Endothelial cell-specific mineralocorticoid receptor activation promotes diastolic dysfunction in diet-induced obese male mice

Abstract

Widespread consumption of diets high in fat and fructose (Western diet, WD) has led to increased prevalence of obesity and diastolic dysfunction (DD). DD is a prominent feature of heart failure with preserved ejection fraction (HFpEF). However, the underlying mechanisms of DD are poorly understood, and treatment options are still limited. We have previously shown that deletion of the cell-specific mineralocorticoid receptor in endothelial cells (ECMR) abrogates DD induced by WD feeding in female mice. However, the specific role of ECMR activation in the pathogenesis of DD in male mice has not been clarified. Therefore, we fed 4-wk-old ECMR knockout (ECMRKO) male mice and littermates (LM) with either a WD or chow diet (CD) for 16 wk. WD feeding resulted in DD characterized by increased left ventricle (LV) filling pressure (E/e′) and diastolic stiffness [E/e′/LV inner diameter at end diastole (LVIDd)]. Compared with CD, WD in LM resulted in increased myocardial macrophage infiltration, oxidative stress, and increased myocardial phosphorylation of Akt, in concert with decreased phospholamban phosphorylation. WD also resulted in focal cardiomyocyte remodeling, characterized by areas of sarcomeric disorganization, loss of mitochondrial electron density, and mitochondrial fragmentation. Conversely, WD-induced DD and associated biochemical and structural abnormalities were prevented by ECMR deletion. In contrast with our previously reported observations in females, WD-fed male mice exhibited enhanced Akt signaling and a lower magnitude of cardiac injury. Collectively, our data support a critical role for ECMR in obesity-induced DD and suggest critical mechanistic differences in the genesis of DD between males and females.

INTRODUCTION

Heart failure with preserved ejection fraction (HFpEF) represents over half of heart failure cases in the United States (1) and is associated with diminished quality of life as well as increased mortality (2). Importantly, pharmacological treatments for this condition are still limited (3, 4). A significant number of patients with HFpEF is also affected by obesity resulting from chronic consumption of diets rich in fat and sucrose (Western diet, WD). HFpEF is characterized by diastolic dysfunction (DD) and increased cardiac oxidative stress and inflammation (5–8). Of note, coronary endothelial cells (ECs) regulate cardiac function through cross talk with cardiomyocytes, fibroblasts, and immune cells (9–12). Conversely, endothelial dysfunction is associated with cardiac inflammation, oxidative stress, hypertrophy, and fibrosis (10). We and others have previously reported improvements in DD by antagonism of the mineralocorticoid receptor (MR) in both genetic and nutritional models of obesity-induced cardiomyopathy (9, 11, 13, 14). Moreover, with the use of tissue-specific MR knockout (KO) mice, the contributions of cell-specific MR activation and ensuing enhanced mineralocorticoid activity have been increasingly recognized in the pathogenesis of cardiovascular disease (CVD). In this regard, we have recently reported that mineralocorticoid receptor in endothelial cells (ECMR) promote downstream activation of the EC sodium channel (EnNaC), in concert with cardiac oxidative stress, proinflammatory macrophage polarization, and fibrosis in obese female mice, all of which contribute to the pathogenesis of DD (15). Of note, sex differences have been well described in the prevalence and clinical manifestations of DD associated with obesity and overnutrition (16–18). Moreover, the role of ECMR in the genesis of DD in males chronically fed a WD remains to be elucidated. Therefore, in the present investigation, we evaluated the impact and underlying mechanisms of ECMR on DD in conditions of chronic WD feeding in males.

RESEARCH DESIGN AND METHODS

Animals

Animal procedures were performed in accordance with the Animal Use and Care Committee (ACUC) at the University of Missouri in Columbia, the Harry S. Truman Memorial Veterans Administration Hospital Subcommittee for Animal Safety, and National Institutes of Health guidelines. To assess the effect of the deletion of MR in endothelial cells we used ECMRKO mice and littermates (LM) as controls. This model has been previously characterized in our laboratory (15, 19). Briefly, mice with loxP sites encompassing exon 5 and 6 of the MR gene were sequentially crossed with Cad-Cre-positive mice (VE-Cadherin promoter driving expression of Cre recombinase) (20) to obtain double-floxed, MR Cre-positive mice (ECMRKO) and double-floxed MR VE-Cadherin Cre-negative mice (LM). The VE-Cadherin Cre recombinase used in this model has been previously shown to be expressed in adult endothelial cells (21). At 4 wk of age, mice were placed on either a WD consisting of 4.65 kcal/g of food, 46% kcal from fat, 36% kcal from carbohydrate [sucrose (17.5%), and high-fructose corn syrup (17.5%) of weight], and 17.6% kcal from protein (Test Diet modified 58Y1; 5APC) or chow diet consisting of 3.31 kcal/g of food, 13% kcal fat, 58% kcal from carbohydrate, 29% kcal from protein (Laboratory Rodent Diet 5001*, Lab Diet, CD) for 16 wk. Mice were provided water ad libitum while cohoused in an environmentally controlled facility maintained at 24°C on a 12-h light:dark cycle from 0700 to 1900. Mice were fasted for 4 h in the morning (5:00 to 9:00 AM) before a blood sample was collected from the tail vein. Blood was analyzed for glucose using an Alpha Trak II glucometer. Fat mass was measured by EchoMRI 500 for quantitative magnetic resonance analysis (Echo Medical Systems, Houston, TX). The composition of diet (including sodium content) is depicted in Table 1.

