Development Of Myocardial Hypertrophy Research Articles

Inhibition of Nuclear Import of Calcineurin Prevents Myocardial Hypertrophy

The time that transcription factors remain nuclear is a major determinant for transcriptional activity. It has recently been demonstrated that the phosphatase calcineurin is translocated to the nucleus with the transcription factor nuclear factor of activated T cells (NF-AT). This study identifies a nuclear localization sequence (NLS) and a nuclear export signal (NES) in the sequence of calcineurin. Furthermore we identified the nuclear cargo protein importinbeta(1) to be responsible for nuclear translocation of calcineurin. Inhibition of the calcineurin/importin interaction by a competitive peptide (KQECKIKYSERV), which mimicked the calcineurin NLS, prevented nuclear entry of calcineurin. A noninhibitory control peptide did not interfere with the calcineurin/importin binding. Using this approach, we were able to prevent the development of myocardial hypertrophy. In angiotensin II-stimulated cardiomyocytes, [(3)H]-leucine incorporation (159%+/-9 versus 111%+/-11; P<0.01) and cell size were suppressed significantly by the NLS peptide compared with a control peptide. The NLS peptide inhibited calcineurin/NF-AT transcriptional activity (227%+/-11 versus 133%+/-8; P<0.01), whereas calcineurin phosphatase activity was unaffected (298%+/-9 versus 270%+/-11; P=NS). We conclude that calcineurin is not only capable of dephosphorylating NF-AT, thus enabling its nuclear import, but the presence of calcineurin in the nucleus is also important for full NF-AT transcriptional activity.

Estrogen receptor alpha up‐regulation and redistribution in human heart failure

Clinical and animal studies suggest that estrogen receptors are involved in the development of myocardial hypertrophy and heart failure. In this study, we investigated whether human myocardial estrogen receptor alpha (ERalpha) expression, localization, and association with structural proteins was altered in end stage-failing hearts. We found a 1.8-fold increase in ERalpha mRNA and protein in end-stage human dilated cardiomyopathy (DCM, n=41), as compared with controls (n=25). ERalpha was visualized by confocal immunofluorescence microscopy and localized to the cytoplasm, sarcolemma, intercalated discs and nuclei of cardiomyocytes. Immunofluorescence studies demonstrated colocalization of ERalpha with beta-catenin at the intercalated disc in control hearts and immunoprecipitation studies confirmed complex formation of both proteins. Interestingly, the ERalpha/beta-catenin colocalization was lost at the intercalated disc in DCM hearts. Thus, the ERalpha/beta-catenin colocalization in the intercalated disc may be of functional relevance and a loss of this association may play a role in the progression of heart failure. The increase of total ERalpha expression may represent a compensatory process to contribute to the stability of cardiac intercalated discs.

ERK expression and activation in myocardial hypertrophy of spontaneously hypertensive rats with different ages

To investigate the expression and activation of extracellular signal-regulated kinases (ERK) in myocardial hypertrophy of spontaneously hypertensive rats (SHR) with different ages. SHRs and Wistar Kyoto (WKY) rats were divided into 4 groups (5-, 8-,14-, 24-week old) respectively. The ratio of left ventricle weight to body weight (LVW/BW) was measured as the indicator of myocardial hypertrophy. The basal ERK expression (b-ERK) and phosphorylated-ERK (p-ERK) in myocardial tissue were examined by Western blot. The blood pressure of SHR was significantly higher than that of age-matched WKY rats from 8-week-old (P<0.001). The ratio of LVW/BW in SHR was increased significantly from 14-week-old (P<0.01). There was no significant difference of b-ERK expression between SHR and age-matched WKY rats. The p-ERK level of 5-week-old SHR was similar to that of 5-week-old WKY rats. The p-ERK level of SHR was significantly higher than that of age-matched WKY rats from 8-week-old to 24-week-old (P<0.01). The ratio of LVW/BW was positively related to p-ERK level but not related to b-ERK. The activation of ERK may play an important role in the development of myocardial hypertrophy caused by hypertension.

