Effects of a Rho kinase inhibitor on pressure overload induced cardiac hypertrophy and associated diastolic dysfunction

The RhoA-Rho kinase (ROCK) signaling pathway has an important role in cardiovascular diseases. However, the effect of Rho kinase inhibition on pressure overload-induced cardiac hypertrophy (POH) and associated diastolic dysfunction has not been evaluated. This study examined the effect of a selective ROCK inhibitor (GSK-576371) in a POH model, induced by suprarenal abdominal aortic constriction. POH rats were divided into the following four groups: 1 (GSK 1, n = 9) or 3 (GSK 3, n = 10) mg/kg bid GSK-576371, 1 mg.kg(-1).day(-1) ramipril (n = 10) or vehicle (n = 11) treatment for 4 wk. Sham animals (n = 11) underwent surgery without banding. Echocardiograms were performed before surgery and posttreatment, and hemodynamic data were obtained at completion of the study. Echocardiography showed an increase in relative wall thickness of the left ventricle (LV) following POH + vehicle treatment compared with sham animals. This was attenuated by both doses of GSK-576371 and ramipril. Vehicle treatment demonstrated abnormal diastolic parameters, including mitral valve (MV) inflow E wave deceleration time, isovolumic relaxation time, and MV annular velocity, which were dose dependently restored toward sham values by GSK-576371. LV end diastolic pressure was increased following POH + vehicle treatment compared with sham (6.9 +/- 0.7 vs. 3.2 +/- 0.7 mmHg, P = 0.008) and was reduced with GSK 3 and ramipril treatment (1.7 +/- 0.7, P < 0.01 and 2.9 +/- 0.6 mmHg, P < 0.01, respectively). Collagen I deposition in the LV was increased following POH + vehicle treatment (32.2%; P < 0.01) compared with sham animals and was significantly attenuated with GSK 1 (21.7%; P < 0.05), GSK 3 (23.8%; P < 0.01), and ramipril (35.5%; P < 0.01) treatment. These results suggest that ROCK inhibition improves LV geometry and reduces collagen deposition accompanied by improved diastolic function in POH.     

Vestibular dysfunction in vitamin D receptor mutant mice

The vitamin D endocrine system is essential for calcium and bone homeostasis. Vitamin D deficits are associated with muscle weakness and osteoporosis, whereas vitamin D supplementation may improve muscle function, body sway and frequency of falls, growth and mineral homeostasis of bones. The loss of muscle strength and mass, as well as deficits in bone formation, lead to poor balance. Poor balance is one of the main causes of falls, and may lead to dangerous injuries. Here we examine balance functions in vitamin D receptor deficient (VDR−/−) mice, an animal model of vitamin D-dependent rickets type II, and in 1α-hydroxylase deficient (1α-OHase−/−) mice, an animal model of pseudovitamin D-deficiency rickets. Recently developed methods (tilting box, rotating tube test), swim test, and modified accelerating rotarod protocol were used to examine whether the absence of functional VDR, or the lack of a key vitamin D-activating enzyme, could lead to mouse vestibular dysfunctions. Overall, VDR−/− mice, but not 1α-OHase−/− mice, showed shorter latency to fall from the rotarod, smaller fall angle in the tilting box test, and aberrant poor swimming. These data suggest that VDR deficiency in mice is associated with decreased balance function, and may be relevant to poorer balance/posture control in humans with low levels of vitamin D.     

Three-dimensional structure-function relationship of vitamin D and vitamin D receptor model

On the basis of conformational analysis of the vitamin D side chain and studies using conformationally restricted synthetic vitamin D analogs, we have suggested the active space region concept of vitamin D: The vitamin D side-chain region was grouped into four regions (A, G, EA and EG) and the A and EA regions were suggested to be important for vitamin D actions. We extended our theory to known highly potent vitamin D analogs and found a new region F. The analogs which occupy the F region have such modifications as 22-oxa, 22-ene, 16-ene and 18-nor. Altogether, the following relationship between the space region and activity was found: Affinity for vitamin D receptor (VDR), EA > A> F > G > EG; Affinity for vitamin D binding protein (DBP), A > G,EA,EG; Target gene transactivation, EA > F > A > EG > or = G; Cell differentiation, EA > F > A > EG > or = G; Bone calcium mobilization, EA > GA > F > or = EG; Intestinal calcium absorption, EA = A > or = G > EG. We modeled the 3D structure of VDR-LBD (ligand binding domain) using hRARgamma as a template, to develop our structure-function theory into a theory involving VDR. 1alpha,25(OH)(2)D(3) was docked into the ligand binding pocket of the VDR with the side chain heading the wide cavity at the H-11 site, the A-ring toward the narrow beta-turn site, and the beta-face of the CD ring facing H3. Amino acid residues forming hydrogen bonds with the 1alpha- and 25-OH groups were specified: S237 and R274 forming a pincer type hydrogen-bond for the 1alpha-OH and H397 for the 25-OH. Mutants of several amino acid residues that are hydrogen-bond candidates were prepared and their biologic properties were evaluated. All of our mutation results together with known mutation data support our VDR model docked with the natural ligand.     

