Endothelial Mineralocorticoid Receptor Deletion Prevents Diet-Induced Cardiac Diastolic Dysfunction in Females

Abstract

Overnutrition and insulin resistance are especially prominent risk factors for the development of cardiac diastolic dysfunction in females. We recently reported that consumption of a Western diet (WD) containing excess fat (46%), sucrose (17.5%), and high fructose corn syrup (17.5%) for 16 weeks resulted in cardiac diastolic dysfunction and aortic stiffening in young female mice and that these abnormalities were prevented by mineralocorticoid receptor blockade. Herein, we extend those studies by testing whether WD-induced diastolic dysfunction and factors contributing to diastolic impairment, such as cardiac fibrosis, hypertrophy, inflammation, and impaired insulin signaling, are modulated by excess endothelial cell mineralocorticoid receptor signaling. Four-week-old female endothelial cell mineralocorticoid receptor knockout and wild-type mice were fed mouse chow or WD for 4 months. WD feeding resulted in prolonged relaxation time, impaired diastolic septal wall motion, and increased left ventricular filling pressure indicative of diastolic dysfunction. This occurred in concert with myocardial interstitial fibrosis and cardiomyocyte hypertrophy that were associated with enhanced profibrotic (transforming growth factor β1/Smad) and progrowth (S6 kinase-1) signaling, as well as myocardial oxidative stress and a proinflammatory immune response. WD also induced cardiomyocyte stiffening, assessed ex vivo using atomic force microscopy. Conversely, endothelial cell mineralocorticoid receptor deficiency prevented WD-induced diastolic dysfunction, profibrotic, and progrowth signaling, in conjunction with reductions in macrophage proinflammatory polarization and improvements in insulin metabolic signaling. Therefore, our findings indicate that increased endothelial cell mineralocorticoid receptor signaling associated with consumption of a WD plays a key role in the activation of cardiac profibrotic, inflammatory, and growth pathways that lead to diastolic dysfunction in female mice.

Keywords: cardiovascular diseases; diet, Western; insulin resistance; obesity; receptors, mineralocorticoid.

Fig.1

WD induced cardiac diastolic dysfunction is prevented in ECMR^−/− female mice. (A) Representative mid-ventricle short-axis cine-MRI images that correspond to end-diastole, end-systole, and early diastole phases of cardiac cycle from control diet (CD) fed ECMR KO mouse (CD ECMR^−/−, the second row), WD fed wild type mouse (WD ECMR^+/+, the third row), and WD fed ECMR KO mouse (WD ECMR^−/−, lower row) compared to the CD fed wild type mouse (CD ECMR^+/+). (B) Left ventricular (LV) diastolic relaxation time, (C) peak filling rate, and (D) LV initial filling rate were derived from in vivo cine-MRI. Representative tissue doppler are shown of the early and late (E′ and A′) motion of the septal annulus during diastole and during early systole (E), myocardial performance index (F), and isovolumic relaxation time (IVRT) (G). (H) Atomic force microscopy nanoindentation measurements of the elastic modulus (stiffness) of acutely-isolated ventricular cardiomyocytes. WD-feeding caused an increase in cardiomyocyte stiffness in wild type mice but not in ECMR KO mice. n=4 to 6 per group. *P<0.01 compared with CD ECMR^+/+; # P<0.05 compared with WD ECMR^+/+.

Fig.2

WD induced cardiac fibrosis is prevented in ECMR^−/− mice. (A) Representative images of left ventricular immunostaining for interstitial fibrosis using picrosirius red with quantification of interstitial collagen deposition by average gray scale intensities. Representative images immunostaining for Col 1 (B), connective tissue growth factor (CTGF) (C), and fibronectin (D) with corresponding measures of average gray scale intensities. Scale bar = 50 μm. (E) Representative blots of TGF-β (F), phosphorylation of Smad 2/3 (G), and Smad7 (H) in left ventricle tissues by using western-blot with corresponding quantitative analysis. Control diet wild type (CD ECMR^+/+), ECMR KO control diet (CD ECMR^−/−), western diet wild type (WD ECMR^+/+), and western diet ECMR KO (WD ECMR^−/−). n=3 to 5 per group. *P<0.05 compared with CD ECMR^+/+; # P<0.05 compared with WD ECMR^+/+.

Fig.3

WD- induced myocardial oxidative stress and adaptive pro-inflammatory cytokines are ameliorated in ECMR^−/− female mice. (A) Representative images of left ventricular sections stained for 3-NT, a marker of oxidant stress from accumulation of oxidant peroxynitrite (ONOO⁻). (B) Expression of pro-inflammatory cytokine MCP-1, IL1, IL 17. (C-D) Expression of M1/M2 macrophage marker CD86, CD11b, CD206, and IL10. ECMR KO increased M2 macrophage marker IL10 and CD206 mRNA expression in WD fed mice as measured by real-time PCR. Control diet wild type (CD ECMR^+/+), ECMR KO control diet (CD ECMR^−/−), western diet wild type (WD ECMR^+/+), and western diet ECMR KO (WD ECMR^−/−). n=3 to 5 per group. *P<0.01 compared with CD ECMR^+/+; # P<0.05 compared with WD ECMR^+/+.

Fig.4

ECMR^−/− prevents WD-impaired insulin metabolic signals resulting in down-regulation of phosphorylation of Akt/eNOS. (A) The expression and activation of S6K, PI3/K, AKT, eNOS were performed with immunoprecipitation (IP) and immunoblotting (IB). (B-F) Representative blots of S6K, PI3/K, AKT, and eNOS in left ventricle tissues by using IB and IP with corresponding quantitative analysis. Control diet wild type (CD ECMR^+/+), ECMR KO control diet (CD ECMR^−/−), western diet wild type (WD ECMR^+/+), and western diet ECMR KO (WD ECMR^−/−). n=3 per group. *P<0.05 compared with CD ECMR^+/+; # P<0.05 compared with WD ECMR^+/+.

Fig.5

WD- induced cardiac hypertrophy is prevented in ECMR^−/− mice. (A) Representative images of myocardial immunostaining for hypertrophy with quantitative analysis of the cardiomyocyte sizes (B) Phosphorylation (p) of ERK 1/2 in left ventricular tissues using western-blot with representative analysis below or the ratio of (p) ERK 1/2 to total. Scale bar = 50 μm. Control diet wild type (CD ECMR^+/+), ECMR KO control diet (CD ECMR^−/−), western diet wild type (WD ECMR^+/+), and western diet ECMR KO (WD ECMR^−/−). n=3 to 5 per group. *P<0.05 compared with CD ECMR^+/+; # P<0.05 compared with WD ECMR^+/+.