- Email: [email protected]
Estrogen receptor beta mediates acute myocardial protection following ischemia
Estrogen receptor beta mediates acute myocardial protection following ischemia Meijing Wang, MD, MS, Paul R. Crisostomo, MD, Troy Markel, MD, Yue Wang, PhD, Keith D. Lillemoe, MD, and Daniel R. Meldrum, MD, Indianapolis, Ind
Background. Gender differences have been noted in acute ischemia/reperfusion injury. Estrogen and the estrogen receptors (ER) appear to play a critical role in cardiovascular gender differences, given that females have improved myocardial functional recovery associated with decreased tissue inflammation. It has been suggested that ER beta plays a part in decreasing myocardial inflammation following hemorrhage. It remains unknown, however, whether ER beta also may be protective following the more severe insult of complete global, normothermic ischemia/reperfusion injury in the isolated mouse heart. Methods. Adult male and female wild-type (WT) and ER beta knockout (ERbKO) mouse hearts were subjected to 20 minutes ischemia and 60 minutes reperfusion (Langendorff model). Myocardial contractile function (±dP/dt) was continuously recorded. Heart tissue was analyzed for tumor necrosis factor, interleukin (IL)-1b, IL-6, and IL-10 levels as determined by enzyme-linked immunosorbent assay. Results. Females had markedly improved functional recovery compared with males following ischemia/ reperfusion. This recovery was associated with lower levels of myocardial tumor necrosis factor, IL-1b, and IL-6 in females. However, ERbKO females exhibited significantly less postischemic functional recovery than WT females and were similar to WT males. Interestingly, increased myocardial production of tumor necrosis factor, IL-1b, and IL-6 was noted in ERbKO female hearts in response to ischemia/ reperfusion. No significant differences were found between male WT and male ERbKO in postischemic functional recovery and proinflammatory cytokine production. Conclusion. ER beta plays a role in the protective effects of estrogen following global, warm ischemia/ reperfusion of the isolated mouse heart. This understanding ultimately may enable the development of pharmaceutical agents that harness such protection with minimal collateral sex hormone effects. (Surgery 2008;144:233-8.) From the Department of Surgery, Indiana University School of Medicine, Indianapolis, Ind
ISCHEMIC HEART DISEASE is the leading cause of death in men and women in the United States. Effective therapy for ischemic heart disease requires restoration of blood flow, which is accompanied by paradoxical ischemia/reperfusion (I/R) injury. Much research has been devoted to minimizing the
Presented at the 3rd Annual Academic Surgical Congress, Huntington Beach, California, February 2008. Supported in part by research grants from the National Institutes of Health (R01GM070628, R01HL085595, K99/R00 HL0876077, and F32HL085982), and an American Heart Association Grant-in-Aid and American Heart Association Post-Doctoral Fellowship (0725663Z). Accepted for publication March 7, 2008. Reprint requests Daniel R. Meldrum, MD, 545 Barnhill Dr., Emerson Hall 215, Indianapolis, IN 46202. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2008 Mosby, Inc. All rights reserved. doi:10.1016/j.surg.2008.03.009
effects of I/R injury.1 Our incomplete understanding of the cellular and molecular mechanisms that may mediate the severity of myocardial I/R injury, however, has led to limited clinical results. One of the prevailing hypotheses is that gender or sex hormones may be involved in regulating the myocardial response to I/R. This is appreciated clinically and experimentally in that females have a lower overall incidence of heart failure, improved survival, and better age-matched cardiac contractility after myocardial I/R compared with males.2,3 Indeed, estrogen has widely been implicated in the cardioprotection found in females.3,4 However, recent negative or neutral outcomes of clinical trials fail to demonstrate cardioprotection of hormone replacement therapy on postmenopausal females.5 This controversy highlights the need to further elucidate the mechanisms of estrogen’s cardioprotection in females. Estrogen initiates its biologic actions by binding to estrogen receptor alpha (ERa) and/or estrogen SURGERY 233
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receptor beta (ERb), both of which are present in myocytes.3,6 Although some investigations have shown that ERb mediates the cardioprotective effects of estrogen on female hearts under hypercontractile conditions,7,8 the role of ERb remains unclear in the response of otherwise normal myocardium to acute I/R. Yet, myocardial inflammation plays a critical role in I/R injury and is characterized by the expression of inflammatory cytokines.9,10 Cannon and Dinarello11 first discussed the effect of gender on inflammation.12 In addition, estrogen has been shown to act as an immunomodulator of inflammation in chronic heart disease. Therefore, it can be postulated that estrogen may mediate cardioprotection through regulation of inflammatory signaling. In fact, our group4 has demonstrated previously that administration of 17-beta-estradiol decreased inflammatory signaling and thereby improved cardiac dysfunction following acute ischemia. However, it is unknown whether ERb mediates myocardial inflammatory cytokine production in response to acute I/R. Therefore, we hypothesize that, in normal female hearts, ERb will provide protection of myocardial function following I/R through a decrease in myocardial inflammatory cytokine production. The purpose of this study was to determine the effect of ERb on postischemic myocardial function and inflammatory signaling using mice with a targeted mutation of ERb. MATERIALS AND METHODS Animals. The estrogen receptor-beta deficiency mouse strain (ERbKO) was of C57BL/6J background and purchased from Taconic Farms, Inc (Hudson, NY). A total of 10 ERbKO mice (16 ± 4 weeks) and 10 wild-type (WT) of both genders were fed a standard diet and acclimated in a quiet quarantine room for more than 1 month prior to the experiments. The animal protocol was reviewed and approved by the Indiana 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 publication No. 85-23, revised, 1985). All isolated mouse hearts were subjected to the same I/R protocol: 15-minute equilibration period, 20-minute global ischemia (37°C), and 60-minute total reperfusion. Mouse hearts were divided into 4 experimental groups: (1) normal males (n = 5); (2) ERbKO males (n = 5); (3) normal females (n = 5); and (4) ERbKO females (n = 5).