Zerumbone prevents pressure overload-induced left ventricular systolic dysfunction by inhibiting cardiac hypertrophy and fibrosis

BackgroundCardiac hypertrophy and fibrosis are hallmarks of cardiac remodeling and are involved functionally in the development of heart failure (HF). However, it is unknown whether Zerumbone (Zer) prevents left ventricular (LV) systolic dysfunction by inhibiting cardiac hypertrophy and fibrosis.PurposeThis study investigated the effect of Zer on cardiac hypertrophy and fibrosis in vitro and in vivo.Study Design/methodsIn primary cultured cardiac cells from neonatal rats, the effect of Zer on phenylephrine (PE)-induced hypertrophic responses and transforming growth factor beta (TGF-β)-induced fibrotic responses was observed. To determine whether Zer prevents the development of pressure overload-induced HF in vivo, a transverse aortic constriction (TAC) mouse model was utilized. Cardiac function was evaluated by echocardiography. The changes of cardiomyocyte surface area were observed using immunofluorescence staining and histological analysis (HE and WGA staining). Collagen synthesis and fibrosis formation were measured by scintillation counter and picrosirius staining, respectively. The total mRNA levels of genes associated with hypertrophy (ANF and BNP) and fibrosis (Postn and α-SMA) were measured by qRT-PCR. The protein expressions (Akt and α-SMA) were assessed by western blotting.ResultsZer significantly suppressed PE-induced increase in cell size, mRNA levels of ANF and BNP, and Akt phosphorylation in cardiomyocytes. The TGF-β-induced increase in proline incorporation, mRNA levels of Postn and α-SMA, and protein expression of α-SMA were decreased by Zer in cultured cardiac fibroblasts. In the TAC male C57BL/6 mice, echocardiography results demonstrated that Zer improved cardiac function by increasing LV fractional shortening and reducing LV wall thickness compared with the vehicle group. ZER significantly reduced the level of phosphorylated Akt both in cultured cardiomyocytes treated with PE and in the hearts of TAC. Finally, Zer inhibited the pressure overload-induced cardiac hypertrophy and cardiac fibrosis.ConclusionZer ameliorates pressure overload-induced LV dysfunction, at least in part by suppressing both cardiac hypertrophy and fibrosis.     

Right ventricular diastolic function in canine models of pressure overload,volume overload,and ischemia

By limiting filling, abnormalities of right ventricular (RV) diastolic function may impair systolic function and affect adaptation to disease. To quantify diastolic RV pressure-volume relations and myocardial compliance (MC), a new sigmoidal model was developed. RV micromanometric and sonomicrometric data in alert dogs at control (n = 16) and under surgically induced subacute (2-5 wk) RV pressure overload (n = 6), volume overload (n = 7), and ischemia (n = 6) were analyzed. The conventional exponential model detected no changes from control in the passive filling pressure-volume (P(pf)-V) relations. The new sigmoidal model revealed significant quantifiable changes in P(pf)-V relations. Maximum RV MC (MC(max)), attained during early filling, is reduced from control in pressure overload (P = 0.0016), whereas filling pressure at maximum MC (P(MCmax)) is increased (P = 0.0001). End-diastolic RV MC increases significantly in volume overload (P = 0.0131), whereas end-diastolic pressure is unchanged. In ischemia, MC(max) is decreased (P = 0.0102), with no change in P(MCmax). We conclude that the sigmoidal model quantifies important changes in RV diastolic function in alert dog models of pressure overload, volume overload, and ischemia.     

Anti-TGF-beta treatment prevents skin and lung fibrosis in murine sclerodermatous graft-versus-host disease: a model for human scleroderma.

