A more recent version of this article appeared on May 25, 2001

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 88/10/1020    most recent hh1001.090858v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Camper-Kirby, D. Articles by Sussman, M. A. Search for Related Content PubMed Articles by Camper-Kirby, D. Articles by Sussman, M. A. Related Collections Other myocardial biology Cell signalling/signal transduction

(Circulation Research. 2001;0:hh1001.090858.)
© 2001 American Heart Association, Inc.

Article

Myocardial Akt Activation and Gender

Increased Nuclear Activity in Females Versus Males

Dreama Camper-Kirby, Sara Welch, Angela Walker, Isao Shiraishi, Kenneth D. R. Setchell, Erik Schaefer, Jan Kajstura, Piero Anversa Mark A. Sussman

From the Divisions of Molecular Cardiovascular Biology (D.C.-K., S.W., A.W., I.S., M.A.S.) and Clinical Mass Spectrometry (K.D.R.S.), The Children’s Hospital Research Foundation, Cincinnati, Ohio; Biosource International (E.S.), Hopkinton, Mass; and Cardiovascular Research Institute (J.K., P.A.), New York Medical College, Valhalla, NY.

Correspondence to Dr Mark A. Sussman, Division of Molecular Cardiovascular Biology, The Children’s Hospital and Research Foundation, Room 3033, 3333 Burnet Ave, Cincinnati, OH 45229. E-mail sussman{at}heart.chmcc.org

Abstract

Abstract—Cardiovascular disease risk is higher in men than women, but the basis for this discrepancy remains controversial. Estrogenic stimulation of the myocardium or isolated cardiomyocytes has been purported to exert multiple beneficial effects associated with inhibition of maladaptive responses to pathogenic insults. This report describes a significant difference between the sexes in myocardial activation of Akt, a protein kinase that regulates a broad range of physiological responses including metabolism, gene transcription, and cell survival. We find that young women possess higher levels of nuclear-localized phospho-Akt⁴⁷³ relative to comparably aged men or postmenopausal women. Both localization of phospho-Akt⁴⁷³ in myocardial nuclei of sexually mature female mice versus males and Akt kinase activity in nuclear extracts of hearts from female mice versus males are elevated. Cytosolic localization of phospho-forkhead, a downstream nuclear target of Akt, is also increased in female relative to male mice, suggesting a potential mechanism for cardioprotective nuclear signaling resulting from Akt activation. Phospho-Akt⁴⁷³ levels and localization at cardiac nuclei are similarly increased in transgenic mice with myocardium-specific expression of insulin-like growth factor I, a proven stimulus for Akt activation. Phospho-Akt⁴⁷³ is also localized to the nucleus of cultured cardiomyocytes after exposure to 17ß-estradiol or genistein (a phytoestrogen in soy protein–based diets), and neonatal exposure of litters to genistein elevated nuclear phospho-Akt⁴⁷³ localization. The activation of Akt in a gender-dependent manner may help explain differences observed in cardiovascular disease risk between the sexes and supports the potential beneficial effects of estrogenic stimulation.

Key Words: Akt • gender • survival • phytoestrogen • insulin-like growth factor-1

The observed gender gap in heart disease has led to considerable speculation regarding the underlying etiology.^{1 2} Compelling evidence from multiple experimental and clinical investigations indicates that estrogen plays a pivotal role in reducing risk for cardiovascular disease.³ Consequently, this evidence has been used for touting estrogen as having a beneficial effect in the recommendation of hormone replacement therapy (HRT) to postmenopausal women in order to ameliorate cardiovascular disease risk. Although the rationale for HRT remains controversial,^{4 5} multiple epidemiological studies show that congestive heart failure is more prevalent in men when compared with age-matched women.⁶ The link with estrogen is therefore quite strong, even though the specific molecular signals explaining why women are more protected from heart failure remain unknown.

One molecular mechanism proven to reduce cytopathic damage associated with myocardial injury involves the activation of the serine/threonine protein kinase called Akt (also known as protein kinase B). Akt lies at the intersection of multiple cellular signaling pathways involved in regulation of glucose metabolism, gene transcription, protein synthesis, the cell cycle, and cell survival.⁷ Akt is the downstream effector molecule for signal transduction initiated by membrane receptors such as the insulin and insulin-like growth factor I (IGF-I) that activate phosphatidylinositol 3-kinase (PI3-K).^{8 9 10 11 12} Signaling mediated by IGF-I or PI3-K exerts multiple beneficial effects in cardiac biology.^{13 14 15 16} The potential for therapeutic relevance of this pathway is supported by the observation that activation of Akt inhibits apoptosis in cultured cardiomyocytes and diminishes ischemia-reperfusion injury in vivo.^{17 18} Most importantly, in the context of our present study, PI3-K–mediated Akt activation resulting from estrogen treatment has been recently demonstrated.^{19 20}

Multiple binding partners and intracellular substrates for Akt have been identified. Interestingly, temporal changes in Akt localization occur after activation, with Akt starting out in the cytoplasm, then moving to a membrane proximal position, and ultimately accumulating in the nucleus.^{21 22} Nuclear translocation of activated Akt after phosphorylation is widely accepted, and published reports suggest that biologically relevant targets of the active, phosphorylated form of Akt are likely to be nuclear. One such substrate of Akt is the proapoptotic transcription factor, forkhead, which is found in the nucleus. After phosphorylation by Akt, forkhead translocates from the nucleus to the cytoplasm.^{23 24 25} Data presented in the current study reveal a gender-specific localization of phospho-Akt⁴⁷³ in female versus male humans and mice that also correlated with the predicted phosphorylation and localization of forkhead transcription factor. Nuclear localization of phospho-Akt⁴⁷³ was also increased in vivo and in vitro by the phytoestrogen genistein, which can act as an agonist at estrogen receptors. These findings may help to explain some of the protective cardiovascular effects reported to be associated with being female or consuming phytoestrogen-enriched diets that contain soy products.

Materials and Methods

Human Samples
Myocardial samples were obtained at autopsy from 9 adults who had died from causes other than cardiovascular disease. The age of the patients and the number of tissue samples obtained from each is indicated in Table 1. To our knowledge, there was no evidence of prior use of HRT by any of the subjects. Formalin-fixed sections of the left ventricular myocardium were deparaffinized and heated in a microwave oven for 10 minutes in citrate buffer, pH 6.0. After washing in PBS, samples were incubated with phospho-Akt⁴⁷³ antibody diluted 1:40 (12.5 µg/mL) in PBS.

View this table: [in this window] [in a new window]   Table 1.

Animals
FVB/N mice bred and raised in the vivaria facility for use in experiments were maintained on a diet with a low phytoestrogen content (2014 diet; Harlan). Syngeneic transgenic mice expressing IGF-I specifically in the myocardium have been previously described.¹³ Neonatal rat cardiomyocyte cultures were prepared as previously described²⁶ and cultured in serum-free medium for 48 hours before estrogenic stimulation. All experiments were conducted in accordance with the Guide for the Use and Care of Laboratory Animals and approved by the Institutional Animal Care and Use Committee.

Reagents for Estrogenic Stimulation
17ß-estradiol and the nonsteroidal estrogen genistein (both obtained from Sigma Biochemicals) were used for the in vitro studies at concentrations of 10^-6 mol/L and 250 µg/mL, respectively. Cultures were treated overnight and prepared for microscopic analysis the following morning. For the in vivo experiments, nursing females were injected daily with 1 mg/day IP of genistein beginning 3 days after birth.

