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. Author manuscript; available in PMC: 2023 Jun 1.
Published in final edited form as: Circ Heart Fail. 2022 May 17;15(6):e008758. doi: 10.1161/CIRCHEARTFAILURE.121.008758

Estrogen protects cardiac function and energy metabolism in dilated cardiomyopathy induced by loss of cardiac IRS1 and IRS2

Hui Yan 1,2,†, Wanbao Yang 1,†, Fenghua Zhou 1, Quan Pan 1, Kimberly Allred 1, Clinton Allred 1, Yuxiang Sun 1, David Threadgill 3, David Dostal 4,5, Carl Tong 4, Shaodong Guo 1,*
PMCID: PMC9675316  NIHMSID: NIHMS1796441  PMID: 35579013

Abstract

Background:

Type 2 diabetes (T2D) is a high-risk factor for incident of cardiovascular diseases (CVD). Women at young ages show a reduced incidence of both T2D and CVD compared to men, but these disparities disappear in postmenopausal women versus age-matched men. Thus, ovaries and ovarian hormones, such as estrogen, are expected to protect from T2D and CVD. In this study, we aimed to investigate the role of ovaries and ovarian hormone estrogen in cardiac function and energy metabolism using the cardiac IRS1 and IRS2 double genes knockout mice that mimic cardiac insulin resistance.

Methods:

Control and heart-specific IRS1/2 double genes knock-out (H-DKO) mice were treated with placebo or 17β-estradiol (E2) pellets, respectively, through subcutaneous implantation. Female mice were subjected to a bilateral ovariectomy (OVX) surgery to remove endogenous E2. The cardiac function and energy metabolism were determined using echocardiography and indirect calorimeter, respectively.

Results:

All male H-DKO mice died of heart failure at 6–8 weeks as we previously described (Qi et al. Diabetes 2013, 62:3887–3900), but all female H-DKO mice survived more than 1 year. Removal of ovaries in H-DKO female mice resulted in cardiac dysfunction, and ultimately animal death. However, E2 supplementation prevented the dilated cardiomyopathy, improved cardiac function and energy metabolism, and enhanced lifespan in both male and OVX female mice deficient for cardiac IRS1 and IRS2 genes, largely owing to the activation of Akt-Foxo1 signaling cascades.

Conclusions:

These results show that estrogen protects mice from cardiac insulin resistance-induced diabetic cardiomyopathy. This may provide a fundamental mechanism for the gender difference for the incidence of both T2D and CVD. This study highlights that estrogen signaling could be a potential target for improving cardiac function and energy metabolism in humans with T2D.

Keywords: estrogen, diabetic cardiomyopathy, insulin resistance, energy metabolism, IRS1/2

Subject Terms: Cardiomyopathy, Metabolic Syndrome

Introduction

Cardiovascular disease (CVD) remains a leading cause of death globally. CVD is the consequences of structural and functional failure in heart, including coronary heart disease, heart failure (HF), cardiomyopathy, and stroke 1. The correlation between abnormal insulin signaling and increased incidence of CVD has been established in the epidemiological studies 2–4. Diabetic patients with insulin resistance exhibit a higher risk of heart disease compared with the non-diabetic patients 5. Coexistence of diabetes mellitus and cardiomyopathy accelerates the development of heart failure 6. Importantly, gender disparities exist in both type 2 diabetes mellitus (T2D) and CVD with reduced incidences in women at an early age, but these disparities disappear during postmenopause when the release of estrogen declines 2, 7, 8. However, hormone replacement therapy (HRT) is less effective in preventing these diseases in clinical trials initiated by the Women’s Health Initiative (WHI) 9–11. Thus, investigating the mechanism of estrogen action in insulin signaling and cardiac dysfunction is important for understanding the gender differences in prevalence of CVD, and may provide new therapeutic strategies for treating the disease.

We previously established a mouse model of heart insulin resistance-induced cardiomyopathy via the ablation of insulin receptor substrate (IRS) 1 and IRS2 genes in the heart (H-DKO: Heart-Double Knock-Out) 12. H-DKO male mice exhibited dilated cardiomyopathy with impaired cardiac metabolism and reduced ATP production, and the mice died of HF at 6–8 weeks-of-age 12. The heart is an energy-consuming organ in which insulin signaling regulates cardiac energetics, metabolism, and growth 13. Dysregulation of insulin signaling disrupts substrate utilization and impairs cardiac energetics, contributing to cardiomyopathy in patients with T2D 14–16. In the ob/ob and db/db mouse models of T2D, insulin resistance causes mitochondrial dysfunction, reduced ATP synthesis, and impaired respiration 17. Genetic deletion of insulin receptors also results in cardiac mitochondrial dysfunction and oxidative stress in the absence of hyperglycemia 18. In our previous study, we found that estrogen improves insulin sensitivity and enhances PI3K-Akt-Foxo1 signaling in an IRS1/2-independent manner 19. Notably, in this study, all female H-DKO mice survived more than 1 year as compared to the dead male H-DKO mice, indicating a protective effect against cardiac dysfunction and cardiomyopathy by steroid hormones. We further investigated the role of steroid hormone estrogen and ovaries in the prevention of heart failure and regulation of energy metabolism upon the loss-of-function of cardiac IRS1 and IRS2 genes. We hypothesize that estrogen prevents cardiac dysfunction and prolongs the survival of H-DKO mice of both sexes via boosting energy metabolism.

Methods

The original datasets in this study are available to other researchers upon reasonable request to the corresponding author.

Animals.