Scleroderma, a debilitating acquired connective tissue disease, is characterized by fibrosis, particularly of the skin and lungs. Monocyte-produced TGF-beta1, a potent stimulus for collagen synthesis, is thought to drive the fibrosis. Here, we thoroughly characterize a murine sclerodermatous graft-vs-host disease (Scl GVHD) model for scleroderma that reproduces important features of scleroderma including skin thickening, lung fibrosis, and up-regulation of cutaneous collagen mRNA, which is preceded by monocyte infiltration and the up-regulation of cutaneous TGF-beta1 mRNA. Most importantly, we can prevent fibrosis in both the skin and lungs of mice with Scl GVHD by inhibiting TGF-beta with neutralizing Abs. The murine Scl GVHD model provides the unique opportunity to study basic immunologic mechanisms that drive fibrosing diseases and GVHD itself and will be useful for testing new therapies for these diseases.     

Invariant NKT cell defects in vitamin D receptor knockout mice prevents experimental lung inflammation

Vitamin D receptor (VDR) deficiency (knockout [KO]) results in a failure of mice to generate an airway hyperreactivity (AHR) response on both the BALB/c and C57BL/6 background. The cause of the failed AHR response is the defective population of invariant NKT (iNKT) cells in the VDR KO mice because wild-type (WT) iNKT cells rescued the AHR response. VDR KO mice had significantly fewer iNKT cells and normal numbers of T cells in the spleen compared with WT mice. In BALB/c VDR KO mice, the reduced frequencies of iNKT cells were not apparent in the liver or thymus. VDR KO and WT Th2 cells produced similar levels of IFN-γ and IL-5. On the BALB/c background, Th2 cells from VDR KO mice produced less IL-13, whereas on the C57BL/6 background, Th2 cells from VDR KO mice produced less IL-4. Conversely, VDR KO iNKT cells were defective for the production of multiple cytokines (BALB/c: IL-4, IL-5, and IL-13; C57BL/6: IL-4 and IL-17). Despite relatively normal Th2 responses, BALB/c and C57BL/6 VDR KO mice failed to develop AHR responses. The defect in iNKT cells as a result of the VDR KO was more important than the highly susceptible Th2 background of the BALB/c mice. Defective iNKT cell responses in the absence of the VDR result in the failure to generate AHR responses in the lung. The implication of these mechanistic findings for human asthma requires further investigation.     

Cardiomyocyte-restricted inhibition of G protein-coupled receptor kinase-3 attenuates cardiac dysfunction after chronic pressure overload

Transgenic mice with cardiac-specific expression of a peptide inhibitor of G protein-coupled receptor kinase (GRK)3 [transgenic COOH-terminal GRK3 (GRK3ct) mice] display myocardial hypercontractility without hypertrophy and enhanced α(1)-adrenergic receptor signaling. A role for GRK3 in the pathogenesis of heart failure (HF) has not been investigated, but inhibition of its isozyme, GRK2, has been beneficial in several HF models. Here, we tested whether inhibition of GRK3 modulated evolving cardiac hypertrophy and dysfunction after pressure overload. Weight-matched male GRK3ct transgenic and nontransgenic littermate control (NLC) mice subjected to chronic pressure overload by abdominal aortic banding (AB) were compared with sham-operated (SH) mice. At 6 wk after AB, a significant increase of cardiac mass consistent with induction of hypertrophy was found, but no differences between GRK3ct-AB and NLC-AB mice were discerned. Simultaneous left ventricular (LV) pressure-volume analysis of electrically paced, ex vivo perfused working hearts revealed substantially reduced systolic and diastolic function in NLC-AB mice (n = 7), which was completely preserved in GRK3ct-AB mice (n = 7). An additional cohort was subjected to in vivo cardiac catheterization and LV pressure-volume analysis at 12 wk after AB. NLC-AB mice (n = 11) displayed elevated end-diastolic pressure (8.5 ± 3.1 vs. 2.9 ± 1.2 mmHg, P < 0.05), reduced cardiac output (3,448 ± 323 vs. 4,488 ± 342 μl/min, P < 0.05), and reduced dP/dt(max) and dP/dt(min) (both P < 0.05) compared with GRK3ct-AB mice (n = 16), corroborating the preserved cardiac structure and function observed in GRK3ct-AB hearts assessed ex vivo. Increased cardiac mass and myocardial mRNA expression of β-myosin heavy chain confirmed the similar induction of cardiac hypertrophy in both AB groups, but only NLC-AB hearts displayed significantly elevated mRNA levels of brain natriuretic peptide and myocardial collagen contents as well as reduced β(1)-adrenergic receptor responsiveness to isoproterenol, indicating increased LV wall stress and the transition to HF. Inhibition of cardiac GRK3 in mice does not alter the hypertrophic response but attenuates cardiac dysfunction and HF after chronic pressure overload.     

