1. Fig. 1. Timeline: Adult (6-month-old) and old (22-month-old) rats were ovariectomized. Half of the animals received immediate E₂ replacement in vivo (ovx + E₂ pellet), while the other half did not. After a recovery period of 9 weeks, myocytes were isolated from the respective animals and analyzed.
From: 17β-Estradiol, Aging, Inflammation, and the Stress Response in the Female Heart
Endocrinology. 2011;152(4): doi: /en
Endocrinology | Copyright © 2011 by The Endocrine Society

2. Fig. 2. Aging and estrogen withdrawal impairs fractional shortening
Fig. 2. Aging and estrogen withdrawal impairs fractional shortening. A, Graphs summarize fractional shortening and LV diastolic dimension 9 weeks postovariectomy. Lower panels are representative m-mode echo images showing wall motion in adult and old animals with and without E₂ replacement. B, Graphs summarize heart to body weight ratios, plasma atrial natriuretic peptide, and β-MHC expression in isolated cardiomyocytes from old ovx and old ovx + E₂ pellet rats (parameters increased in congestive heart failure and/or LV hypertrophy). β-MHC was normalized to GAPDH expression. Representative Western blots are shown on the right. Lanes: 1, old ovx; 2, old ovx + E₂ pellet. n = 4–7 per group. *, P < 0.05 vs. adult ovx and adult ovx + E₂ pellet.
From: 17β-Estradiol, Aging, Inflammation, and the Stress Response in the Female Heart
Endocrinology. 2011;152(4): doi: /en
Endocrinology | Copyright © 2011 by The Endocrine Society

3. Fig. 3. NF-κB activation and HSP 72 expression in myocytes from adult and old rats treated with E₂in vitro. A, Graph summarizes the results of NF-κB activation in adult and old rats treated with 5 and 100 nm E₂ for 15 min in vitro. B, Graph summarizes ERK 1/2 expression, normalized to β-actin, in the isolated cardiomyocytes. Upper panel, A representative Western blot for ERK 1/2 and β-actin. Lanes: 1, adult ovx; 2, adult ovx + E₂ pellet; 3, old ovx; 4, old ovx + E₂ pellet. *, P < 0.05 vs. adult ovx. C, Graph summarizes the results of HSP 72 expression in adult and old rats treated with 5 and 100 nm E₂ for 12 h in vitro. HSP 72 was normalized to GAPDH expression and to adult ovx. A representative Western blot is shown to the right. n = 5–6 per group and 7–12 plates per group. *, P < 0.05 vs. control (ctl); ^#, P < 0.05 vs. all other ctls.
From: 17β-Estradiol, Aging, Inflammation, and the Stress Response in the Female Heart
Endocrinology. 2011;152(4): doi: /en
Endocrinology | Copyright © 2011 by The Endocrine Society

4. Fig. 5. Differences in HSF-1 activation and expression with age
Fig. 5. Differences in HSF-1 activation and expression with age. A, A representative blot for HSF-1 and GAPDH expression in cardiomyocytes in adult and ovx rats with and without E₂ replacement untreated or treated with 100 nm E₂in vitro. Graph on right summarizes HSF-1 expression normalized to GAPDH and to adult ovx ctl. B, EMSA showing HSF-1 binding in myocytes from adult and old rats with and without E₂ replacement untreated or treated with 100 nm E₂in vitro. The image is representative of five independent experiments. C, Graph summarizes the results of immunoprecipitated HSF-1 blotted for acetylation and phospho Ser^303/307 and normalized to total HSF-1. The immunoprecipitated samples were untreated cardiac myocytes from adult and old ovx rats with and without E₂ replacement. Representative Western blots are shown on left. n = 7–10 animals and plates per group. *, P < 0.05 vs. adult ovx.
From: 17β-Estradiol, Aging, Inflammation, and the Stress Response in the Female Heart
Endocrinology. 2011;152(4): doi: /en
Endocrinology | Copyright © 2011 by The Endocrine Society

5. Fig. 6. Hypoxia-reoxygenation induced release of lactate dehydrogenase (LDH), a marker of cell necrosis. A, LDH release after myocyte hypoxia (12 h) followed by reoxygenation (12 h). B, Graphs summarize HSP 25, 32, and 72 expression in adult and old myocytes with and without E₂ replacement under normoxic and hypoxic conditions. All HSP expression was normalized to GAPDH and to adult ovx. Representative Western blots for HSP 25, 32, 72, and GAPDH are shown on the right. Lanes: 1, adult ovx; 2, adult ovx + E₂ pellet; 3, old ovx; 4, old ovx + E₂ pellet. n = 6 animals and 6–11 plates per group. *, P < 0.05 vs. ctl; ^#, P < 0.05 vs. all other groups.
From: 17β-Estradiol, Aging, Inflammation, and the Stress Response in the Female Heart
Endocrinology. 2011;152(4): doi: /en
Endocrinology | Copyright © 2011 by The Endocrine Society

6. Fig. 7. Inflammatory and oxidative stress is increased in old ovx cardiac myocytes. A, Graph summarizes IL-6 and TNF-α levels in the plasma of old rats with and without E₂ replacement. n = 6–11 animals per group. Subsequent studies focused on the isolated cardiac myocytes. B, Graph summarizes the relative mRNA expression of IL-6 and TNF-α in isolated myocytes from old ovx and old ovx + E₂ pellet rats treated with or without 100 nm E₂ pretreatment in vitro. Cytokine expression was normalized to GAPDH. n = 5 animals and plates per group. ^&, P < 0.05 vs. all other groups. C, Graph summarizes the increase in oxidative stress after treating myocytes from old ovx and old ovx + E₂ pellet rats with 100 μm H₂O₂. A subset of cardiac myocytes were pretreated with 5 and 100 nm E₂ for 6 h before insult with H₂O₂. n = 3 animals and 9 plates per group. *, P < 0.05 vs. ctl; ^#, P < 0.05 vs. ctl and identical group in old ovx. D, Representative Westerns and a graph summarizing the expression of catalase, Cu/Zn-SOD, and Mn-SOD, basally and with 100 nm E₂ treatment for 24 h. Blots were normalized to GAPDH and to old ovx. Graph summarizing the results are shown to the right. n = 4–5 animals and plates per group.
From: 17β-Estradiol, Aging, Inflammation, and the Stress Response in the Female Heart
Endocrinology. 2011;152(4): doi: /en
Endocrinology | Copyright © 2011 by The Endocrine Society

7. Fig. 4. ER, HSP 25, 32, and 90 expression in adult and old myocytes
Fig. 4. ER, HSP 25, 32, and 90 expression in adult and old myocytes. A, Graph summarizes the basal ER-α and β expression in cardiac myocytes from adult and old rats with or without in vivo E₂ replacement. ER expression was normalized to GAPDH expression and to adult ovx. Right panel, Representative western blots. n = 3 animals and 3–5 plates per group. Ctl, control. B, Representative Western blots of HSP 25, 32, 90, and GAPDH expression in adult and old rats treated with 5 and 100 nm E₂ for 12 h in vitro. n = 6 animals and 6–11 plates per group. P = n.s., data not shown.
From: 17β-Estradiol, Aging, Inflammation, and the Stress Response in the Female Heart
Endocrinology. 2011;152(4): doi: /en
Endocrinology | Copyright © 2011 by The Endocrine Society