Animal procedures were approved by the institutional animal care and use committee (IACUC) at Texas A&M University. All the investigation and procedures conformed to the NIH Guide for the Care and Use of Laboratory Animals. Heart-specific IRS1 and IRS2 double knockout (H-DKO) mice were generated by breeding floxed IRS1L/L::IRS2L/L mice with αMHC-Cre mice, as previously described 12. Heart-specific IRS1, IRS2, and TGFβ triple knockout (H-TGFTKO) mice were generated by breeding floxed IRS1L/L::IRS2L/L::TGFβL/L mice with αMHC-Cre mice. Tamoxifen-inducible heart-specific IRS1 and IRS2 double knockout (iH-DKO) mice were generated by breeding floxed IRS1L/L::IRS2L/L mice with αMHC-merCREmer mice followed by a tamoxifen administration as previously described 20. All mice were on the C57BL/6J background. Cre+ and Cre- littermates were used throughout the experiments, in which Cre- littermates were used as Control mice. All mice were fed a standard chow diet (54% calories from carbohydrate, 14% from fat, and 32% from protein, 3.0 kcal/g; Envigo Teklad Diet) ad libitum. Euthanasia of animals in the home cage was performed through carbon dioxide inhalation. Carbon dioxide was delivered from a pressurized tank into a cage at a rate of 2 L/min until the cessation of respiration of mice. Mice were remained in cages for an additional 3 minutes; cervical dislocation was then performed to guarantee death.

Tamoxifen induction of IRS1/2 knockout.

Tamoxifen was dissolved in corn oil to a final concentration of 20 mg/ml. Mice at the age of 9-weeks were intraperitoneally injected with 100 mg/kg body weight tamoxifen daily for 5 consecutive days, as previously described 21.

Tamoxifen acts as a selective estrogen receptor modulator, so we conducted a preliminary trial and confirmed that tamoxifen had no prolonged influence on the body’s weight gain, blood glucose levels, and expressions of heart mitochondria-related genes after 5-week injection (Figure S1). Thus, we performed the estrogen replacement after 5 weeks of injection. To further rule out the modulation of estrogen signaling by tamoxifen, both control and iH-DKO mice were administrated with tamoxifen solution.

Ovariectomy and estrogen replacement.

Mice were randomly assigned to experimental groups (n = 6–10). All mice were subjected to the subcutaneous implantation of placebo or 17β-estradiol (E2) pellet (0.05 mg/pellet, 60-day release; Innovative Research of America, Sarasota, FL) using cholesterol as the vehicle, as previously described 19. For H-DKO male mice, the placebo or E2 pellet was administrated at age of 3-weeks. For tamoxifen induced-H-DKO mice, the placebo or E2 pellet was implanted 5 weeks after tamoxifen injection. Female mice underwent a bilateral ovariectomy (OVX) surgery (except for intact control mice) at the time of placebo or E2 pellet implantation.

Microarray analysis.

A total of 100 ng RNA from hearts of control and H-DKO mice was biotin-labeled and hybridized to Affymetrix Genechip Mouse 1.0 ST Array, which was performed at the University of Texas Southwestern Microarray Core facility. The raw intensity values were transformed using the robust multi-array average analysis and normalized. Transcriptome Analysis Console 4.1 (Applied Biosystems) was used for further analysis, as previously described 22.

Echocardiography.

Echocardiograms were performed on conscious mice with hand restraint using a VisualSonics Vevo 3100 system (Fujifilm VisualSonics Inc.), with a probe providing 30-µm spatial resolution. The chest hair was removed by using hair removal lotion before the measurement. Ultrasound transmission gel was applied to the exposed chest. Left ventricular anterior wall (LVAW), posterior wall (LVPW), left ventricular internal diameter (LVID), and IVS at end-diastole and end-systole were measured from M-mode recordings. Left ventricular volumes (LV Vol) at end-diastole and end-systole were measured from B-mode for the calculation of ejection fraction (EF), fractional shortening (FS), stroke volume (SV), and cardiac output (CO).

Mitochondrial function measurements.

Mitochondrial function in HL-1 cardiac muscle cell line was determined using the XFe96 Analyzer. HL-1 cells (20,000 cells/well) were seeded into the XF96 microplates. Cells were pretreated with Foxo1 inhibitor, AS1842856, for 3 h, followed by 100 nM E2 treatment for 1 h prior to the measurement of oxygen consumption rate (OCR) in the Analyzer. Primary hepatocytes (8000 cells/well) were seeded into the XF96 microplates. Cells were treated with 100 nM E2 for 1 h prior to the measurement of oxygen consumption rate (OCR) in the Analyzer. During the measurement, oligomycin, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), and antimycin A/Rotenone were added to calculate basal respiration, maximal respiration, ATP production, and spare respiration. All measurements were conducted according to the manufacturer’s instructions.

Western blotting.

Total proteins were isolated from the hearts of mice. Equal amounts of protein were loaded into SDS-PAGE gel for Western blotting. Antibodies against Foxo1, phosphorylated AKT at S473, total AKT, HO1, TFAM, Cytochrome c, COX1, β-actin, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA).

Serum E2 Measurements.

Serum was extracted from the mice at the end of the experiments and serum E2 levels were measured using ELISA kit (Cayman, Ann Arbor, MI) according to the manufacturer’s instructions.

Indirect calorimetric measurements.

Control and iH-DKO mice were admitted to TSE Phenomaster cages (TSE system Inc. Chesterfield, MO) after 4 weeks of the E2 implant, with free access to water and food. Animals were acclimatized in individual metabolic cages for 2 days before measurements. Gas exchange (VO2 consumption and VCO2 output), food intake, and physical activities were recorded in successive 40- to 60-min cycles for 2 consecutive days. The gas exchange data were used to calculate respiratory exchange ratio (RER = VCO2:VO2), energy expenditure (EE = [3.815 + 1.232 x RER] x VO2), fatty acid oxidation (FAO = EE x [1-RER]/0.3), and contribution of FAO to EE ratio 23. Values were normalized by body weight to the power of 0.75.

Histological analysis.

Hearts were fixed in 10% formaldehyde and then processed for paraffin-embedding. Samples were sectioned to a thickness of 5 μm and stained with hematoxylin and eosin (H&E) and Masson trichrome staining for morphometric and fibrosis analyses, as previously described 22. Masson trichrome staining result was quantified using ImageJ.

RNA isolation and quantitative PCR analysis.

RNA from hearts was extracted with Trizol reagent (Invitrogen, Carlsbad, CA) and used for cDNA synthesis via iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA). Quantitative PCR analysis was performed with Real-time PCR system (Bio-Rad, Hercules, CA) using primers as previously described 22.

Statistical analysis.

