Estrogen Inhibits Cardiomyocyte Hypertrophy in Vitro

Evidence from in vivo studies suggests that some inputs to cardiac hypertrophy are opposed by the actions of estrogen. However, the mechanisms of E2 action in this respect are mainly unknown. An important pathway that is utilized by multiple hypertrophic stimuli involves the activation of the tyrosine phosphatase, calcineurin (PP2B). Here we show that 17β-estradiol (E2) significantly prevents angiotensin II (AngII)- or endothelin-1 (ET-1)-induced new protein synthesis, skeletal muscle actin expression, and increased surface area in cultured rat cardiomyocytes. ET-1 stimulated calcineurin phosphatase activity, resulting in new protein synthesis, and both were prevented by E2. E2 induced the MCIP1 gene, an inhibitor of calcineurin activity, via phosphatidylinositol 3-kinase, transcriptional, and mRNA stability mechanisms. Small interfering RNA for MCIP1 significantly reversed both the E2 restraint of protein synthesis and the inhibition of AngII-induced calcineurin activity. AngII-induced the translocation of the hypertrophic transcription factor, NF-AT, to the nucleus of the cardiomyocyte and stimulated NF-AT transcriptional activity. Both were prevented by E2. AngII also stimulated the activation of ERK and protein kinase C, contributing to cardiac hypertrophy. E2 inhibited these pathways, related to the stimulation of atrial natriuretic peptide production and secretion. Thus, restraint of calcineurin and kinase signaling to the hypertrophic program underlie these important effects of E2. Evidence from in vivo studies suggests that some inputs to cardiac hypertrophy are opposed by the actions of estrogen. However, the mechanisms of E2 action in this respect are mainly unknown. An important pathway that is utilized by multiple hypertrophic stimuli involves the activation of the tyrosine phosphatase, calcineurin (PP2B). Here we show that 17β-estradiol (E2) significantly prevents angiotensin II (AngII)- or endothelin-1 (ET-1)-induced new protein synthesis, skeletal muscle actin expression, and increased surface area in cultured rat cardiomyocytes. ET-1 stimulated calcineurin phosphatase activity, resulting in new protein synthesis, and both were prevented by E2. E2 induced the MCIP1 gene, an inhibitor of calcineurin activity, via phosphatidylinositol 3-kinase, transcriptional, and mRNA stability mechanisms. Small interfering RNA for MCIP1 significantly reversed both the E2 restraint of protein synthesis and the inhibition of AngII-induced calcineurin activity. AngII-induced the translocation of the hypertrophic transcription factor, NF-AT, to the nucleus of the cardiomyocyte and stimulated NF-AT transcriptional activity. Both were prevented by E2. AngII also stimulated the activation of ERK and protein kinase C, contributing to cardiac hypertrophy. E2 inhibited these pathways, related to the stimulation of atrial natriuretic peptide production and secretion. Thus, restraint of calcineurin and kinase signaling to the hypertrophic program underlie these important effects of E2. Cardiac hypertrophy develops in response to hypertension and is consequent to 80% of all myocardial infarctions. Hypertrophy is an independent risk factor for the development of ischemia, arrhythmia, and sudden death (1Dunn F.G. Pfeffer M.A. N. Engl. J. Med. 1999; 340: 1279-1280Crossref PubMed Scopus (23) Google Scholar, 2Saxon L.A. De Marco T. Card. Electrophysiol. Rev. 2002; 6: 18-25Crossref PubMed Scopus (10) Google Scholar). The most important vascular hormone that contributes to the development of hypertrophy is angiotensin II (AngII) 1The abbreviations used are: AngII, angiotensin II; E2, 17-β-estradiol; ET-1, endothelin-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siRNA, small interfering RNA; ET-1, endothelin-1; PI3K, phosphatidylinositol 3-kinase; ERK, extracellular signal-regulated kinase; PKC, protein kinase C; DRB, 5,6-dichlorobenzimidazole; RT, reverse transcription; BNP, brain natriuretic peptide; ANP, atrial natriuretic peptide; GFP, green fluorescent protein; ER, estrogen receptor; HRT, hormone replacement therapy; (3Wagenaar L.J. Voors A.A. Buikema H. van Gilst W.H. Can. J. Cardiol. 