Postischemic application of estrogen ameliorates myocardial damage in an in vivo mouse model

j o u r n a l o f s u r g i c a l r e s e a r c h  n o v e m b e r 2 0 1 8 ( 2 3 1 ) 3 6 6 e3 7 2

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Postischemic application of estrogen ameliorates myocardial damage in an in vivo mouse model Yang Yang, MD,a I-Wen Wang, MD, PhD,a,b Mark Turrentine, MD,a and Meijing Wang, MDa,* a b

Division of Cardiothoracic Surgery, Department of Surgery, Indiana University School of Medicine, Indianapolis, Indiana Methodist Hospital, IU Health, Indianapolis, Indiana

article info

abstract

Article history:

Background: Cardioprotection provided by estrogen has been recognized for many years. It

Received 28 February 2018

is noteworthy that most of these studies employ a means of preinjury application in

Received in revised form

experimental research and the preventive usage in clinical studies. Compared to pre-

27 April 2018

treatment, postischemic administration of estrogen will be more practical in treating

Accepted 31 May 2018

myocardial ischemia. On the other hand, defect in circadian clock gene period2 (Per2) has

Available online xxx

been shown to aggravate ischemia-induced heart damage. Given that Per2 expression decreases as a consequence of menopause, in this study, we aim to determine (1) potential

Keywords:

improvement of myocardial function by postischemic administration of 17b-estradiol (E2)

Myocardial ischemia and

using an in vivo mouse myocardial ischemia/reperfusion (I/R) model and (2) the role of E2 in

reperfusion Acute postischemic treatment of estrogen

regulating myocardial Per2 expression following I/R. Methods: Thirty-minute occlusion of left anterior descending artery followed by 24-h reperfusion was performed on adult C57BL ovariectomized female mice. Groups (n ¼ 3-6/

Circadian clock gene Per2

group) were as follows: (1) Sham, (2) I/R þ vehicle, and (3) I/R þ E2. Vehicle or 0.5 mg/kg of E2

Myocardial inflammatory response

was subcutaneously injected right after 30-min ischemia. Following 24-h reperfusion,

Coronary artery occlusion

myocardial function was determined. Heart tissue was collected for analysis of cleaved caspase-3 and Per2 expression by Western blotting, as well as proinflammatory cytokine production (IL-1b, IL-6, and TNF-a) by enzyme-linked immunosorbent assay. Results: I/R significantly impaired left ventricular function and increased myocardial levels of active caspase-3, IL-1b, and IL-6. Importantly, postischemic treatment of E2 markedly restored I/R-depressed myocardial function, reduced caspase-3 activation, and decreased proinflammatory cytokine production (IL-1b, IL-6, and TNF-a). Intriguingly, a trend of the decreased Per2 level was observed in ovariectomized female hearts subjected to I/R, whereas E2 treatment upregulated myocardial Per2 expression. Conclusions: Our study represents the initial evidence that postischemic administration of E2 effectively preserves the myocardium against I/R injury and this protective effect of E2 may involve upregulation of Per2 in ischemic heart. ª 2018 Elsevier Inc. All rights reserved.

13th Annual Academic Surgical Congress, January 30-February 1, 2018, Jacksonville, FL. * Corresponding author. Indiana University School of Medicine, Surgery, 980 West Walnut Street, R2 E319, Indianapolis, IN 46202. Tel./fax: þ317 274 0827. E-mail address: [email protected] (M. Wang). 0022-4804/$ e see front matter ª 2018 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jss.2018.05.076