Results were presented as the mean ± SD. Data were analyzed by one-way ANOVA to determine the significance of the model. Differences between groups were determined by Tukey post-hoc test or Student t-test, as appropriate. P<0.05 was considered statistically significant. Log-rank tests were used to determine the significance of Kaplan-Meier survival curve of mice, as previously described 22.

Results

Estrogen improves cardiac dysfunction and prolongs the survival in H-DKO male mice.

Insulin signaling has been involved in the regulation of cardiac function. Loss-of-function of IRS1 and IRS2 genes in the heart caused the early death of H-DKO male mice at the age of 6- to 8-weeks. Interestingly, all female H-DKO mice survived more than one year before menopause, suggesting a protective effect of steroid hormones, such as estrogen, on the survival of H-DKO mice (Figure 1A). In order to assess whether estrogen prevents the death of male H-DKO mice and promotes cardiac function, male mice were treated with estrogen through subcutaneous implantation of an E2 pellet. E2 implantation significantly prolonged the lifespan of H-DKO males to the age of 9- to 11-weeks (P < 0.0001) (Figure 1B). H-DKO male mice exhibited systolic dysfunction, which was partially attenuated by the E2 implantation, as measured by echocardiography (Figure 1C). Specifically, systolic parameters ejection fraction (EF) and fractional shortening (FS) were reduced by 70% (P<0.0001) and 86% (P=0.0004) in H-DKO male mice as compared with WT males, respectively, while estrogen improved EF by 44% (P=0.023) and FS by 106% (P=0.028) in H-DKO males (Figure 1D). Hearts of H-DKO males exhibited dilated cardiomyopathy with an increase in left ventricular internal diameter (LVID) by 1.3-fold at end-diastole (P=0.001) and by 3.3-fold at end-systole (P=0.0004) (Figure 1D). E2 attenuated H-DKO-induced dilated cardiomyopathy as shown by a decrease in H-DKO mice of LVID by 35% at end-diastole (P=0.038) and by 31% at end-systole (P=0.036) (Figure 1D). H-DKO mice exhibited no significant changes in heart rate (HR) compared to other groups. Stroke volume (SV) and cardiac output (CO) directly represent the capacity of the heart to pump blood. H-DKO mice had significant reductions in both SV and CO compared to control mice, while E2 supplementation restored the SV and CO in H-DKO mice (Figure 1D).

Figure 1. Estrogen impacts the loss of cardiac IRS1- and IRS2-induced death and cardiac dysfunction in male mice.

Figure 1.

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A: Kaplan-Meier curve shows the survival of H-DKO male (n = 18) and female mice (n = 8). B-D: Three-week-old male control and H-DKO mice were subjected to E2 or placebo subcutaneous implantation. Kaplan-Meier curve shows the survival of mice (n = 8–13) (B). Cardiac function of mice was measured at 6-weeks-of-age by echocardiography with representative M-mode images (C) and cardiac parameters (n = 4) (D). Data are presented as the mean ± SD. Statistical differences were calculated by two-way ANOVA with Sidak’s correction for multiple comparisons. #P < 0.05 vs. H-DKO mice with placebo. E-H: Nine-week-old male control and iH-DKO mice were intraperitoneally injected with tamoxifen (100 mg/kg body weight) daily for 5 consecutive days. E2 or placebo pellet was subcutaneous implanted to the mice 5 weeks after tamoxifen induction. Kaplan-Meier curve shows the survival of mice (n = 6–12) (E). All mice were euthanized and sampled at 28-weeks-of-age to record fasting body weight (F), heart or liver weight (G), and heart/BW or liver/BW ratio (H) (n = 4–6). Data are presented as the mean ± SD. Statistical differences were calculated by one-way ANOVA with Sidak’s correction for multiple comparisons. *P < 0.05 vs. control mice with placebo; #P < 0.05 vs. iH-DKO mice with placebo. H-DKO indicates heart-specific IRS1 and IRS2 double knockout; WT, wild type; EF, ejection fraction; FS, fractional shortening; LVID;s, left ventricular internal diameter at end-systole; LVID;d, left ventricular internal diameter at end-diastole; BPM, beats per minute; iH-DKO, tamoxifen-inducible heart-specific IRS1 and IRS2 double knockout; and BW, body weight.

We further investigated the protective effect of E2 on survival in conditional heart IRS1 and IRS2 double knockout mice. Conditional deletion of IRS1/2 in the heart still caused the death of male mice. iH-DKO male mice started dying after 7 weeks of tamoxifen induction, while E2 supplement delayed the death of iH-DKO starting at 13 weeks of tamoxifen induction (Figure 1E). When half of the mice were dead, the rest of the mice were sacrificed for samples collection, as a result of which it was found that E2 only had a tendency to improve the survival of iH-DKO mice. In addition, iH-DKO mice showed a decrease in body weight (BW), but the effect of E2 on BW was not significant (Figure 1F). Heart weight and heart/BW ratio were not significantly changed among different groups. H-DKO mice showed decreases in liver weight and liver/BW ratio, which were alleviated by E2 supplementation (Figure 1G, H).

Collectively, estrogen shows the protective effect on male mice upon deletion of IRS1/2 by delaying death and attenuating dilated cardiomyopathy.

Estrogen prevents cardiac dysfunction induced by the removal of ovaries in iH-DKO female mice.