2002; 18: 1331-1339PubMed Google Scholar). Myocardial hypertrophy frequently develops in older humans. Women have a lower overall incidence of left ventricular hypertrophy than men, but left ventricular hypertrophy in post-menopausal women exceeds the incidence in age-matched males (4Agabiti-Rosei E. Muiesan M.L. J. Hypertens. 2002; 20: S34-S38Google Scholar). The latter can be reversed in postmenopausal women by hormone replacement therapy (5Miya Y. Sumino H. Ichikawa S. Nakamura T. Kanda T. Kumakura H. Takayama Y. Mizunuma H. Sakamaki T. Kurabayashi M. Hypertens. Res. 2002; 25: 153-159Crossref PubMed Scopus (38) Google Scholar). Animal studies support a possible anti-hypertrophic action of estrogen in the heart. In ovariectomized mice, estrogen supplementation causes a 30% reduction in pressure overload-induced hypertrophy (6van Eickels M. Grohe C. Cleutjens J.P. Janssen B.J. Wellens H.J. Doevendans P.A. Circulation. 2001; 104: 1419-1423Crossref PubMed Scopus (263) Google Scholar). Although the basis is not known, one mechanism could be related to intracellular calcium. In this regard, the FKBP12.6 gene encodes a protein that modulates the intracellular ryanodine receptor and calcium store release. Disruption of this gene results in severe cardiac hypertrophy only in male mice. However, when female FKBP12.6 null mice are treated with tamoxifen, a specific estrogen receptor (ER) antagonist, hypertrophic changes similar to those in the male mice are observed (7Xin H.B. Senbonmatsu T. Cheng D.S. Wang Y.X. Copello J.A. Ji G.J. Collier M.L. Deng K.Y. Jeyakumar L.H. Magnuson M.A. Inagami T. Kotlikoff M.I. Fleischer S. Nature. 2002; 416: 334-338Crossref PubMed Scopus (260) Google Scholar). Perhaps estrogen dampens either the unregulated intracellular calcium sparking or resulting events that cause cardiac hypertrophy. The responsible mechanism for this important steroid action is obscure. In general, the actions of estrogen are mediated through nuclear ER by transcribing genes that encode proteins that lead to the biological effects. Additional actions of estrogen are mediated by plasma membrane ER-initiated steroid signaling. Such signaling impacts both transcriptional (8Pedram A. Razandi M. Aitkenhead M. Hughes C.C.W. Levin E.R. J. Biol. Chem. 2002; 277: 50768-50775Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar) and nontranscriptional effects of the sex steroid. We postulated that genes up-regulated by membrane ER signaling could oppose the development of cardiac hypertrophy and impact intracellular calcium signaling to this disorder. Here we report that estradiol (E2) limits vascular hormone-induced cardiomyocyte hypertrophy. In isolated cardiomyocytes, E2 inhibits the important hypertrophic pathway involving the calcium-sensitive protein phosphatase, calcineurin, as induced by the vascular peptides AngII or endothelin-1. We also found that AngII stimulates additional hypertrophic signaling involving ERK and PKC. E2 inhibits this signaling, dependent upon the ability of the sex steroid to up-regulate atrial natriuretic peptide (ANP) production and secretion. Thus, E2 modulates multiple signal inputs to prevent cardiomyocyte hypertrophy in vitro. Isolation and Culture of Rat Neonatal Cardiomyocytes—Myocytes were isolated from the hearts of 1-to-3-day-old rats or pregnant female rats (Charles River Laboratories) using a cardiomyocyte isolation kit (Worthington), according to the manufacturer's instructions. The cells were incubated in Dulbecco's modified Eagle's medium/F-12 supplemented with 10% fetal bovine serum, 1× ITS (insulin/transferrin/selenium) (Sigma) antibiotic and antimycotic, and 10 μg/ml fibronectin (to aid adherence). Measurement of Protein Synthesis, Protein Secretion, and Cell Area—After 24 h in media without serum, the cells were treated with either 100 nmol/liter AngII or 10 nmol/liter ET-1 or no treatment, in the presence or