Antibodies and Fluorescent Conjugates
Phosphorylation site–specific antibodies to phosphoserine⁴⁷³ on Akt (Biosource International, Camarillo, Calif) and phosphoserine²⁵⁶ on forkhead (Cell Signaling Technologies, Beverly, Mass) were used for both microscopy and immunoblotting experiments. Human myocytes were identified by labeling with mouse monoclonal anticardiac myosin heavy-chain ß antibody (monoclonal antibody 1548, Chemicon, Temecula, Calif) and Cy5-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, Pa). Nuclei were labeled with propidium iodide (Molecular Probes, Eugene, Oreg) at 10 µg/mL. Mouse myofibrils were labeled with antibody to sarcomeric -actinin (Sigma Immunochemicals, St Louis, Mo). An antibody to histone 4, which was used to normalize sample loading in immunoblots, was the generous gift of Dr Bruce Aronow (Childrens Hospital Research Foundation, Cincinnati, Ohio).

Microscopic Analyses
Sections were prepared from hearts that were fixed in 4% paraformaldehyde/PBS overnight at 4°C. The next day, hearts were subjected to a progressive sucrose gradient of 10%, 20%, and then 30% at 4°C, allowing 1 hour at each step to achieve equilibration of the heart with the sucrose solution. Sucrose-infiltrated hearts were prepared for confocal microscopy as previously described.²⁷ Nuclei that could be clearly identified were counted for the analysis of phospho-Akt⁴⁷³ localization. Any staining for nuclei with coincident ambiguous phospho-Akt⁴⁷³ labeling was considered negative.

Biochemical Analyses
Preparation of nuclear and cytosolic extract fractions was performed according to the method of Liew et al²⁸ with some modifications for use with mice. Details of this protocol are available for viewing in the online data supplement at http://www.circresaha.org.

Immunoblots
Samples were assayed using standard techniques as described in the online supplement. Kinase assays were performed using an Akt kinase activity kit (Cell Signaling Technologies) as recommended by the manufacturer.

Statistics
All determinations for significance were performed by Student t test analysis of sample populations using Microsoft Excel. Values of P<0.01 were considered significant.

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

Results

Nuclear Accumulation of Phospho-Akt⁴⁷³ Is Higher in Women Versus Men and Decreases in Women After Menopause
Myocardial sections were obtained from young men as well as from premenopausal and postmenopausal women (Table 1). Sections were stained with antibody to phospho-Akt⁴⁷³ and propidium iodide to label nuclei. This analysis clearly identified the presence of anti–phospho-Akt⁴⁷³ antibody staining in the nucleus (Figure 1). Quantification of nuclear phospho-Akt⁴⁷³ labeling revealed a significant (P=0.0002) increase in adult premenopausal women (34.3%±4.5) versus men (5.9%±1.5). In contrast to the tissue samples from younger women, phospho-Akt⁴⁷³ nuclear labeling was low in sections from postmenopausal women (1.9%±1.1). The level of nuclear phospho-Akt⁴⁷³ labeling was not significantly different in tissue samples from postmenopausal women and men (P=0.29). Declining phospho-Akt⁴⁷³ level in older women could be accounted for by diminished estrogenic stimulation, but the possibility of an age-associated reduction in Akt activity independent of estrogen should also be considered.

View larger version (149K): [in this window] [in a new window]   Figure 1. Nuclear phospho-Akt473 localization is increased in women relative to men but decreases after menopause. Shown are examples of labeling in sections from 2 young women (fem 1 and fem 2), 1 man (male), and 1 postmenopausal woman (fem 3). Arrows indicate nuclei staining for phospho-Akt473 (green), together with all nuclei (blue) and antibody to myosin heavy chain ß to show distribution of cardiomyocytes (red). Frequency of nuclear phospho-Akt473 reactivity is increased in sections from young female hearts (see Table 1). Magnification x550 for all sections.

Nuclear Accumulation of Phospho-Akt⁴⁷³ Is Higher in Female Relative to Male Mice
Nuclear extracts were prepared from pooled hearts of young adult mice at 45 days after birth. For this analysis, females were housed separately from males after weaning to prevent alteration of hormonal balance resulting from pregnancy. Immunoblots using anti–phospho-Akt⁴⁷³ antibody show a 1.98±0.53-fold increase in immunoreactivity with female relative to male samples (Figure 2, top left), which was statistically significant (P<0.01, n=8 experiments). Minor variations in the loading of nuclear preparation samples for immunoblot analysis were corrected by standardization relative to histone protein (H4) level. Similarly, assessment of Akt kinase activity in immunoprecipitates from nuclear extracts showed a significantly higher level in females than in males (1.5±0.3-fold, n=5, P=0.01) as measured by substrate phosphorylation of glycogen synthase kinase 3 (GSK; Figure 3). These differences did not result from changes in Akt protein levels, which were comparable between the 2 immunoprecipitated nuclear extracts (Figure 3). Total Akt protein levels in unfractionated heart lysates were also shown to be equivalent regardless of gender (data not shown), indicating that sexual differences in phospho-Akt⁴⁷³ and Akt kinase activity are unrelated to accumulation of protein. These findings were corroborated by confocal microscopy, which showed nuclear phospho-Akt⁴⁷³ localization in myocardial sections of 28.5±5.7% for females compared with only 14.3±2.7% in males (Figure 2, left; Table 2). Statistical analyses comparing the different groups are presented in Table 2. These results demonstrate that young adult female mice possess higher levels of nuclear phospho-Akt⁴⁷³ than comparably aged male mice.

View larger version (92K): [in this window] [in a new window]   Figure 2. Nuclear phospho-Akt473 localization is increased in female mice relative to males and is induced by treatment with IGF-I or genistein. Shown are representative immunoblots (top) and confocal micrographs (bottom) of mouse hearts. The figure is organized by experimental comparison, with individual sets grouped according to gender (left), TIGFO mice (center), and genistein-treated mice (right). Protein loading was normalized to histone protein (H4) level. Bottom, Confocal microscopy of phospho-Akt473 labeling in hearts from 6-week-old (left) and 3-week-old mice (center and right) used for quantification in Table 2. Each set of micrographs was acquired at identical settings for illumination and sensitivity. Nuclei considered negative for Akt immunoreactivity show staining for propidium iodide alone in red (examples indicated by arrowheads), whereas nuclei with coincident Akt label appear green/yellow (examples indicated by arrows). Female mice show a preponderance of nuclear phospho-Akt473 reactivity compared with age-matched males, as do TIGFO mice (center) and genistein-treated litters (right) analyzed at 3 weeks after birth compared with age-matched nontransgenic controls. Bars in photomicrographs are 10, 20, or 40 µm as indicated.

View larger version (18K): [in this window] [in a new window]   Figure 3. Akt activity is increased in nuclear extracts of female vs male mouse hearts. Immunoblot analysis shows relative Akt activity measured by phosphorylation of a GSK substrate and measured by binding of anti–phospho-GSK (p-GSK) antibody. Total Akt was immunoprecipitated from either male or female extracts separately and used for phosphorylation of GSK. Top, Densitometric analysis shows a 1.5-fold increase in phospho-GSK signal in the female vs the male sample. Bottom, Probing the same samples shows the total amount of Akt protein was similar in both samples, indicating a higher level of Akt specific activity in the female sample rather than elevation of Akt protein level.