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Isolated heart preparation (Langendorff). Experiments were performed with the use of a Langendorff apparatus, as described previously for use in mouse hearts.13 Briefly, mice were anesthetized (sodium pentobarbital, 60 mg/kg intraperitoneally) and heparinized (500 U intraperitoneally). Hearts were rapidly excised via median sternotomy and placed in 4°C Krebs-Henseleit solution. The aorta was cannulated, and the heart was perfused with oxygenated (95% O2/5% CO2) Krebs-Henseleit solution (37°C). A pulmonary arteriotomy and left atrial resection were performed before insertion of a water-filled latex balloon through the left atrium into the left ventricle. The preload volume (balloon volume) was held constant during the entire experiment to allow continuous recording of the left ventricular developed pressure. A 3-way stopcock above the aortic root was used to create global ischemia, during which the heart was placed in a 37°C degassed organ bath. Hearts were paced at 400 beats per minute except during ischemia, and pacing was reinitiated within 3 minutes of reperfusion. Coronary flow was measured by collecting pulmonary artery effluent. Data were continuously recorded using a PowerLab 8 preamplifier/digitizer (AD Instruments Inc, Milford, Mass) and an Apple G4 PowerPC computer (Apple Computer Inc, Cupertino, Calif). The maximal positive and negative values of the first derivative of pressure (+dP/dt and dP/dt) were calculated using PowerLab software. Myocardial cytokine expression. Heart tissue was homogenized individually in cold buffer containing 20 mM Tris (pH 7.5); 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride and then centrifuged at 12000 rpm for 10 minutes. Myocardial tumor necrosis factor-alpha (TNF), interleukin (IL)-1b, IL-6, and IL-10 in the cardiac tissue were determined by enzyme-linked immunosorbent assay (ELISA) using a commercially available ELISA kit (BD Opt EIA ELISA set; BD Biosciences Pharmingen, San Diego, Calif and Duo set ELISA Development System; R&D Systems Inc, Minneapolis, Minn). ELISA was performed according to the manufacturer’s instructions. All samples and standards were measured in duplicate. Presentation of data and statistical analysis. All reported values are mean ± SEM. Data were compared using 2-way analysis of variance with post hoc Bonferroni test or the Student t test (male WT vs female WT; male WT vs male ERbKO; and female WT vs female ERbKO). A 2-tailed probability value > .05 was considered statistically significant.
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Fig 1. Myocardial function (±dP/dt) following ischemia/reperfusion in male wild-type (M WT) and male estrogen receptor-beta knockout (M ERbKO) mouse hearts. A, +dP/dt maximum (% of equilibration); B, dP/dt maximum (% of equilibration). Results are mean ± SEM.
RESULTS Myocardial function. Ischemia/reperfusion injury impaired myocardial function as exhibited by the depression of +dP/dt and the elevation of dP/dt in both male and female hearts (Figs 1 and 2). Deficiency of the ERb gene had no significant effects on I/R-induced myocardial dysfunction (±dP/dt) in male hearts (Fig 1), whereas ERbKO resulted in increased depression of +dP/ dt and elevation of dP/dt in female hearts compared with female WT (Fig 2). In addition, gender differences have been noted in I/R-depressed myocardial function with improved ±dP/dt in female WT hearts compared with male WT hearts (Fig 3). However, ablation of the ERb gene neutralized gender differences in myocardial function following I/R, as similar impairments of contractility and compliance in male WT, male ERbKO, and female ERbKO hearts were noted (Fig 3).
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Fig 2. Myocardial function (±dP/dt) following ischemia/reperfusion in female wild-type (F WT) and female estrogen receptor-beta knockout (F ERbKO) mouse hearts. A, +dP/dt maximum (% of equilibration); B, dP/dt maximum (% of equilibration). Results are mean ± SEM. *P < .05. **P < .01. ***P < .001 vs F WT at the corresponding time.
Myocardial inflammatory response to ischemia/ reperfusion. Myocardial production of TNF, IL-1b, IL-6, and IL-10 in heart tissue was measured via ELISA. Compared with male WT hearts, female WT hearts demonstrated a trend of decreased levels of proinflammatory cytokines (TNF, IL-1b, and IL-6) and exhibited a trend of increased IL10 production, an antiinflammatory cytokine, after I/R. Interestingly, ablation of the ERb gene significantly increased myocardial proinflammatory cytokine production (TNF, IL-1b, and IL-6), and likely decreased antiinflammatory IL-10 expression in female hearts, but not in males, following I/R (Fig 4). DISCUSSION In the present study, we found that, in animals with a higher baseline of endogenous estrogen (females but not males), ERb mediates acute myocardial functional protection following I/R and reduces myocardial TNF, IL-1b, and IL-6
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Fig 3. Recovery of myocardial function after ischemia/ reperfusion at end-reperfusion in male wild-type (M WT), male estrogen receptor-beta knockout (M ERbKO), female wild-type (F WT) and female estrogen receptor-beta knockout (F ERbKO) mouse hearts. A, Recovery of +dP/dt (% of equilibration). B, Recovery of dP/dt (% of equilibration). Results are mean ± SEM. *P < .05, F WT vs M WT. #P < .05, F ERbKO vs F WT.