Alogliptin prevents diastolic dysfunction and preserves left ventricular mitochondrial function in diabetic rabbits

Background

There are increasing evidence that left ventricle diastolic dysfunction is the initial functional alteration in the diabetic myocardium. In this study, we hypothesized that alogliptin prevents diastolic dysfunction and preserves left ventricular mitochondrial function and structure in diabetic rabbits.

Methods

A total of 30 rabbits were randomized into control group (CON, n?=?10), alloxan-induced diabetic group (DM, n?=?10) and alogliptin-treated (12.5 mg/kd/day for 12 weeks) diabetic group (DM-A, n?=?10). Echocardiographic and hemodynamic studies were performed in vivo. Mitochondrial morphology, respiratory function, membrane potential and reactive oxygen species (ROS) generation rate of left ventricular tissue were assessed. The serum concentrations of glucagon-like peptide-1, insulin, inflammatory and oxidative stress markers were measured. Protein expression of TGF-β1, NF-κB p65 and mitochondrial biogenesis related proteins were determined by Western blotting.

Results

DM rabbits exhibited left ventricular hypertrophy, left atrial dilation, increased E/e′ ratio and normal left ventricular ejection fraction. Elevated left ventricular end diastolic pressure combined with decreased maximal decreasing rate of left intraventricular pressure (??dp/dtmax) were observed. Alogliptin alleviated ventricular hypertrophy, interstitial fibrosis and diastolic dysfunction in diabetic rabbits. These changes were associated with decreased mitochondrial ROS production rate, prevented mitochondrial membrane depolarization and improved mitochondrial swelling. It also improved mitochondrial biogenesis by PGC-1α/NRF1/Tfam signaling pathway.

Conclusions

The DPP-4 inhibitor alogliptin prevents cardiac diastolic dysfunction by inhibiting ventricular remodeling, explicable by improved mitochondrial function and increased mitochondrial biogenesis.

     

Characterization of heart size and blood pressure in the vitamin D receptor knockout mouse

Our previous studies showed vitamin D deficiency results in increased cardiac contractility, hypertrophy and fibrosis and has profound effects on heart proteomics, structure and function in rat. In this study we found that the heart in vitamin D receptor knockout (VDR-KO) mice is hypertrophied. Six homozygous VDR knockout (−/−), six wild type (+/+) and six heterozygous (+/−) mice were fed a diet containing 2% Ca, 1.25% P and 20% lactose to maintain normal blood calcium and phosphate levels for 12 months. Tail-cuff blood pressure was performed on all mice. Blood pressure determinations showed no differences in systolic or mean blood pressure in WT (+/+), KO (−/−) or HETERO (+/−) mice at 3 and 6 months. However, decreased systolic BP in the KO mice relative to WT at 9 months of age was observed. ECG analysis showed no significant differences in the intact KO, HETERO or WT mice. The mice were killed at 12 months. Heart weight/body weight ratio was 41% (P < .003) greater in the KO mice versus WT and HETERO was 19% (P < .05) increased versus WT. Other VDR-KO tissues did not display hypertrophy. Cross sectional and longitudinal analysis of the heart myofibrils showed highly significant cellular hypertrophy in VDR-KO mice. Trichrome staining of heart tissue showed marked increase in fibrotic lesions in the KO mice. Analysis of plasma renin activity, angiotensin II (AII) and aldosterone levels showed elevated but not significantly different renin activity in KO versus WT and no significant differences in AII or aldosterone levels. Our data do not support the concept that the renin-angiotensin system or hypertension are the factors that elicit these changes. Data presented here reveal that ablation of the VDR signaling system results in profound changes in heart structure. We propose that calcitriol acts directly on the heart as a tranquilizer by blunting cardiomyocyte hypertrophy.     