Table 1.

Comparison of control and Western diet compositions

	Control Diet Chow		Western Diet Chow
Dietary Component	% Diet	% Energy	% Diet	% Energy
Fat	5.1	13.4	24	46.4
Carbohydrates	48.7	57.9	41.8	36
Sucrose	3.15		17.5
High-fructose corn syrup	0		17.5
Protein	24.1	28.7	20.5	17.6
Sodium	0.39		0.14
Fiber	3.3%		5.0

Fat consists of porcine animal fat, essential fatty acids, corn, and soybean oil. Carbohydrate consists of starch, maltodextrin, and powdered cellulose.

Two-Dimensional Echocardiography

Doppler ultrasound studies were performed using a Vevo 2100 (Transonic, Fujifilms Ontario, Canada) at the Small Animal Ultrasound Imaging Center at the Harry S. Truman Veterans Affairs Research Center on isoflurane-anesthetized mice (1.75%–2% in a 100% oxygen stream) utilizing an MS400 (18–38 MHz) echo probe near the end of the intervention (15). Two-dimensional echocardiograms were performed in the apical four-chamber view. A small sample volume was positioned within the mitral inflow stream just proximal to the mitral leaflets to acquire early (E) and late (A) diastolic blood flow velocities in pulse-wave (PW) Doppler mode. From the PW spectra, we determined isovolumic relaxation time (IVRT), isovolumic contraction time (IVCT), and ejection times, parameters required to calculate the myocardial performance index (MPI), also known as the Tie index. MPI was calculated as the sum of isovolumic contraction and relaxation times divided by ejection time. B- and M-mode images of the left ventricle and septum in short-axis view were acquired at the level of the papillary muscles. Left ventricular anterior and posterior wall thicknesses at end systole and diastole, luminal diameters, and ejection fraction (EF) were determined offline in M-mode. Next, tissue Doppler imaging was performed in the apical four-chamber view by placing a sample volume at the septal annulus to acquire early (e′) and late (a′) septal annular velocities. All data were acquired and analyzed offline by a single blinded observer.

Ultrastructure Analysis Using Transmission Electron Microscopy

LV heart tissue was thinly sliced (2 mm squares) and placed immediately in primary EM fixative (2% glutaraldehyde, 2% paraformaldehyde in 0.1 M sodium cadodylate buffer, pH 7.35). A Pelco 3440 laboratory microwave was utilized for secondary fixation, with acetone dehydration and Epon-Spurr’s resin infiltration. Specimens were placed on a rocker overnight at room temperature, embedded the following morning, and polymerized at 60°C for 24 h. A Leica Ultracut UCT microtome with a 45° Diatome diamond knife was used to prepare 85-nm thin sections. The specimens were then stained with 5% uranyl acetate and Sato’s triple lead stain. A JOEL 1400-EX transmission electron microscopy (TEM) (Joel, Tokyo, Japan) was used to view three fields randomly chosen per mouse to obtain three ×2,500 images per LV (15).

Evaluation of Cardiac Hypertrophy and Cardiac Fibrosis

A portion of the LV myocardium was obtained at harvest and fixed in 3% paraformaldehyde, dehydrated in ethanol, and paraffin-embedded. To assess interstitial fibrosis, 5 µm sections were stained with picrosirius red as previously described (22). After slides were scanned, Aperio ImageScope (Leica Biosystems, Buffalo Grove IL) was used to quantify 10 interstitial images (×20) of the most fibrotic regions per sample. The built-in algorithm Positive Pixel Count (V9) was used with the following parameters to determine percent fibrosis (Hue value: 0.6875, Hue width: 0.4, Color saturation threshold: 0.0). Finally, positivity (positive/total pixels) was averaged over all regions from a single group to determine interstitial fibrosis. Hypertrophy was evaluated by hematoxylin and eosin staining of 5 µm sections from 3% paraformaldehyde-fixed specimens. Two ×40 images were captured and 20 cells from each image were counted for evaluation of cardiomyocyte size using ImageJ analysis by a blinded observer.

Immunohistochemistry

To assess production of reactive oxidative species, sections of LV myocardium were fixed as described for hypertrophy and fibrosis, then incubated with a 1:150 rabbit polyclonal anti-3-nitrotyrosine (3-NT) antibody (No. ab5411 RRID AB_177459 Millipore, MA) by automated staining. An Aperio ImageScope (Leica Biosystems, Buffalo Grove IL) was used for scanning and quantifying 10 images (×20) corresponding to the 10 interstitial images used for evaluation of fibrosis. The built-in algorithm Positive Pixel Count (V9) was used with the following parameters to quantitate brown-colored staining (Hue value: 0.6875, Hue width: 0.4, Color saturation threshold: 0.0). Intensity of the staining was averaged over all regions from a single group to determine the extent of 3-NT accumulation.

Accumulation of myocardial macrophages was determined by staining using F4/80 antibody, a macrophage marker. Five micron sections of the LV free wall were initially quenched of endogenous peroxidase and incubated with 1:200 rabbit polyclonal anti-F4/80 antibody (No. ab100790 RRID AB_10675322, Abcam, MA) overnight. Sections were then washed and incubated with a secondary antibody and signals were visualized using the diaminobenzidine (DAB) chromogen system (DAKO). For F4/80-positive macrophages, the Aperio ImageScope was used to identify F4/80 macrophages in 10 sites at ×40 magnification. Analysis was performed in a blinded manner.