production. Estrogen has been widely recognized as a cardioprotective agent in females. The biologic function of estrogen is conducted through the estrogen receptors. Two ER subtypes have been identified: ERa and ERb, both of which belong to the nuclear receptor gene family of transcription factors and are expressed in the heart.6 Ablation of the ERb gene has been reported to reduce myocardial functional recovery in female hearts under conditions of enhanced contractility following I/R injury.8 Most recently, Nikolic et al7 found that a selective ERb agonist, 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), restored estrogenmediated cardioprotection in ovariectomized female subjected to myocardial I/R combined with hypercontractile condition. In addition, Dr Chaudry’s group demonstrated that administration of an ERb-selective agonist (DPN), but not an ERa agonist, improved trauma-hemorrhage–depressed cardiac function in males.14,15 In the present study, our results are in agreement with previous observations. Most importantly,
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we found that, under baseline conditions, female ERbKO hearts demonstrated a statistically significant decrease in functional recovery of contractility (+dP/dT) and compliance ( dP/dT) compared with female WT hearts following I/R. This result extended the previous findings, which demonstrated that ERb protected myocardial function in the female heart associated with other pathophysiological status, to our current finding that ERb can mediate cardioprotection in normal females. However, deficiency of the ERb gene did not affect myocardial functional recovery in male hearts (with presumably lower estrogen levels) after acute I/R. With respect to ERb expressed in the male heart,15 our data suggest that ERb-mediated cardioprotection may require a certain level of estrogen in the circulating and/or local tissue/organ. ERb may modulate the myocardial dysfunction induced by I/R injury at several levels. ERb has been reported to regulate expression of metabolism genes. Gabel et al8 found that female mice lacking ERb demonstrated poorer postischemic myocardial functional recovery compared with female WT, which was associated with decreased cardiac expression of SPOT 14 (a gene involved in lipid metabolism). In addition, upregulation of 6phosphofructo 2-kinase/fructose-2,6-bisphosphatase expression also has been noted in DPN-treated ovariectomized female hearts following I/R.7 Hsieh et al14 reported that stimulation of ERb with DPN restored trauma-hemorrhage–decreased cardiac PGC-1a and ATP levels and, therefore, improved myocardial function in males. In addition, elevated cardiac expression of HSP70 has also been observed in DPN-treated ovariectomized female hearts following I/R7 and in male rats supplemented with DPN after trauma-hemorrhage.15 Moreover, ERb has been shown to mediate cardiac electrophysiology through upregulation of Kv4.3 channel expression and calcium-handling protein expression in chronic heart failure after myocardial infarction.16,17 Thus, it is possible that ERb mediates protected cardiac contractility and compliance in females via improved metabolism and modulation of ion channels. There is no doubt myocardial injury can result from inflammatory cytokine production, such as TNF, IL-1b, and IL-6. A large number of studies have demonstrated that the myocardium can produce substantial amounts of TNF, IL-1b, and IL-6 in response to acute I/R, resulting in significant myocardial dysfunction.10,18,19 To date, little information exists regarding the role of ERb in myocardial inflammation following acute I/R. In fact, estrogen plays an important role in modulating
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inflammation in chronic heart disease. Deshpande et al20 demonstrated that estradiol decreased lipopolysaccharide-induced IL-1, IL-6, and TNF production and also attenuated NFkB-binding activity. Mizushima et al21 reported that male trauma patients had increased IL-6 levels, and estradiol treatment after trauma-hemorrhage in animals reduced the increased IL-6. Previously, we have indicated that exogenous 17-beta-estradiol, a nonselective ER ligand, improved I/R-induced myocardial dysfunction and decreased myocardial inflammatory cytokine production (TNF, IL-1b, and IL-6) following acute ischemia in ovariectomized females as well as males. Our results of this study further demonstrate that ablation of ERb increases myocardial production of TNF, IL-1b, and IL-6 in female hearts in response to I/R insult. Additionally, decreased IL-10 levels were noted in female ERbKO hearts compared to female WT. IL-10, an antiinflammatory cytokine, has been reported to suppress the production of TNF, IL1b, and IL-6.22 Deficiency of IL-10 also has been shown to enhance inflammatory response in a murine model of myocardial infarction.23 Therefore, this study provides direct evidence that ERb mediates cardioprotection in females likely through a reduction in cardiac TNF, IL-1b, and IL-6 expression and an elevation of antiinflammatory IL-10 production. In another study,24 we found that ERa mediates improved myocardial function in females via upregulation of the protective ERK½ activation and downregulation of the proapoptotic Jun N-terminal kinase activation during myocardial ischemia. Here, a similar finding is noted in that cardiac protection in females is also mediated by ERb following I/R. This raises the following question: Which ER(s) or ER signaling mediates the predominant cardioprotection in response to acute I/R? Hamada et al25 recently reported that both ERa and ERb are responsible for the estradiol-mediated endothelial progenitor cell function, which improved cardiac function after myocardial infarction. Yet, ERa and ERb are capable of forming ERa/ERb heterodimers to stimulate downstream signals in addition to forming ERa or ERb homodimers.6 Therefore, it is possible that both ERa
= Fig 4. Myocardial production of TNF (A), IL-1b (B), IL-6 (C), and IL-10 (D) after end-reperfusion in male wild-type (M WT), male estrogen receptor-beta knockout (M ERbKO), female wild-type (F WT) and female estrogen receptor-beta knockout (F ERbKO) mouse hearts. Results are mean ± SEM. *P < .05, F WT vs M WT. #P < .05, F ERbKO vs F WT.