View this table: [in this window] [in a new window]   Table 2. Nuclear Localization of Phospho-Akt473 in Mice by Confocal Microscopy

Nuclear Accumulation of Phospho-Akt⁴⁷³ in Transgenic Mice That Produce IGF-I in the Myocardium
IGF-I is known to stimulate Akt, so transgenic IGF-I–overexpressing (TIGFO) mice producing IGF under control of a cardiac-specific promoter activated at or shortly after birth were used to assess potential correlation between Akt activation and nuclear localization in vivo. For this analysis, nuclear extracts were prepared from pooled hearts of sexually immature mice at 3 weeks after birth. This age was chosen to avoid differences in Akt stimulation resulting from gender (see Table 2) that would have otherwise complicated the interpretation of IGF-I–mediated effects. Nuclear localization of phospho-Akt⁴⁷³ in juvenile mice was similar in 3-week-old male and female mice (data not shown), confirming our contention that gender differences would not be a confounding variable at this premature age. In comparison, TIGFO mice at 3 weeks of age (Figure 2, center) showed levels of nuclear phospho-Akt⁴⁷³ staining that were significantly higher than in age-matched nontransgenic controls (2.6±1.0-fold increase, P<0.01, n=5 experiments). Nuclear phospho-Akt⁴⁷³ localization in TIGFO mice was also >2-fold above that of normal female adult mice (Table 2), consistent with the enhanced cardioprotective phenotype of TIGFO mice compared with nontransgenic controls.¹³ These results demonstrate that constitutive IGF-I production in TIGFO mice promotes myocardial Akt phosphorylation and nuclear localization in vivo, under conditions in which a gender-independent cardioprotective effect is also observed.

Forkhead Protein Shows Increased Phosphorylation in Female Mice Consistent With Akt Activation
Phospho-forkhead²⁵⁶ protein levels were compared between 6-week-old male versus female samples by immunoblot and confocal microscopy analyses (Figure 4). Phospho-forkhead²⁵⁶ protein level was significantly increased 1.7±0.2-fold (P=0.01; n=4) in the cytosol of female samples relative to age-matched males. Confocal microscopy also shows increased immunoreactivity in female heart sections relative to males, in agreement with the immunoblotting results. In addition, the nonnuclear localization of phospho-forkhead²⁵⁶ immunoreactivity is consistent with previous studies illustrating the translocation of the forkhead protein from the nucleus to the cytosol on phosphorylation.^{23 24 25} Elevated phosphorylation of forkhead protein phosphorylation at serine²⁵⁶, a known target site for Akt, in female hearts relative to males illustrates a potential key gender difference in Akt-associated cardiac signaling.

View larger version (64K): [in this window] [in a new window]   Figure 4. Increased phosphorylation of forkhead protein in male vs female mice. Shown are a representative immunoblot (top) and confocal microscopy (bottom) of phospho-forkhead (p-FKHD) in mouse hearts. Top, Hearts were separated by gender, and 3 hearts for each gender were combined to create the immunoblot samples. Minor variations in loading of cytosolic samples for immunoblot analysis were corrected by normalizing relative to GAPDH. Quantification reveals a 1.7±0.2-fold increase in phospho-forkhead in female relative to male samples. Bottom, Confocal microscopy of phospho-forkhead (green) immunoreactivity in heart sections from 6-week-old mice. Female sample shows higher phospho-forkhead labeling than the age-matched male. Propidium iodide–labeled nuclei are shown in red. Both scans shown for microscopic analysis were acquired at identical settings for illumination and sensitivity. Bar=20 µm for both photomicrographs.

Nuclear Accumulation of Phospho-Akt⁴⁷³ and Cytoplasmic Phospho-Forkhead²⁵⁶ Reactivity Is Induced by Estrogenic Stimulation in Cultured Cardiomyocytes
Cultured cardiomyocytes allow for the use of defined medium and circumvent concerns related to contributory effects of paracrine signaling mechanisms in vivo. Thus, neonatal rat cardiomyocyte cultures were incubated overnight with 17ß-estradiol and then examined for localization of phospho-Akt⁴⁷³ and phospho-forkhead²⁵⁶ the next day. The morphological phenotype of 17ß-estradiol–treated cells was similar to that of untreated controls, with no evidence of remodeling such as hypertrophic enlargement. Confocal microscopy shows that exposure to 17ß-estradiol increased nuclear reactivity for phospho-Akt⁴⁷³ (Figure 5, left), as well as cytoplasmic immunoreactivity for phospho-forkhead²⁵⁶ (Figure 5, right), consistent with the postulate that estrogen can activate Akt in cardiomyocytes.

View larger version (59K): [in this window] [in a new window]   Figure 5. Nuclear phospho-Akt473 and cytosolic phospho-forkhead256 localization is stimulated in cultured cardiomyocytes by administration of 17ß-estradiol or genistein. Cells were treated with either 10-8 mol/L 17ß-estradiol or 250 µmol/L genistein overnight and processed for confocal microscopy the following morning. Cells were labeled with antibodies to phospho-Akt473 (left side; green) or phospho-forkhead256 (right side; green) and -actinin (blue), as well as propidium iodide to stain nuclei (red). Treatment with either estrogenic stimulus results in accumulation of phospho-Akt473 in the nucleus and increased cytoplasmic reactivity for phospho-forkhead256. Nuclear phospho-forkhead256 labeling showed a punctate pattern with variable intensity between individual cells in treated cultures that may reflect differences in the efficiency of the phospho-forkhead256 protein export. Bar=20 µm for all phospho-Akt473 photomicrographs and 10 µm for all phospho-forkhead256 photomicrographs as indicated.

Genistein, a naturally occurring dietary phytoestrogen that is a member of the selective estrogen receptor modulator (SERM) family, is a partial estrogen agonist both in vitro²⁹ and in vivo.^{30 31} To determine whether this SERM could induce nuclear phospho-Akt⁴⁷³ accumulation in the neonatal cardiomyocyte cultures, cells were incubated overnight with genistein and examined by confocal microscopy the next day. As with 17ß-estradiol–treated cells, the morphology of genistein-treated cultures was similar to that of untreated controls, and there was no evidence of remodeling such as hypertrophic enlargement. Confocal microscopy revealed the accumulation of phospho-Akt⁴⁷³ at the nucleus of cardiomyocytes after exposure to genistein (Figure 5, left), similar to results obtained using 17ß-estradiol. Cytoplasmic immunoreactivity for phospho-forkhead²⁵⁶ was also increased (Figure 5, right), consistent with that previously observed after 17ß-estradiol treatment. Collectively, these results demonstrate that 2 different estrogen agonists can induce nuclear accumulation of phospho-Akt⁴⁷³ and cytosolic immunoreactivity for phospho-forkhead²⁵⁶ in cultured cardiomyocytes.

Genistein Mediates Nuclear Accumulation of Phospho-Akt⁴⁷³ In Vivo
Because genistein stimulates nuclear phospho-Akt⁴⁷³ accumulation in vitro (Figure 5), experiments were performed to determine the effect of genistein administration on phospho-Akt⁴⁷³ distribution in vivo. For this analysis, genistein was administered to lactating females beginning at 3 days after birth and continued for the duration of the experiment. Offspring were allowed to suckle ad libitum as a means for delivering genistein to the young. Nuclear extracts were prepared from pooled hearts obtained from 3-week-old sexually immature suckling mice to avoid differences in Akt activity in older animals resulting from gender (see Table 2). Suckling pups showed significantly increased levels of circulating genistein (6.6-fold over vehicle-treated controls, P=0.0012) as determined by mass spectrometric analysis of serum samples (see online data supplement available at http://www.circresaha.org). Serum levels of 2 other isoflavones, daidzein and its metabolite equol, were also significantly elevated in litters from treated females relative to vehicle-injected controls (6.8- and 108.9-fold, respectively; P<0.0001). Sections of myocardium from genistein-exposed pups showed marked nuclear localization of phospho-Akt⁴⁷³ as determined by confocal microscopy. Anti–phospho-Akt⁴⁷³ nuclear staining levels were 8-fold higher in genistein-treated mice (84.1±4.6%) compared with age-matched untreated juvenile controls (10.2±2.2%; Figure 2, bottom right; Table 2). Immunoblots of the corresponding nuclear extracts showed a less dramatic, although still significantly different (P<0.01, n=5 experiments), 1.34±0.16-fold increase in phospho-Akt⁴⁷³ with genistein-treated mice relative to untreated control samples (Figure 2, top right). These results demonstrate that genistein, alone or in combination with other isoflavone metabolites, induces accumulation of nuclear myocardial phospho-Akt⁴⁷³ in vivo. However, the modest increase in immunoreactivity detected by Western blotting coupled with the high percentage of nuclear labeling as seen by confocal microscopy suggests a lower average level of phospho-Akt⁴⁷³ per labeled nucleus than that observed in mature females or TIGFO mice, which may be consistent with the partial estrogen agonist effects of genistein.