Western Blotting

Frozen sections of LV myocardium were thawed and lysed in homogenization buffer containing 250 mM sucrose, 50 mM Tris·HCl, protease and phosphatase inhibitor cocktail, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, and 150 mM NaCl. Lysates were sonicated and centrifuged at 13,000 g for 10 min. After determination of protein concentration, lysates were mixed with 5× SDS sample buffer and boiled for 5 min except for the immunoblotting of phospholamban (PLB). Subsequently, 20–50 µg of each protein lysate was electrophoretically separated on an SDS-polyacrylamide gel and transferred to nitrocellulose membranes. After being blocked in 5% bovine serum albumin in PBS for 1 h, the membrane was incubated with appropriate antibodies. After the membrane was washed, horseradish peroxidase-conjugated secondary antibodies were incubated with membranes followed by developing of blots using a chemiluminescence kit (Amersham, Arlington Heights, IL). Protein levels were quantified by densitometry and normalized to GAPDH for Akt and mammalian target of rapamycin (mTOR) signaling, and to ponceau S staining for phospholamban. The antibodies used in the present study have been extensively used in our laboratory (9, 15, 17, 22). Furthermore, the specificity of the antibodies has been examined by their respective manufacturers and validated by either knockout, inhibitor studies for phosphorylated forms, or silencing strategies, (23–27) (Supplemental Table S1; see https://doi.org/10.6084/m9.figshare.21561684.v1). Antibodies used were: Phospho-Akt serine (Ser⁴⁷³) (No. 9271, RRIDAB_329825, Cell Signaling, MA), Phospho-Akt threonine (Thr³⁰⁸) (No. 4056, RRID AB_331163, Cell Signaling), Akt (No. 9272 RRID AB329827, Cell Signaling), Phospho-mTOR (Ser²⁴⁸¹) (RRID AB 2262884, Cell Signaling), Phospho-mTOR (Ser²⁴⁴⁸) (No. 2976, RRID_490932, Cell Signaling), m-TOR (No. 2972, AB_ 330978, Cell Signaling), GAPDH (sc-20356, RRIDAB_641103 Santa Cruz, CA), SERCA 2 (No. ab2861 RRID AB_2061425, Abcam, MA), Phospho-phospholamban (No. ab15000 RRID AB_301562, Abcam), and phospholamban (No. ab2865 RRID AB_ 2167905, Abcam). Primary antibodies were used in 1:1,000 dilution and secondary antibodies were diluted 1:30,000.

Isolation of ECs and Measurement of Sodium Currents by Patch Clamp

Lung ECs were isolated from a subgroup of male mice fed a WD using a commercial kit (Miltenyi Biotec, Inc., Auburn, CA) as previously described (12). Purity of isolated ECs was confirmed by CD31 staining. After ECs were cultured for 5 to 8 days, at 37°C, 5% CO₂, whole cell Na⁺ currents were recorded by patch clamp using an EPC-10 amplifier (Heka) and Patchmaster and analyzed using Igor Pro software (28). EnNaC currents were confirmed by inhibition with the Na⁺ channel inhibitor, amiloride (1 µM).

Statistical Analysis

Results are reported as the means ± SE. Differences in outcomes were determined using two-way ANOVA and Tukey post hoc test. Results were considered significant when P < 0.05. Unpaired t test was for the statistical analysis of sodium currents in ECs from LM mice and ECMR KO mice fed with WD. The sample size for the power analysis was based on our previously published results (9, 15). All statistical analyses were performed using GraphPad (Prism v.9.0).

RESULTS

ECMR Deletion Prevents WD-Induced Cardiac Diastolic Dysfunction

Sixteen weeks of WD feeding resulted in significant increases in body weight, fat mass, and fasting blood glucose levels. ECMR deletion did not impact any of these measurements (Table 2). Cardiac function parameters evaluated by echocardiography are presented in Table 3 and Fig. 1. Compared with CD-fed mice, WD feeding in LM was associated with increases in LV filling pressure (E/e′ ratio) and diastolic stiffness [E/e′/LV inner diameter at end diastole (LVIDd)], results that indicate impaired diastolic function (Fig. 1, A and B). ECMR deletion was associated with improvements in WD-induced diastolic abnormalities (Fig. 1, A and B). WD also caused impairments in the isovolumetric contraction time (IVCT) and myocardial performance index (MPI); however, these abnormalities were not improved by ECMR deletion (Table 3).

Table 2.

Body composition and fasting glucose values in the different cohorts

	CD-LM	CD-KO	WD-LM	WD-KO
Body weight, g	29.7 + 0.86 (9)	29.6 + 0.63 (8)	35.7 + 1.22* (12)	36.7 + 0.69 (15)
Fat mass, g	5.30 + 1.78 (3)	3.89 + 0.72 (5)	10.5 + 0.66** (6)	11.44 + 1.08 (6)
Blood glucose, mg/dL	155 + 9 (6)	172 + 6 (6)	208 + 16*** (7)	196 + 7 (5)

Values are represented as means ± SE, number of mice per cohort is stated in parenthesis. CD, control diet; ECMRKO, ECMR knockout; LM, littermate; WD, Western diet. Two-way ANOVA and Tukey post hoc test were used for statistical analysis.

*P = 0.0002 vs. CD-LM; **P = 0.02 vs. CD-LM; ***P = 0.019 vs. CD-LM.

Table 3.