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and ERb contribute to cardioprotection in females under normal contractile conditions in response to acute I/R. However, further studies are required to determine this hypothesis by using ERa and ERb double-knockout mice. This study provides direct evidence that the protective effects of ERb on female myocardial function is correlated with ERb-reduced cardiac inflammatory markers TNF, IL-1b, and IL-6 in female hearts subjected to I/R. Further understanding the effects of gender, estrogen, and the estrogen receptors may allow us to advance therapeutic manipulations in menopausal females and potentially males. REFERENCES 1. Niagara MI, Haider H, Jiang S, Ashraf M. Pharmacologically preconditioned skeletal myoblasts are resistant to oxidative stress and promote angiomyogenesis via release of paracrine factors in the infarcted heart. Circ Res 2007;100:545-55. 2. Tunstall-Pedoe H, Morrison C, Woodward M, Fitzpatrick B, Watt G. Sex differences in myocardial infarction and coronary deaths in the Scottish MONICA population of Glasgow 1985 to 1991. Presentation, diagnosis, treatment, and 28day case fatality of 3991 events in men and 1551 events in women. Circulation 1996;93:1981-92. 3. Mendelsohn ME, Karas RH. The protective effects of estrogen on the cardiovascular system. N Engl J Med 1999;340:1801-11. 4. Wang M, Tsai BM, Reiger KM, Brown JW, Meldrum DR. 17beta-Estradiol decreases p38 MAPK-mediated myocardial inflammation and dysfunction following acute ischemia. J Mol Cell Cardiol 2006;40:205-12. 5. Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML, et al. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women’s Health Initiative randomized controlled trial. JAMA 2002;288:321-33. 6. Babiker FA, De Windt LJ, van Eickels M, Grohe C, Meyer R, Doevendans PA. Estrogenic hormone action in the heart: regulatory network and function. Cardiovasc Res 2002;53:709-19. 7. Nikolic I, Liu D, Bell JA, Collins J, Steenbergen C, Murphy E. Treatment with an estrogen receptor-beta-selective agonist is cardioprotective. J Mol Cell Cardiol 2007;42:769-80. 8. Gabel SA, Walker VR, London RE, Steenbergen C, Korach KS, Murphy E. Estrogen receptor beta mediates gender differences in ischemia/reperfusion injury. J Mol Cell Cardiol 2005;38:289-97. 9. Meldrum DR. Tumor necrosis factor in the heart. Am J Physiol 1998;274(3 Pt 2):R577-95. 10. Cain BS, Meldrum DR, Dinarello CA, Meng X, Joo KS, Banerjee A, Harken AH. Tumor necrosis factor-alpha and interleukin-1beta synergistically depress human myocardial function. Crit Care Med 1999;27:1309-18. 11. Cannon JG, Dinarello CA. Increased plasma interleukin-1 activity in women after ovulation. Science 1985;227:1247-9.
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12. Lynch EA, Dinarello CA, Cannon JG. Gender differences in IL-1 alpha, IL-1 beta, and IL-1 receptor antagonist secretion from mononuclear cells and urinary excretion. J Immunol 1994;153:300-6. 13. Wang M, Tsai BM, Crisostomo PR, Meldrum DR. Tumor necrosis factor receptor 1 signaling resistance in the female myocardium during ischemia. Circulation 2006;114(1 Suppl):I282-9. 14. Hsieh YC, Choudhry MA, Yu HP, Shimizu T, Yang S, Suzuki T, et al. Inhibition of cardiac PGC-1alpha expression abolishes ERbeta agonist-mediated cardioprotection following trauma-hemorrhage. Faseb J 2006;20:1109-17. 15. Yu HP, Shimizu T, Choudhry MA, Hsieh YC, Suzuki T, Bland KI, et al. Mechanism of cardioprotection following traumahemorrhagic shock by a selective estrogen receptor-beta agonist: up-regulation of cardiac heat shock factor-1 and heat shock proteins. J Mol Cell Cardiol 2006;40:185-94. 16. Korte T, Fuchs M, Arkudas A, Geertz S, Meyer R, Gardiwal A, et al. Female mice lacking estrogen receptor beta display prolonged ventricular repolarization and reduced ventricular automaticity after myocardial infarction. Circulation 2005;111:2282-90. 17. Pelzer T, Loza PA, Hu K, Bayer B, Dienesch C, Calvillo L, et al. Increased mortality and aggravation of heart failure in estrogen receptor-beta knockout mice after myocardial infarction. Circulation 2005;111:1492-8. 18. Meldrum DR, Dinarello CA, Cleveland JC Jr, Cain BS, Shames BD, Meng X, et al. Hydrogen peroxide induces tumor necrosis factor alpha-mediated cardiac injury by a P38 mitogen-activated protein kinase-dependent mechanism. Surgery 1998;124:291-6; discussion 297. 19. Yang S, Hu S, Choudhry MA, Rue LW III, Bland KI, Chaudry IH. Anti-rat soluble IL-6 receptor antibody down-regulates cardiac IL-6 and improves cardiac function following trauma-hemorrhage. J Mol Cell Cardiol 2007;42: 620-30. 20. Deshpande R, Khalili H, Pergolizzi RG, Michael SD, Chang MD. Estradiol down-regulates LPS-induced cytokine production and NFkB activation in murine macrophages. Am J Reprod Immunol 1997;38:46-54. 21. Mizushima Y, Wang P, Jarrar D, Cioffi WG, Bland KI, Chaudry IH. Estradiol administration after trauma-hemorrhage improves cardiovascular and hepatocellular functions in male animals. Ann Surg 2000;232:673-9. 22. Lacraz S, Nicod LP, Chicheportiche R, Welgus HG, Dayer JM. IL-10 inhibits metalloproteinase and stimulates TIMP1 production in human mononuclear phagocytes. J Clin Invest 1995;96:2304-10. 23. Yang Z, Zingarelli B, Szabo C. Crucial role of endogenous interleukin-10 production in myocardial ischemia/reperfusion injury. Circulation 2000;101:1019-26. 24. Wang M, Crisostomo P, Wairiuko GM, Meldrum DR. Estrogen receptor-alpha mediates acute myocardial protection in females. Am J Physiol Heart Circ Physiol 2006;290:H2204-9. 25. Hamada H, Kim MK, Iwakura A, Ii M, Thorne T, Qin G, et al. Estrogen receptors alpha and beta mediate contribution of bone marrow-derived endothelial progenitor cells to functional recovery after myocardial infarction. Circulation 2006;114:2261-70.