To assess the role of the ovary and ovarian hormone E2 in the protection of survival and cardiac function in iH-DKO female mice, we performed a bilateral OVX surgery followed by subcutaneous E2 implantation. The E2 level in intact female mice was around 6.7 pg/ml, and ovariectomy significantly reduced the E2 level to 2 pg/ml by about 70%. E2 pellet implantation significantly increased serum E2 levels in ovariectomized mice, but the E2 levels in mice with E2 pellets were still within the physiological range (Figure S2). Generally, removal of ovaries had no significant influences on survival rate, cardiac morphology, heart function, BW, and organ weight in control female mice (Figure 2A–H). In iH-DKO female mice, intact mice survived for more than 12 weeks after deletion of cardiac IRS1 and IRS2 induced by tamoxifen. However, iH-DKO females started dying at 2 weeks and 60% of mice died at 6 weeks after removal of ovaries. E2 implantation improved the survival rate to 60% but failed to totally prevent death (P=0.041) (Figure 2A). OVX iH-DKO mice exhibited global chamber dilatation, while intact or E2-treated OVX iH-DKO mice had similar cardiac morphology as control mice (Figure 2B). The cardiac muscle fibers of iH-DKO mice in different groups were loose and out-of-order arranged compared with control mice. In iH-DKO mice, removal of ovaries further exacerbated the muscle fiber disorder and caused fibrosis, which were attenuated by E2 supplementation (Figure 2C). Consistently, in echocardiography analysis, OVX iH-DKO mice showed significant increases in left ventricular internal diameter (LVID) and left ventricular volume (LV Vol) as well as decreases in thickness of interventricular septum (IVS), left ventricular anterior wall (LVAW), and posterior wall (LVPW) at both end-diastole and end-systole, compared with other groups. However, intact or E2-treated iH-DKO mice were protected from enlargement of left ventricle and thinned cardiac muscle walls induced by OVX iH-DKO (Figure 2D, E). Moreover, OVX reduced EF, FS, and cardiac output by 76% (P=0.0002), 90% (P<0.0001), and 38% (P=0.039), respectively, in iH-DKO female mice as compared with control mice. In contrast, intact or E2-treated iH-DKO mice showed attenuated cardiac dysfunction with improvement in these cardiac parameters as compared with OVX iH-DKO females (Figure 2D, E). Only OVX iH-DKO mice showed a significant decrease in BW compared with control intact mice, and E2 supplementation could prevent the BW decrease (Figure 2F). In iH-DKO female mice, removal of ovaries significantly increased heart weight and heart/BW ratio, indicating a heart hypertrophy, while E2 supplementation partially alleviated the heart hypertrophy and resulted in a similar heart weight and heart/BW ratio to the intact group (Figure 2G, H).

Figure 2. Estrogen impacts the conditional loss of cardiac IRS1- and IRS2-induced death and cardiac dysfunction in female mice.

Figure 2.

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Nine-week-old control and iH-DKO female mice were i.p. injected with tamoxifen (100 mg/kg body weight) continuously for 5 days. After induction of the conditional IRS1 and IRS2 double knockout, all mice were subjected to a bilateral OVX surgery (except intact mice) and implantation of placebo or E2 at 14-weeks-of-age. A: Kaplan-Meier curve shows the survival of the mice (n=5–9). B, C: Mice were euthanized at 20-weeks-of-age to collect heart samples for histomorphology analysis. Representative H&E staining images show the cardiac morphology in ventricular chamber sections of the heart (B). Representative Masson-Trichrome staining images show the cardiac interstitial fibrosis of the hearts (n=3–4) (C). D, E: Cardiac function of mice was measured at 18-weeks-of-age by echocardiography with representative M-mode images (D) and cardiac parameters (n = 3) (E). F-H：At 20-weeks-of-age, fasting body weight (F), heart or liver weight (G), and heart/BW or liver/BW ratio (H) were recorded (n = 3–5). Data are presented as the mean ± SD. Statistical differences were calculated by one-way ANOVA with Sidak’s correction for multiple comparisons. *P < 0.05 vs. control intact mice with placebo; #P < 0.05 vs. iH-DKO OVX mice with placebo; ****P<0.0001. OVX indicates ovariectomy; iH-DKO, tamoxifen-inducible heart-specific IRS1 and IRS2 double knockout; EF, ejection fraction; FS, fractional shortening; IVS;d, interventricular septum at end-diastole; LVAW;d, left ventricular anterior wall at end-diastole; LVPW;d, left ventricular posterior wall at end-diastole; IVS;s, interventricular septum at end-systole; LVAW;d, left ventricular anterior wall at end-systole; LVPW;d, left ventricular posterior wall at end-systole; LVID;s, left ventricular internal diameter at end-systole; LVID;d, left ventricular internal diameter at end-diastole; LV Vol;d, left ventricular volume at end-diastole; LV Vol;s, left ventricular volume at end-systole; BMP, beats per minute; and BW, body weight.

Taken together, ovaries and ovarian hormone estrogen protect female mice from death, cardiac dysfunction, and cardiac muscle fibrosis induced by loss of cardiac IRS1 and IRS2.

Loss of cardiac IRS1 and IRS2 induces remodeling of genes associated with cardiac metabolism

We further investigated the transcription changes in response to the deletion of IRS1/2 in the heart. The transcriptional analysis was performed in hearts isolated from wild-type (WT) and H-DKO male mice at 5-weeks-of-age by using Affymetrix microarray platform. A total of 65,956 genes were investigated, including 26,336 coding genes. There were 505 coding genes differentially expressed in H-DKO heart as compared with WT (P < 0.05) (Figure 3A). The Kegg pathway analysis identified top signaling pathways with the number of genes more than 2-fold change in response to the deletion of cardiac IRS1 and IRS2 (P < 0.05) (Figure 3B). H-DKO hearts exhibited the down-regulation of genes associated with energy metabolism-related pathways, including oxidative phosphorylation/fatty acid β-oxidation/electron transport chain (ETC). In contrast, H-DKO up-regulated genes associated with inflammation/muscle contraction/focal adhesion (Figure 3B).

Figure 3. Affymetrix microarray gene profiling of hearts from control and H-DKO mice.

Figure 3.

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Transcriptomic analysis of total RNA extracted from the hearts of control and H-DKO male mice. A: Summary of Affymetrix microarray analysis. B: Pathway analysis in response to the deletion of cardiac IRS1 and IRS2 with the number of genes with more than 2-fold changes. Blue bars represent down-regulated genes and red bars represent up-regulated genes in H-DKO mice. C: Heatmap of genes with more than 10-fold changes in the hearts of H-DKO mice. D: Heatmap of genes in pathway analysis (B) in response to the deletion of cardiac IRS1 and IRS2 with the number of genes with more than 2-fold changes. The blue color represents low expression levels, and the red color represents high expression levels. WT indicates wild-type and H-DKO, heart-specific IRS1 and IRS2 double knockout.

