Quantification correction for free-breathing myocardial T1ρ mapping in mice using a recursively derived description of a T1ρ* relaxation pathway.

BACKGROUND: Fast and accurate T1ρ mapping in myocardium is still a major challenge, particularly in small animal models. The complex sequence design owing to electrocardiogram and respiratory gating leads to quantification errors in in vivo experiments, due to variations of the T1ρ relaxation pathway. In this study, we present an improved quantification method for T1ρ using a newly derived formalism of a T1ρ* relaxation pathway. METHODS: The new signal equation was derived by solving a recursion problem for spin-lock prepared fast gradient echo readouts. Based on Bloch simulations, we compared quantification errors using the common monoexponential model and our corrected model. The method was validated in phantom experiments and tested in vivo for myocardial T1ρ mapping in mice. Here, the impact of the breath dependent spin recovery time Trec on the quantification results was examined in detail. RESULTS: Simulations indicate that a correction is necessary, since systematically underestimated values are measured under in vivo conditions. In the phantom study, the mean quantification error could be reduced from - 7.4% to - 0.97%. In vivo, a correlation of uncorrected T1ρ with the respiratory cycle was observed. Using the newly derived correction method, this correlation was significantly reduced from r = 0.708 (p < 0.001) to r = 0.204 and the standard deviation of left ventricular T1ρ values in different animals was reduced by at least 39%. CONCLUSION: The suggested quantification formalism enables fast and precise myocardial T1ρ quantification for small animals during free breathing and can improve the comparability of study results. Our new technique offers a reasonable tool for assessing myocardial diseases, since pathologies that cause a change in heart or breathing rates do not lead to systematic misinterpretations. Besides, the derived signal equation can be used for sequence optimization or for subsequent correction of prior study results.

Fast myocardial T1ρ mapping in mice using k-space weighted image contrast and a Bloch simulation-optimized radial sampling pattern.

PURPOSE: T1ρ dispersion quantification can potentially be used as a cardiac magnetic resonance index for sensitive detection of myocardial fibrosis without the need of contrast agents. However, dispersion quantification is still a major challenge, because T1ρ mapping for different spin lock amplitudes is a very time consuming process. This study aims to develop a fast and accurate T1ρ mapping sequence, which paves the way to cardiac T1ρ dispersion quantification within the limited measurement time of an in vivo study in small animals. METHODS: A radial spin lock sequence was developed using a Bloch simulation-optimized sampling pattern and a view-sharing method for image reconstruction. For validation, phantom measurements with a conventional sampling pattern and a gold standard sequence were compared to examine T1ρ quantification accuracy. The in vivo validation of T1ρ mapping was performed in N = 10 mice and in a reproduction study in a single animal, in which ten maps were acquired in direct succession. Finally, the feasibility of myocardial dispersion quantification was tested in one animal. RESULTS: The Bloch simulation-based sampling shows considerably higher image quality as well as improved T1ρ quantification accuracy (+ 56%) and precision (+ 49%) compared to conventional sampling. Compared to the gold standard sequence, a mean deviation of - 0.46 ± 1.84% was observed. The in vivo measurements proved high reproducibility of myocardial T1ρ mapping. The mean T1ρ in the left ventricle was 39.5 ± 1.2 ms for different animals and the maximum deviation was 2.1% in the successive measurements. The myocardial T1ρ dispersion slope, which was measured for the first time in one animal, could be determined to be 4.76 ± 0.23 ms/kHz. CONCLUSION: This new and fast T1ρ quantification technique enables high-resolution myocardial T1ρ mapping and even dispersion quantification within the limited time of an in vivo study and could, therefore, be a reliable tool for improved tissue characterization.

Mitofusin 2 Is Essential for IP3-Mediated SR/Mitochondria Metabolic Feedback in Ventricular Myocytes.

Aim: Endothelin-1 (ET-1) and angiotensin II (Ang II) are multifunctional peptide hormones that regulate the function of the cardiovascular and renal systems. Both hormones increase the intracellular production of inositol-1,4,5-trisphosphate (IP3) by activating their membrane-bound receptors. We have previously demonstrated that IP3-mediated sarcoplasmic reticulum (SR) Ca2+ release results in mitochondrial Ca2+ uptake and activation of ATP production. In this study, we tested the hypothesis that intact SR/mitochondria microdomains are required for metabolic IP3-mediated SR/mitochondrial feedback in ventricular myocytes. Methods: As a model for disrupted mitochondrial/SR microdomains, cardio-specific tamoxifen-inducible mitofusin 2 (Mfn2) knock out (KO) mice were used. Mitochondrial Ca2+ uptake, membrane potential, redox state, and ATP generation were monitored in freshly isolated ventricular myocytes from Mfn2 KO mice and their control wild-type (WT) littermates. Results: Stimulation of ET-1 receptors in healthy control myocytes increases mitochondrial Ca2+ uptake, maintains mitochondrial membrane potential and redox balance leading to the enhanced ATP generation. Mitochondrial Ca2+ uptake upon ET-1 stimulation was significantly higher in interfibrillar (IFM) and perinuclear (PNM) mitochondria compared to subsarcolemmal mitochondria (SSM) in WT myocytes. Mfn2 KO completely abolished mitochondrial Ca2+ uptake in IFM and PNM mitochondria but not in SSM. However, mitochondrial Ca2+ uptake induced by beta-adrenergic receptors activation with isoproterenol (ISO) was highest in SSM, intermediate in IFM, and smallest in PNM regions. Furthermore, Mfn2 KO did not affect ISO-induced mitochondrial Ca2+ uptake in SSM and IFM mitochondria; however, enhanced mitochondrial Ca2+ uptake in PNM. In contrast to ET-1, ISO induced a decrease in ATP levels in WT myocytes. Mfn2 KO abolished ATP generation upon ET-1 stimulation but increased ATP levels upon ISO application with highest levels observed in PNM regions. Conclusion: When the physical link between SR and mitochondria by Mfn2 was disrupted, the SR/mitochondrial metabolic feedback mechanism was impaired resulting in the inability of the IP3-mediated SR Ca2+ release to induce ATP production in ventricular myocytes from Mfn2 KO mice. Furthermore, we revealed the difference in Mfn2-mediated SR-mitochondrial communication depending on mitochondrial location and type of communication (IP3R-mRyR1 vs. ryanodine receptor type 2-mitochondrial calcium uniporter).