Background. Gender differences have been noted in acute ischemia/reperfusion injury. Estrogen and the estrogen receptors (ER) appear to play a critical role in cardiovascular gender differences, given that females have improved myocardial functional recovery associated with decreased tissue inflammation. It has been suggested that ER beta plays a part in decreasing myocardial inflammation following hemorrhage. It remains unknown, however, whether ER beta also may be protective following the more severe insult of complete global, normothermic ischemia/reperfusion injury in the isolated mouse heart. Methods. Adult male and female wild-type (WT) and ER beta knockout (ERbKO) mouse hearts were subjected to 20 minutes ischemia and 60 minutes reperfusion (Langendorff model). Myocardial contractile function (±dP/dt) was continuously recorded. Heart tissue was analyzed for tumor necrosis factor, interleukin (IL)-1b, IL-6, and IL-10 levels as determined by enzyme-linked immunosorbent assay. Results. Females had markedly improved functional recovery compared with males following ischemia/ reperfusion. This recovery was associated with lower levels of myocardial tumor necrosis factor, IL-1b, and IL-6 in females. However, ERbKO females exhibited significantly less postischemic functional recovery than WT females and were similar to WT males. Interestingly, increased myocardial production of tumor necrosis factor, IL-1b, and IL-6 was noted in ERbKO female hearts in response to ischemia/ reperfusion. No significant differences were found between male WT and male ERbKO in postischemic functional recovery and proinflammatory cytokine production. Conclusion. ER beta plays a role in the protective effects of estrogen following global, warm ischemia/ reperfusion of the isolated mouse heart. This understanding ultimately may enable the development of pharmaceutical agents that harness such protection with minimal collateral sex hormone effects. (Surgery 2008;144:233-8.) From the Department of Surgery, Indiana University School of Medicine, Indianapolis, Ind
ISCHEMIC HEART DISEASE is the leading cause of death in men and women in the United States. Effective therapy for ischemic heart disease requires restoration of blood flow, which is accompanied by paradoxical ischemia/reperfusion (I/R) injury. Much research has been devoted to minimizing the
Presented at the 3rd Annual Academic Surgical Congress, Huntington Beach, California, February 2008. Supported in part by research grants from the National Institutes of Health (R01GM070628, R01HL085595, K99/R00 HL0876077, and F32HL085982), and an American Heart Association Grant-in-Aid and American Heart Association Post-Doctoral Fellowship (0725663Z). Accepted for publication March 7, 2008. Reprint requests Daniel R. Meldrum, MD, 545 Barnhill Dr., Emerson Hall 215, Indianapolis, IN 46202. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2008 Mosby, Inc. All rights reserved. doi:10.1016/j.surg.2008.03.009
effects of I/R injury.1 Our incomplete understanding of the cellular and molecular mechanisms that may mediate the severity of myocardial I/R injury, however, has led to limited clinical results. One of the prevailing hypotheses is that gender or sex hormones may be involved in regulating the myocardial response to I/R. This is appreciated clinically and experimentally in that females have a lower overall incidence of heart failure, improved survival, and better age-matched cardiac contractility after myocardial I/R compared with males.2,3 Indeed, estrogen has widely been implicated in the cardioprotection found in females.3,4 However, recent negative or neutral outcomes of clinical trials fail to demonstrate cardioprotection of hormone replacement therapy on postmenopausal females.5 This controversy highlights the need to further elucidate the mechanisms of estrogen’s cardioprotection in females. Estrogen initiates its biologic actions by binding to estrogen receptor alpha (ERa) and/or estrogen SURGERY 233
234 Wang et al
receptor beta (ERb), both of which are present in myocytes.3,6 Although some investigations have shown that ERb mediates the cardioprotective effects of estrogen on female hearts under hypercontractile conditions,7,8 the role of ERb remains unclear in the response of otherwise normal myocardium to acute I/R. Yet, myocardial inflammation plays a critical role in I/R injury and is characterized by the expression of inflammatory cytokines.9,10 Cannon and Dinarello11 first discussed the effect of gender on inflammation.12 In addition, estrogen has been shown to act as an immunomodulator of inflammation in chronic heart disease. Therefore, it can be postulated that estrogen may mediate cardioprotection through regulation of inflammatory signaling. In fact, our group4 has demonstrated previously that administration of 17-beta-estradiol decreased inflammatory signaling and thereby improved cardiac dysfunction following acute ischemia. However, it is unknown whether ERb mediates myocardial inflammatory cytokine production in response to acute I/R. Therefore, we hypothesize that, in normal female hearts, ERb will provide protection of myocardial function following I/R through a decrease in myocardial inflammatory cytokine production. The purpose of this study was to determine the effect of ERb on postischemic myocardial function and inflammatory signaling using mice with a targeted mutation of ERb. MATERIALS AND METHODS Animals. The estrogen receptor-beta deficiency mouse strain (ERbKO) was of C57BL/6J background and purchased from Taconic Farms, Inc (Hudson, NY). A total of 10 ERbKO mice (16 ± 4 weeks) and 10 wild-type (WT) of both genders were fed a standard diet and acclimated in a quiet quarantine room for more than 1 month prior to the experiments. The animal protocol was reviewed and approved by the Indiana 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 publication No. 85-23, revised, 1985). All isolated mouse hearts were subjected to the same I/R protocol: 15-minute equilibration period, 20-minute global ischemia (37°C), and 60-minute total reperfusion. Mouse hearts were divided into 4 experimental groups: (1) normal males (n = 5); (2) ERbKO males (n = 5); (3) normal females (n = 5); and (4) ERbKO females (n = 5).
