Estrogen attenuates left ventricular and cardiomyocyte hypertrophy by an estrogen receptor-dependent pathway that increases calcineurin degradation

Abstract

Left ventricular (LV) hypertrophy commonly develops in response to chronic hypertension and is a significant risk factor for heart failure and death. The serine-threonine phosphatase calcineurin (Cn)A plays a critical role in the development of pathological hypertrophy. Previous experimental studies in murine models show that estrogen limits pressure overload-induced hypertrophy; our purpose was to explore further the mechanisms underlying this estrogen effect. Wild-type, ovariectomized female mice were treated with placebo or 17beta-estradiol (E2), followed by transverse aortic constriction (TAC), to induce pressure overload. At 2 weeks, mice underwent physiological evaluation, immediate tissue harvest, or dispersion of cardiomyocytes. E2 replacement limited TAC-induced LV and cardiomyocyte hypertrophy while attenuating deterioration in LV systolic function and contractility. These E2 effects were associated with reduced abundance of CnA. The primary downstream targets of CnA are the nuclear factor of activated T-cell (NFAT) family of transcription factors. In transgenic mice expressing a NFAT-activated promoter/luciferase reporter gene, E2 limited TAC-induced activation of NFAT. Moreover, the inhibitory effects of E2 on LV hypertrophy were absent in CnA knockout mice, supporting the notion that CnA is an important target of E2-mediated inhibition. In cultured rat cardiac myocytes, E2 inhibited agonist-induced hypertrophy while also decreasing CnA abundance and NFAT activation. Agonist stimulation also reduced CnA ubiquitination and degradation that was prevented by E2; all in vitro effects of estrogen were reversed by an estrogen receptor (ER) antagonist. These data support that E2 reduces pressure overload induced hypertrophy by an ER-dependent mechanism that increases CnA degradation, unveiling a novel mechanism by which E2 and ERs regulate pathological LV and cardiomyocyte growth.

Figure 1. Estrogen Promotes Concentric Remodeling Following TAC

A. PV Loop Analyses: (Also see Table 1). Top: Representative steady state PV loops from sham, placebo-TAC and E2-TAC mice. The placebo-TAC heart exhibits a rightward shift in the PV loop consistent with LV dilation which is reversed by E2 replacement. Bottom Left: LV end diastolic volume (EDV); Middle: LV end systolic volume (ESV); Right: LV Ejection Fraction (EF). *p<0.01 vs. Shams; † p<0.05 vs. Placebo-TAC. B. Myocyte Morphometry Results: Top: Representative images of dispersed myocytes from a Sham, Placebo-TAC and E2-TAC heart. Bottom Left: Myocyte 2-dimensional area; Middle: Myocyte Length; Right: Myocyte Width. TAC significantly increased myocyte area, length, and width in the placebo-treated mice. E2 replacement limited myocyte enlargement by attenuating the increase in myocyte length with no effect on myocyte width. N=9 Shams, 6 Placebo-TAC and 8 E2-TAC mice. Equivalent TAC gradients were confirmed by pulse wave Doppler measurements during echocardiography. * p<0.05 vs. Shams; † p<0.05 vs. Placebo-TAC.

Figure 1. Estrogen Promotes Concentric Remodeling Following TAC

A. PV Loop Analyses: (Also see Table 1). Top: Representative steady state PV loops from sham, placebo-TAC and E2-TAC mice. The placebo-TAC heart exhibits a rightward shift in the PV loop consistent with LV dilation which is reversed by E2 replacement. Bottom Left: LV end diastolic volume (EDV); Middle: LV end systolic volume (ESV); Right: LV Ejection Fraction (EF). *p<0.01 vs. Shams; † p<0.05 vs. Placebo-TAC. B. Myocyte Morphometry Results: Top: Representative images of dispersed myocytes from a Sham, Placebo-TAC and E2-TAC heart. Bottom Left: Myocyte 2-dimensional area; Middle: Myocyte Length; Right: Myocyte Width. TAC significantly increased myocyte area, length, and width in the placebo-treated mice. E2 replacement limited myocyte enlargement by attenuating the increase in myocyte length with no effect on myocyte width. N=9 Shams, 6 Placebo-TAC and 8 E2-TAC mice. Equivalent TAC gradients were confirmed by pulse wave Doppler measurements during echocardiography. * p<0.05 vs. Shams; † p<0.05 vs. Placebo-TAC.