In these differentially expressed genes, there were 18 genes with more than 10-fold change (Figure 3C). We noticed that Tgfβ was up-regulated by 13-fold in H-DKO hearts. TGFβ (transforming growth factor β) is a master regulator that drives fibrosis in multiple cells 24. Given the fibrosis of heart muscle in OVX H-DKO mice observed in this study, the loss of IRS1/2 might cause cardiac dysfunction through the induction of TGFβ. However, the deletion of TGFβ in IRS1/2::TGFβ triple knockout mice (T-DKO) mice did not improve the survival rate of male mice (Figure S3), indicating that TGFβ-mediated fibrosis is not the leading cause of death in H-DKO mice.

Transcriptional analysis of individual genes represented as heat maps supported the pathway analysis, down-regulation of energy metabolic markers, and up-regulation of genes associated with inflammation/focal adhesion/contraction (Figure 3D). Hearts from H-DKO mice showed decreased expression of genes related to fatty acid transport (Cpt2, CD36, Slc25a20, and Slc27a1), fatty acid β-oxidation markers (Acsl1, Crat, Decr1, Acadvl, Hadha, Hadhb, and Acadm), and ETC (Ndufa3, Ndufs8, Ndufa9, Ndufs1, Sdhc, Uqcrc1, Uqcrq, Cox5b, Cox6a, and Cox7a1) (Figure 3D). The higher expression of genes involved in extracellular matrix remodeling and fibrosis (Col3a1, Col5a2, Col1a1, Col1a2, Fn1, Lamc2, Thbs1, Thbs4, Tnc, Actn1, Tln1, Igf1r, Itga5, Itgb1, Itgb5, Itgb6, and Bcl2) and muscle contraction (Mybpc2, Myh7, Vim, Actn4, Des, Tnni1, Tpm2, and Acta1) were observed in the hearts of H-DKO mice (Figure 3D).

Collectively, these data indicate that loss of cardiac IRS1 and IRS2 results in a gene transcriptional profile associated with impaired cardiac energy metabolism, which may be the leading cause of cardiac dysfunction.

Estrogen restores the transcriptional profile of cardiac energy metabolism and promotes mitochondrial function in a Foxo1-dependent manner.

We next identified the role of E2 supplementation in the regulation of the cardiac function markers and energy metabolic markers in the hearts of male mice. H-DKO placebo male hearts exhibited mRNA expression profiles of heart failure with increased ANP and β-MHC by 28- and 4.4-fold, respectively, and decreased α-MHC by 70%, compared to control placebo group. Expression of fibrosis markers TGFβ and Collagen1 were significantly increased by 60% and 4.4-fold in H-DKO placebo males, respectively (Figure 4A). In contrast, E2 reduced the expression of ANP, β-MHC, TGFβ, and Collagen1 by 51%, 53%, 66%, and 40%, respectively, and increased α-MHC expression by 75% (Figure 4A) in the hearts of H-DKO mice. We measured expression of genes related to energy metabolism. Deletion of IRS1 and IRS2 in heart significantly decreased mRNA levels of fatty acid oxidation genes CPT1α, Acsl1, Hadha, and Acadm by about 70% to 80%, and reduced expression of ETC genes Sdha, Uqcrc1, ND6, Cox1, Atp5α by around 85%. Moreover, mRNA levels of mitochondrial biosynthesis markers Tfam and NRF-1 were significantly reduced by 75%, and Hmox-1 that catalyzes the degradation of heme was increased by 40%. However, in H-DKO male mice hearts, E2 significantly restored the expression of these energy metabolism-associated markers by about 75% to 170%. (Figure 4A).

Figure 4. Estrogen impacts the loss of IRS1- and IRS2-disrupted gene expression, cardiac dysfunction, and mitochondrial function-related genes in the heart.

Figure 4.

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A-B: Nine-week-old male control and iH-DKO mice were intraperitoneally injected with tamoxifen (100 mg/kg body weight) daily for 5 consecutive days. E2 or placebo pellet was subcutaneous implanted to the mice 5 weeks after tamoxifen induction. All mice were euthanized at 28-weeks-of-age for RNA extraction and gene expression analysis in the heart (n = 4–6) (A). Foxo1 and pAKT-S473 protein levels were detected in the hearts of male control and iH-DKO mice (n=3–4) (B). Data are presented as the mean ± SD. Statistical differences were calculated by one-way ANOVA with Sidak’s correction for multiple comparisons. *P < 0.05 vs. control mice with placebo; #P < 0.05 vs. iH-DKO mice with placebo. C-D: Nine-week-old control and iH-DKO female mice were i.p. injected with tamoxifen (100 mg/kg body weight) continuously for 5 days. After induction of the conditional IRS1 and IRS2 double knockout, all mice were subjected to a bilateral OVX surgery (except intact mice) and implantation of placebo or E2 at 14-weeks-of-age. All mice were euthanized at 20-weeks-of-age for RNA extraction and gene expression analysis in the heart (n = 3–4) (C). Foxo1 and pAKT-S473 protein levels were detected in the hearts of male control and iH-DKO mice (n=3) (D). Data are presented as the mean ± SD. Statistical differences were calculated by one-way ANOVA with Sidak’s correction for multiple comparisons. *P < 0.05 vs. control intact mice with placebo; #P < 0.05 vs. iH-DKO OVX mice with placebo. E. Basal respiration, maximal respiration, ATP production, and spare respiration capacity were calculated by OCR in HL1 cells treated with E2 and Foxo1 inhibitor, AS1842856 (n=7–9). Data are presented as the mean ± SD. Statistical differences were calculated by one-way ANOVA with Sidak’s correction for multiple comparisons. *P < 0.05, **P<0.01, ***P<0.001. OVX indicates ovariectomy; iH-DKO, tamoxifen-inducible heart-specific IRS1 and IRS2 double knockout; OCR, oxygen consumption rate and FCCP, carbonyl-p-trifluoromethoxyphenylhydrazone.