Discussion

To our knowledge, this study is the first demonstration of nuclear signal transduction in the myocardium that could account for prior observations of gender- and diet-associated differences in myocardial disease risk. Our demonstration of increased nuclear phospho-Akt⁴⁷³ in females versus males indicates that gender influences signal transduction in the myocardium.

A wide variety of factors including IGF-I, ß-adrenergic or G protein–coupled receptor agonists, and stimulation of protease-activated receptors can induce Akt activation in cardiomyocytes. Experiments with cultured vascular endothelial cells^{19 20} or cardiomyocytes (Figure 5) indicate that estrogenic stimulation can also activate Akt. Convergent signaling of these diverse pathways on Akt presumably reflects important cross talk between signal transduction mechanisms, as has been reported for estrogen-mediated stimulation of the IGF-I receptor pathway,³² activation of the IGF-I receptor by estrogen-mediated stimulation of PI3-K,³³ and increased Akt activity in vitro after estradiol or IGF-I treatment of carcinoma cells.³⁴ Akt activation by estrogen could also be the basis for inhibition of apoptosis induced by staurosporine treatment of cultured cardiomyocytes³⁵ (J. Molkentin, personal communication, January 2001), especially given that cardiomyocytes possess functional estrogen receptors.^{36 37} Functional benefits for the heart provided by estrogenic stimulation include cardioprotection from ischemia-reperfusion injury in ovariectomized rats,³⁸ inhibition of pressure overload–induced hypertrophy (L. DeWindt, personal communication, January 2001), and phenotypic rescue of transgenic mouse models of dilated cardiomyopathy (M. Sussman, unpublished results, 2000). Reports of gender-associated differences in murine cardiomyopathic phenotypes^{39 40} could potentially have a mechanistic basis via differences in phospho-Akt⁴⁷³ levels observed in this study.

Within the nucleus, targets for Akt-mediated phosphorylation include members of the forkhead-related transcription factor family,^{23 24 25} cAMP-responsive element binding protein (CREB),⁴¹ nuclear factor-B,^{42 43} and an S6 kinase–related kinase.⁴⁴ Akt exerts opposing inhibitory (forkhead-related) versus stimulatory (nuclear factor-B and CREB) gene transcription, but all of these actions are postulated to promote cell survival. Lethal dilated cardiomyopathy resulting from cardiac-specific expression of dominant-negative CREB in transgenic mice is consistent with this idea.⁴⁵ Increased phospho-forkhead²⁵⁶ levels (Figure 3), which have been linked to antiapoptotic effects in non-cardiac cells,²⁴ could account for decreased apoptosis in the human female failing heart relative to males.⁴⁶

Because immunofluorescence is not quantitative and intensity of nuclear labeling can be variable, in the current study, we routinely used immunoblotting to verify increased amounts of phosphoproteins from mouse myocardium. By immunoblotting, levels of both phospho-Akt⁴⁷³ and phospho-forkhead²⁵⁶ were shown to be elevated in samples from female mice relative to male mice. Variation in the level of cytoplasmic Akt activity, the intensity of Akt activation within individual nuclei, and the percentage of nuclei showing Akt immunoreactivity could all contribute as potential modulators leading to variation in phenotypic consequences of Akt activation.

The effect of estrogenic stimulation of Akt and its potential role in ameliorating cardiovascular disease has a potentially important connection to nutrition. Anecdotal evidence that cardiovascular disease risk may be lowered by estrogenic stimulation can be found in literature related to HRT (reviewed in Reference 3³ ) and to diets that are rich in phytoestrogens.^{47 48 49} Although most studies have been concerned with vascular effects and serum lipid levels,⁵⁰ it is reasonable to postulate that beneficial effects may also extend to the myocardium. These plant-based compounds include the isoflavones genistein and daidzein, which are naturally highly enriched in soybean products.^{51 52} Genistein acts as a partial estrogen agonist and shows selective binding to estrogen receptor ß that is highly localized in the vascular tree.^{30 53 54} Genistein has a half-life of almost 8 hours and attains extremely high concentrations in serum when soy foods are consumed.⁵² We have demonstrated that genistein treatment induces nuclear accumulation of phospho-Akt⁴⁷³ both in cultured cardiomyocytes (Figure 5) and in the myocardium of mice (Figure 2). Therefore, estrogenic effects of a phytoestrogen-enriched diet could significantly influence myocardial signaling, because most commercially available rodent chow is highly enriched in genistein and other isoflavones that could result in daily intakes of up to 2.5 mg of dietary genistein for an adult mouse.⁵⁵ Increased Akt activation has been observed in the myocardium of mice fed chow enriched for phytoestrogens relative to age-matched mice fed a diet with negligible phytoestrogen content (data not shown). Genistein administration is well tolerated, has high bioavailability via oral intake, and inhibits pathogenesis in transgenic mouse models of dilated cardiomyopathy (M. Sussman, unpublished results, 2000), suggesting a potential novel therapeutic approach for treatment of dilation. However, it is important to point out that the integrated consequences resulting from genistein versus estrogen exposure are likely to have distinct differences in the activation of signal transduction pathways. Regardless of the signaling pathway(s) involved, the implications for this observation are profound, as the answer could potentially impact all researchers studying myocardial signal transduction affected by either estrogen or crossover signaling in the myocardium between the estrogen and IGF receptors. Experiments are underway to determine whether the gender- and estrogen-associated differences described in this study are absent from mice lacking functional PI3-K, the functional intermediate in the estrogen pathway leading to Akt activation in endothelial cells.

Acknowledgments

This research was funded by grants to M.A.S. from the National Institutes of Health (Grant HL58224-02) as well as Grant-in-Aid and Established Investigator Awards from the American Heart Association National Organization (Grants 9750638N and 0040051N). We are grateful to Jeffrey Molkentin and Gary Schwartzbauer for helpful discussions.

Footnotes

Original received December 19, 2000; revision received March 30, 2001; accepted March 30, 2001.

References

1. Rosano GM, Panina G. Cardiovascular pharmacology of hormone replacement therapy. Drugs Aging. 1999;15:219–234.

2. Weinberg EO, Theinelt CD, Katz SE, Bartunek J, Tajima M, Rohrbach S, Douglas PS, Lorell BH. Gender differences in molecular remodeling in pressure overload hypertrophy. J Am Coll Cardiol. 1999;34:264–273.

3. Hayward CS, Kelly RP, Collins P. The roles of gender, the menopause and hormone replacement on cardiovascular function. Cardiovasc Res. 2000;46:28–49.

4. Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B, Vittinghoff E. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. JAMA. 1998;280:605–612.

5. Blumenthal RS, Zacur HA, Reis SE, Post WS. Beyond the null hypothesis: do the HERS results disprove the estrogen/coronary heart disease hypothesis? Am J Cardiol. 2000;85:1015–1017.

6. Gottdiener JS, Arnold AM, Aurigemma GP, Polak JF, Tracy RP, Kitzman DW, Gardin JM, Rutledge JE, Boineau RC. Predictors of congestive heart failure in the elderly: the cardiovascular health study. J Am Coll Cardiol. 2000;35:1628–1637.