yang et al  postischemic single dose e2 in heart i/r

Introduction Despite significant advances in current therapy, ischemic heart disease remains the leading cause of mortality in developed countries for both men and women.1 Notably, sexual dimorphisms have been reported to affect the outcomes following myocardial ischemia (MI) with better recovery in the female myocardium as compared with the male myocardium.2-5 Besides, MI occurs uncommonly in premenopausal women, whereas this risk increases in the postmenopausal age group,6,7 suggesting that estrogen may protect the heart from ischemic injury. However, controversial results from clinical trials have shown that hormone replacement therapy did not convey cardioprotective effects on postmenopausal females.8-12 This led to a recognition that estrogen protection appears more complicated than originally thought. On the other hand, the majority of studies employ the approach of preinjury treatment of estrogen in animal experiments5,13-16 and its preventive usage in clinical research.812 Considering therapeutic potential in the treatment of acute MI, postinjury administration of estrogen will be more practical compared with pretreatment. In this study, we will evaluate therapeutic effects of postischemic administration of 17b-estradiol (E2) on myocardial function, apoptotic signaling, and inflammatory response using an in vivo mouse myocardial ischemia/reperfusion (I/R) model. Our previous work from animal experiments has shown that females exhibited decreased myocardial inflammation, reduced apoptotic signaling, increased prosurvival pathways, and enhanced myocardial expression of growth factors compared with males following acute myocardial I/R4,17-19; these salutary effects were mediated via the rapid action of estrogen in the ischemic heart.19-21 However, the detailed molecular and cellular mechanisms require more investigation. Of note, accumulating evidence has demonstrated that clock regulators are intimately implicated in cellular functions other than circadian rhythm, thereby influencing cellular metabolism, cell cycle, and mitochondrial activities.22-24 Particularly, the circadian clock gene period2 (Per2) is reported to modulate heart energy homeostasis following MI. The Per2 knockout (KO) mouse has disturbed lipid metabolisms and impaired cardiac function after myocardial infarction,25,26 implying the importance of Per2 in heart pathophysiological processes. More importantly, Per2 expression decreases as a consequence of menopause,27 suggesting a potential link between estrogen level and Per2 expression. Therefore, in this study, we will investigate Per2 levels in the heart subjected to I/R injury and determine the role of postischemic administration of E2 in regulating myocardial Per2 expression.

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animals from Jackson Laboratories (Bar Harbor, ME). These animals were acclimated and raised with a standard diet for an additional 8-10 wk before experiments. The animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Indiana University. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, revised 1996).

Mouse in vivo myocardial I/R model The OVX female mice were placed supinely after anesthetized with inhalation of isoflurane and mechanically ventilated using a TOPO small animal ventilator (Kent Scientific Corporation, Torrington, CT) set at 110-120 per min of respiration rate and 18-20 cm H2O of inspiration pressure. The heart was exposed through a left-sided mini-thoracotomy, and an 8-0 Prolene suture was passed underneath the left anterior descending coronary artery (LAD) at 2w3 mm distal to its origin between the left auricle and conus arteriosus. A suture loop around the artery and tubing (a 2-mm-long piece of PE-10 tubing) were tightened to ensure the LAD occlusion, which was maintained for 30 min. Buprenorphine SR (1 mg/kg body weight) was injected subcutaneously during the ischemic period. After ischemia, the tubing was removed, and the LAD was reopened for reperfusion. Vehicle or 0.5 mg/kg body weight of E2 (E8875; Sigma-Aldrich, Urbana, IL) in corn oil28 was subcutaneously injected right after 30-min ischemia. The animals were allowed to awaken on a warm pad and kept in a cage with free access to wet food and water during the reperfusion period. At 24-h reperfusion, the animals were anesthetized, intubated, and ventilated. Myocardial function (left ventricular pressure [LVP ¼ LV systolic pressure  end diastolic pressure], heart rate, rate pressure product [RPP ¼ LVP * heart rate], and dP/dT) was then detected using the Millar Mikro-Tip pressure catheter (SPR-671; Millar Inc, Houston, TX) inserted into the LV. Data were recorded using a PowerLab 8 data acquisition digitizer (ADInstruments Inc, Colorado Springs, CO).

Experimental groups The OVX female mice (13- to 16-wk-olds) were randomly divided into three groups (Fig. 1A): (1) Sham (with operation but no I/R, n ¼ 3); (2) 30-min LAD occlusion followed by 24-h reperfusion þ vehicle (I/R, n ¼ 6); and (3) I/R þ E2 (n ¼ 6). After recording the LV function at 24 h after surgery, the hearts were harvested with snap-freezing in liquid nitrogen and saved in a 80 C freezer for further analysis.