Antihypertensive Therapy: Role of Aldosterone Antagonists

The interest for the therapeutic potential of aldosterone antagonists in essential hypertension comes from the recently discovered nonclassical pathways of aldosterone actions, above all the presence of extra-adrenal aldosterone production and the discovery of aldosterone's proinflammatory and profibrotic actions. The review begins with the discussion of experimental studies on animals, demonstrating the role of aldosterone in cardiovascular remodeling and the effects of aldosterone blockade on hypertensive target organ damage. Then recent clinical studies are presented, that confirm in humans the deleterious role of aldosterone, in particular in the development of myocardial hypertrophy, cardiovascular fibrosis and arterial stiffness. After a brief description of the new selective aldosterone antagonist, eplerenone, compared to the well-known non-selective aldosterone antagonist, spironolactone, the results of studies on essential hypertensive patients are discussed, evaluating the efficacy of aldosterone antagonists in lowering blood pressure, but, more important, in protecting against target organ damage.

Increased Collagen Deposition and Diastolic Dysfunction but Preserved Myocardial Hypertrophy After Pressure Overload in Mice Lacking PKCε

Overexpression and activation of protein kinase C-epsilon (PKCepsilon) results in myocardial hypertrophy. However, these observations do not establish that PKCepsilon is required for the development of myocardial hypertrophy. Thus, we subjected PKCepsilon-knockout (KO) mice to a hypertrophic stimulus by transverse aortic constriction (TAC). KO mice show normal cardiac morphology and function. TAC caused similar cardiac hypertrophy in KO and wild-type (WT) mice. However, KO mice developed more interstitial fibrosis and showed enhanced expression of collagen Ialpha1 and collagen III after TAC associated with diastolic dysfunction, as assessed by tissue Doppler echocardiography (Ea/Aa after TAC: WT 2.1+/-0.3 versus KO 1.0+/-0.2; P<0.05). To explore underlying mechanisms, we analyzed the left ventricular (LV) expression pattern of additional PKC isoforms (ie, PKCalpha, PKCbeta, and PKCdelta). After TAC, expression and activation of PKCdelta protein was increased in KO LVs. Moreover, KO LVs displayed enhanced activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), whereas p42/p44-MAPK activation was attenuated. Under stretch, cultured KO fibroblasts showed a 2-fold increased collagen Ialpha1 (col Ialpha1) expression, which was prevented by PKCdelta inhibitor rottlerin or by p38 MAPK inhibitor SB 203580. In conclusion, PKCepsilon is not required for the development of a pressure overload-induced myocardial hypertrophy. Lack of PKCepsilon results in upregulation of PKCdelta and promotes activation of p38 MAPK and JNK, which appears to compensate for cardiac hypertrophy, but in turn, is associated with increased collagen deposition and impaired diastolic function.

Changes in Myocardial Blood Supply during Experimental Hypertension Treated with Verapamil in Rabbits

Rabbit hearts were studied at different periods of experimental arterial hypertension and its treatment with verapamil. Morphological study revealed a progressive decrease in the number of capillaries per unit mass during the development of myocardial hypertrophy in hypertensive hearts. In hypertensive animals treated with verapamil the density of capillaries in both ventricles did not decrease, but verapamil injected to intact animals sharply decreased this parameter.

Myocardial activation of the β-Adrenergic Receptor Kinase (βARK1) by Protein Kinase C (PKC) in vivo

Body. Chronic stimulation of Gq-coupled receptors in the heart leads to PKC activation and myocyte hypertrophy, a precursor to heart failure. βARK1 is a G-protein-coupled receptor kinase (GRK) which phosphorylates and desensitizes agonist-occupied β-adrenergic receptors (βARs), which are critical for cardiac function. Our objective was to determine if myocardial PKC can activate βARK1 in vivo. We studied βARK1 expression by protein immunoblotting and βARK1 activity in the hearts of transgenic mice with cardiac-specific expression of a peptide which increases αPKC activity in the heart by 50% (αPKCact) versus non-transgenic controls. Data are expressed as mean ± SEM. Results were compared using Student’s t-test. Protein expression of βARK1 in myocardial extracts was not significantly different between αPKCact and control: n = 7 in each group, P > 0.05. In contrast, βARK1 activity, as measured by its ability to phosphorylate rhodopsin, was elevated 3-fold in cardiac membrane preparations from αPKCact mice versus control: 3100 ± 214 versus 1058 ± 149 densitometry units, P < 0.05, n = 6 in each group. Based on these data, it appears that αPKC can activate βARK1 in the heart. This may be an important mechanism of βAR dysfunction in the development of myocardial hypertrophy as βARK1 is the primary GRK expressed in the heart.

