Mineralocorticoid receptor antagonism treats obesity-associated cardiac diastolic dysfunction

Abstract

Patients with obesity and diabetes mellitus exhibit a high prevalence of cardiac diastolic dysfunction (DD), an independent predictor of cardiovascular events for which no evidence-based treatment exists. In light of renin-angiotensin-aldosterone system activation in obesity and the cardioprotective action of mineralocorticoid receptor (MR) antagonists in systolic heart failure, we examined the hypothesis that MR blockade with a blood pressure-independent low-dose spironolactone (LSp) would treat obesity-associated DD in the Zucker obese (ZO) rat. Treatment of ZO rats exhibiting established DD with LSp normalized cardiac diastolic function, assessed by echocardiography. This was associated with reduced cardiac fibrosis, but not reduced hypertrophy, and restoration of endothelium-dependent vasodilation of isolated coronary arterioles via a nitric oxide-independent mechanism. Further mechanistic studies revealed that LSp reduced cardiac oxidative stress and improved endothelial insulin signaling, with no change in arteriolar stiffness. Infusion of Sprague-Dawley rats with the MR agonist aldosterone reproduced the DD noted in ZO rats. In addition, improved cardiac function in ZO-LSp rats was associated with attenuated systemic and adipose inflammation and an anti-inflammatory shift in cardiac immune cell mRNAs. Specifically, LSp increased cardiac markers of alternatively activated macrophages and regulatory T cells. ZO-LSp rats had unchanged blood pressure, serum potassium, systemic insulin sensitivity, or obesity-associated kidney injury, assessed by proteinuria. Taken together, these data demonstrate that MR antagonism effectively treats established obesity-related DD via blood pressure-independent mechanisms. These findings help identify a particular population with DD that might benefit from MR antagonist therapy, specifically patients with obesity and insulin resistance.

Keywords: aldosterone; echocardiography; immune marker; metabolic syndrome X.

Figure 1. MR antagonism treats obesity-associated cardiac diastolic dysfunction

Indices of cardiac diastolic function in Zucker rats, specifically E′/A′ (A), Vp (B), IVRT (C), and MPI (D). ZL, Zucker lean; ZO, Zucker obese; ZO-LSp, ZO treated with spironolactone; E′/A′, early-to-late diastolic septal annulus motion ratio; IVRT, isovolumic relaxation time; MPI, myocardial performance index; Vp, propagation velocity of mitral inflow. Values are mean±SE; n=8–10; **p<0.05 versus all other groups, §p=0.06 versus ZL.

Figure 2. Increased cardiac fibrosis, not hypertrophy, in ZO rats is resolved by MR antagonism

Cardiac weights, assessed by left ventricle-to-tibia length (LV:TL) ratio, cardiomyocyte area (A), and cardiac interstitial/periarterial fibrosis (B) in Zucker rats. Interstitial collagen and periarterial fibrosis assessed by picrosirius red (PR) and Verhoeff-Van Gieson (VVG) staining, respectively. Representative images in lower panel, arrows indicate coronary arteries. GSI, gray scale intensity; ZL, Zucker lean; ZO, Zucker obese; ZO-LSp, ZO treated with spironolactone. Values are mean±SE; n=5–10; *p<0.05 versus ZL, **p<0.05 versus all other groups, †p<0.05 versus ZO, §p=0.07 versus ZL.

Figure 3. Impaired coronary arteriolar endothelial function in ZO rats is restored by MR antagonism

(A) Vasodilator responses of coronary arterioles from Zucker rats to endothelium-dependent dilators acetylcholine and insulin and (B) insulin-stimulated Akt (Thr³⁰⁸) phosphorylation in endothelial lysates from Zucker aortas. ZL, Zucker lean; ZO, Zucker obese; ZO-LSp, ZO treated with spironolactone. Values are mean±SE; n=5–8; *p<0.05 versus ZL or unstimulated, **p<0.05 versus all other groups, ‡p<0.05 versus ZL.

Figure 4. Elevated cardiac oxidative stress in ZO rats is ameliorated by MR antagonism

(A) Cardiac reactive oxygen species (ROS) production and NADPH oxidase activity in Zucker rats. (B) Cardiac 3-nitrotryrosine (3-NT) staining in Zucker rats. Representative 3-NT images in lower panel. GSI, gray scale intensity; ZL, Zucker lean; ZO, Zucker obese; ZO-LSp, ZO treated with spironolactone. Values are mean±SE; n=3–10; **p<0.05 versus all other groups, §p=0.07 versus ZL.

Figure 5. Systemic and adipose inflammatory markers are elevated in ZO rats and attenuated by MR antagonism

(A) Plasma levels of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and thiobarbituric acid reactive substances (TBARS) in Zucker rats. (B) Gene expression of inflammatory (MCP-1, TNF-α, VCAM-1, ICAM-1) and oxidative stress (NADPH oxidase subunits p22phox/p47phox) genes in epididymal adipose from Zucker rats. (C) Expression of macrophage (F4/80, CD11c) and T cell (CD4, FoxP3) mRNAs in epididymal adipose of Zucker rats. ZL, Zucker lean; ZO, Zucker obese; ZO-LSp, Zucker obese treated with spironolactone; VCAM-1, vascular cell adhesion molecule-1; ICAM-1, intracellular adhesion molecule-1. Values are mean±SE; n=4–6; *p<0.05 versus ZL, †p<0.05 versus ZO, §p=0.07 versus ZL, ¥p=0.08 versus ZL.

Figure 6. MR antagonism increases markers of anti-inflammatory immune cells in the heart of ZO rats

Gene expression of markers for total macrophages (F4/80), M1 macrophages (CD11c), M2 macrophages (CD163), T cells (CD4), and regulatory T cells (FoxP3) in cardiac tissue from Zucker rats. ZL, Zucker lean; ZO, Zucker obese; ZO-LSp, Zucker obese treated with spironolactone. Values are mean±SE; n=5–6; **p<0.05 versus all groups.