7. Kandel ES, Hay N. The regulation and activities of the multifunctional serine/threonine kinase Akt/PKB. Exp Cell Res. 1999;253:210–229.

8. Burgering BM, Coffer PJ. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature. 1995;376:599–602.

9. Franke TF, Kaplan DR, Cantley LC. PI3K: downstream AKTion blocks apoptosis. Cell. 1997;88:435–437.

10. Franke TF, Kaplan DR, Cantley LC, Toker A. Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science. 1997;275:665–668.

11. Marte BM, Downward J. PKB/Akt: connecting phosphoinositide 3-kinase to cell survival and beyond. Trends Biochem Sci. 1997;22:355–358.

12. Vanhaesebrock B, Alessi DR. The PI3K-PDK1 connection: more than just a road to PKB. Biochem J. 2000;346:561–567.

13. Li Q, Li B, Wang X, Leri A, Jana KP, Liu Y, Kajstura J, Baserga R, Anversa P. Overexpression of insulin-like growth factor-1 in mice protects from myocyte death after infarction, attenuating ventricular dilation, wall stress, and cardiac hypertrophy. J Clin Invest. 1997;100:1991–1999.

14. Lee WL, Chen JW, Ting CT, Ishiwata T, Lin SJ, Korc M, Wang PH. Insulin-like growth factor I improves cardiovascular function and suppresses apoptosis of cardiomyocytes in dilated cardiomyopathy. Endocrinology. 1999;140:4831–4840.

15. Wang L, Ma W, Markovich R, Chen J-W, Wang PH. Regulation of cardiomyocyte apoptotic signaling by insulin-like growth factor I. Circ Res. 1998;83:516–522.

16. Otani H, Yamamura T, Nakao Y, Hattori R, Kawaguchi H, Osako M, Imamura H. Insulin-like growth factor-I improves recovery of cardiac performance during repurfusion in isolated rat heart by a wortmannin-sensitive mechanism. J Cardiovasc Pharmacol. 2000;35:275–281.

17. Matsui T, Li L, del Monte F, Fukui Y, Franke TF, Hajjar RJ, Rosenzweig A. Adenoviral gene transfer of activated phosphatidylinositol 3'-kinase and Akt inhibits apoptosis of hypoxic cardiomyocytes in vitro. Circulation. 1999;100:2373–2379.

18. Fujio Y, Nguyen T, Wencker D, Kitsis RN, Walsh K. Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart. Circ Res. 2000;101:660–667.

19. Simoncini T, Hafezi-Moghadam A, Brazil DP, Ley K, Chin WW, Liao JK. Interaction of the oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature. 2000;407:538–541.

20. Haynes MP, Sinha D, Russel KS, Collinge M, Fulton D, Morales-Ruiz M, Sessa WC, Bender JR. Membrane receptor estrogen engagement activates endothelial nitric oxide synthase via the PI3-K-Akt pathway in human endothelial cells. Circ Res. 2000;87:677–682.

21. Andjelkovic M, Alessi DR, Meier R, Fernandez A, Lamb NJ, Frech M, Cron P, Cohen P, Lucocq JM, Hemmings BA. Role of translocation in the activation and function of protein kinase B. J Biol Chem. 1997;272:31515–31524.

22. Ferrigno P, Silver PA. Regulated nuclear localization of stress-responsive factors: how the nuclear trafficking of protein kinases and transcription factors contributes to cell survival. Oncogene. 1999;18:6129–6134.

23. Biggs WH III, Meisenhelder J, Hunter T, Cavenee WK, Arden KC. Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1. Proc Nat Acad Sci U S A. 1999;96:7421–7426.

24. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME. Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor. Cell. 1999;96:857–868.

25. Nakae J, Park B-C, Accili D. Insulin stimulates phosphorylation of the forkhead transcription factor FKHR on serine 253 through a wortmannin-sensitive pathway. J Biol Chem. 1999;274:15982–15985.

26. Sussman MA, Baqué S, Uhm C-S, Daniels MP, Price B, Simpson D, Terracio L, Kedes L. Altered expression of tropomodulin in cardiomyocytes disrupts the sarcomeric structure of myofibrils. Circ Res. 1998;82:94–105.

27. Sussman MA, Welch S, Gude N, Khoury PR, Daniels SR, Kirkpatrick D, Walsh RA, Price RL, Lim HW, Molkentin JD. Pathogenesis of dilated cardiomyopathy: molecular, structural, and population analyses in tropomodulin-overexpressing transgenics. Am J Pathol. 1999;155:2101–2113.

28. Liew CC, Jackowski G, Ma T, Sloe MJ. Nonenzymatic separation of myocardial cell nuclei from whole heart tissue. Am J Physiol. 1983;244:C3–C10.

29. Finking G, Wohlfrom M, Lenz C, Wolkenhauer M, Eberle C, Hanke H. The phytoestrogens genistein and daidzein and 17 ß-estradiol inhibit development of neointima in aortas from male and female rabbits in vitro after injury. Coron Artery Dis. 1999;10:607–615.

30. Hilakivi-Clarke L, Cho E, Clarke R. Maternal genistein exposure mimics the effects of estrogen on mammary gland development in female mouse offspring. Oncol Rep. 1998;5:609–616.

31. Strauss L, Makela S, Joshi S, Huhtaniemi I, Santti R. Genistein exerts estrogen-like effects in male mouse reproductive tract. Mol Cell Endocrinol. 1998;144:83–93.

32. Kahlert S, Nuedling S, van Eickels M, Vetter H, Meyer R, Grohe C. Estrogen receptor rapidly activates the IGF-1 receptor pathway. J Biol Chem. 2000;275:18447–18453.

33. Richards RG, Walker MP, Sebastian J, DiAugustine RP. Insulin-like growth factor-1 (IGF-1) receptor-insulin receptor substrate complexes in the uterus: altered signaling response to estradiol in the IGF-1 (m/m) mouse. J Biol Chem. 1998;273:11962–11969.

34. Ahmad S, Singh N, Glazer RI. Role of AKT1 in 17ß-estradiol- and insulin-like growth factor I (IGF-I)-dependent proliferation and prevention of apoptosis in Mcf-7 breast carcinoma cells. Biochem Pharmacol. 1999;58:425–430.

35. Pelzer T, Schumann M, Neumann M, deJager T, Stimpel M, Serfling E, Neyses L. 17ß-Estradiol prevents programmed cell death in cardiac myocytes. Biochem Biophys Res Commun. 2000;268:192–200.

36. Grohe C, Kahlert S, Lobbert K, Stimpel M, Karas RH, Vetter H, Neyses L. Cardiac myocytes contain functional estrogen receptors. FEBS Lett. 2000;416:107–112.

37. Nuedling S, Kahlert S, Loebbert K, Meyer R, Vetter H, Grohe C. Differential effects of 17 ß-estradiol on mitogen-activated protein kinase pathways in rat cardiomyocytes. FEBS Lett. 1999;454:271–276.

38. Zhai P, Eurell TE, Cotthaus R, Jeffrey EH, Bahr JM, Gross DR. Effect of estrogen on global myocardial ischemia-reperfusion injury in female rats. Am J Physiol Heart Circ Physiol. 2000;279:H2766–H2775.

39. Berul CI, Christe ME, Aronovitz MJ, Maguire CT, Seidman CE, Seidman JG, Mendelsohn ME. Familial hypertrophic cardiomyopathy mice display gender differences in electrophysiological abnormalities. J Interv Card Electrophysiol. 1998;2:7–14.

40. Djouadi F, Weinheimer CJ, Saffitz JE, Pitchford C, Bastin J, Gonzalez FJ, Kelly DP. A gender-related defect in lipid metabolism and glucose homeostasis in peroxisome proliferator-activated receptor -deficient mice. J Clin Invest. 1998;102:1083–1091.