Methods and materials

Enzyme-linked immunosorbent assay

Animals

Heart tissue below the LAD occlusion level was homogenized in cold RIPA buffer (Sigma, Saint Louis, MO) and centrifuged at 12,000 rpm for 10 min. Protein levels of IL-1b, IL-6, and TNF-a in cardiac tissue were determined

Female C57BL/6J mice were ovariectomized (OVX) at 5- to 6wk of age and were purchased as surgically modified

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j o u r n a l o f s u r g i c a l r e s e a r c h  n o v e m b e r 2 0 1 8 ( 2 3 1 ) 3 6 6 e3 7 2

Fig. 1 e (A) The schematic shows experimental design and groups. (B) Mouse hearts without or with I/R injury. (B1) Normal heart and ECG before injury. (B2) The pale LV anterior wall and ST elevation (arrow) following LAD ligation. (B3) Returned pink color of LV anterior wall and returned ST during reperfusion. (Color version of figure is available online.)

by enzyme-linked immunosorbent assay (ELISA) using commercially available ELISA kits (cat# DY401 [IL-1b], DY406 [IL-6], DY410 [TNF-a]; R&D Systems Inc, Minneapolis, MN) based on the manufacturer’s instructions. All samples and standards were measured in duplicate.

Statistical analysis

Western blotting

Results

Cardiac protein samples were obtained by homogenizing heart tissue below the LAD occlusion level in cold RIPA buffer containing Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA). Protein concentration was measured with BCA protein assay reagent (Bio-Rad, Hercules, CA). The protein extracts (25 mg/sample) were electrophoresed in NuPAGE 4%-12% Bis-Tris protein gels (Thermo Fisher Scientific). The gel was then transferred to a nitrocellulose membrane. After blocking in 5% dry milk for 1 h at room temperature, the membrane was incubated with the primary antibody against cleaved caspase-3 (1:1000, cat# 9664; Cell Signaling Technology, Danvers, MA), Per2 (5 mg/mL, cat# PA523339; Thermo Fisher Scientific) or GAPDH (1:1000, cat#5174; Cell Signaling Technology) at 4 C with gentle shaking overnight. After washing with three times of 0.1% PBST, the membrane was incubated with goat anti-rabbit IgG secondary antibody (1:2000; Thermo Fisher Scientific) and detected using SuperSignal West Pico Stable Peroxide Solution (Thermo Fisher Scientific). The densitometry data were analyzed using the Image J software.

Postischemic administration of E2 preserved myocardial function following I/R injury

All reported results are means  SEM. Data were evaluated using one-way ANOVA with multiple comparisons test. Difference was considered statistically significant when P < 0.05.

Based on the studies,29,30 30-min ischemia induced by LAD ligation has significantly damaged LV function and resulted in pathophysiological changes. In addition, the first 24 h after acute MI is crucial for treatment. Clinically, one-third of patients will die within the first 24 h of myocardial infarction. Therefore, in this study, we selected 30-min ischemia and followed by 24-h reperfusion to evaluate the therapeutic effects of postischemic usage of estrogen on acute stage of MI. Successful ligation of the LAD was confirmed by myocardial blanching and abnormal movement of the anterior wall, as well as electrocardiography evidence (the elevated ST) (Fig. 1B2). Reperfusion was verified by returned color (pinkred) of the LV anterior wall and decreased ST elevation (Fig. 1B3). Thirty-minute LAD occlusion followed by 24-h reperfusion significantly damaged LV function, as demonstrated by decreased LVP (Fig. 2A), RPP (Fig. 2C), þdP/dt (Fig. 2D), and impaired dP/dt (Fig. 2E) when compared with the sham group. Intriguingly, postischemic treatment of E2

yang et al  postischemic single dose e2 in heart i/r

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Fig. 2 e Left ventricular function detected after 24-h reperfusion. (A) LVP [ LV systolic pressureeend diastolic pressure. (B) Heart rate (BPM) among groups of Sham, I/R, and I/R D E2. (C) Rate pressure product (RPP [ LVP 3 BPM). (D) The maximum value of the first derivative (dP/dt); and (E) the maximum negative value of the first derivative (LdP/dt). Postischemic treatment of E2 (single dose) markedly ameliorates I/R-damaged myocardial function. Mean ± SEM, *P < 0.05, **P < 0.01 (One-way ANOVA with Tukey’s multiple comparisons test).

markedly restored myocardial function following I/R (Fig. 2), indicating the effective protection of postischemic E2 administration on I/R-induced myocardial dysfunction. Comparable heart rate was observed among the groups (Fig. 2B).