absence of 10 nmol/liter E2. New protein synthesis, a marker of hypertrophy, was determined using 3Hleucine uptake. The cells were cultured as above with the addition of 1 μCi/ml 3Hleucine for 48 h. After 48 h, the cells were rinsed in Dulbecco's modified Eagle's medium/F-12 and lysed with Trizol. The protein and RNA were extracted per the manufacturer's instructions. The protein fraction was counted in a scintillation counter, and the mean ± S.E. of the counts was were for and the results were by of was protein the was determined 24 h of with AngII ± E2 or E2 The was and using was Cell surface area was determined and by was of the area was by to the of using of a mean ± S.E. was for the surface area in The and of cells in this respect were determined and of gene in the was using was with for hypertrophic of cardiac hypertrophy in vivo and in or was used as a The were as skeletal muscle actin and reverse and GAPDH, and ANP, and and BNP, and and NF-AT was determined using a kit per the manufacturer's instructions. were treated as above for 24 h and and the proteins were was determined by of the in the presence of and this from the phosphatase activity. were for phosphatase and for and the were of the NF-AT transcription factor was determined by with a to a with NF-AT from of using the without NF-AT was determined by a were to and a The are the mean of NF-AT translocation from the to the nucleus was determined by Cell were by as M. A. J. Levin E.R. 2002; PubMed Scopus Google Scholar). and mRNA studies were as were by The were to with and The RNA was isolated to a with of for and were in and in were MCIP1 mRNA by the E2 for h and transcription h with nmol/liter RNA was isolated h the addition of the using specific to MCIP1 was used to the of mRNA were used as an The of the MCIP1 were to and for of the RNA were using and with cells were for 24 h and treated with hypertrophic estrogen as in gene was the RNA and or protein 48 h of the of protein were to of 48 h of for not and and were determined the by (8Pedram A. Razandi M. Aitkenhead M. Hughes C.C.W. Levin E.R. J. Biol. Chem. 2002; 277: 50768-50775Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, M. A. J. Levin E.R. 2002; PubMed Scopus Google Scholar). was determined by protein with 24 h of the with AngII ± E2. the of the kinase protein most were used for an in as for activity. proteins were the as was as the of to by the as we M. A. T. Levin E.R. J. Google Scholar). of ER in of ER were determined in cells by using we M. A. J. Levin E.R. 2002; PubMed Scopus Google Scholar). The presence of ER the plasma membrane of was determined by membrane proteins by as we M. A. Levin E.R. 18: PubMed Scopus Google Scholar). for and were from and and the cardiac was from AngII or of with the hypertrophic peptides AngII or ET-1 stimulated of protein with 10 E2 as as an 80% of the stimulated protein The inhibition by sex steroid in a a as as 1 E2, and was reversed by an ER We also determined the of a hypertrophic skeletal muscle actin a in actin mRNA in cells treated with AngII or However, in cells treated with either peptide E2, the in mRNA were significantly hypertrophy in vitro. We found that the mean area was stimulated by the hypertrophic peptides and was in cells ET-1 induced an in and actin with and both were reversed by E2 E2 no these not similar results were observed of the hypertrophic AngII or and AngII hypertrophy in through ET-1 Y. M. J. PubMed Scopus Google studies were with one of the and have that E2 stimulates gene expression, to sex steroid of hypertrophy De L.J. van Eickels M. Grohe C. van M. Doevendans P.A. Circulation. PubMed Scopus Google Scholar, M. J. S. A. 2001; PubMed Scopus Google Scholar). is also that and in the is up-regulated by a of hypertrophic and the natriuretic peptides are anti-hypertrophic J. J. 2002; Scholar). Thus, natriuretic peptide stimulation be a response that limits the of cardiac severe hypertrophy is We E2 stimulates and BNP, and this have the effects of E2. We determined the effects of AngII, E2, or both