The vitamin D receptor: a primitive steroid receptor related to thyroid hormone receptor

We have previously reported the cloning and sequencing of both the chicken and human vitamin D3 receptor cDNAs. A comparison of their deduced amino acid sequence with that of the other classic steroid hormone receptors and the receptor for thyroid hormone indicates that there are two regions of conservation between these molecules. The first is a 70 amino acid, cysteine-rich sequence (C1), the second region (C2) is a 62 amino acid region located towards the carboxyl terminus of the proteins. In other systems the former has been identified as a region responsible for DNA binding activity, whereas the latter represents the NH2-terminal boundary of the hormone binding domain. We present here evidence utilizing eucaryotic expression of cDNA encoding the hVDR C1 domain, followed by a DNA cellulose chromatography assay, which confirms that the DNA binding activity resides in this region of the receptor for vitamin D3. Additionally, the vitamin D3 receptor contains a 60 amino acid portion at its carboxyl terminus (C3) which exhibits homology with the receptor for thyroid hormone. Conservation in this region of the molecule is found only between homologous or closely related receptors. This indicates a relationship between the vitamin D3 receptor and the receptor for thyroid hormone and may suggest that they evolved from a single primordial gene.     

Propylthiouracil prevents cutaneous and pulmonary fibrosis in the reactive oxygen species murine model of systemic sclerosis

Introduction

Recent advances suggest that the cellular redox state may play a significant role in the progression of fibrosis in systemic sclerosis (SSc). Another, and as yet poorly accounted for, feature of SSc is its overlap with thyroid abnormalities. Previous reports demonstrate that hypothyroidism reduces oxidant stress. The aim of this study was therefore to evaluate the effect of propylthiouracil (PTU), and of the hypothyroidism induced by it, on the development of cutaneous and pulmonary fibrosis in the oxidant stress murine model of SSc.

Methods

Chronic oxidant stress SSc was induced in BALB/c mice by daily subcutaneous injections of hypochlorous acid (HOCl) for 6 weeks. Mice (n = 25) were randomized into three arms: HOCl (n = 10), HOCl plus PTU (n = 10) or vehicle alone (n = 5). PTU administration was initiated 30 minutes after HOCl subcutaneous injection and continued daily for 6 weeks. Skin and lung fibrosis were evaluated by histologic methods. Immunohistochemical staining for alpha-smooth muscle actin (α-SMA) in cutaneous and pulmonary tissues was performed to evaluate myofibroblast differentiation. Lung and skin concentrations of vascular endothelial growth factor (VEGF), extracellular signal-related kinase (ERK), rat sarcoma protein (Ras), Ras homolog gene family (Rho), and transforming growth factor (TGF) β were analyzed by Western blot.

Results

Injections of HOCl induced cutaneous and lung fibrosis in BALB/c mice. PTU treatment prevented both dermal and pulmonary fibrosis. Myofibroblast differentiation was also inhibited by PTU in the skin and lung. The increase in cutaneous and pulmonary expression of VEGF, ERK, Ras, and Rho in mice treated with HOCl was significantly prevented in mice co-administered ////with PTU.

Conclusions

PTU, probably through its direct effect on reactive oxygen species or indirectly through thyroid function inhibition, prevents the development of cutaneous and pulmonary fibrosis by blocking the activation of the Ras-ERK pathway in the oxidant-stress animal model of SSc.     

Longitudinal arrhythmogenic remodelling in a mouse model of longstanding pressure overload

Introduction. Sudden arrhythmogenic cardiac death is a major cause of mortality in patients with congestive heart failure due to adverse electrical remodelling. To establish whether abnormal conduction is responsible for arrhythmogenic remodelling in progressed stages of heart failure, we have monitored functional, structural and electrical remodelling in a murine model of heart failure, induced by longstanding pressure overload. Methods. Mice were subjected to transverse aortic constriction (TAC; n=18) or sham operated (n=19) and monitored biweekly by echocardiography and electrocardiography. At the 16-week endpoint, electrical mapping was performed to measure epicardial conduction velocity and susceptibility to arrhythmias. Finally, tissue sections were stained for Cx43 and fibrosis. Results. In TAC mice, fractional shortening decreased gradually and was significantly lower compared with sham at 16 weeks. Left ventricular hypertrophy was significant after six weeks. TAC mice developed PQ prolongation after 12 weeks, QT prolongation after 16 weeks and QRS prolongation after two weeks. Right ventricular conduction velocity was slowed parallel to fibre orientation. In 8/18 TAC hearts, polymorphic ventricular tachyarrhythmias were provoked and none in sham hearts. TAC mice had more interstitial fibrosis than sham. Immunohistology showed that Cx43 levels were similar but highly heterogeneous in TAC mice. All parameters were comparable in TAC mice with and without arrhythmias, except for Cx43 heterogeneity, which was significantly higher in arrhythmogenic TAC mice. Conclusion. Chronic pressure overload resulted in rapid structural and electrical remodelling. Arrhythmias were related to heterogeneous expression of Cx43. This may lead to functional block and unstable reentry, giving rise to polymorphic ventricular tachyarrhythmias. (Neth Heart J 2010;18:509-15.)     