Postischemic treatment of E2 decreased myocardial damage and reduced proinflammatory cytokine production following I/R

more practical potential in treating acute MI. In the present study, we clearly demonstrated that postischemic treatment with E2 significantly protected the myocardium from I/R injury, as shown by improved LV function, reduced active/ cleaved caspase-3, and decreased proinflammatory cytokine production compared with untreated counterparts in an in vivo mouse myocardial I/R model. More importantly, the

Apoptosis occurs during MI. To investigate whether using E2 can reduce myocardial apoptotic signaling, we detected active/cleaved caspase-3 protein levels in the OVX female heart subjected to I/R. We observed that I/R significantly increased active/cleaved caspase-3 levels, whereas E2 treatment decreased I/R-induced caspase-3 activation (Fig. 3). Similarly, the increased proinflammatory cytokine production (IL-1b and IL-6) was noticed in OVX female hearts following I/ R, whereas postischemic treatment with E2 significantly reduced these cytokine production (IL-1b, IL-6, and TNF-a) (Fig. 4).

Effect of postischemic administration of E2 on myocardial Per2 expression We further determined myocardial Per2 expression following I/R. A trend of decreased Per2 expression was observed in the I/R group compared with the sham hearts. Postischemic E2 treatment significantly upregulated myocardial Per2 levels (Fig. 5), indicating a role of E2 in regulating myocardial Per2 expression in response to I/R.

Discussion Given a clinical setting in which the ischemic damage is already present, postischemic administration of estrogen has

Fig. 3 e Myocardial cleaved/active caspase-3 in the hearts of Sham, I/R, and I/R D E2. (A) Representative Western blots (3 lanes/group); (B) Densitometry bar graph of cleaved caspase-3/GAPDH. I/R increases myocardial cleaved caspase-3 levels, whereas E2 treatment significantly reduces active caspase-3. Mean ± SEM, N [ 3-6 hearts/ group, **P < 0.01 (One-way ANOVA with Tukey’s multiple comparisons test).

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Fig. 4 e Myocardial production of IL-1b (A), IL-6 (B), and TNF-a (C) in groups of Sham, I/R, and I/R D E2 by enzyme-linked immunosorbent assay. Postischemic single dose of E2 significantly decreases myocardial production of IL-1b, IL-6, and TNFa following I/R. All results shown as mean ± SEM, N [ 3-6 hearts/group, *P < 0.05 (One-way ANOVA with Tukey’s multiple comparisons test).

beneficial effect of estrogen on the ischemic myocardium was associated with upregulated myocardial Per2 expression. Apoptosis has shown to engage in pathophysiological processes during MI. The treatment aimed at reducing apoptosis is associated with decreased myocardial lesion in ischemia,31,32 supporting the ischemia-mediated pathologic apoptosis. Accumulated evidence has demonstrated that estrogen can activate survival pathways and suppress apoptosis to protect the ischemic heart. Our previous study has indicated that chronic estrogen supplementation (preischemic usage) decreased proapoptotic signaling in the hearts subjected to I/R injury. Similarly, in the present study, postischemic treatment of E2 reduced myocardial apoptotic protein, thus protecting the myocardium from I/R-induced damage. In addition, MI leads to local inflammation with the production of a variety of proinflammatory cytokines. These cytokines contribute to induction of other detrimental molecules, initiation of cell death process, and aggravation of myocardial dysfunction. In this study, we observed that postischemic administration of E2 significantly decreased myocardial IL-1b, IL-6, and TNF-a production. Taking together, our data indicate that postischemic administration of E2 reduces myocardial apoptotic signaling and decreases inflammatory response, thus protecting myocardial function following I/R injury. However, we did not notice the significantly increased TNF-a levels in OVX female hearts subjected to I/R herein. TNF-a has been shown to rapidly release in the cardiac tissue after MI and such increased TNF-a at the early time of ischemia acts as an initiator to influence other effector cells.33 In fact, based on our previous studies,34-36 TNF-a is able to induce proinflammatory and inflammatory cytokine release including IL-1b and IL-6 during myocardial I/ R. Therefore, it is not surprising to observe the significantly increased production of IL-1b and IL-6, but not TNF-a, in the hearts following I/R. Very possibly, we missed the right timing for analysis of peak levels of I/R-induced TNF-a in this study. The effects of estrogen are mediated mainly through estrogen receptor (ER)a and/or ERb, which are expressed in the heart. Our previous studies have demonstrated that both ERa and ERb are involved in estrogen-mediated cardioprotection following acute I/R by using ERaKO and ERbKO