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Isolated heart preparation (Langendorff). Experiments were performed with the use of a Langendorff apparatus, as described previously for use in mouse hearts.13 Briefly, mice were anesthetized (sodium pentobarbital, 60 mg/kg intraperitoneally) and heparinized (500 U intraperitoneally). Hearts were rapidly excised via median sternotomy and placed in 4°C Krebs-Henseleit solution. The aorta was cannulated, and the heart was perfused with oxygenated (95% O2/5% CO2) Krebs-Henseleit solution (37°C). A pulmonary arteriotomy and left atrial resection were performed before insertion of a water-filled latex balloon through the left atrium into the left ventricle. The preload volume (balloon volume) was held constant during the entire experiment to allow continuous recording of the left ventricular developed pressure. A 3-way stopcock above the aortic root was used to create global ischemia, during which the heart was placed in a 37°C degassed organ bath. Hearts were paced at 400 beats per minute except during ischemia, and pacing was reinitiated within 3 minutes of reperfusion. Coronary flow was measured by collecting pulmonary artery effluent. Data were continuously recorded using a PowerLab 8 preamplifier/digitizer (AD Instruments Inc, Milford, Mass) and an Apple G4 PowerPC computer (Apple Computer Inc, Cupertino, Calif). The maximal positive and negative values of the first derivative of pressure (+dP/dt and dP/dt) were calculated using PowerLab software. Myocardial cytokine expression. Heart tissue was homogenized individually in cold buffer containing 20 mM Tris (pH 7.5); 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride and then centrifuged at 12000 rpm for 10 minutes. Myocardial tumor necrosis factor-alpha (TNF), interleukin (IL)-1b, IL-6, and IL-10 in the cardiac tissue were determined by enzyme-linked immunosorbent assay (ELISA) using a commercially available ELISA kit (BD Opt EIA ELISA set; BD Biosciences Pharmingen, San Diego, Calif and Duo set ELISA Development System; R&D Systems Inc, Minneapolis, Minn). ELISA was performed according to the manufacturer’s instructions. All samples and standards were measured in duplicate. Presentation of data and statistical analysis. All reported values are mean ± SEM. Data were compared using 2-way analysis of variance with post hoc Bonferroni test or the Student t test (male WT vs female WT; male WT vs male ERbKO; and female WT vs female ERbKO). A 2-tailed probability value > .05 was considered statistically significant.
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Fig 1. Myocardial function (±dP/dt) following ischemia/reperfusion in male wild-type (M WT) and male estrogen receptor-beta knockout (M ERbKO) mouse hearts. A, +dP/dt maximum (% of equilibration); B, dP/dt maximum (% of equilibration). Results are mean ± SEM.
RESULTS Myocardial function. Ischemia/reperfusion injury impaired myocardial function as exhibited by the depression of +dP/dt and the elevation of dP/dt in both male and female hearts (Figs 1 and 2). Deficiency of the ERb gene had no significant effects on I/R-induced myocardial dysfunction (±dP/dt) in male hearts (Fig 1), whereas ERbKO resulted in increased depression of +dP/ dt and elevation of dP/dt in female hearts compared with female WT (Fig 2). In addition, gender differences have been noted in I/R-depressed myocardial function with improved ±dP/dt in female WT hearts compared with male WT hearts (Fig 3). However, ablation of the ERb gene neutralized gender differences in myocardial function following I/R, as similar impairments of contractility and compliance in male WT, male ERbKO, and female ERbKO hearts were noted (Fig 3).
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Fig 2. Myocardial function (±dP/dt) following ischemia/reperfusion in female wild-type (F WT) and female estrogen receptor-beta knockout (F ERbKO) mouse hearts. A, +dP/dt maximum (% of equilibration); B, dP/dt maximum (% of equilibration). Results are mean ± SEM. *P < .05. **P < .01. ***P < .001 vs F WT at the corresponding time.
Myocardial inflammatory response to ischemia/ reperfusion. Myocardial production of TNF, IL-1b, IL-6, and IL-10 in heart tissue was measured via ELISA. Compared with male WT hearts, female WT hearts demonstrated a trend of decreased levels of proinflammatory cytokines (TNF, IL-1b, and IL-6) and exhibited a trend of increased IL10 production, an antiinflammatory cytokine, after I/R. Interestingly, ablation of the ERb gene significantly increased myocardial proinflammatory cytokine production (TNF, IL-1b, and IL-6), and likely decreased antiinflammatory IL-10 expression in female hearts, but not in males, following I/R (Fig 4). DISCUSSION In the present study, we found that, in animals with a higher baseline of endogenous estrogen (females but not males), ERb mediates acute myocardial functional protection following I/R and reduces myocardial TNF, IL-1b, and IL-6
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Fig 3. Recovery of myocardial function after ischemia/ reperfusion at end-reperfusion in male wild-type (M WT), male estrogen receptor-beta knockout (M ERbKO), female wild-type (F WT) and female estrogen receptor-beta knockout (F ERbKO) mouse hearts. A, Recovery of +dP/dt (% of equilibration). B, Recovery of dP/dt (% of equilibration). Results are mean ± SEM. *P < .05, F WT vs M WT. #P < .05, F ERbKO vs F WT.