Ultrasound-derived cardiac parameters

Parameters	CD-LM (6)	CD-KO (5)	WD-LM (8)	WD-KO (10)
Ejection fraction, %	60 ± 3.0	63 ± 2.4	65 ± 2.9	67.4 ± 2.8
Cardiac output, mL·min−1	38 ± 4	36 ± 2	32 ± 3	34 ± 2
Stroke volume, µL	88 ± 7	87 ± 4	72 ± 4*	77 ± 4
IVCT, ms	6.2 ± 0.4	7.9 ± 0.8	10.7 ± 0.6*	9.3 ± 0.5
E, mm·s−1	675 ± 21	754 ± 74	726 ± 34	718 ± 31
A, mm·s−1 late	470 ± 30	480 ± 62	578 ± 28*	523 ± 29
E/A ratio	1.46 ± 0.07	1.62 ± 0.15	1.23 ± 0.03	1.39 ± 0.05
e′, mm·s−1	32.5 ± 2.9	31.8 ± 4.2	22.8 ± 2.9*	28.6 ± 2.2
a′, mm·s−1	76 ± 4	69 ± 10	51 ± 3	76 ± 6
e′/a′ ratio	1.33 ± 0.05	1.28 ± 0.18	0.95 ± 0.09*	0.75 ± 0.0
E/e′ (LV filling pressure)	21.6 ± 1.9	24.5 ± 2.1	33.9 ± 2.4*	26.0 ± 1.4#
Diastolic stiffness (E/e′/LVIDd)	5.18 ± 0.4	6.16 ± 0.6	8.74 ± 0.65*	6.96 ± 0.36#
IVRT, ms	12.2 ± 0.7	12.6 ± 0.6	15.4 ± 1.0*	13.4 ± 1.1
MPI	0.39 ± 0.02	0.46 ± 0.4	0.59 ± 0.03	0.52 ± 0.03
LA/Ao ratio	1.09 ± 0.04	0.99 ± 0.05	1.23 ± 0.0.05	1.03 ± 0.04#
LV mass, mg	91.6 ± 3.3	93.7 ± 4.3	98.7 ± 6.1	95.2 ± 45
AWTd, mm	0.78 ± 0.04	0.79 ± 0.02	0.90 ± 0.04	0.84 ± 0.04
PWTd, mm	0.72 ± 0.03	0.77 ± 0.03	0.81 ± 0.04	0.80 ± 0.04
LVIDd, mm	4.15 ± 0.11	4.00 ± 0.06	3.89 ± 0.15	3.76 ± 0.15
LVIDs, mm	2.83 ± 0.13	2.61 ± 0.16	2.54 ± 0.18	2.38 ± 0.16

Values are represented as means ± SE. Sample sizes are shown in parentheses. a, late mitral flow velocity; Ao, aorta diameter; AWTd, anterior wall thickness at end diastole; CD, control diet; e′, early septal wall velocity during diastole; a′, late septal wall velocity during diastole; E, early mitral flow velocity; E/E′, LV filling pressure; ECMR, endothelial cells mineralocorticoid receptor; ECMRKO, ECMR knockout, IVCT, isovolumic contraction time; IVRT, isovolumic relaxation time; LA, left atrium diameter; LM, littermate; LV, left ventricle; LVIDd, LV inner diameter at end diastole; LVIDs, LV inner diameter at end systole; MPI, myocardial performance index; PWTd, posterior wall thickness at end diastole; s′, peak septal wall velocity during systole; WD, Western diet. Two-way ANOVA and Tukey post hoc test were used for statistical analysis.

*P < 0.05 vs. CD-LM; #P < 0.05 vs. WD.

Figure 1.

Endothelial cell mineralocorticoid receptor (ECMR) deletion prevents Western diet (WD)-induced diastolic dysfunction in male mice. A: E/E′ ratio (left ventricle, LV filling pressure) [#P = 0.001 vs. control diet fed littermates (CD-LM); ϕP = 0.02 vs. WD-LM]. B: diastolic stiffness (E/e′/LV inner diameter at end diastole (LVIDd)] (#P = 0.0004 vs. CD-LM; ϕP = 0.05 vs. WD-LM). ECMRKO, ECMR knockout. Data are expressed as means ± SE. Two-way ANOVA and Tukey post hoc test were used for statistical analysis. Sample size: 6 for CD-LM, 5 for CD-KO, 8 for WD-LM, and 10 for WD-KO.

ECMR Deletion Ameliorates Myocardial Tissue WD-Induced Akt and mTOR Activation

Akt is involved in multiple molecular pathways, including insulin signaling, glucose homeostasis, protein synthesis, as well as cell growth and survival (29), and it is also known to participate in the pathogenesis of CVD and heart failure (30). We have previously shown in myocardial tissue of male Zucker obese rats with established DD an enhanced Akt phosphorylation/activation (22). Therefore, in the present investigation, we examined Akt and downstream mammalian target of rapamycin (mTOR) phosphorylation/activation in the myocardium of our experimental groups by Western immune blot analysis (Fig. 2A). Relative to CD-fed mice, WD feeding in LM mice was associated with enhanced Akt activation assessed by Thr³⁰⁸ and Ser⁴⁷³ phosphorylation, whereas ECMR deletion resulted in significantly reduced Akt phosphorylation/activation in WD-fed mice (Fig. 2, B–F). The ratio of phosphorylated Akt (Thr³⁰⁸ and Ser⁴⁷³) to total Akt protein also showed a significant increase in WD-fed mice and was significantly lower in ECMRKO animals (Fig. 2, D and F). We further studied downstream signaling via mTOR (Fig. 3, A–F). Overall, there were no differences in total mTOR expression or mTOR Ser²⁴⁸¹ in both the treatment groups (Fig. 3, B–D). However, WD-fed LM mice exhibited increased mTOR Ser²⁴⁴⁸ phosphorylation/activation, which was significantly reduced in ECMRKO mice fed a WD (Fig. 3E). The ratio of phosphorylated mTOR (Ser²⁴⁴⁸) to total mTOR was also increased in WD-fed LM mice and significantly reduced in ECMRKO mice fed a WD (Fig. 3F).