41. Du K, Montminy M. CREB is a regulatory target for the protein kinase Akt/PKB. J Biol Chem. 1998;273:32377–32379.

42. Kane LP, Shapiro VS, Stokoe D, Weiss A. Induction of NF-B by the Akt/PKB kinase. Curr Biol. 1999;9:601–604.

43. Madrid LV, Wang C-Y, Guttridge DC, Schottelius AJG, Baldwin AS, Mayo M. Akt suppresses apoptosis by stimulating the transactivation potential of the RelA/p65 subunit of NF-B. Mol Cell Biol. 2000;20:1626–1638.

44. Koh H, Jee K, Lee B, Kim J, Kim D, Yun Y-H, Kim JW, Choi H-S, Chung J. Cloning and characterization of a nuclear S6 kinase, S6 kinase-related kinase (SRK): a novel nuclear target of Akt. Oncogene. 1999;18:5115–5119.

45. Fentzke RC, Kocarz CE, Lang RM, Lin H, Leiden JM. Dilated cardiomyopathy in transgenic mice expressing a dominant negative CREB transcription factor in the heart. J Clin Invest. 1998;101:2415–2426.

46. Guerra S, Leri A, Wang X, Finato N, Di Loreto C, Beltrami CA, Kajstura J, Anversa P. Myocyte death in the failing human heart is gender dependent. Circ Res. 1999;85:856–866.

47. Anderson JW, Smith BM, Washnock CS. Cardiovascular and renal benefits of dry bean and soybean intake. Am J Clin Nutr. 1999;70(suppl):464S–474S.

48. Cassidy A, Griffin B. Phyto-oestrogens. a potential role in the prevention of CHD? Proc Nutr Soc. 1999;58:193–199.

49. Lissin LW, Cooke JP. Phytoestrogens and cardiovascular health. J Am Coll Cardiol. 2000;35:1403–1410.

50. Herrington DM, Reboussin DM, Brosnihan KB, Sharp PC, Shumaker SA, Snyder TE, Furberg CD, Kowalchuk GJ, Stuckey TD, Rogers WJ, Givens DH, Waters D. Effects of estrogen replacement on the progression of coronary-artery atherosclerosis. N Engl J Med. 2000;343:522–529.

51. Kim H, Peterson G, Barnes S. Mechanism of action for the soy isoflavone genistein: emerging role for its effects via transforming growth factor ß signaling pathways. Am J Clin Nutr. 1998;68(suppl):1418S–1425S.

52. Setchell KDR. Phytoestrogens: the biochemistry, physiology, and implications for human health of soy isoflavones. Am J Clin Nutr. 1998;68(suppl):1333S–1346S.

53. Barkhem T, Carlsson B, Nilsson Y, Enmark E, Gustafsson J-A, Nilsson S. Differential response of estrogen receptor and estrogen receptor ß to partial estrogen agonists/antagonists. Mol Pharmacol. 1998;54:105–112.

54. Kuiper GG, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, van der Burg TP, Gustafsson JA. Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor ß. Endocrinology. 1998;139:4252–4263.

55. Thigpen JE, Setchell KDR, Ahlmark KB, Locklear J, Spahr T, Caviness GF, Goelz MF, Haseman JK, Newbold RR, Forsythe DB. Phytoestrogen content of purified, open- and closed-formula laboratory animal diets. Lab Anim Sci. 1999;49:530–536.

This article has been cited by other articles:

				P. Hojman, M. Pedersen, A. R. Nielsen, R. Krogh-Madsen, C. Yfanti, T. Akerstrom, S. Nielsen, and B. K. Pedersen Fibroblast Growth Factor-21 Is Induced in Human Skeletal Muscles by Hyperinsulinemia Diabetes, December 1, 2009; 58(12): 2797 - 2801. [Abstract] [Full Text] [PDF]

				B. Ostadal, I. Netuka, J. Maly, J. Besik, and I. Ostadalova Gender Differences in Cardiac Ischemic Injury and Protection--Experimental Aspects Experimental Biology and Medicine, September 1, 2009; 234(9): 1011 - 1019. [Abstract] [Full Text] [PDF]

				A. A. Kazi, K. H. Molitoris, and R. D. Koos Estrogen Rapidly Activates the PI3K/AKT Pathway and Hypoxia-Inducible Factor 1 and Induces Vascular Endothelial Growth Factor A Expression in Luminal Epithelial Cells of the Rat Uterus Biol Reprod, August 1, 2009; 81(2): 378 - 387. [Abstract] [Full Text] [PDF]

				S. Miyamoto, M. Rubio, and M. A. Sussman Nuclear and mitochondrial signalling Akts in cardiomyocytes Cardiovasc Res, May 1, 2009; 82(2): 272 - 285. [Abstract] [Full Text] [PDF]

				F. Princen, E. Bard, F. Sheikh, S. S. Zhang, J. Wang, W. M. Zago, D. Wu, R. D. Trelles, B. Bailly-Maitre, C. R. Kahn, et al. Deletion of Shp2 Tyrosine Phosphatase in Muscle Leads to Dilated Cardiomyopathy, Insulin Resistance, and Premature Death Mol. Cell. Biol., January 15, 2009; 29(2): 378 - 388. [Abstract] [Full Text] [PDF]

				J. R. Bell, E. R. Porrello, C. E. Huggins, S. B. Harrap, and L. M. D. Delbridge The intrinsic resistance of female hearts to an ischemic insult is abrogated in primary cardiac hypertrophy Am J Physiol Heart Circ Physiol, April 1, 2008; 294(4): H1514 - H1522. [Abstract] [Full Text] [PDF]

				A. Gonzalez, M. Rota, D. Nurzynska, Y. Misao, J. Tillmanns, C. Ojaimi, M. E. Padin-Iruegas, P. Muller, G. Esposito, C. Bearzi, et al. Activation of Cardiac Progenitor Cells Reverses the Failing Heart Senescent Phenotype and Prolongs Lifespan Circ. Res., March 14, 2008; 102(5): 597 - 606. [Abstract] [Full Text] [PDF]

				P. D. Metcalfe, J. A. Leslie, M. T. Campbell, D. R. Meldrum, K. L. Hile, and K. K. Meldrum Testosterone exacerbates obstructive renal injury by stimulating TNF-{alpha} production and increasing proapoptotic and profibrotic signaling Am J Physiol Endocrinol Metab, February 1, 2008; 294(2): E435 - E443. [Abstract] [Full Text] [PDF]

				J. D. Gardner, G. L. Brower, T. G. Voloshenyuk, and J. S. Janicki Cardioprotection in female rats subjected to chronic volume overload: synergistic interaction of estrogen and phytoestrogens Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H198 - H204. [Abstract] [Full Text] [PDF]

				Z. Cao, L. Liu, W. Packwood, M. Merkel, P. D. Hurn, and D. M. Van Winkle Sex differences in the mechanism of Met5-enkephalin-induced cardioprotection: role of PI3K/Akt Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H302 - H310. [Abstract] [Full Text] [PDF]

				J. P. Konhilas and L. A. Leinwand The Effects of Biological Sex and Diet on the Development of Heart Failure Circulation, December 4, 2007; 116(23): 2747 - 2759. [Full Text] [PDF]

				J.-T. Hsu, W.-H. Kan, C.-H. Hsieh, M. A. Choudhry, M. G. Schwacha, K. I. Bland, and I. H. Chaudry Mechanism of estrogen-mediated attenuation of hepatic injury following trauma-hemorrhage: Akt-dependent HO-1 up-regulation J. Leukoc. Biol., October 1, 2007; 82(4): 1019 - 1026. [Abstract] [Full Text] [PDF]