Figure 2. Influence of E2 Replacement on Calcineurin Signaling Following TAC

A. Left: Calcineurin A protein expression increased following TAC in placebo-treated mice but not those treated with E2. Right: MCIP1.4 gene expression. TAC increased the mRNA level of MCIP1.4 that is prevented by E2 replacement. p<0.05 vs. Shams; † p<0.05 vs. Placebo-TAC. B. Myocardial Luciferase activity from NFAT-Luc Transgenic Mice 2 weeks following TAC. TAC led to a significant 3.2 fold increase in myocardial luciferase activity that is attenuated by E2 replacement. * p<0.05 vs. Shams; † p=0.05 vs. Placebo-TAC. C. E2 Reduces LV hypertrophy in Wild Type but not in CnAβ knockout mice. LV mass indexed to tibial length 4 weeks post-TAC in ovariectomized, female CnA beta KO mice (Right) and wild type littermates (Left). * p<0.01 vs. Sham; † p<0.05 vs. placebo-TAC.

Figure 2. Influence of E2 Replacement on Calcineurin Signaling Following TAC

A. Left: Calcineurin A protein expression increased following TAC in placebo-treated mice but not those treated with E2. Right: MCIP1.4 gene expression. TAC increased the mRNA level of MCIP1.4 that is prevented by E2 replacement. p<0.05 vs. Shams; † p<0.05 vs. Placebo-TAC. B. Myocardial Luciferase activity from NFAT-Luc Transgenic Mice 2 weeks following TAC. TAC led to a significant 3.2 fold increase in myocardial luciferase activity that is attenuated by E2 replacement. * p<0.05 vs. Shams; † p=0.05 vs. Placebo-TAC. C. E2 Reduces LV hypertrophy in Wild Type but not in CnAβ knockout mice. LV mass indexed to tibial length 4 weeks post-TAC in ovariectomized, female CnA beta KO mice (Right) and wild type littermates (Left). * p<0.01 vs. Sham; † p<0.05 vs. placebo-TAC.

Figure 2. Influence of E2 Replacement on Calcineurin Signaling Following TAC

A. Left: Calcineurin A protein expression increased following TAC in placebo-treated mice but not those treated with E2. Right: MCIP1.4 gene expression. TAC increased the mRNA level of MCIP1.4 that is prevented by E2 replacement. p<0.05 vs. Shams; † p<0.05 vs. Placebo-TAC. B. Myocardial Luciferase activity from NFAT-Luc Transgenic Mice 2 weeks following TAC. TAC led to a significant 3.2 fold increase in myocardial luciferase activity that is attenuated by E2 replacement. * p<0.05 vs. Shams; † p=0.05 vs. Placebo-TAC. C. E2 Reduces LV hypertrophy in Wild Type but not in CnAβ knockout mice. LV mass indexed to tibial length 4 weeks post-TAC in ovariectomized, female CnA beta KO mice (Right) and wild type littermates (Left). * p<0.01 vs. Sham; † p<0.05 vs. placebo-TAC.

Figure 3. Estrogen Inhibits Agonist Induced Cardiomyocyte Hypertrophy in vitro

A. Myocyte Cell Size: Neonatal rat ventricular myocytes (NRVMs) were plated in serum containing media for 24 hours. 17 β-estradiol (E2, 10nM), ICI 182,780 (ICI, 1.0uM), both, or vehicle control were added in serum free media overnight. Cells were then stimulated with phenylephrine (50uM) for 48 hours. Pretreatment with E2 limited PE-induced cardiomyocyte enlargement that was reversed by the ER antagonist, ICI. These data are representative of 6 independent experiments. * p<0.01 vs. control cells; † p<0.05 vs. PE. B. ANP Gene Expression. Total cellular RNA was harvested from 2.5×10⁶ cells plated on 10cm dishes and real time rtPCR performed to quantify the level of ANP expression. E2 (10nM) had no effect on basal levels of ANP expression but significantly limited the PE-induced increase in ANP mRNA that was reversed by ICI (0.5uM). * p<0.01 vs. control cells; † p<0.05 vs. PE.