Figure 2.

Effect of endothelial cell mineralocorticoid receptor (ECMR) deletion on Western diet (WD)-induced myocardial Akt activation in male mice. A: Western blot images. B: phosphorylation of Akt at the Thr³⁰⁸ residue [&P = 0.005 vs. control diet fed littermates (CD-LM); #P < 0.001 vs. CD-LM; ϕP = 0.001 vs. Western diet fed littermates (WD-LM)]. C: total Akt (#P = 0.04 vs. CD-LM). D: ratio of phosphorylation of Akt at the Thr³⁰⁸ residue to total Akt (&P = 0.001 vs. CD-LM; #P = <0.001 vs. CD-LM; ϕP < 0.001 vs. WD-LM). E: phosphorylation of Akt at Ser⁴⁷³(#P = 0.003 vs. CD-LM; ϕP = 0.001 vs. WD-LM). F: ratio of phosphorylation of Akt at the Ser⁴⁷³ residue to total Akt (#P < 0.001 vs. CD-LM; ϕP = 0.003 vs. WD-LM). ECMRKO, ECMR knockout. Data are expressed as means ± SE. Two-way ANOVA and Tukey post hoc test were used for statistical analysis. Sample size: 6 for CD-LM, 5 for CD-KO, 6 for WD-LM, and 7 for WD-KO.

Figure 3.

Effect of endothelial cell mineralocorticoid receptor (ECMR) deletion on Western diet (WD)-induced myocardial mammalian target of rapamycin (mTOR) activation in male mice. A: Western blot image. B: phosphorylation of mTOR at the Ser²⁴⁸¹ residue. C: total mTOR. D: ratio of phosphorylation of mTOR at Ser²⁴⁸¹ to total mTOR. E: phosphorylation of mTOR at Ser²⁴⁴⁸ [#P = 0.004 vs. control diet fed littermates (CD-LM); ϕP = 0.02 vs. Western diet fed littermates (WD-LM)]. F: ratio of phosphorylation of mTOR at Ser²⁴⁴⁸ residue to total mTOR (#P = 0.009 vs. CD-LM; P = 0.05 vs. WD-LM). ECMRKO, ECMR knockout. Data are expressed as means ± SE. Two-way ANOVA and Tukey post hoc test were used for statistical analysis. Sample size: 6 for CD-LM, 5 for CD-KO, 6 for WD-LM, and 7 for WD-KO.

ECMR Deletion Prevents Myocardial Tissue WD-Induced Suppression of Phosphorylation of Phospholamban

We have previously shown changes in the expression of key calcium-handling proteins in a mouse model of DD (31). In this regard, phospholamban (PLB) is a critical regulator of cardiac contractility, as it regulates calcium uptake in the sarcoplasmic reticulum (SR) by modulating the activity of SR calcium ATPase (SERCA) (31). Thus, we evaluated changes in the phosphorylation state of PLB and determined the levels of SERCA2 protein (Fig. 4, A–D). Neither WD feeding nor ECMRKO was associated with changes in SERCA2 protein levels (Fig. 4B). However, WD feeding resulted in a significant decrease in phosphorylation of PLB (5 kDa monomeric form) in LM mice (Fig. 4C), and there was a trend toward increased phosphorylation in the ECMRKO cohort (P = 0.06) (Fig. 4C). Total protein and the phosphorylation state of the 25-kDa multimeric form of PLB was not significantly different in any of the cohorts (Fig. 4A). Total PLB was marginally increased in ECMRKO (Fig. 4D); however, the ratio of phosphorylated 5 kDa monomeric PLB to total phospholamban did not significantly increase in ECMRKO mice (data not shown).

Figure 4.

Effect of endothelial cell mineralocorticoid receptor (ECMR) deletion on myocardial phospholamban (PLB) phosphorylation and SERCA2 expression in male mice. A: Western blot image. B: SERCA2 expression. C: phosphorylation of phospholamban 5 kDa [#P < 0.014 vs. control diet fed littermates (CD-LM); ϕP = 0.06 vs. Western diet fed littermates (WD-LM)]. D: total phospholamban 5 kDa. ECMRKO, ECMR knockout. Data are expressed as means ± SE. Two-way ANOVA and Tukey post hoc test were used for statistical analysis. Sample size: 6 for CD-LM, 5 for CD-KO, 6 for WD-LM, and 7 for WD-KO.

Effects of ECMR Deletion on Myocardial Oxidative Stress, Inflammatory Responses, Fibrosis, and Hypertrophy

3-Nitrotyrosine (3-NT) staining, a marker of oxidative stress, was increased in cardiomyocytes following WD feeding in LM controls (Fig. 5A). Conversely, 3-NT accumulation in the WD-fed ECMRKO cohort was significantly decreased compared with LM (Fig. 5A). As we have previously reported development of maladaptive macrophage immune inflammatory responses associated with enhanced oxidative stress in female mice fed WD and its abrogation by ECMR deletion (15), we examined F4/80 as a marker of macrophage infiltration. Myocardial accumulation of macrophages was increased in WD-fed mice and was prevented by ECMR deletion (Fig. 5B). In contrast, neither the WD feeding nor ECMR deletion had a significant effect on markers of myocardial fibrosis or cardiomyocyte size (Fig. 5, C and D).