Functional consequence of serotonin/5-HT2B receptor signaling in heart: role of mitochondria in transition between hypertrophy and heart failure?

Para- and/or autocrine factors that interact with receptors coupled to G proteins of the Gq/11 families are implicated in the development of myocardial hypertrophy and apoptosis. Whether hypertrophy renders cardiac myocytes more sensitive or resistant to apoptosis is still controversial. A delicate balance may exist among hypertrophy, apoptosis, and heart failure. However, the factors involved in the transition from compensatory hypertrophy to heart failure have not been elucidated yet in the context of Gq-coupled pathways. Overexpressing serotonin Gq–coupled 5-HT2B receptor in the mouse heart induces mitochondrial proliferation associated with hypertrophy, whereas ablation of this receptor leads to mitochondrial structural and functional impairments associated with myofibrillar breakdown and dilated cardiomyopathy. In this review, we discuss a novel role of mitochondria in the transition from hypertrophic to dilated cardiomyopathy that involves the Gq-coupled 5-HT2BR signaling pathway. ### Role of Serotonin in the Heart Serotonin (5-hydroxytryptamine [5-HT]) was first isolated from blood and found to function as a vasoconstrictor. 5-HT is found in the following 3 main areas of the body: the intestinal wall (where it causes increased gastrointestinal motility), blood vessels (where it causes constriction of large vessels), and the central nervous system. Several lines of evidence suggest that 5-HT regulates cardiovascular function during embryogenesis and adulthood. 5-HT is secreted from enterochromaffin cells into the blood and stored in the platelets. Circulating 5-HT can also be taken up by sympathetic neurons and vascular endothelial cells and can be co-released. The effects of 5-HT in the cardiovascular system are complex: for example, 5-HT has been associated with bradycardia or tachycardia, hypotension or hypertension, and vasodilatation or vasoconstriction. Recent pharmacological studies suggested that compounds acting on 5-HT receptors could be of therapeutic use in the treatment of migraine, hypertension, and heart and vascular diseases.1,2 The various biological actions of 5-HT are mediated by numerous cognate receptors. It …

183 Role of the ubiquitin-proteasome system in the development of myocardial hypertrophy

183 Role of the ubiquitin-proteasome system in the development of myocardial hypertrophy

Release of Preformed Ang II from Myocytes Mediates Angiotensinogen and ET-1 Gene Overexpression In Vivo via AT1 Receptor

P. A. Modesti, S. Zecchi-Orlandini, S. Vanni, G. P olidori, I. Bertolozzi, A. M. Perna, L. Formigli, I. Cecioni, M. Coppo, M. Boddi and G. G. Neri Serneri. Release of Preformed Ang II from Myocytes Mediates Angiotensinogen and ET-1 Gene Overexpression in vivo via AT1 Receptor. Journal of Molecular and Cellular Cardiology (2002) 34, 1491–1500. The role of angiotensin II in pressure overload is still debated because notwithstanding its effects on myocyte contractility angiotensin II is not an obligatory factor for the development of hypertrophy. To define the role of angiotensin II in acute pressure overload we studied the effects of AT1 blockade (valsartan 80mg per day) on myocardial contractility, cardiac growth factor gene expression, and myocardial hypertrophy in aortic banded (60mmHg) pigs. Acute pressure overload caused an abrupt reduction of myocardial contractility, measured by the end-systolic stiffness constant, and a sharp increase in end-systolic stress which rapidly normalized (within 12h) in the placebo group. In AT1-blocked animals end-systolic stiffness constant remained significantly depressed up to 24h and end-systolic stress was still elevated up to 48h (both P<0.05 vs placebo). In both groups confocal microscopy revealed that granular staining of angiotensin II in cardiomyocyte cytoplasm disappeared after 30min of pressure overload. AT1 blockade abolished following cardiac overexpression of angiotensinogen and endothelin-1 genes as shown in RT-PCR studies and the consequent angiotensin II and endothelin-1 release in the coronary circulation. Conversely, insulin-like growth factor-I and ACE mRNA overexpression, as well as the onset of left ventricular mass increase, were not significantly affected by AT1 blockade. In conclusion: (1) mechanical stress releases preformed angiotensin II from myocyte in vivo; (2) the AT1 blockade abolishes cardiac angiotensin II and endothelin-1 production with delayed recovery of myocardial contractility; whereas (3) the overexpression of insulin-like growth factor-I gene and the development of myocardial hypertrophy are not angiotensin II-mediated effects.