Figure 3. Estrogen Inhibits Agonist Induced Cardiomyocyte Hypertrophy in vitro

A. Myocyte Cell Size: Neonatal rat ventricular myocytes (NRVMs) were plated in serum containing media for 24 hours. 17 β-estradiol (E2, 10nM), ICI 182,780 (ICI, 1.0uM), both, or vehicle control were added in serum free media overnight. Cells were then stimulated with phenylephrine (50uM) for 48 hours. Pretreatment with E2 limited PE-induced cardiomyocyte enlargement that was reversed by the ER antagonist, ICI. These data are representative of 6 independent experiments. * p<0.01 vs. control cells; † p<0.05 vs. PE. B. ANP Gene Expression. Total cellular RNA was harvested from 2.5×10⁶ cells plated on 10cm dishes and real time rtPCR performed to quantify the level of ANP expression. E2 (10nM) had no effect on basal levels of ANP expression but significantly limited the PE-induced increase in ANP mRNA that was reversed by ICI (0.5uM). * p<0.01 vs. control cells; † p<0.05 vs. PE.

Figure 4. Calcineurin Signaling Pathway as a Target of E2-mediated inhibition in vitro

A. Left: Calcineurin Protein Expression. NRVMs were harvested 24 hours following PE stimulation and CnA expression was measured by Western blotting using an antibody that recognizes both the α and β isoforms of CnA. PE (50uM) caused a significant rise in CnA protein expression that was limited by E2 pretreatment and reversed by the ER antagonist, ICI. These data represent 5 independent experiments. Right: NFAT activation: NRVMs were transfected with an NFAT promoter-luciferase reporter plasmid along with an L7RH-β-galactosidase plasmid for signal normalization. Upon serum deprivation, cells were pretreated with E2 (10nM) or vehicle. PE was added on the following day and cells harvested 24 hours later. PE (50uM) induced a more than 3 fold rise in NFAT activation that was significantly limited by E2 and CSA pretreatment. * p<0.01 vs. control cells; † p<0.05 vs. PE. B. A constitutively active mutant of CnA (caCnA) blocks the inhibition by E2 of cardiomyocyte growth and NFAT-activation. Left: Cell size data in NRVMs infected with Adv-GFP as a control, or Adv-caCnA. E2 pretreatment inhibits hypertrophy of GFP expressing cells stimulated with PE, but has no effect on NRVM hypertrophy stimulated by caCnA. Similar loss of E2 effect was seen in ARVMs expressing caCnA. Right: Similar results are seen with activation of NFAT in which the inhibition by E2 of PE-induced NFAT activation is maintained in GFP expressing cells but is lost in cells expressing caCnA. Both graphs are representative of 3 independent experiments. * p<0.01 vs. control cells; † p<0.05 vs. PE. C. E2 has no additive effects in NRVMs pretreated with the CnA inhibitor, Cyclosporine (500ng/ml). Following 24 hours of growth in serum containing media, NRVMs were serum deprived overnight in the presence of vehicle, E2, cyclosporine, or their combination and stimulated with PE for an additional 48 hours. * p<0.01 vs. control cells; † p<0.05 vs. PE. D. PE reduces CnA degradation that is normalized by E2 pretreatment. NRVMs were treated with E2, E2 plus ICI, or vehicle upon serum deprivation. On the following day, cells were pretreated with cycloheximide (40ug/ml-based on preliminary experiments demonstrating complete inhibition of GFP-tagged ERα translation delivered by adenoviral transfection). 2 hours later, cells were stimulated with PE and harvested at 6, and 24 hours. CnA expression was normalized to β-actin. The line graph (left) shows quantified CnA levels. PE significantly prevents CnA degradation at 24 hours which is normalized by E2 replacement and reversed by the ER antagonist, ICI. A representative western blot of the control and 24 hour samples is shown to the right. These data are representative of 4 independent experiments. * p<0.01 vs. control cells; † p<0.05 vs. PE.