Figure 5.

Effect of endothelial cell mineralocorticoid receptor (ECMR) deletion on oxidative stress, macrophage accumulation, myocardial fibrosis, and cardiac hypertrophy in male mice. A: 3-nytrotyrosine staining of left ventricle (LV) for oxidative stress [#P < 0.03 vs. control diet fed littermates (CD-LM); ϕP = 0.02 vs. Western diet fed littermates (WD-LM)]; B: LV myocardial F4/80 staining for macrophage accumulation (#P < 0.001 vs. CD-LM; ϕP = 0.004 vs. WD-LM). C: quantification of LV myocardial fibrosis by picrosirius red staining. D: hematoxylin and eosin staining for cardiomyocyte size measurement. ECMRKO, ECMR knockout. Data are expressed as means ± SE. Two-way ANOVA and Tukey post hoc test were used for statistical analysis. Sample size: 6 for CD-LM, 5 for CD-KO, 6 for WD-LM, and 7 for WD-KO. Scale bar: 50 µm.

Mitochondrial Abnormalities Caused by WD Feeding Were Abrogated by ECMR Deletion

Ultrastructural examination of the left ventricle in WD-fed mice showed focal cardiomyocyte remodeling, characterized by areas of normal appearance interspersed with sections of sarcomeric disorganization (Fig. 6, A–D). WD feeding also resulted in mitochondrial disorganization with loss of mitochondrial matrix electron density and cristae resulting in hyperlucency, as well as crista fragmentation (Fig. 6C). These changes were attenuated in ECMRKO mice fed a WD (Fig. 6D). Quantitative analysis of mitochondrial abnormalities also showed significant mitochondrial injury in WD diet-fed LM mice that were significantly improved by deletion of ECMR (Fig. 6E). Mitochondrial density was decreased in WD feeding in LM mice, which was improved in ECMR KO mice fed WD (Fig. 6F). However, neither mitochondrial area nor mitochondrial diameter was affected in both the treatment groups (Fig. 6, G and H).

Figure 6.

Endothelial cell mineralocorticoid receptor (ECMR) deletion prevents disorganization of sarcomeres and accumulation of aberrant mitochondria in male mice. A (CD): illustrates the orderly nature of rows of sarcomeres (S) alternating with intermyofibrillar mitochondria (IMF Mt). (Magnification: ×2,500, scale bar = 1 µm). Note that there is usually only one to three layers of Mt within the IMF Mt regions in the control diet (CD.) Inset depicts a higher magnification of IMF Mt ×10,000; scale bar = 0.2 µm. B (CD ECMR-KO): demonstrates similar findings as in the control diet. C (WD): markedly disordered sarcomeres and the accumulation of aberrant Mt with loss of electron dense Mt matrix and fragmentation of crista in the Western model (double arrows); note insets in each image (each inset ×10,000; scale bar = 0.2 µm). D (WD-ECMR-KO): electron dense Mt and prevention of disordered sarcomeres and the aberrant IMF Mt in the WD (C). Endothelial cell mineralocorticoid receptor knockout models appear similar to the control models in A. Ld, lipid droplets; S, sarcomere; Z, Z lines. Quantitative data are shown in E–H. E: Mito injury [#P = 0.0001 vs. control diet fed littermates (CD-LM); ϕP = 0.0008 vs. Western diet fed littermates (WD-LM)]; F: mitochondrial density (#P = 0.043 vs. CD-LM, ϕP = 0.0144 vs. WD-LM). G: mitochondria area. H: mitochondrial diameter. For quantitative data 10 mitochondria from three different sections from each mice was used (30 mitochondria for each sample). Two-way ANOVA and Tukey post hoc test were used for statistical analysis. Sample size: 3/group.

ECMR Deletion Prevents WD-Induced Endothelial Epithelial Sodium Channel Activation

Endothelial epithelial sodium channel (EnNaC) is a downstream target of ECMR activation (19, 32). Our group has shown that deletion of the α subunit of EnNaC in endothelial cells decreases cardiomyocyte stiffness and prevents WD-induced DD (12). We have also recently reported that WD induces EnNaC activation in endothelial cells (28). Therefore, in this study, we examined whether ECMRKO results in decreased EnNaC activation in endothelial cells in the WD-fed mice. As expected, WD did lead to increased inward sodium current in endothelial cells. Conversely, ECMRKO resulted in decreased inward sodium current, as shown in Fig. 7. These results suggest that ECMRKO prevents WD-induced activation of EnNaC in endothelial cells.

Figure 7.

Endothelial cell mineralocorticoid receptor (ECMR) deletion prevents Western diet (WD)-induced endothelial epithelial sodium channel activation in male mice. A: representative tracings of whole cell Na⁺ currents as assessed by patch clamp. Currents were recorded at membrane potentials between −80 and +80 mV at 40 mV intervals. B: graph showing group data, at −80 mV, from 16 cells from three WD-fed mice and 19 cells from 5 ECMRKO mice fed the WD. Data are expressed as means ± SE. Unpaired T test was for the statistical analysis #P = 0.04 vs. WD-LM.