Calibrated histochemistry applied to oxygen supply and demand in hypertrophied rat myocardium.

Oxygen supply and demand of individual cardiomyocytes during the development of myocardial hypertrophy is studied using calibrated histochemical methods. An oxygen diffusion model is used to calculate the critical extracellular oxygen tension (PO(2,crit)) required by cardiomyocytes to prevent hypoxia during hypertrophic growth, and determinants of PO(2,crit) are estimated using calibrated histochemical methods for succinate dehydrogenase activity, cardiomyocyte cross-sectional area, and myoglobin concentration. The model calculation demonstrates that it is essential to calibrate the histochemical methods, so that absolute values for the relevant parameters are obtained. The succinate dehydrogenase activity, which is proportional to the maximum rate of oxygen consumption, and the myoglobin concentration hardly change while the cardiomyocytes grow. The cross-sectional area of the cardiomyocytes, which increases up to threefold in the right ventricular wall due to pulmonary hypertension in monocrotaline-treated rats, is the most important determinant of PO(2,crit) in this model of myocardial hypertrophy. The relationship between oxygen supply and demand at the level of the cardiomyocyte can be investigated using paired determinations of spatially integrated succinate dehydrogenase activity and capillary density. Hypoxia-inducible factor 1alpha can be demonstrated by immunohistochemistry in cardiomyocytes with high PO(2,crit) and increased spatially integrated succinate dehydrogenase activity, indicating that limited oxygen supply affects gene expression in these cells. We conclude that a mismatch of oxygen supply and demand may develop during hypertrophic growth, which can play a role in the transition from myocardial hypertrophy to heart failure.

40 Development of myocardial hypertrophy in normotensive WKY rats

40 Development of myocardial hypertrophy in normotensive WKY rats

Echocardiographic assessment of LV hypertrophy and function in aortic-banded mice: necropsy validation.

We characterized the time course of the left ventricular (LV) geometric and functional changes after aortic banding, validated them by necropsy, and investigated the sensitivity of echocardiographic findings on LV hypertrophy. C57BL/6 mice were subjected to transverse aortic constriction (TAC) or sham operation; echocardiographic assessments were performed before or at 2, 4, 6, and 11 wk after surgery; and some of the mice were euthanized at the corresponding time points. There was a progressive increase in diastolic posterior wall thickness and LV systolic dimension; the percentage of LV fractional shortening (LV%FS) decreased progressively at 4 wk, whereas these parameters remained stable in sham-operated mice. Echo LV mass and LV%FS correlated well with actual whole heart mass and ratio of lung weight to body weight, respectively (r = 0.765 and -0.749, respectively; P < 0.0001). These results suggest that the development of myocardial hypertrophy and systolic dysfunction is a time-dependent process. Echocardiographic assessment of myocardial hypertrophy and functional changes correlate well with the actual heart mass and lung mass. Echocardiography is sensitive enough to assess myocardial hypertrophy and heart functional changes induced by pressure overload in mice.

Adrenergic activation of cardiac phospholipase D: role of alpha(1)-adrenoceptor subtypes.