Figure 4. Calcineurin Signaling Pathway as a Target of E2-mediated inhibition in vitro

A. Left: Calcineurin Protein Expression. NRVMs were harvested 24 hours following PE stimulation and CnA expression was measured by Western blotting using an antibody that recognizes both the α and β isoforms of CnA. PE (50uM) caused a significant rise in CnA protein expression that was limited by E2 pretreatment and reversed by the ER antagonist, ICI. These data represent 5 independent experiments. Right: NFAT activation: NRVMs were transfected with an NFAT promoter-luciferase reporter plasmid along with an L7RH-β-galactosidase plasmid for signal normalization. Upon serum deprivation, cells were pretreated with E2 (10nM) or vehicle. PE was added on the following day and cells harvested 24 hours later. PE (50uM) induced a more than 3 fold rise in NFAT activation that was significantly limited by E2 and CSA pretreatment. * p<0.01 vs. control cells; † p<0.05 vs. PE. B. A constitutively active mutant of CnA (caCnA) blocks the inhibition by E2 of cardiomyocyte growth and NFAT-activation. Left: Cell size data in NRVMs infected with Adv-GFP as a control, or Adv-caCnA. E2 pretreatment inhibits hypertrophy of GFP expressing cells stimulated with PE, but has no effect on NRVM hypertrophy stimulated by caCnA. Similar loss of E2 effect was seen in ARVMs expressing caCnA. Right: Similar results are seen with activation of NFAT in which the inhibition by E2 of PE-induced NFAT activation is maintained in GFP expressing cells but is lost in cells expressing caCnA. Both graphs are representative of 3 independent experiments. * p<0.01 vs. control cells; † p<0.05 vs. PE. C. E2 has no additive effects in NRVMs pretreated with the CnA inhibitor, Cyclosporine (500ng/ml). Following 24 hours of growth in serum containing media, NRVMs were serum deprived overnight in the presence of vehicle, E2, cyclosporine, or their combination and stimulated with PE for an additional 48 hours. * p<0.01 vs. control cells; † p<0.05 vs. PE. D. PE reduces CnA degradation that is normalized by E2 pretreatment. NRVMs were treated with E2, E2 plus ICI, or vehicle upon serum deprivation. On the following day, cells were pretreated with cycloheximide (40ug/ml-based on preliminary experiments demonstrating complete inhibition of GFP-tagged ERα translation delivered by adenoviral transfection). 2 hours later, cells were stimulated with PE and harvested at 6, and 24 hours. CnA expression was normalized to β-actin. The line graph (left) shows quantified CnA levels. PE significantly prevents CnA degradation at 24 hours which is normalized by E2 replacement and reversed by the ER antagonist, ICI. A representative western blot of the control and 24 hour samples is shown to the right. These data are representative of 4 independent experiments. * p<0.01 vs. control cells; † p<0.05 vs. PE.

Figure 4. Calcineurin Signaling Pathway as a Target of E2-mediated inhibition in vitro

