Combined effects of 17β-estradiol and exercise training on cardiac apoptosis in ovariectomized rats

Figures

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  Fig 1. Effect of 17β-estradiol treatment and exercise training on cardiac histopathological and TUNEL-positive apoptotic changes. Representative histopathological analyses of cardiac sections from the left ventricles in the sham-operated (Sham), bilateral ovariectomized (OVX), OVX treated with 17β-estradiol (OVX-E2) and OVX rats treated with 17β-estradiol and exercise (OVX-E2-EX) groups were performed with (A) hematoxylin-eosin staining (interstitial space: wide, arrows indicated) and (B) Masson’s trichrome staining (fibrosis: blue color, arrows indicated). The images of the myocardial architecture are magnified 200×; (C) Representative stained apoptotic cells of cardiac sections were performed by staining with 4,6-diamidino-2-phenylindole (DAPI) staining (top, blue spots) and the terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) assay (bottom, green spots, arrows indicated) The images of myocardial architecture are magnified 400×; (D) The bar represents the percentage of the blue area to the field area in Masson’s trichrome staining, and the percentage of TUNEL apoptosis was expressed as the ratio of TUNEL-positive cells relative to total DAPI-stained nuclei…

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  Fig 2. Effects of the 17β-estradiol treatment and exercise training on the cardiac estrogen receptor and IGF-1-related pro-survival pathways. (A) The representative protein products of estrogen receptor-α (ERα), estrogen receptor-β (ERβ), IGF-1, IGF-1R, p-PI3K and p-Akt extracted from the left ventricles of excised hearts of sham-operated (Sham), bilateral ovariectomized (OVX), OVX treated with 17β-estradiol (OVX-E2) and OVX rats treated with 17β-estradiol and exercise (OVX-E2-EX) were measured by Western Blotting analysis. (B) The bars represent the relative protein quantification of ERα, ERβ, IGF-1, IGF-1R, p-PI3K and p-Akt on the basis of α-tubulin and indicate the mean values ± SD (n = 8 in each group). * P<0.05, ** P<0.01 indicates significant differences from the Sham group. # P<0.05 and ## P<0.01 indicates significant differences from the OVX group. †† P<0.01, indicates significant differences between OVX-E2 and OVX-E2-EX group.

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  Fig 3. Effects of the 17β-estradiol treatment and exercise training on the cardiac anti-apoptotic pathway. (A) The representative protein products of Bcl2, Bcl-xL and p-Bad were extracted from the left ventricles of excised hearts of sham-operated (Sham), bilateral ovariectomized (OVX), OVX treated with 17β-estradiol (OVX-E2) and OVX rats treated with 17β-estradiol and exercise (OVX-E2-EX) were measured by Western Blotting analysis. (B) The bars represent the relative protein quantification of Bcl2, Bcl-xL and p-Bad on the basis of α-tubulin and indicate the mean values ± SD (n = 8 in each group). ** P<0.01 indicate significant differences from the Sham group. ## P<0.01 indicate significant differences from the OVX group. †† P<0.01, indicate significant differences between the OVX-E2 and OVX-E2-EX group.

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  Fig 4. Effect of 17β-estradiol treatment and exercise training on cardiac mitochondria–dependent apoptotic pathway. (A) The representative protein products of t-Bid, Bad, Bax, Cytochrome c, activated caspase 9 and activated caspase 3 were extracted from the left ventricles of excised hearts of the sham-operated (Sham), bilateral ovariectomized (OVX), OVX treated with 17β-estradiol (OVX-E2) and OVX rats treated with 17β-estradiol and exercise (OVX-E2-EX) were measured by Western Blotting analysis. (B) The bars represent the relative protein quantification of t-Bid, Bad, Bax, and Cytochrome c, activated caspase 9 and activated caspase 3 on the basis of α-tubulin and indicate mean values ± SD (n = 8 in each group). **P<0.01, indicate significant differences from the Sham group. ##P<0.01, indicate significant differences from the OVX group. †P<0.05, †† P<0.01, indicate significant differences between the OVX-E2 and OVX-E2-EX group.

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  Fig 5. Effect of 17β-estradiol treatment and exercise training on cardiac Fas receptor–dependent apoptotic pathway. (A) The representative protein products of TNF-α, Fas ligand, Fas receptors, Fas-associated death domain (FADD) and activated caspase 8 were extracted from the left ventricles of excised hearts of the sham-operated (Sham), bilateral ovariectomized (OVX), OVX treated with 17β-estradiol (OVX-E2) and OVX rats treated with 17β-estradiol and exercise (OVX-E2-EX) were measured by Western Blotting analysis. (B) The bars represent the relative protein quantification of TNF-α, Fas ligand, Fas receptors, FADD and activated caspase 8 on the basis of α-tubulin and indicate the mean values ± SD (n = 8 in each group). **P<0.01, indicate significant differences from the Sham group. #P<0.05, ##P<0.01, indicate significant differences from the OVX group. †P<0.05, ††P<0.01, indicate significant differences between the OVX-E2 and OVX-E2-EX group.

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  Fig 6. Proposed hypothesized diagram. Our hypothesis proposed that ovariectomy attenuated the estrogen receptor related survival pathway (ERα, ERβ, IGF-1, IGF-1R, p-PI3K, p-Akt, Bcl-2, Bcl-xL and p-Bad) and activated the cardiac mitochondria-dependent apoptotic pathway (t-Bid, Bad, Bax, Cytochrome c, activated caspase-9, and activated caspase-3), as well as activated the cardiac Fas receptor-dependent apoptotic pathway (TNF-α, Fas ligand, Fas receptors, activated caspase-8, and activated caspase-3) after an ovariectomy. However, the estrogen receptor enhanced the related survival pathway and suppressed the cardiac Fas receptor-dependent as well as the mitochondria-dependent apoptotic pathway which were observed in either the 17β-estradiol (E2) treatment or E2 treatment combined with exercise intervention. Furthermore, the protein levels of ERβ, IGF-1, IGF-1R, Bcl-2 and Bcl-xL were further enhanced when combined with E2 and exercise training. The protein levels of t-Bid, Cytochrome c, Fas receptors, FADD, activated caspase-8, activated caspase-9 and activated caspase-3 were further decreased when combined with E2 and exercise training.