production. Estrogen has been widely recognized as a cardioprotective agent in females. The biologic function of estrogen is conducted through the estrogen receptors. Two ER subtypes have been identified: ERa and ERb, both of which belong to the nuclear receptor gene family of transcription factors and are expressed in the heart.6 Ablation of the ERb gene has been reported to reduce myocardial functional recovery in female hearts under conditions of enhanced contractility following I/R injury.8 Most recently, Nikolic et al7 found that a selective ERb agonist, 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), restored estrogenmediated cardioprotection in ovariectomized female subjected to myocardial I/R combined with hypercontractile condition. In addition, Dr Chaudry’s group demonstrated that administration of an ERb-selective agonist (DPN), but not an ERa agonist, improved trauma-hemorrhage–depressed cardiac function in males.14,15 In the present study, our results are in agreement with previous observations. Most importantly,
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we found that, under baseline conditions, female ERbKO hearts demonstrated a statistically significant decrease in functional recovery of contractility (+dP/dT) and compliance ( dP/dT) compared with female WT hearts following I/R. This result extended the previous findings, which demonstrated that ERb protected myocardial function in the female heart associated with other pathophysiological status, to our current finding that ERb can mediate cardioprotection in normal females. However, deficiency of the ERb gene did not affect myocardial functional recovery in male hearts (with presumably lower estrogen levels) after acute I/R. With respect to ERb expressed in the male heart,15 our data suggest that ERb-mediated cardioprotection may require a certain level of estrogen in the circulating and/or local tissue/organ. ERb may modulate the myocardial dysfunction induced by I/R injury at several levels. ERb has been reported to regulate expression of metabolism genes. Gabel et al8 found that female mice lacking ERb demonstrated poorer postischemic myocardial functional recovery compared with female WT, which was associated with decreased cardiac expression of SPOT 14 (a gene involved in lipid metabolism). In addition, upregulation of 6phosphofructo 2-kinase/fructose-2,6-bisphosphatase expression also has been noted in DPN-treated ovariectomized female hearts following I/R.7 Hsieh et al14 reported that stimulation of ERb with DPN restored trauma-hemorrhage–decreased cardiac PGC-1a and ATP levels and, therefore, improved myocardial function in males. In addition, elevated cardiac expression of HSP70 has also been observed in DPN-treated ovariectomized female hearts following I/R7 and in male rats supplemented with DPN after trauma-hemorrhage.15 Moreover, ERb has been shown to mediate cardiac electrophysiology through upregulation of Kv4.3 channel expression and calcium-handling protein expression in chronic heart failure after myocardial infarction.16,17 Thus, it is possible that ERb mediates protected cardiac contractility and compliance in females via improved metabolism and modulation of ion channels. There is no doubt myocardial injury can result from inflammatory cytokine production, such as TNF, IL-1b, and IL-6. A large number of studies have demonstrated that the myocardium can produce substantial amounts of TNF, IL-1b, and IL-6 in response to acute I/R, resulting in significant myocardial dysfunction.10,18,19 To date, little information exists regarding the role of ERb in myocardial inflammation following acute I/R. In fact, estrogen plays an important role in modulating
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inflammation in chronic heart disease. Deshpande et al20 demonstrated that estradiol decreased lipopolysaccharide-induced IL-1, IL-6, and TNF production and also attenuated NFkB-binding activity. Mizushima et al21 reported that male trauma patients had increased IL-6 levels, and estradiol treatment after trauma-hemorrhage in animals reduced the increased IL-6. Previously, we have indicated that exogenous 17-beta-estradiol, a nonselective ER ligand, improved I/R-induced myocardial dysfunction and decreased myocardial inflammatory cytokine production (TNF, IL-1b, and IL-6) following acute ischemia in ovariectomized females as well as males. Our results of this study further demonstrate that ablation of ERb increases myocardial production of TNF, IL-1b, and IL-6 in female hearts in response to I/R insult. Additionally, decreased IL-10 levels were noted in female ERbKO hearts compared to female WT. IL-10, an antiinflammatory cytokine, has been reported to suppress the production of TNF, IL1b, and IL-6.22 Deficiency of IL-10 also has been shown to enhance inflammatory response in a murine model of myocardial infarction.23 Therefore, this study provides direct evidence that ERb mediates cardioprotection in females likely through a reduction in cardiac TNF, IL-1b, and IL-6 expression and an elevation of antiinflammatory IL-10 production. In another study,24 we found that ERa mediates improved myocardial function in females via upregulation of the protective ERK½ activation and downregulation of the proapoptotic Jun N-terminal kinase activation during myocardial ischemia. Here, a similar finding is noted in that cardiac protection in females is also mediated by ERb following I/R. This raises the following question: Which ER(s) or ER signaling mediates the predominant cardioprotection in response to acute I/R? Hamada et al25 recently reported that both ERa and ERb are responsible for the estradiol-mediated endothelial progenitor cell function, which improved cardiac function after myocardial infarction. Yet, ERa and ERb are capable of forming ERa/ERb heterodimers to stimulate downstream signals in addition to forming ERa or ERb homodimers.6 Therefore, it is possible that both ERa
= Fig 4. Myocardial production of TNF (A), IL-1b (B), IL-6 (C), and IL-10 (D) after end-reperfusion in male wild-type (M WT), male estrogen receptor-beta knockout (M ERbKO), female wild-type (F WT) and female estrogen receptor-beta knockout (F ERbKO) mouse hearts. Results are mean ± SEM. *P < .05, F WT vs M WT. #P < .05, F ERbKO vs F WT.