A. Left: Calcineurin Protein Expression. NRVMs were harvested 24 hours following PE stimulation and CnA expression was measured by Western blotting using an antibody that recognizes both the α and β isoforms of CnA. PE (50uM) caused a significant rise in CnA protein expression that was limited by E2 pretreatment and reversed by the ER antagonist, ICI. These data represent 5 independent experiments. Right: NFAT activation: NRVMs were transfected with an NFAT promoter-luciferase reporter plasmid along with an L7RH-β-galactosidase plasmid for signal normalization. Upon serum deprivation, cells were pretreated with E2 (10nM) or vehicle. PE was added on the following day and cells harvested 24 hours later. PE (50uM) induced a more than 3 fold rise in NFAT activation that was significantly limited by E2 and CSA pretreatment. * p<0.01 vs. control cells; † p<0.05 vs. PE. B. A constitutively active mutant of CnA (caCnA) blocks the inhibition by E2 of cardiomyocyte growth and NFAT-activation. Left: Cell size data in NRVMs infected with Adv-GFP as a control, or Adv-caCnA. E2 pretreatment inhibits hypertrophy of GFP expressing cells stimulated with PE, but has no effect on NRVM hypertrophy stimulated by caCnA. Similar loss of E2 effect was seen in ARVMs expressing caCnA. Right: Similar results are seen with activation of NFAT in which the inhibition by E2 of PE-induced NFAT activation is maintained in GFP expressing cells but is lost in cells expressing caCnA. Both graphs are representative of 3 independent experiments. * p<0.01 vs. control cells; † p<0.05 vs. PE. C. E2 has no additive effects in NRVMs pretreated with the CnA inhibitor, Cyclosporine (500ng/ml). Following 24 hours of growth in serum containing media, NRVMs were serum deprived overnight in the presence of vehicle, E2, cyclosporine, or their combination and stimulated with PE for an additional 48 hours. * p<0.01 vs. control cells; † p<0.05 vs. PE. D. PE reduces CnA degradation that is normalized by E2 pretreatment. NRVMs were treated with E2, E2 plus ICI, or vehicle upon serum deprivation. On the following day, cells were pretreated with cycloheximide (40ug/ml-based on preliminary experiments demonstrating complete inhibition of GFP-tagged ERα translation delivered by adenoviral transfection). 2 hours later, cells were stimulated with PE and harvested at 6, and 24 hours. CnA expression was normalized to β-actin. The line graph (left) shows quantified CnA levels. PE significantly prevents CnA degradation at 24 hours which is normalized by E2 replacement and reversed by the ER antagonist, ICI. A representative western blot of the control and 24 hour samples is shown to the right. These data are representative of 4 independent experiments. * p<0.01 vs. control cells; † p<0.05 vs. PE.

Figure 4. Calcineurin Signaling Pathway as a Target of E2-mediated inhibition in vitro

A. Left: Calcineurin Protein Expression. NRVMs were harvested 24 hours following PE stimulation and CnA expression was measured by Western blotting using an antibody that recognizes both the α and β isoforms of CnA. PE (50uM) caused a significant rise in CnA protein expression that was limited by E2 pretreatment and reversed by the ER antagonist, ICI. These data represent 5 independent experiments. Right: NFAT activation: NRVMs were transfected with an NFAT promoter-luciferase reporter plasmid along with an L7RH-β-galactosidase plasmid for signal normalization. Upon serum deprivation, cells were pretreated with E2 (10nM) or vehicle. PE was added on the following day and cells harvested 24 hours later. PE (50uM) induced a more than 3 fold rise in NFAT activation that was significantly limited by E2 and CSA pretreatment. * p<0.01 vs. control cells; † p<0.05 vs. PE. B. A constitutively active mutant of CnA (caCnA) blocks the inhibition by E2 of cardiomyocyte growth and NFAT-activation. Left: Cell size data in NRVMs infected with Adv-GFP as a control, or Adv-caCnA. E2 pretreatment inhibits hypertrophy of GFP expressing cells stimulated with PE, but has no effect on NRVM hypertrophy stimulated by caCnA. Similar loss of E2 effect was seen in ARVMs expressing caCnA. Right: Similar results are seen with activation of NFAT in which the inhibition by E2 of PE-induced NFAT activation is maintained in GFP expressing cells but is lost in cells expressing caCnA. Both graphs are representative of 3 independent experiments. * p<0.01 vs. control cells; † p<0.05 vs. PE. C. E2 has no additive effects in NRVMs pretreated with the CnA inhibitor, Cyclosporine (500ng/ml). Following 24 hours of growth in serum containing media, NRVMs were serum deprived overnight in the presence of vehicle, E2, cyclosporine, or their combination and stimulated with PE for an additional 48 hours. * p<0.01 vs. control cells; † p<0.05 vs. PE. D. PE reduces CnA degradation that is normalized by E2 pretreatment. NRVMs were treated with E2, E2 plus ICI, or vehicle upon serum deprivation. On the following day, cells were pretreated with cycloheximide (40ug/ml-based on preliminary experiments demonstrating complete inhibition of GFP-tagged ERα translation delivered by adenoviral transfection). 2 hours later, cells were stimulated with PE and harvested at 6, and 24 hours. CnA expression was normalized to β-actin. The line graph (left) shows quantified CnA levels. PE significantly prevents CnA degradation at 24 hours which is normalized by E2 replacement and reversed by the ER antagonist, ICI. A representative western blot of the control and 24 hour samples is shown to the right. These data are representative of 4 independent experiments. * p<0.01 vs. control cells; † p<0.05 vs. PE.