Adrenergic stimulation of the heart leads to activation of the phospholipase D signal transduction pathway with formation of the intracellular second messengers phosphatidic acid and diacylglycerol, which may play a role in the development of myocardial hypertrophy by activating mitogen-activated protein kinases and protein kinase C. So far, the adrenergic receptor subtypes mediating activation of cardiac phospholipase D are not known. We developed an assay for determination of phospholipase D activity in the isolated perfused rat heart. Utilizing the phospholipase D specific transphosphatidylation reaction the stable product phosphatidylethanol (PEtOH) is formed in rat hearts perfused in the presence of 1% ethanol. Myocardial PEtOH formation was used as a marker of phospholipase D activity and was determined by HPLC and evaporative light-scattering detection (PEtOH microg/mg myocardial protein). Basal PEtOH formation in unstimulated hearts was 0.06+/-0.01 microg/mg. Stimulation of the hearts with norepinephrine resulted in a concentration-dependent phospholipase D activation with a maximum formation of PEtOH (0.17+/-0.01 microg/mg) at 100 micromol/l norepinephrine. The norepinephrine-induced increase in PLD activity was completely blocked by the alpha(1)-adrenoceptor antagonist prazosin and was unaffected by the beta-adrenoceptor antagonist propranolol. Further characterisation of alpha(1)-adrenoceptor subtypes with selective alpha(1)-adrenoceptor antagonists demonstrated a complete inhibition of the norepinephrine-induced phospholipase D activation by WB 4101 (alpha(1A)-selective: 0.06+/-0.01 microg/mg) and by BMY 7378 (alpha(1D)-selective: 0.07+/-0.01 microg/mg). In contrast, the alpha(1B)-adrenoceptor antagonist chloroethylclonidine had no inhibitory effect on norepinephrine-stimulated phospholipase D activity (0.14+/-0.01 microg/mg). Adrenergic activation of the cardiac phospholipase D signal transduction pathway is mediated by alpha(1)-adrenoceptors. Here, the alpha(1A)-adrenoceptor subtype, but not the alpha(1B)-adrenoceptor are coupled to activation of cardiac phospholipase D.

Regression of cardiomyocyte hypertrophy in SHR following chronic inhibition of the Na(+)/H(+) exchanger.

Experiments were performed to examine the effect of chronic inhibition of the Na(+)/H(+) exchanger isoform-1 (NHE-1) on cardiac hypertrophy of spontaneously hypertensive rats (SHR). SHR were orally treated during 1 month with two different doses (0.3 and 3.0 mg/kg/day) of the NHE-1 inhibitor, cariporide, or nifedipine (10.0 mg/kg/day). The two doses of cariporide did not differ in their effects after 1 month of treatment, since both induced a slight decrease in systolic blood pressure (SBP) of approximately 6 mmHg and regression of the heart weight to body weight ratio (mg/g) from 3.28+/-0.05 to 3.04+/-0.05 (0.3 mg) and 2.99+/-0.10 (3.0 mg, P<0.05). Nifedipine, given for the same period, produced similar reduction in the hypertrophy index (3.03+/-0.05), but with a much greater decrease in arterial pressure (35.6+/-7.4 mmHg). Chronic treatment with cariporide induced a complete regression of the augmented cross sectional area of left ventricular myocytes without significant changes in collagen content, serum procollagen 1 propeptide levels or myocardial distensibility. NHE inhibition represents a novel approach to induce regression of pathological hypertrophy of the heart. The finding can be rationalized mechanistically by previous in vitro studies suggesting a role of the NHE in the development of myocardial hypertrophy.

Correlations of the renin-angiotensin-system (RAS) gene polymorphisms with cardiac growth factors endothelin-1 and angiotensin II in high performance athletes

Background: The regulation of myocardial mass in athletes involves complex interactions between physical stress, cardiac growth factors (GF), and the individual genetic background. Studies suggest the renin-angiotensin-system (RAS) plays a key role in the development of myocardial hypertrophy. Genetic polymorphisms of the RAS, associated with an activation, have been shown to correlate with left ventricular mass (LVM) in athletes. It is not clear whether these polymorphisms influence angiotensin (A) II levels directly or only markers coprecipitating with other factors modifying LVM. Endothelin (ET)-1 acts synergistically to AII in the induction of cardiac hypertrophy. The role of these GF in an athlete's heart is not yet clarified. Objectives: To analyze the correlation of RAS-gene polymorphisms with AII and ET-1 plasma concentrations in high performance athletes. Methods: In 83 male Caucasian, mainly endurance trained, elite athletes, intensity and duration of exercise were assessed by questionnaires; LVM was determined by 2-dimensional echocardiography. Plasma concentrations of AII and ET-1 were determined by radioimmunoassay. Results: LV/VMi significantly above reported average levels in sedentary persons was observed. AII plasma restingconcentrations correlated with the ACE gene polymorphism (p = .039), finding the highest levels in DD-athletes. There were no further correlations between the AII levels and the angiotensinogen gene polymorphisms, and the combined polymorphisms (p =.585, p = .162). ET-1 plasma levels did not correlate with any of the investigated parameters. No GF correlates with LV/VMi. Conclusions: The highest AII plasma resting concentrations were found in athletes homozygous to DD. This finding suggests a functional effect of the ACE I/D genotypes, possibly influencing cardiac growth in high performance athletes. The absence of a correlation with ET-1 plasma levels does not rule out its influence; local cardiac effects remain elusive. Further longitudinal studies under controlled exercise conditions are necessary.

