling.

Keywords: cardiac remodeling; chymase; estrogen; mast cell.

Figure 1

Transverse aortic constriction (TAC) or sham (Sh) surgery was performed in 7-wk-old rats with either ovaries intact (Int) or bilateral ovariectomy (OVX). Changes in the ratio of left ventricular weight (LV) to tibia length (TL), myocyte cross sectional area (CSA) and LV interstitial collagen volume fraction (CVF) after 18 days post TAC or Sh surgery are shown in Panels A, B and D, respectively. Representative images of myocardium stained with collagen specific picrosirius red are depicted for the four study groups in Panel C (x 100 magnifications). All values are mean ± SEM, N=6–7, *p<0.05 vs. Int-Sh, +p<0.05 vs. OVX-Sh, #p<0.05 vs. Int-TAC.

Figure 2

Transverse aortic constriction (TAC) or sham surgery (Sh) was performed in 7-wk-old rats with ovaries intact (Int) or bilateral ovariectomy (OVX). Eighteen days after TAC or Sh surgery, Left ventricle (LV) tissue chymase protein content was measured by Western blot (panels A and B). Plasma chymase levels were measured using an ELISA kit (panel C). LV mast cell density and degranulation were measured using toluidine blue stained tissue (panels E and F). All values are mean ± SEM, N=6–7, *p<0.05 vs. Int-Sh and OVX-Sh, +p<0.05 vs. all other groups. Presented in Panel D is a representative image of chymase immunohistochemistry stained LV tissue depicting its localization in mast cells and external mast cell granules.

Figure 3

Treatment with estrogen (E2), nedocromil (Ne) and chymostatin (Ch) started 3 days prior to transverse aortic constriction (TAC) in ovariectomized (OVX) rats and continued for 3 wks. Depicted respectively in panels A, B and D are the ratio of left ventricle weight (LV) to tibia length (TL), myocyte cross sectional area (CSA), and LV interstitial collagen volume fraction (CVF) with and without treatment. Shown in Panel C are the representative photomicrographs of LV tissue for the untreated and treated groups stained for collagen with picrosirius red (x 100 magnifications). All values are mean ± SEM, N=6–7, *p<0.05 vs OVX-TAC.

Figure 4

Treatment with estrogen (E2), nedocromil (Ne) and chymostatin (Ch) started 3 days prior to transverse aortic constriction (TAC) in ovariectomized (OVX) rats and continued for 3 wks. Left ventricle (LV) tissue chymase protein content as measured by Western blot is presented in panels A and B and ELISA-determined plasma chymase levels in panel C. Also shown are LV mast cell density (panel D) and degranulation (panel E). All values are mean ± SEM, N=6–7, *p<0.05 vs OVX-TAC.

Figure 5

Panels A and B - Active TGF- 1 levels in rats 18 days following transverse aortic constriction (TAC) or sham (Sh) surgery with ovaries intact (Int) or removed (OVX). Panels C and D - Active TGF- 1 levels in OVX rats treated with estrogen (E2), nedocromil (Ne) or chymostatin (Ch) started 3 days prior to 18 days of TAC. All values are mean ± SEM, N=6–7, *p<0.05 vs. Int-Sh, OVX-Sh and Int-TAC; +p<0.05 vs. OVX-TAC.

Figure 6

A schematic of the mast cell-chymase-TGF- 1 pathway responsible for adverse myocardial remodeling in response to transverse aortic constriction (TAC). In response to TAC, mast cell activators are released from myocardial cells to activate cardiac mast cells. Upon activation, cardiac mast cells release chymase into the interstitium which then activates TGF-β1. Active TGF-β1 stimulates fibroblast and myofibroblast cells to synthesize and secrete collagen as well as promote cardiomyocyte hypertrophy. Estrogen and nedocromil prevent mast cell chymase release and chymostatin inhibits chymase activity. As a consequence they prevent the TAC-induced increase in TGF-β1 and the resulting adverse cardiac remodeling.