				G. Han, H. Ma, R. Chintala, K. Miyake, D. J. R. Fulton, S. A. Barman, and R. E. White Nongenomic, endothelium-independent effects of estrogen on human coronary smooth muscle are mediated by type I (neuronal) NOS and PI3-kinase-Akt signaling Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H314 - H321. [Abstract] [Full Text] [PDF]

				C. F. Deschepper and B. Llamas Hypertensive Cardiac Remodeling in Males and Females: From the Bench to the Bedside Hypertension, March 1, 2007; 49(3): 401 - 407. [Full Text] [PDF]

				J. C. Hunter, J. C. Kostyak, J. L. Novotny, A. M. Simpson, and D. H. Korzick Estrogen deficiency decreases ischemic tolerance in the aged rat heart: roles of PKC{delta}, PKC{epsilon}, Akt, and GSK3beta Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2007; 292(2): R800 - R809. [Abstract] [Full Text] [PDF]

				I. Shiojima and K. Walsh Regulation of cardiac growth and coronary angiogenesis by the Akt/PKB signaling pathway Genes & Dev., December 15, 2006; 20(24): 3347 - 3365. [Abstract] [Full Text] [PDF]

				M. A. Sovershaev, E. M. Egorina, T. V. Andreasen, A. K. Jonassen, and K. Ytrehus Preconditioning by 17beta-estradiol in isolated rat heart depends on PI3-K/PKB pathway, PKC, and ROS Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1554 - H1562. [Abstract] [Full Text] [PDF]

				N. Gude, J. Muraski, M. Rubio, J. Kajstura, E. Schaefer, P. Anversa, and M. A. Sussman Akt Promotes Increased Cardiomyocyte Cycling and Expansion of the Cardiac Progenitor Cell Population Circ. Res., August 18, 2006; 99(4): 381 - 388. [Abstract] [Full Text] [PDF]

				Y. Tsujita, J. Muraski, I. Shiraishi, T. Kato, J. Kajstura, P. Anversa, and M. A. Sussman Nuclear targeting of Akt antagonizes aspects of cardiomyocyte hypertrophy PNAS, August 8, 2006; 103(32): 11946 - 11951. [Abstract] [Full Text] [PDF]

				J. Kim, I. S. Kil, Y. M. Seok, E. S. Yang, D. K. Kim, D. G. Lim, J.-W. Park, J. V. Bonventre, and K. M. Park Orchiectomy Attenuates Post-ischemic Oxidative Stress and Ischemia/Reperfusion Injury in Mice: A ROLE FOR MANGANESE SUPEROXIDE DISMUTASE J. Biol. Chem., July 21, 2006; 281(29): 20349 - 20356. [Abstract] [Full Text] [PDF]

				A. F. Ceylan-Isik, K. H. LaCour, and J. Ren Sex difference in cardiomyocyte function in normal and metallothionein transgenic mice: the effect of diabetes mellitus J Appl Physiol, May 1, 2006; 100(5): 1638 - 1646. [Abstract] [Full Text] [PDF]

				I. G. Poornima, P. Parikh, and R. P. Shannon Diabetic Cardiomyopathy: The Search for a Unifying Hypothesis Circ. Res., March 17, 2006; 98(5): 596 - 605. [Abstract] [Full Text] [PDF]

				K. Nishimura, W. Li, Y. Hoshino, T. Kadohama, H. Asada, S. Ohgi, and B. E. Sumpio Role of AKT in cyclic strain-induced endothelial cell proliferation and survival Am J Physiol Cell Physiol, March 1, 2006; 290(3): C812 - C821. [Abstract] [Full Text] [PDF]

				S. Bae and L. Zhang Gender Differences in Cardioprotection against Ischemia/Reperfusion Injury in Adult Rat Hearts: Focus on Akt and Protein Kinase C Signaling J. Pharmacol. Exp. Ther., December 1, 2005; 315(3): 1125 - 1135. [Abstract] [Full Text] [PDF]

				A. Leri, J. Kajstura, and P. Anversa Cardiac Stem Cells and Mechanisms of Myocardial Regeneration Physiol Rev, October 1, 2005; 85(4): 1373 - 1416. [Abstract] [Full Text] [PDF]

				P. Anversa Aging and Longevity: The IGF-1 Enigma Circ. Res., September 2, 2005; 97(5): 411 - 414. [Full Text] [PDF]

				A. Kher, K. K. Meldrum, M. Wang, B. M. Tsai, J. M. Pitcher, and D. R. Meldrum Cellular and molecular mechanisms of sex differences in renal ischemia-reperfusion injury Cardiovasc Res, September 1, 2005; 67(4): 594 - 603. [Abstract] [Full Text] [PDF]

				A. Ascensao, J. Magalhaes, J. M. C. Soares, R. Ferreira, M. J. Neuparth, F. Marques, P. J. Oliveira, and J. A. Duarte Moderate endurance training prevents doxorubicin-induced in vivo mitochondriopathy and reduces the development of cardiac apoptosis Am J Physiol Heart Circ Physiol, August 1, 2005; 289(2): H722 - H731. [Abstract] [Full Text] [PDF]

				S. Bae, R. D. Gilbert, C. A. Ducsay, and L. Zhang Prenatal cocaine exposure increases heart susceptibility to ischaemia-reperfusion injury in adult male but not female rats J. Physiol., May 15, 2005; 565(1): 149 - 158. [Abstract] [Full Text] [PDF]

				K. M. Park, J. I. Kim, Y. Ahn, A. J. Bonventre, and J. V. Bonventre Testosterone Is Responsible for Enhanced Susceptibility of Males to Ischemic Renal Injury J. Biol. Chem., December 10, 2004; 279(50): 52282 - 52292. [Abstract] [Full Text] [PDF]

				J. P. Konhilas, A. H. Maass, S. W. Luckey, B. L. Stauffer, E. N. Olson, and L. A. Leinwand Sex modifies exercise and cardiac adaptation in mice Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2768 - H2776. [Abstract] [Full Text] [PDF]

				R. D. Patten, I. Pourati, M. J. Aronovitz, J. Baur, F. Celestin, X. Chen, A. Michael, S. Haq, S. Nuedling, C. Grohe, et al. 17{beta}-Estradiol Reduces Cardiomyocyte Apoptosis In Vivo and In Vitro via Activation of Phospho-Inositide-3 Kinase/Akt Signaling Circ. Res., October 1, 2004; 95(7): 692 - 699. [Abstract] [Full Text] [PDF]

				V. P.M van Empel and L. J De Windt Myocyte hypertrophy and apoptosis: a balancing act Cardiovasc Res, August 15, 2004; 63(3): 487 - 499. [Abstract] [Full Text] [PDF]

				X.-J. Du Gender modulates cardiac phenotype development in genetically modified mice Cardiovasc Res, August 15, 2004; 63(3): 510 - 519. [Abstract] [Full Text] [PDF]

				K. A. Webster Aktion in the Nucleus Circ. Res., April 16, 2004; 94(7): 856 - 859. [Full Text] [PDF]

				I. Shiraishi, J. Melendez, Y. Ahn, M. Skavdahl, E. Murphy, S. Welch, E. Schaefer, K. Walsh, A. Rosenzweig, D. Torella, et al. Nuclear Targeting of Akt Enhances Kinase Activity and Survival of Cardiomyocytes Circ. Res., April 16, 2004; 94(7): 884 - 891. [Abstract] [Full Text] [PDF]

				A. Huang, D. Sun, Z. Wu, C. Yan, M. A. Carroll, H. Jiang, J. R. Falck, and G. Kaley Estrogen Elicits Cytochrome P450--Mediated Flow-Induced Dilation of Arterioles in NO Deficiency: Role of PI3K-Akt Phosphorylation in Genomic Regulation Circ. Res., February 6, 2004; 94(2): 245 - 252. [Abstract] [Full Text] [PDF]