We further investigated the role of ovaries and E2 in the transcriptional regulation in the heart of WT or iH-DKO female mice. Similarly, loss of cardiac IRS1/2 significantly influenced expression levels of heart failure markers in female mice, as evidenced by the results that both iH-DKO intact and OVX mice exhibited increases in ANP and β-MHC as well as a decrease in α-MHC expression, as compared with control intact mice (Figure 4C). E2 prevented OVX iH-DKO-induced changes in expression levels of α-MHC, β-MHC, and ANP (Figure 4C), showing protection of the heart at transcription level. E2 significantly decreased expression of fibrosis marker genes TGFβ and Collagen1 by 70% and 90%, respectively, in OVX iH-DKO mice (Figure 4C); this is consistent with the attenuated interstitial fibrosis in E2-treated groups (Figure 2C). In intact female mice, cardiac loss of IRS1 and IRS2 significantly reduced expression of fatty acid oxidation genes CPT1α, Acsl1, Hadha, and Acadm by about 30% to 55%, and decreased mRNA levels of ETC genes Sdha, Cox1, Uqcrc1, and Atp5α by around 20% to 70%, indicating impaired cardiac energy metabolism. iH-DKO hearts also exhibited suppression of Tfam and NRF-1 levels and induction of Hmox-1 expression, suggesting a mitochondrial dysfunction (Figure 4C). Moreover, removal of ovaries further decreased mRNA levels of these metabolic genes by 50% to 70% in iH-DKO mice, whereas E2 increased expression of these markers by 80% to 130% in OVX iH-DKO females, and almost restored the levels to those of intact iH-DKO mice (Figure 4C).

In the previous study, we found E2 can activate Akt and lead to Foxo1 degradation even without IRS1/2 in hepatocytes 19. Given that Foxo1 activation mediates the loss of cardiac IRS1/2-induced death 20, E2 may restore heart function and protect H-DKO mice from death through the regulation of Akt-Foxo1 signaling. In this study, we found that the mRNA level of Foxo1 was not significantly changed by deletion of IRS1/2, OVX surgery, or E2 by implantation in both genders (Figure 4A and 4C). We further performed the Western-blot analysis showing the abundance of Foxo1, pAkt, and total Akt in hearts of the male and OVX female mice w/o estrogen implants. Loss of IRS1/2 in both genders significantly decreased the abundance of phosphorylated Akt and increased Foxo1 protein levels (Figure 4B and 4D). As mentioned, activation of Akt by phosphorylation leads to Foxo1 degradation, and impaired IRS1/2-Akt signaling causes uncontrolled Foxo1 activation; this impairs mitochondrial function. Our data indicate that estrogen improves cardiac and mitochondrial dysfunction in H-DKO mice, potentially through the activation of Akt-Foxo1 signaling.

As shown above, estrogen supplement attenuated cardiac IRS1 and IRS2 deficiency-induced down-regulation of energy metabolism markers and inactivation of Akt-Foxo1 signaling. Specifically, E2 increased the expression levels of mitochondrial function-associated genes. We further investigated the role of E2 in the regulation of mitochondrial function using the Seahorse XFe96 Analyzer in HL-1 cells, a heart muscle cell line. Either inhibition of Foxo1 by AS1842856 or E2 supplement significantly increased basal respiration, maximal respiration, spare respiration capacity, and ATP production. However, when Foxo1 was inhibited, the promotion of mitochondrial function by E2 disappeared (Figure 4E). Additionally, we found that E2 treatment or deletion of Foxo1 significantly increased the basal respiration, maximal respiration, spare respiration capacity, and ATP production in hepatocytes. E2 treatment did not further promote mitochondrial function in Foxo1 knockout cells (Figure S4). Taken together, E2 increases mitochondrial function in a Foxo1-dependent manner.

Estrogen restores the suppression of energy metabolism induced by deletion of cardiac IRS1 and IRS2 in mice of both sexes

Loss of IRS1/2 leads to the down-regulation of energy metabolism-related signaling pathways, while E2 could restore the gene profile of energy metabolism and mitochondrial function. Thus, we investigated the roles of ovaries and E2 in the regulation of global energy metabolism. Cardiac IRS1 and IRS2 deficiency significantly decreased diurnal and nocturnal O2 consumption and energy expenditure (EE) in male mice (Figure 5A, C), but had no significant effect on intact female mice (Figure 6A, C). However, upon removal of ovaries, iH-DKO effectively reduced O2 consumption and EE in female mice, as compared with control mice. Moreover, E2 implantation significantly increased O2 consumption and EE in both male and OVX female iH-DKO mice (Figure 5A, C and 6A, C). Given that O2 consumption represents heart function, these data support that E2 prevents iH-DKO induced cardiac dysfunction.

Figure 5. Estrogen improves metabolic phenotype in iH-DKO male mice.

Figure 5.

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Control and iH-DKO male mice were i.p. injected with tamoxifen at 9-weeks-of-age continuously for 5 days. After induction of the conditional IRS1 and IRS2 double knockout, all mice were subjected to subcutaneous implants of placebo or E2 at 14-weeks-of-age. Metabolic phenotypes of mice were determined using TSE Phenomaster cages at 18-weeks-of-age. Oxygen consumption (VO2) (A), Respiratory exchange ratio (RER) (B), energy expenditure (EE) (C), fatty acid oxidation (FAO) (D), contribution of FAO to total EE (E), food intake (F), and physical activity (G) during 12 h light and dark cycles were recorded and calculated as the average of 3-day measurement after acclimatization. Data are presented as the mean ± SD, n= 4–5. Statistical differences were calculated by one-way ANOVA with Sidak’s correction for multiple comparisons. *P < 0.05 vs. control intact mice with placebo; #P < 0.05 vs. iH-DKO OVX mice with placebo. iH-DKO indicates tamoxifen-inducible heart-specific IRS1 and IRS2 double knockout; VO2, oxygen consumption; and RER, respiratory exchange ratio.

Figure 6. Estrogen improves the metabolic phenotype in iH-DKO female mice.

Figure 6.

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Control and iH-DKO female mice were i.p. injected with tamoxifen at 9-weeks-of-age continuously for 5 days. After induction of the conditional IRS1 and IRS2 double knockout, all mice were subjected to a bilateral OVX surgery (except intact mice) and implants of placebo or E2 at 14-weeks-of-age. Metabolic phenotypes of mice were determined at 18-weeks-of-age using TSE Phenomaster cages. Oxygen consumption (VO2) (A), Respiratory exchange ratio (RER) (B), energy expenditure (EE) (C), fatty acid oxidation (FAO) (D), contribution of FAO to total EE (E), food intake (F), and physical activity (G) during 12 h light and dark cycles were recorded and calculated as the average of 3-day measurement after acclimatization. All data are presented as the mean ± SD, n= 4–5. Statistical differences were calculated by one-way ANOVA with Sidak’s correction for multiple comparisons. *P < 0.05 vs. control intact mice with placebo; #P < 0.05 vs. iH-DKO OVX mice with placebo. OVX indicates ovariectomy; iH-DKO, tamoxifen-inducible heart-specific IRS1 and IRS2 double knockout; VO2, oxygen consumption; and RER, respiratory exchange ratio.