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and ERb contribute to cardioprotection in females under normal contractile conditions in response to acute I/R. However, further studies are required to determine this hypothesis by using ERa and ERb double-knockout mice. This study provides direct evidence that the protective effects of ERb on female myocardial function is correlated with ERb-reduced cardiac inflammatory markers TNF, IL-1b, and IL-6 in female hearts subjected to I/R. Further understanding the effects of gender, estrogen, and the estrogen receptors may allow us to advance therapeutic manipulations in menopausal females and potentially males. REFERENCES 1. Niagara MI, Haider H, Jiang S, Ashraf M. Pharmacologically preconditioned skeletal myoblasts are resistant to oxidative stress and promote angiomyogenesis via release of paracrine factors in the infarcted heart. Circ Res 2007;100:545-55. 2. Tunstall-Pedoe H, Morrison C, Woodward M, Fitzpatrick B, Watt G. Sex differences in myocardial infarction and coronary deaths in the Scottish MONICA population of Glasgow 1985 to 1991. Presentation, diagnosis, treatment, and 28day case fatality of 3991 events in men and 1551 events in women. Circulation 1996;93:1981-92. 3. Mendelsohn ME, Karas RH. The protective effects of estrogen on the cardiovascular system. N Engl J Med 1999;340:1801-11. 4. Wang M, Tsai BM, Reiger KM, Brown JW, Meldrum DR. 17beta-Estradiol decreases p38 MAPK-mediated myocardial inflammation and dysfunction following acute ischemia. J Mol Cell Cardiol 2006;40:205-12. 5. Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML, et al. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women’s Health Initiative randomized controlled trial. JAMA 2002;288:321-33. 6. Babiker FA, De Windt LJ, van Eickels M, Grohe C, Meyer R, Doevendans PA. Estrogenic hormone action in the heart: regulatory network and function. Cardiovasc Res 2002;53:709-19. 7. Nikolic I, Liu D, Bell JA, Collins J, Steenbergen C, Murphy E. Treatment with an estrogen receptor-beta-selective agonist is cardioprotective. J Mol Cell Cardiol 2007;42:769-80. 8. Gabel SA, Walker VR, London RE, Steenbergen C, Korach KS, Murphy E. Estrogen receptor beta mediates gender differences in ischemia/reperfusion injury. J Mol Cell Cardiol 2005;38:289-97. 9. Meldrum DR. Tumor necrosis factor in the heart. Am J Physiol 1998;274(3 Pt 2):R577-95. 10. Cain BS, Meldrum DR, Dinarello CA, Meng X, Joo KS, Banerjee A, Harken AH. Tumor necrosis factor-alpha and interleukin-1beta synergistically depress human myocardial function. Crit Care Med 1999;27:1309-18. 11. Cannon JG, Dinarello CA. Increased plasma interleukin-1 activity in women after ovulation. Science 1985;227:1247-9.
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12. Lynch EA, Dinarello CA, Cannon JG. Gender differences in IL-1 alpha, IL-1 beta, and IL-1 receptor antagonist secretion from mononuclear cells and urinary excretion. J Immunol 1994;153:300-6. 13. Wang M, Tsai BM, Crisostomo PR, Meldrum DR. Tumor necrosis factor receptor 1 signaling resistance in the female myocardium during ischemia. Circulation 2006;114(1 Suppl):I282-9. 14. Hsieh YC, Choudhry MA, Yu HP, Shimizu T, Yang S, Suzuki T, et al. Inhibition of cardiac PGC-1alpha expression abolishes ERbeta agonist-mediated cardioprotection following trauma-hemorrhage. Faseb J 2006;20:1109-17. 15. Yu HP, Shimizu T, Choudhry MA, Hsieh YC, Suzuki T, Bland KI, et al. Mechanism of cardioprotection following traumahemorrhagic shock by a selective estrogen receptor-beta agonist: up-regulation of cardiac heat shock factor-1 and heat shock proteins. J Mol Cell Cardiol 2006;40:185-94. 16. Korte T, Fuchs M, Arkudas A, Geertz S, Meyer R, Gardiwal A, et al. Female mice lacking estrogen receptor beta display prolonged ventricular repolarization and reduced ventricular automaticity after myocardial infarction. Circulation 2005;111:2282-90. 17. Pelzer T, Loza PA, Hu K, Bayer B, Dienesch C, Calvillo L, et al. Increased mortality and aggravation of heart failure in estrogen receptor-beta knockout mice after myocardial infarction. Circulation 2005;111:1492-8. 18. Meldrum DR, Dinarello CA, Cleveland JC Jr, Cain BS, Shames BD, Meng X, et al. Hydrogen peroxide induces tumor necrosis factor alpha-mediated cardiac injury by a P38 mitogen-activated protein kinase-dependent mechanism. Surgery 1998;124:291-6; discussion 297. 19. Yang S, Hu S, Choudhry MA, Rue LW III, Bland KI, Chaudry IH. Anti-rat soluble IL-6 receptor antibody down-regulates cardiac IL-6 and improves cardiac function following trauma-hemorrhage. J Mol Cell Cardiol 2007;42: 620-30. 20. Deshpande R, Khalili H, Pergolizzi RG, Michael SD, Chang MD. Estradiol down-regulates LPS-induced cytokine production and NFkB activation in murine macrophages. Am J Reprod Immunol 1997;38:46-54. 21. Mizushima Y, Wang P, Jarrar D, Cioffi WG, Bland KI, Chaudry IH. Estradiol administration after trauma-hemorrhage improves cardiovascular and hepatocellular functions in male animals. Ann Surg 2000;232:673-9. 22. Lacraz S, Nicod LP, Chicheportiche R, Welgus HG, Dayer JM. IL-10 inhibits metalloproteinase and stimulates TIMP1 production in human mononuclear phagocytes. J Clin Invest 1995;96:2304-10. 23. Yang Z, Zingarelli B, Szabo C. Crucial role of endogenous interleukin-10 production in myocardial ischemia/reperfusion injury. Circulation 2000;101:1019-26. 24. Wang M, Crisostomo P, Wairiuko GM, Meldrum DR. Estrogen receptor-alpha mediates acute myocardial protection in females. Am J Physiol Heart Circ Physiol 2006;290:H2204-9. 25. Hamada H, Kim MK, Iwakura A, Ii M, Thorne T, Qin G, et al. Estrogen receptors alpha and beta mediate contribution of bone marrow-derived endothelial progenitor cells to functional recovery after myocardial infarction. Circulation 2006;114:2261-70.