The flavonoid apigenin suppresses vitamin D receptor expression and vitamin D responsiveness in normal human keratinocytes

Apigenin, a flavonoid with chemopreventive properties, induces cellular growth arrest, with concomitant inhibition of intracellular signaling cascades and decreased proto-oncogene expression. We report that apigenin potently inhibited vitamin D receptor (VDR) mRNA and protein expression in human keratinocytes without changes in VDR mRNA half-life. Concurrently, downregulation of retinoid X receptor alpha, a dramatic loss of c-myc mRNA, and upregulation of p21(WAF1) took place. Furthermore, a nearly complete suppression of vitamin D responsiveness was observed as estimated by induction of 24-hydroxylase mRNA. The apigenin effect on VDR expression was shared by some other (quercetine and fisetine) but not all tested flavonoids. Interestingly, the apigenin-mediated VDR suppression was counteracted by the NFkappaB inhibitors sodium salicylate and caffeic acid phenethyl ester. The presented results propose suppression of nuclear receptor levels as a novel mechanism whereby flavonoids exert their pleiotropic effects. This study may also contribute to the understanding of the regulation of VDR expression in epidermal keratinocytes.     

Heart size-independent analysis of myocardial function in murine pressure overload hypertrophy

Pressure overload cardiac hypertrophy may be a compensatory mechanism to normalize systolic wall stress and preserve left ventricular (LV) function. To test this concept, we developed a novel in vivo method to measure myocardial stress (sigma)-strain (epsilon) relations in normal and hypertrophied mice. LV volume was measured using two pairs of miniature omnidirectional piezoelectric crystals implanted orthogonally in the endocardium and one crystal placed on the anterior free wall to measure instantaneous wall thickness. Highly linear sigma-epsilon relations were obtained in control (n = 7) and hypertrophied mice produced by 7 days of transverse aortic constriction (TAC; n = 13). Administration of dobutamine in control mice significantly increased the load-independent measure of LV contractility, systolic myocardial stiffness. In TAC mice, systolic myocardial stiffness was significantly greater than in control mice (3,156 +/- 1,433 vs. 1,435 +/- 467 g/cm(2), P < 0.01), indicating enhanced myocardial contractility with pressure overload. However, despite the increased systolic performance, both active (time constant of LV pressure decay) and passive (diastolic myocardial stiffness constant) diastolic properties were markedly abnormal in TAC mice compared with control mice. These data suggest that the development of cardiac hypertrophy is associated with a heightened contractile state, perhaps as an early compensatory response to pressure overload.     

Mineralocorticoid receptor blockade improves diastolic function independent of blood pressure reduction in a transgenic model of RAAS overexpression

There is emerging evidence that aldosterone can promote diastolic dysfunction and cardiac fibrosis independent of blood pressure effects, perhaps through increased oxidative stress and inflammation. Accordingly, this investigation was designed to ascertain if mineralocorticoid receptor blockade improves diastolic dysfunction independently of changes in blood pressure through actions on myocardial oxidative stress and fibrosis. We used young transgenic (mRen2)27 [TG(mRen2)27] rats with increases in both tissue ANG II and circulating aldosterone, which manifests age-related increases in hypertension and cardiac dysfunction. Male TG(mRen2)27 and age-matched Sprague-Dawley rats were treated with either a low dose (～1 mg·kg(-1)·day(-1)) or a vasodilatory, conventional dose (～30 mg·kg(-1)·day(-1)) of spironolactone or placebo for 3 wk. TG(mRen2)27 rats displayed increases in systolic blood pressure and plasma aldosterone levels as well as impairments in left ventricular diastolic relaxation without changes in systolic function on cine MRI. TG(mRen2)27 hearts also displayed hypertrophy (left ventricular weight, cardiomyoctye hypertrophy, and septal wall thickness) as well as fibrosis (interstitial and perivascular). There were increases in oxidative stress in TG(mRen2)27 hearts, as evidenced by increases in NADPH oxidase activity and subunits as well as ROS formation. Low-dose spironolactone had no effect on systolic blood pressure but improved diastolic dysfunction comparable to a conventional dose. Both doses of spironolactone caused comparable reductions in ROS/3-nitrotryosine immunostaining and perivascular and interstitial fibrosis. These data support the notion mineralocorticoid receptor blockade improves diastolic dysfunction through improvements in oxidative stress and fibrosis independent of changes in systolic blood pressure.