Absence of pressure overload induced myocardial hypertrophy after conditional inactivation of Galphaq/Galpha11 in cardiomyocytes.

Myocardial hypertrophy is an adaptational response of the heart to increased work load, but it is also associated with a high risk of cardiac mortality due to its established role in the development of cardiac failure, one of the leading causes of death in developed countries. Multiple growth factors and various downstream signaling pathways involving, for example, ras, gp-130 (ref. 4), JNK/p38 (refs. 5,6) and calcineurin/NFAT/CaM-kinase have been implicated in the hypertrophic response. However, there is evidence that the initial phase in the development of myocardial hypertrophy involves the formation of cardiac para- and/or autocrine factors like endothelin-1, norepinephrine or angiotensin II (refs. 7,8), the receptors of which are coupled to G-proteins of the Gq/11-, G12/13- and Gi/o-families. Cardiomyocyte-specific transgenic overexpression of alpha1-adrenergic or angiotensin (AT1)-receptors as well as of the Gq alpha-subunit, Galphaq, results in myocardial hypertrophy. These data demonstrate that chronic activation of the Gq/G11-family is sufficient to induce myocardial hypertrophy. In order to test whether Gq/G11 mediate the physiological hypertrophy response to pressure overload, we generated a mouse line lacking both Galphaq and Galpha11 in cardiomyocytes. These mice showed no detectable ventricular hypertrophy in response to pressure-overload induced by aortic constriction. The complete lack of a hypertrophic response proves that the Gq/G11-mediated pathway is essential for cardiac hypertrophy induced by pressure overload and makes this signaling process an interesting target for interventions to prevent myocardial hypertrophy.

Possible mechanism of regression of myocardial hypertrophy.

Structure of the myocardium was studied in rabbits with renovascular hypertension during the development of myocardial hypertrophy and its regression under the effect of beta-adrenoceptor antagonist lopressor. Myocardial hypertrophy was associated with ultrastructural changes in cardiomyocytes, while lopressor therapy led to their regression and normalization of cardiomyocyte ultrastructure. Regression of hypertrophic changes was accompanied by a marked increase in the number of extracellular nuclei, which indicated enhanced apoptosis of cardiomyocytes.

Congestive Heart Failure: Fifty Years of Progress

Congestive Heart Failure: Fifty Years of Progress

Gender-differences in myocardial adaptation to afterload in normotensive and hypertensive rats.

Echocardiographic studies suggest that women appear to exhibit a greater degree of myocardial hypertrophy in response to increased afterload than men. Therefore, gender differences and the role of estrogen and testosterone in the development of myocardial hypertrophy were studied in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. Male and female rats were either surgically neutered or underwent a sham operation at 21 days of age. A subgroup of neutered females of each strain received 17beta-estradiol replacement. At 6 months, the heart weight-to-body weight ratio was assessed and correlated with systemic blood pressure. Compared with males, females had significantly smaller body and heart weights in both normotensive and hypertensive strains. Despite this, females consistently had significantly greater heart weight-to-body weight ratios. In females, neutering significantly lowered the heart weight-to-body weight ratio in WKY rats, which was returned to intact levels with estrogen replacement. Female SHR showed similar, but not statistically significant, responses. In males, neutering appeared to result in a higher heart weight-to-body weight ratio in WKY rats, but the opposite was seen in SHR. In addition, there was a significant correlation between arterial blood pressure and heart weight-to-body weight ratio (systolic r=0.45, P=0.0015: diastolic r=0.52, P=0.0002) in intact males and females of both strains, and for a given diastolic pressure, females always exhibited a greater heart weight-to-body weight ratio than males. Thus, a greater degree of myocardial hypertrophy in females appears to be related to the presence of estrogen in both normotensive and hypertensive rats. Females show a stronger relationship between heart/body weight and blood pressure than males, which occurred independent of the presence of estrogen.