				R. M.A. Henry, O. Kamp, P. J. Kostense, A. M.W. Spijkerman, J. M. Dekker, R. van Eijck, G. Nijpels, R. J. Heine, L. M. Bouter, and C. D.A. Stehouwer Left Ventricular Mass Increases With Deteriorating Glucose Tolerance, Especially in Women: Independence of Increased Arterial Stiffness or Decreased Flow-Mediated Dilation: The Hoorn Study Diabetes Care, February 1, 2004; 27(2): 522 - 529. [Abstract] [Full Text] [PDF]

				S. Mabuchi, M. Ohmichi, A. Kimura, Y. Nishio, E. Arimoto-Ishida, N. Yada-Hashimoto, K. Tasaka, and Y. Murata Estrogen Inhibits Paclitaxel-Induced Apoptosis via the Phosphorylation of Apoptosis Signal-Regulating Kinase 1 in Human Ovarian Cancer Cell Lines Endocrinology, January 1, 2004; 145(1): 49 - 58. [Abstract] [Full Text] [PDF]

				R. Liew, M. A Stagg, J. Chan, P. Collins, and K. T MacLeod Gender determines the acute actions of genistein on intracellular calcium regulation in the guinea-pig heart Cardiovasc Res, January 1, 2004; 61(1): 66 - 76. [Abstract] [Full Text] [PDF]

				P. H. Sugden Ras, Akt, and Mechanotransduction in the Cardiac Myocyte Circ. Res., December 12, 2003; 93(12): 1179 - 1192. [Abstract] [Full Text] [PDF]

				I. Adini, I. Rabinovitz, J. F. Sun, G. C. Prendergast, and L. E. Benjamin RhoB controls Akt trafficking and stage-specific survival of endothelial cells during vascular development Genes & Dev., November 1, 2003; 17(21): 2721 - 2732. [Abstract] [Full Text] [PDF]

				J. Lindenfeld, J. K. Ghali, H. J. Krause-Steinrauf, S. Khan, K. Adams Jr, S. Goldman, M. A. Peberdy, C. Yancy, S. Thaneemit-Chen, R. L. Larsen, et al. Hormone replacement therapy is associated with improved survival in women with advanced heart failure J. Am. Coll. Cardiol., October 1, 2003; 42(7): 1238 - 1245. [Abstract] [Full Text] [PDF]

				X.-M. Gao, A. Agrotis, D. J. Autelitano, E. Percy, E. A. Woodcock, G. L. Jennings, A. M. Dart, and X.-J. Du Sex Hormones and Cardiomyopathic Phenotype Induced by Cardiac {beta}2-Adrenergic Receptor Overexpression Endocrinology, September 1, 2003; 144(9): 4097 - 4105. [Abstract] [Full Text] [PDF]

				J. Ren, K. K. Hintz, Z. K. F. Roughead, J. Duan, P. B. Colligan, B. H. Ren, K. J. Lee, and H. Zeng Impact of estrogen replacement on ventricular myocyte contractile function and protein kinase B/Akt activation Am J Physiol Heart Circ Physiol, May 1, 2003; 284(5): H1800 - H1807. [Abstract] [Full Text] [PDF]

				M. K. Rutter, H. Parise, E. J. Benjamin, D. Levy, M. G. Larson, J. B. Meigs, R. W. Nesto, P. W.F. Wilson, and R. S. Vasan Impact of Glucose Intolerance and Insulin Resistance on Cardiac Structure and Function: Sex-Related Differences in the Framingham Heart Study Circulation, January 28, 2003; 107(3): 448 - 454. [Abstract] [Full Text] [PDF]

				D. Wang, J. Gutkowska, M. Marcinkiewicz, G. Rachelska, and M. Jankowski Genistein supplementation stimulates the oxytocin system in the aorta of ovariectomized rats Cardiovasc Res, January 1, 2003; 57(1): 186 - 194. [Abstract] [Full Text] [PDF]

				M. A. Sussman, A. McCulloch, and T. K. Borg Dance Band on the Titanic: Biomechanical Signaling in Cardiac Hypertrophy Circ. Res., November 15, 2002; 91(10): 888 - 898. [Abstract] [Full Text] [PDF]

				D. M. Brownson, N. G. Azios, B. K. Fuqua, S. F. Dharmawardhane, and T. J. Mabry Flavonoid Effects Relevant to Cancer J. Nutr., November 1, 2002; 132(11): 3482S - 3489. [Abstract] [Full Text] [PDF]

				J. R. Freshour, S. E. Chase, and K. L. Vikstrom Gender differences in cardiac ACE expression are normalized in androgen-deprived male mice Am J Physiol Heart Circ Physiol, November 1, 2002; 283(5): H1997 - H2003. [Abstract] [Full Text] [PDF]

				A. C. Childs, S. L. Phaneuf, A. J. Dirks, T. Phillips, and C. Leeuwenburgh Doxorubicin Treatment in Vivo Causes Cytochrome c Release and Cardiomyocyte Apoptosis, As Well As Increased Mitochondrial Efficiency, Superoxide Dismutase Activity, and Bcl-2:Bax Ratio Cancer Res., August 15, 2002; 62(16): 4592 - 4598. [Abstract] [Full Text] [PDF]

				I. Shiojima and K. Walsh Role of Akt Signaling in Vascular Homeostasis and Angiogenesis Circ. Res., June 28, 2002; 90(12): 1243 - 1250. [Abstract] [Full Text] [PDF]

				T. Matsui, L. Li, J. C. Wu, S. A. Cook, T. Nagoshi, M. H. Picard, R. Liao, and A. Rosenzweig Phenotypic Spectrum Caused by Transgenic Overexpression of Activated Akt in the Heart J. Biol. Chem., June 14, 2002; 277(25): 22896 - 22901. [Abstract] [Full Text] [PDF]

				S. Welch, D. Plank, S. Witt, B. Glascock, E. Schaefer, S. Chimenti, A. M. Andreoli, F. Limana, A. Leri, J. Kajstura, et al. Cardiac-Specific IGF-1 Expression Attenuates Dilated Cardiomyopathy in Tropomodulin-Overexpressing Transgenic Mice Circ. Res., April 5, 2002; 90(6): 641 - 648. [Abstract] [Full Text] [PDF]

				F. A Babiker, L. J De Windt, M. van Eickels, C. Grohe, R. Meyer, and P. A Doevendans Estrogenic hormone action in the heart: regulatory network and function Cardiovasc Res, February 15, 2002; 53(3): 709 - 719. [Abstract] [Full Text] [PDF]

				K. D. R. Setchell Soy Isoflavones--Benefits and Risks from Nature's Selective Estrogen Receptor Modulators (SERMs) J. Am. Coll. Nutr., October 1, 2001; 20(90005): 354S - 362. [Abstract] [Full Text]

				G. Schwartzbauer and J. Robbins Matters of Sex: Sex Matters Circulation, September 18, 2001; 104(12): 1333 - 1335. [Full Text] [PDF]

				S. Welch, D. Plank, S. Witt, B. Glascock, E. Schaefer, S. Chimenti, A. M. Andreoli, F. Limana, A. Leri, J. Kajstura, et al. Cardiac-Specific IGF-1 Expression Attenuates Dilated Cardiomyopathy in Tropomodulin-Overexpressing Transgenic Mice Circ. Res., April 5, 2002; 90(6): 641 - 648. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 88/10/1020    most recent hh1001.090858v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Camper-Kirby, D. Articles by Sussman, M. A. Search for Related Content PubMed Articles by Camper-Kirby, D. Articles by Sussman, M. A. Related Collections Other myocardial biology Cell signalling/signal transduction