Figure 5. Proteasome inhibition abolishes the inhibitory effect of E2 on CnA expression and cardiomyocyte hypertrophy

A. Lactacystin (10uM) prevents both the agonist-induced increase in CnA and the inhibitory effects of E2. Representative western blot of CnA with corresponding bar graph. B. Lactacystin abolishes the E2-mediated inhibition of NRVM hypertrophy induced by PE. * p<0.01 vs. control cells; † p<0.05 vs. PE. ‡ p<0.05 vs. Lactacystin alone. C. Phenylephrine diminishes CnA ubiquitination that is restored by E2 pretreatment. NRVMs were pretreated overnight with E2 upon serum deprivation, and stimulated with PE. Cells were harvested 4 hours later and IPs of CnA were performed and analyzed by SDS-PAGE. Negative controls consisted of non-immune mouse IgG precipitated in exactly the same way as CnA. PE stimulation reduced CnA ubiquitination that is restored by E2 pretreatment. The bar graph represents data from 5 separate experiments. * p<0.01 vs. Control; † p<0.05 vs. PE.

Figure 5. Proteasome inhibition abolishes the inhibitory effect of E2 on CnA expression and cardiomyocyte hypertrophy

A. Lactacystin (10uM) prevents both the agonist-induced increase in CnA and the inhibitory effects of E2. Representative western blot of CnA with corresponding bar graph. B. Lactacystin abolishes the E2-mediated inhibition of NRVM hypertrophy induced by PE. * p<0.01 vs. control cells; † p<0.05 vs. PE. ‡ p<0.05 vs. Lactacystin alone. C. Phenylephrine diminishes CnA ubiquitination that is restored by E2 pretreatment. NRVMs were pretreated overnight with E2 upon serum deprivation, and stimulated with PE. Cells were harvested 4 hours later and IPs of CnA were performed and analyzed by SDS-PAGE. Negative controls consisted of non-immune mouse IgG precipitated in exactly the same way as CnA. PE stimulation reduced CnA ubiquitination that is restored by E2 pretreatment. The bar graph represents data from 5 separate experiments. * p<0.01 vs. Control; † p<0.05 vs. PE.

Figure 5. Proteasome inhibition abolishes the inhibitory effect of E2 on CnA expression and cardiomyocyte hypertrophy

A. Lactacystin (10uM) prevents both the agonist-induced increase in CnA and the inhibitory effects of E2. Representative western blot of CnA with corresponding bar graph. B. Lactacystin abolishes the E2-mediated inhibition of NRVM hypertrophy induced by PE. * p<0.01 vs. control cells; † p<0.05 vs. PE. ‡ p<0.05 vs. Lactacystin alone. C. Phenylephrine diminishes CnA ubiquitination that is restored by E2 pretreatment. NRVMs were pretreated overnight with E2 upon serum deprivation, and stimulated with PE. Cells were harvested 4 hours later and IPs of CnA were performed and analyzed by SDS-PAGE. Negative controls consisted of non-immune mouse IgG precipitated in exactly the same way as CnA. PE stimulation reduced CnA ubiquitination that is restored by E2 pretreatment. The bar graph represents data from 5 separate experiments. * p<0.01 vs. Control; † p<0.05 vs. PE.

Figure 6. Influence of TAC and E2 Replacement on Myocardial E3 Ubiquitin Ligase Expression

Total cellular RNA was isolated from myocardial samples 2 weeks following TAC or Sham procedure and ubiquitin ligase mRNA expression quantified by real time rtPCR. TAC significantly reduced the expression of Atrogin 1 (left), Mouse double minute 2 (MDM2 – middle) and muscle-specific ring finger protein 1 (MuRF1 – right). E2 replacement normalized MDM2 and MuRF1 but had no effect on atrogin1 mRNA abundance. * p<0.01 vs. Sham; † p<0.05 vs. placebo-TAC.

Figure 7. Proposed model of estrogen and ER-mediated inhibition of cardiomyocyte hypertrophy