and gene 24 h. We found that AngII and E2 stimulated the gene of and We also found that AngII and E2 stimulated and the We the ability of E2 to production the effects of the sex steroid. We determined in the of AngII, E2, or with or without to the in E2 inhibited AngII-induced protein was prevented by a of that the anti-hypertrophic of the ANP, 100 also AngII-induced hypertrophy involves calcineurin, NF-AT translocation to the nucleus J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We found that the calcineurin a in results that hypertrophic effects of ET-1 are mediated in through a possible calcineurin and E2, we to overall phosphatase and calcineurin treated with ET-1 in both phosphatase and calcineurin In cells treated with ET-1 and E2, was a small reduction in overall phosphatase and a reduction in calcineurin activity. suggests that the E2 is specific to this phosphatase and not to overall stimulated by The ER antagonist, inhibited the of E2 but no action by latter is not a action of E2. events in the calcineurin pathway were by the and of the transcription and We determined that to the nucleus of to AngII, and the for an additional The of this transcription factor was and not suggests that a small but important of NF-AT in the hypertrophic We also determined that E2 significantly the AngII-induced in translocation to the nucleus results were found for not results that E2 AngII-induced transcriptional activity. were with a to the or the and treated with AngII E2. cells increased activity, with cells the or to cells with the NF-AT but were not to angiotensin in was observed when cells were treated with E2 In cells treated with both AngII and E2, of the increased observed in the AngII cells was Thus, E2 prevents the stimulation of NF-AT transcriptional activity, related in to nuclear translocation of the transcription MCIP1 by to E2 oppose the hypertrophic Perhaps this involves the calcineurin inhibitor MCIP1 A. S. A. PubMed Scopus Google Scholar, J. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). we treated with AngII or ET-1 of MCIP1 expression, above However, in MCIP1 were and AngII or ET-1 not The inhibitor the increased MCIP1 in of estrogen. results in cells (8Pedram A. Razandi M. Aitkenhead M. Hughes C.C.W. Levin E.R. J. Biol. Chem. 2002; 277: 50768-50775Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar) and that this estrogen action in in through of PI3K, activation a in estrogen of this we determined the ability of AngII, E2, or both to activation by We found that of either E2 or AngII stimulated and was no from both However, 24 h the AngII E2 an activation of and the steroid significantly inhibited AngII-induced The effects of E2 MCIP1 and of the hypertrophic were determined 24 h, is that E2 is through this we the effects of In this we also not a MCIP1 results that signaling to not through We E2 stimulates MCIP1 gene by transcriptional or by the stability of the we nuclear and mRNA stability from treated with or without estrogen were and the new RNA was used to a of for or with RNA from treated with E2 significantly increased of with cells were similar from both of results that E2 stimulates MCIP1 gene of the the cells were treated with E2 as but transcription was h by with μg/ml addition of the transcriptional mRNA was isolated and for the of addition of the DRB, MCIP1 were in the cells with the the of cells of mRNA isolated from the cells MCIP1 by h from the of addition of the estrogen stimulates both the transcription of and the stability of the MCIP1 E2 of Cardiac is important to the anti-hypertrophic effects of E2 were significantly related to we and these cardiomyocytes. to were not to protein and was cells treated with or MCIP1 with cells or cells with a MCIP1 a reduction in RNA The MCIP1 no We interfering RNA for MCIP1 cardiomyocyte protein stimulated by AngII was no in the of either or E2 significantly in cells incubated with both the steroid and AngII in the presence of However, prevented this anti-hypertrophic of E2 by by of results that is important in the inhibition of hypertrophy by E2. the of we treated with and for calcineurin activity. In E2 calcineurin activity. However, in cells treated with the in calcineurin of E2 was an important of MCIP1 to of calcineurin and related hypertrophy. of through ERK and by that AngII and ET-1 hypertrophic signaling in through ERK and activation H. M. S. J. J. PubMed Scopus Google Scholar, Wang C. Wang Y. A.