mouse hearts, as well as selective agonists of ERa or ERb.21,37,38 ERa and ERb are capable of initiating prosurvival signaling, reducing cell death, and inhibiting inflammatory responses, thus mediating estrogen-induced cardioprotection.18,37-42 Collectively, it is reasonable to postulate that postischemic treatment of E2 protects the myocardium through ERa and/or ERb. In fact, by using an isolated heart perfusion system (Langendorff), we have previously shown that postischemic infusion of selective ERa agonist PPT or ERb agonist DPN significantly improved myocardial functional recovery following I/R.21 However, it is noteworthy that G proteinecoupled receptor 30 is also able to mediate estrogen-initiated acute cardiac protection following I/R according to recent evidence.43 Therefore, further study is required to elucidate by which receptor(s)activated signaling estrogen conveys its protection when administered postischemically in the future. Emerging evidence has shown that in addition to controlling circadian rhythm, the clock genes are implicated in

Fig. 5 e Myocardial Per2 expression in groups of Sham, I/R, and I/R D E2. (A) Representative Western blots (3 lanes/ group). (B) Densitometry bar graph of Per2/GAPDH. Postischemic administration of E2 upregulates myocardial Per2 expression after I/R. Mean ± SEM, N [ 3-6 hearts/ group, *P < 0.05 (One-way ANOVA with Tukey’s multiple comparisons test).

yang et al  postischemic single dose e2 in heart i/r

regulating cellular functions.22-24 Among circadian clock family, the circadian protein Per2 is an important mediator to impact cellular metabolism.24,25,44 Increased oxygen consumption and impaired glycolysis have been observed in Per2KO mice in response to MI, associated with increased myocardial infarct size.25 Of note, circadian clock genes are not only regulated by the light cycle but also altered by serum factors and environmental signals that influence nuclear receptor ligands.45-48 In fact, estrogen-related receptor has been reported to control metabolic clock outputs.49 In addition, Per2 expression decreases as a consequence of menopause,27 suggesting a role of estrogen in modulating Per2 expression. In the present study, for the first time, we indicated that postischemic administration of E2-upregulated Per2 expression in the heart subjected to I/R. Importantly, high-throughput gene array analysis for studying gene regulation during reperfusion has shown that only proinflammatory genes are differentially regulated “Per2 genes” in Per2KO mice following myocardial I/R injury.26 In parallel, we did observe that E2-increased Per2 expression was associated with decreased myocardial proinflammatory cytokine production after I/R in the present study. However, further investigation is needed to determine the role of Per2 in estrogen protection using cardiac-restricted deletion of Per2 mice in the future. Hormone replacement therapy, as a preventive measure, has been tested as to protecting postmenopause women from development of heart disease. Although there have been reports showing increased breast cancer risk in association with persistent estrogen usage,50 estrogen should not be ruled out as a short-term therapeutic agent to alleviate acute symptoms related to MI. Estrogen, as a natural steroid hormone, has nonharmful side effects when administered in a short-term fashion. In our future study, we will further determine the optimal dose of postinjury administration of estrogen in reducing cardiomyocyte death and in decreasing myocardial inflammation in response to I/R. In summary, postischemic treatment of 17b-estradiol ameliorates I/R-induced myocardial dysfunction and reduces apoptotic signaling and inflammation in an in vivo mouse model. These results add evidence in support of using estrogen postischemically as a therapeutic intervention for acute MI. More intriguingly, our data regarding E2-upregulated Per2 expression shed light on a novel link between circadian clock genes and sex hormone treatment in regulating myocardial responses following I/R injury. Elucidation of interaction between estrogen and myocardial Per2 in the ischemic heart requires future study.

Acknowledgment This study is partially supported by the Showalter Trust Fund to M.W. and by an award from the Methodist Health Foundation. Authors’ contribution: Y.Y. involved in conducting experiments and acquisition and analysis of the data; I.-W.W. and M.T. involved in interpretation of the data and reviewing the

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work; and M.W. involved in designing the work; acquisition, analysis, and interpretation of the data; and drafting the work.

Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article.

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