DISCUSSION

In this study, we have examined the role of MR activation in endothelial cells in the development of obesity-associated DD in male mice fed a WD. We provide novel evidence demonstrating that ECMR deletion and downstream decreases in EnNaC activity protect against development of DD in males chronically fed a WD. This was associated with suppression of Akt/mTOR activation and enhancement in the phosphorylation status of the calcium-handling protein PLB, which is a key regulator of cardiac contractility (31). The amelioration in DD was also associated with a reduction in WD-induced oxidative stress and macrophage infiltration. Furthermore, ECMR deletion also resulted in decreased focal ultrastructural mitochondrial abnormalities and sarcomere disorganization induced by WD.

Echocardiographic analysis after 16 wk of WD feeding in the control cohort demonstrated DD characterized by an increase in E/e′ ratio, a surrogate marker of LV filling pressure and diastolic stiffness, which was prevented by ECMR deletion. The E/e′ ratio has been reported to predict adverse cardiovascular outcomes, including all-cause mortality, cardiovascular death, and heart failure hospitalizations in various disease states (33, 34). Endothelial dysfunction is often present in clinical conditions such as obesity and type 2 diabetes (35–37), and this dysfunction is typically accompanied by augmented oxidative stress as well as inflammation. Importantly, during the pathogenesis of endothelial dysfunction, ECMR activation plays a critical role by promoting oxidative stress, inflammation, and fibrosis (12, 15, 28, 38, 39). In this study, we did not observe cardiac hypertrophy or fibrosis in WD-fed mice. However, DD can occur independently of cardiac hypertrophy or fibrosis (40–42). These findings suggest that other signaling pathways not resulting in hypertrophy and/or fibrosis contribute to WD-induced DD and the improvements following ECMR deletion.

In this regard, we examined whether increased phosphorylation/activation of Akt and aberrant SERCA2/PLB signaling were implicated. We found significant increases in myocardial Akt phosphorylation, which was significantly reduced in the ECMRKO mice. Excessive activation of Akt can occur as a result of inappropriate activation of renin-angiotensin-aldosterone system, as typically happens in conditions of chronic WD feeding derived from chronic WD feeding (20, 22, 43, 44). Of note, activation of Akt has been observed in patients with type 2 diabetes mellitus and LV dysfunction (44–47). Furthermore, in ob/ob mice, a genetic model for obesity-associated DD, the levels of phosphorylated Akt (Ser⁴⁷³) are also increased (45). Importantly, acute activation of Akt appears to result in beneficial effects on myocardial function, whereas persistent activation of Akt has been associated with increased fatty acid uptake and altered mitochondrial function, thereby having impact on metabolic burden on cardiomyocytes (46). Mitochondrial structural and functional abnormalities mediated by persistent activation of Akt occur independently of cardiac hypertrophy, although hypertrophy may accompany persistent activation of Akt (48). We also observed enhanced phosphorylation of Akt (Thr³⁰⁸) in the absence of phosphorylation/activation of mTOR. In this regard, activation of Akt at Thr³⁰⁸ residues may occur directly by phospho-phosphoinositide-dependent kinase-1, which in turn may enhance phosphorylation of mTOR2 (49) and warrants further additional exploration in further studies.

DD is characterized by impaired myocardial relaxation resulting from impaired calcium handling (50–52). SERCA2 function is partly dependent on the expression level and activity of PLB, as well as its interaction with its inhibitor PLB (53). Phosphorylation of PLB abrogates the negative regulation on SERCA2, thus leading to enhanced calcium sequestration in the SR and enhanced diastolic relaxation (54). SERCA2 protein levels were unaffected in both WD-fed mice and WD-fed mice with ECMR deletion. However, WD feeding resulted in decreased phosphorylation of PLB, which tended to be prevented by ECMR deletion (P = 0.06). These data suggest that ECMR contributes to DD via dysregulation of calcium handling in conditions of WD feeding.

Oxidative stress is a recognized determinant of the development and progression of DD (43, 55). We have observed a significant increase in oxidative stress in the heart caused by WD feeding that was suppressed by ECMR deletion. Impairments in mitochondrial structure and function are also considered to be important contributors to DD, particularly during early stages after high-fat diet feeding (56, 57). We observed mitochondrial and sarcomere disorganization and mitochondrial fragmentation in WD-fed male mice, which were abrogated in ECMRKO mice. Oxidative stress has also shown to be associated with increased macrophage infiltration (15). Accordingly, we also observed accumulation of macrophages in the myocardium in WD-fed mice that was improved by ECMR deletion.

Importantly, we have recently shown that EnNaC promotes WD-induced DD, which is accompanied by oxidative stress and increased intrinsic cardiomyocyte stiffness (12). In the current study, we found that suppression of WD induced increases in inward endothelial cell sodium currents in the ECMRKO mice fed a WD. Although we did not measure EnNaC activity in CD-fed mice in this study, our data in WD-fed mice are comparable with our previous reports, in which we demonstrated lower inward sodium currents in CD relative to WD-fed mice (28). Therefore, ECMR promotion of WD-induced DD may in part relate to downstream activation of EnNaC as excessive activation of EnNaC has been implicated in the stiffening of cardiovascular tissues (12). Although high salt intake in the rodent diet could contribute to our findings (58), the sodium content of the WD use in this investigation was comparatively lower than the chow diet (Table 1).