The nocturnal decreases in RER were observed in iH-DKO mice of males and OVX females (Figure 5B and Figure 6B), resulting in significant increases in fatty acid oxidation (FAO) (Figure 5D and Figure 6D) and the contribution of FAO to EE (FAO/EE) ratio nocturnally (Figure 5E and Figure 6E). These nocturnal decreases in RER were associated with ~40% reduction in nocturnal food intake (FI) (Figure 5F and Figure 6F). Most chronic or severe diseases cause anorexia 25. Thus, the decrease in food intake was the result of heart dysfunction in iH-DKO mice. Notably, E2 treatment significantly increased RER, FI, and physical activity (PA), and decreased FAO and FAO/EE ratio in both male and OVX female iH-DKO in the dark phase. E2 almost restored these indexes to the levels of control mice or intact iH-DKO females, suggesting the protective effect on energy metabolism. Additionally, both male and OVX female iH-DKO mice exhibited ~50% decrease in nocturnal PA, which was improved upon E2 implantation (Figure 5G and Figure 6G). The reduced PA was also a sign of cardiac dysfunction in iH-DKO males and OVX females.

These data indicate that the loss of IRS1/2-induced cardiac function is associated with impaired whole-body energy metabolism. Ovaries or E2 supplementation enhance cardiac function as shown by increased O2 consumption as well as increased the energy expenditure, suggesting their protective effect on heart function and energy metabolism.

Discussion

Cardiovascular disease is the major cause of morbidity and mortality for diabetic patients 3, 5. Both CVDs and T2D exhibit a lower incidence in premenopausal women than in age-matched men, but their incidences rise sharply after female menopause with reduced circulating E2 2, 7, 8. Our previous studies reported that impaired cardiac insulin signaling with loss of IRS1 and IRS1 leads to the death of male mice12, 22. In this study, we present 4 important findings: 1) all female H-DKO mice survive more than a year; 2) Ovaries protect female mice from H-DKO induced cardiac dysfunction and death, and ovarian hormone E2 prolongs the lifespans of male and OVX female H-DKO mice and prevents dilated cardiomyopathy induced by loss of cardiac IRS1 and IRS2; 3) Cardiac loss of IRS1 and IRS2 disrupts cardiac energy metabolism and mitochondrial function-associated gene expression and whole-body energy metabolism, which are restored by the E2 implant; and 4) E2 regulates Akt-Foxo1 activation and promotes mitochondrial function in a Foxo1-dependent manner.

About 90–95% of type 2 diabetes patients suffer from insulin resistance, which is a risk factor for the development of heart failure 15, 26. Insulin action is mediated through binding to the insulin receptors, which then interact with IRS proteins to activate a network of intracellular signaling pathways, such as PI3K-Akt and MAP kinase. Global or cardiac-specific disruptions of several proteins in insulin signaling cascades have been associated with mitochondrial dysfunction, dilated or hypertrophic cardiomyopathy, contractile dysfunction, impaired glucose and fatty acid metabolism, and LV remodeling 15. In this study, H-DKO mice developed dilated cardiomyopathy, and microarray analysis revealed significant down-regulation of genes associated with fatty acid transport, fatty acid oxidation, oxidation phosphorylation, and ETC in H-DKO mice hearts; this implies impaired mitochondrial function and energy metabolism. Type 2 diabetes and insulin-resistant patients exhibit mitochondrial dysfunction; this includes lower oxidative enzyme activities, reduced NADH:O2 oxidoreductase activity, and decreased lipid metabolism 27–29. The mechanism of insulin signaling that mediates mitochondrial function is still incompletely understood, especially when estrogen signaling is involved. We previously reported that insulin modulates mitochondrial biogenesis and oxidative capacity via Irs-PI3K-Foxo1 signaling 30. Loss of IRS1 and IRS2 fails to activate PI3K and Akt, resulting in activation of Foxo1. Foxo1 promotes loss of mitochondrial function by inducing Hmox-1 expression 30. Hmox-1 downregulates NRF-1 and Tfam, key genes for mitochondrial biosynthesis 31; it also catalyzes the degradation of Heme, an essential component of ETC complex III/IV in hepatocytes, as we reported 30. Our results showed the upregulation of Hmox-1, the down-regulation of NRF-1 and Tfam, the inactivation of Akt, and the increased Foxo1 abundance in H-DKO mice hearts; this suggests the involvement of Foxo1 activation in cardiac dysfunction. Importantly, our previous data revealed that the deletion of Foxo1 in the heart can prevent the development of diabetic cardiomyopathy and prolong the lifespan of H-DKO mice 20, confirming that Foxo1 activation is the leading cause of death in H-DKO mice. In addition to the regulation of mitochondrial function, Foxo1 is also reported to promote cell apoptosis, fibrosis, and contractile function in cardiomyocytes 20, 32, 33. Moreover, in this study, H-DKO promotes cardiac remodeling and fibrosis with up-regulation of correspondent markers, such as Tgfβ and collagen I. Given that Foxo1 directly regulates and induces Tgfβ expression 34, TGFβ-mediated fibrosis may cause cardiac dysfunction in H-DKO mice. However, deletion of TGFβ did not rescue the death of H-DKO mice, indicating that the rescue of H-DKO mice by deletion of Foxo1 was not mediated by TGFβ. Foxo1-mediated mitochondrial dysfunction may play an important role in heart failure progression. Collectively, cardiac IRS1/2-Akt-Foxo1 signaling and its regulation of energy metabolism are crucial for sustaining survival of animals.