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Both the AngII and ET-1 are and signaling the activation of ERK and PKC, resulting in cardiac hypertrophy Res. 2002; PubMed Scopus Google Scholar, van J. Cell Cardiol. Full Text PDF PubMed Scopus Google Scholar, J. J. Cardiol. Full Text Full Text PDF PubMed Scopus Google Scholar). is possible that E2 AngII-induced ERK and activation in the cardiomyocytes. We ERK and be to AngII-induced hypertrophy. in the ability of AngII to was significantly by E2, or by Thus, multiple signaling to the overall hypertrophic effects of We E2 modulates AngII-induced kinase activity. We found that AngII significantly stimulated activity, as was prevented by E2 and in reversed by We found that and prevented AngII-induced activity. suggests a from calcineurin to results were found for ERK the ability of E2 to AngII-induced ERK was only 24 h, when hypertrophic were determined in of After only both AngII and E2 stimulated ERK kinase activity. Thus, the of E2 is to kinase in a with anti-hypertrophic effects. the ability of E2 to MCIP1 impact the of and we an to MCIP1 or the cardiomyocytes, the cells 24 h, and incubated the cells with AngII ± E2 for 24 h. in MCIP1 not significantly reverse the ability of AngII to or E2 to either kinase activity. suggests that E2 of MCIP1 to ERK and activity. that inhibits and AngII-induced ERK and activation PubMed Scopus Google Scholar, T. Y. H. N. M. H. 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M. and E. mechanism for E2 action can be postulated from and by studies of mice. of the FKBP12.6 gene in the in vivo cardiomyocyte to calcium sparking through the ryanodine receptor (7Xin H.B. Senbonmatsu T. Cheng D.S. Wang Y.X. Copello J.A. Ji G.J. Collier M.L. Deng K.Y. Jeyakumar L.H. Magnuson M.A. Inagami T. Kotlikoff M.I. Fleischer S. Nature. 2002; 416: 334-338Crossref PubMed Scopus (260) Google Scholar). In these mice, only the males cardiac hypertrophy. However, female mice severe hypertrophy when the ER is from that estrogen modulates the of intracellular calcium and events that the hypertrophic We that FKBP12.6 calcineurin and cardiac hypertrophy (7Xin H.B. Senbonmatsu T. Cheng D.S. Wang Y.X. Copello J.A. Ji G.J. Collier M.L. Deng K.Y. Jeyakumar L.H. Magnuson M.A. Inagami T. Kotlikoff M.I. Fleischer S. 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Full Text PDF PubMed Scopus Google Scholar, J. J. Cardiol. Full Text Full Text PDF PubMed Scopus Google Scholar). We report that AngII-induced kinase activation contributes to overall hypertrophic and that E2 inhibits this signaling. We found that kinase activation by AngII from by calcineurin to ERK and activation is prevented by or Thus, the ability of E2 to calcineurin impacts hypertrophic signaling through ERK and PKC. to ERK and signaling have to in the of cardiomyocyte hypertrophic genes via mechanisms B.J. Biol. 25: PubMed Scopus Google Scholar). We not that the E2 inhibition of ERK and through we found that mediated the inhibition of ERK and PKC. of the overall E2 of hypertrophic signaling is in in E2 of hypertrophic signaling is with PubMed Scopus Google Scholar, T. Y. H. N. M. H. J. 1999; PubMed Scopus Google Scholar) that inhibits ET-1 and AngII signaling through ERK and PKC. possible from to calcineurin be was A. De L.J. H. A. M. J. Biol. Chem. 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Nature. 2002; 416: 334-338Crossref PubMed Scopus (260) Google Scholar) could in mediated through ER cells or In E2 important mechanisms of cardiomyocyte hypertrophy utilized by hypertrophic The effects of E2 a the of in a that could be in women hormone replacement the