Sex-related differences in the pathogenesis of DD are increasingly being recognized (9, 15). We have shown, that in the setting of obesity, females develop DD earlier than males (17), and that prolonged consumption of WD results in more severe DD in WD females with associated oxidative stress and fibrosis (15). We further showed that either systemic MR blockade using a low dose of spironolactone or ECMR deletion prevented WD-induced DD, oxidative stress, hypertrophy, and fibrosis in WD-fed female mice (9, 15). In the present study, we also observed modest increases in oxidative stress but without any significant increase in fibrosis. Moreover, we evidenced augmented Akt activation in myocardium of males fed a WD, whereas decreased Akt signaling was previously seen in WD-fed females (15). This suggests that Akt activation may not only be deleterious but may also represent a compensatory stage in response to WD feeding. Furthermore, this is consistent with our earlier studies, in which we have demonstrated impaired Akt signaling in females at 8 wk of WD feeding but normal Akt signaling in males with normal diastolic function (17). Herein, we present data showing that prolonged WD feeding for 16 wk in males results in DD with increased Akt activation, which is also found in both humans and other preclinical models of ventricular dysfunction (30, 59). We have summarized in Table 4 the findings associated with WD feeding and ECMR deletion in male and females as they are related to DD. The effective prevention of DD despite either increase or decrease in Akt signaling by ECMR deletion in male as well as female WD-fed mice MR targeting is novel target in the management of diastolic dysfunction in both males and females.

Table 4.

Comparison of cardiac remodeling and signaling pathways in male and female mice fed with Western diet with or without ECMR deletion

	Males		Females (15)
	WD-LM	WD-ECMR-KO	WD-LM	WD-ECMR KO
Diastolic dysfunction	Significant but modest	Improved	Significant but marked	Improved
Hypertrophy	Absent	No change	Significant	Improved
Fibrosis	Not significant		Significant	Improved
Oxidative stress	Significant but modest	Improved	Significant but marked	Improved
Akt signaling	Upregulated	Improved	Downregulated	Improved
Mitochondrial abnormalities	Present	Improved	ND	ND
Phospholamban phosphorylation	Decreased	Improved	ND	ND
EnNaC activation	Increased sodium currents	Improved	ND	ND

ECMR, endothelial cell mineralocorticoid receptor; EnNaC, endothelial cell sodium channel; KO, knockout; LM, littermate; ND, not determined; WD, Western diet.

Perspectives and Significance

Our investigation has some limitations. First, we and others have reported the development of DD by consumption of WD (15, 17). However, some studies have not demonstrated high-fat diet induced DD after long-term feeding (60). We speculate that the presence of high fructose in addition to high fat in WD as used in our investigation plays a critical role in the development of DD, and warrants additional exploration. Second, our study does not specifically demonstrate a mitochondrial source of oxidative stress since we utilized accumulation of 3-nitrotyrosine for our analysis. However, previous studies on DD related to WD have shown the role of enhanced mitochondrial oxidative stress (56, 57), which includes increases in reduced nicotinamide adenine dinucleotide (NADH)-dependent production of reactive oxygen species in heart tissue, due to accumulation of advanced glycation end products (61). Finally, we did not record daily food intake to determine the impact of either excessive calories ingestion, or the nutrient composition of the WD. In this regard, we have previously reported food consumption in male mice with control diet or high-fat/high-fructose diet (62). In these studies, we have not observed significant increases in the amount of food consumed but instead we confirmed increased food density from the fat and sucrose content of WD, further supporting a deleterious impact of high fat and fructose.

In conclusion, we present evidence for the role of EC-specific MR in the promotion of WD-induced DD in male mice. ECMR mediated oxidative stress, dysregulation of Akt and mTOR signaling, calcium handling, and focal mitochondrial abnormalities all contribute to WD-induced DD in male mice. Compared with female mice fed a WD, the injury is milder and Akt signaling is distinct in male mice fed with WD. Further studies are required to elucidate the precise cellular and molecular mechanisms underlying ECMR-mediated DD in the setting of WD-induced obesity.

DATA AVAILABILITY

Data will be made available upon reasonable request.

SUPPLEMENTAL DATA

Supplemental Table S1: https://doi.org/10.6084/m9.figshare.21561684.v1.

GRANTS

G.L. received funding from the Veterans Affairs Merit System 5101BX001981. G.L. also received funding from the National Institutes of Health under Grant KO 5K08HL132012-02. A.T.W.-C. and C.M.-A. received funding from the Veterans Affairs Merit System under Grant BX003391. C.M.-A. received funding from HL142770. G.J. received funding from the National Institute of Diabetes and Digestive and Kidney Diseases under Grant DK124329. C.H. received funding from the National Institutes of Health R01HL124155. V.G.D. was supported by the Truman Veterans Affairs Medical Research Foundation.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

G.J., M.A.H., J.R.S., C.M.-A., A.T.W.C., A.A. and G.L., conceived and designed research; A.A., V.G.D., G.J., Y.Y., N.S., H.N., C.H., and M.R.H. performed experiments; A.A., V.G.D., G.J., Y.Y., N.S., H.N., C.H., M.R.H., J.R.S., C.M.-A., and G.L. analyzed data; A.A., V.G.D., A.T.W.-C., G.J., N.S., H.N., C.H., M.R.H., M.A.H., J.R.S., C.M.-A., and G.L. interpreted results of experiments; A.A., N.S., H.N., C.H., J.R.S., C.M.-A., and G.L. prepared figures; A.A., J.R.S., C.M.-A., and G.L. drafted manuscript; A.A., V.G.D., A.T.W.-C., G.J., N.S., H.N., C.H., M.R.H., M.A.H., J.R.S., C.M.-A., and G.L. edited and revised manuscript; A.A., V.G.D., A.T.W.-C., G.J., Y.Y., N.S., H.N., C.H., M.R.H., M.A.H., J.R.S., C.M.-A., and G.L. approved final version of manuscript.