This study demonstrated that E2 rescues loss of IRS1- and IRS2-impaired cardiac energy metabolism and mitochondrial function. Studies in various cell types indicate that estrogen improves mitochondrial biogenesis through its receptors: estrogen receptor (ER)α, ERβ, and G protein-coupled estrogen receptor 1 (GPER) 35–37. Direct interactions of ERα with several proteins in insulin signaling cascades have been reported. ERα binds to p85α catalytic subunit of PI3K in HEK293 and MCF7 cells 37. ERα negatively regulates PTEN activity, resulting in activation of Akt 38. A direct binding of ERα to Foxo1 was reported to suppress Foxo1 transactivation in MCF7 cell lines 39. ERα- and GPER-mediated activation of PI3K, Akt, and Erk1/2 40–42 has also been reported, but whether E2 enhances mitochondrial function via the activation of PI3K-Akt-Foxo1 signaling to compensate the loss of IRS1 and IRS2 is still elusive. This study indicated that E2 activates ERα-Akt-Foxo1 signaling independent of IRS1 and IRS2. Overexpression of ERα enhances both E2- and insulin-induced Akt phosphorylation 19. Moreover, we found that E2 enhances mitochondrial function in a Foxo1-dependent manner. These results suggest the Foxo1 is the target for E2 to enhance mitochondrial function. In this study, the increases in NRF-1 and Tfam expressions by E2 also indicate the inhibition of Foxo1. Consequently, E2 induces transcription of mitochondrial genes and increases expression of complex IV (cytochrome c oxidase), thereby increasing ETC activity and promoting mitochondrial function. Although ERs were reported to be localized to the mitochondria in many tissues in both males and females and regulate mitochondrial gene expression43, the involvement of Foxo1 in mediating the action of E2 makes mitochondrial ERs less important.

Metabolic remodeling has been associated with most cardiac diseases 44. According to the Fick equation, cardiac output and oxygen consumption show a strong linear relationship with a positive correlation. In this study, the reduced O2 consumption confirmed the cardiac dysfunction in both male and OVX female iH-DKO mice, thus resulting in a decrease in whole-body EE. Dilated cardiomyopathy upon deletion of cardiac IRS1 and IRS2 resulted in down-regulation of FAO and ETC gene expression, which is paralleled with the reduced total EE in both sexes. However, whole-body FAO, and the contribution of FAO to total EE during the dark phase, were significantly increased in both male and OVX female iH-DKO mice compared with correspondent compartments. Although metabolic caging analysis reveals that RER and FA oxidation, which are the indicators for energy substrate preference, the whole-body energy metabolism may not accurately represent the cardiac metabolism. We noticed that iH-DKO mice of both sexes exhibited decreases in nocturnal FI and PA as signs of illness. The reduced FI with limited glucose intake accounted for the decreased RER and the increased FAO. Moreover, E2 treatment completely restored the loss of cardiac IRS1- and IRS2- impaired whole-body energy metabolism, indicating its protective effect on cardiac function. However, the role of E2 in the regulation of cardiac energy metabolism and energy substrate utilization remains elusive.

In summary, the loss of cardiac IRS1/2 causes cardiac fibrosis, mitochondrial dysfunction, and disruption of energy metabolism in cardiomyocytes, which may result from Akt inactivation and Foxo1 activation. E2 shows protective effects on cardiac dysfunction induced by the loss of cardiac IRS1/2, potentially through the activation of the ERα-Akt-Foxo1 signaling pathway.

Supplementary Material

Supplemental Publication Material

What is New?

Severe cardiac insulin resistance induced by the loss of IRS1/2 in the heart causes dilated cardiomyopathy and death in male mice, while female mice were protected. Removal of ovaries leads to the death of female cardiac IRS1/2 double genes knockout mice. Sex hormone estrogen enhances cardiac function, promotes energy metabolism, prevents dilated cardiomyopathy, and prolongs survival in both male and OVX female cardiac IRS1/2 double genes knockout mice, largely owing to the regulation of Akt-Foxo1 signaling-mediated mitochondrial function.

What are the Clinical Implications?

This study provides evidence for the gender difference for the incidence of cardiovascular disease and implies that estrogen replacement therapy is feasible for the treatment of diabetic cardiomyopathy through enhancement of mitochondrial function and energy metabolism. This study also reveals that Foxo1 and estrogen signaling pathways are potential therapeutic targets for the prevention or treatment of cardiovascular diseases in patients with type 2 diabetes.

Acknowledgments

We would like to thank Jennifer DeLuca for technical assistance for the OVX surgery and Selene Howe for the Echocardiographic measurement. We also thank Mike Honig who provided English editing for the manuscript.

Sources of Funding

This work was supported by National Institutes of Health grants (R01DK095118, R01 DK120968, and R01DK124588), American Diabetes Association Career Development Award (1-15-CD-09), American Heart Association grant (BGIA-7880040), Faculty Start-up funds from Texas A&M AgriLife Research, and USDA National Institute of Food and Agriculture grant (Hatch 1010958) to S.G (PI). S. G. is recipient of the 2015 American Diabetes Association Research Excellence Thomas R. Lee Award. This work was also partially supported by the National Institutes of Health (R01DK118334 and R01AG064869) to Y.S. (PI) and S.G. (co-I).

Non-standard Abbreviations and Acronyms

BW

Body weight

CO

Cardiac output

CVD

Cardiovascular disease

EE

Energy expenditure

EF

Ejection fraction

FAO

Fatty acid oxidation

FS

Fractional shortening

H-DKO

Heart-specific IRS1 and IRS2 double genes knockout

HF

Heart failure

HR

Heart rate

HRT

Hormone replacement therapy

iH-DKO

Tamoxifen-inducible heart-specific IRS1 and IRS2 double genes knockout

IRS

Insulin receptor substrate

IVS

Interventricular septum

LVAW

Left ventricular anterior wall

LVPW

Left ventricular posterior wall

LVID

Left ventricular internal diameter

LV Vol

Left ventricular volume

OCR

Oxygen consumption rate

OVX

Ovariectomy

RER

Respiratory exchange ratio

SV

Stroke volume

T2D

Type 2 diabetes mellitus

Footnotes

Disclosures

None.

Supplemental Materials:

Figures S1–S4

References

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