Inositol 1,4,5-trisphosphate-mediated sarcoplasmic reticulum-mitochondrial crosstalk influences adenosine triphosphate production via mitochondrial Ca2+ uptake through the mitochondrial ryanodine receptor in cardiac myocytes.

AIMS: Elevated levels of inositol 1,4,5-trisphosphate (IP3) in adult cardiac myocytes are typically associated with the development of cardiac hypertrophy, arrhythmias, and heart failure. IP3 enhances intracellular Ca(2+ )release via IP3 receptors (IP3Rs) located at the sarcoplasmic reticulum (SR). We aimed to determine whether IP3-induced Ca(2+ )release affects mitochondrial function and determine the underlying mechanisms. METHODS AND RESULTS: We compared the effects of IP3Rs- and ryanodine receptors (RyRs)-mediated cytosolic Ca(2+ )elevation achieved by endothelin-1 (ET-1) and isoproterenol (ISO) stimulation, respectively, on mitochondrial Ca(2+ )uptake and adenosine triphosphate (ATP) generation. Both ET-1 and isoproterenol induced an increase in mitochondrial Ca(2+ )(Ca(2 +) m) but only ET-1 led to an increase in ATP concentration. ET-1-induced effects were prevented by cell treatment with the IP3 antagonist 2-aminoethoxydiphenyl borate and absent in myocytes from transgenic mice expressing an IP3 chelating protein (IP3 sponge). Furthermore, ET-1-induced mitochondrial Ca(2+) uptake was insensitive to the mitochondrial Ca(2+ )uniporter inhibitor Ru360, however was attenuated by RyRs type 1 inhibitor dantrolene. Using real-time polymerase chain reaction, we detected the presence of all three isoforms of IP3Rs and RyRs in murine ventricular myocytes with a dominant presence of type 2 isoform for both receptors. CONCLUSIONS: Stimulation of IP3Rs with ET-1 induces Ca(2+ )release from the SR which is tunnelled to mitochondria via mitochondrial RyR leading to stimulation of mitochondrial ATP production.

Development and Characterization of an Inducible Rat Model of Chronic Thromboembolic Pulmonary Hypertension.

Chronic thromboembolic pulmonary hypertension (CTEPH) is an entity of PH that not only limits patients quality of life but also causes significant morbidity and mortality. The treatment of choice is pulmonary endarterectomy. However numerous patients do not qualify for pulmonary endarterectomy or present with residual vasculopathy post pulmonary endarterectomy and require specific vasodilator treatment. Currently, there is no available specific small animal model of CTEPH that could serve as tool to identify targetable molecular pathways and to test new treatment options. Thus, we generated and standardized a rat model that not only resembles functional and histological features of CTEPH but also emulates thrombi fibrosis. The pulmonary embolism protocol consisted of 3 sequential tail vein injections of fibrinogen/collagen-covered polystyrene microspheres combined with thrombin and administered to 10-week-old male Wistar rats. After the third embolism, rats developed characteristic features of CTEPH including elevated right ventricular systolic pressure, right ventricular cardiomyocyte hypertrophy, pulmonary artery remodeling, increased serum brain natriuretic peptide levels, thrombi fibrosis, and formation of pulmonary cellular-fibrotic lesions. The current animal model seems suitable for detailed study of CTEPH pathophysiology and permits preclinical testing of new pharmacological therapies against CTEPH.

Metformin Protects Kidney Cells From Insulin-Mediated Genotoxicity In Vitro and in Male Zucker Diabetic Fatty Rats.

Hyperinsulinemia is thought to enhance cancer risk. A possible mechanism is induction of oxidative stress and DNA damage by insulin, Here, the effect of a combination of metformin with insulin was investigated in vitro and in vivo. The rationales for this were the reported antioxidative properties of metformin and the aim to gain further insights into the mechanisms responsible for protecting the genome from insulin-mediated oxidative stress and damage. The comet assay, a micronucleus frequency test, and a mammalian gene mutation assay were used to evaluate the DNA damage produced by insulin alone or in combination with metformin. For analysis of antioxidant activity, oxidative stress, and mitochondrial disturbances, the cell-free ferric reducing antioxidant power assay, the superoxide-sensitive dye dihydroethidium, and the mitochondrial membrane potential-sensitive dye 5,5',6,6'tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbocyanine iodide were applied. Accumulation of p53 and pAKT were analyzed. As an in vivo model, hyperinsulinemic Zucker diabetic fatty rats, additionally exposed to insulin during a hyperinsulinemic-euglycemic clamp, were treated with metformin. In the rat kidney samples, dihydroethidium staining, p53 and pAKT analysis, and quantification of the oxidized DNA base 8-oxo-7,8-dihydro-2'-deoxyguanosine were performed. Metformin did not show intrinsic antioxidant activity in the cell-free assay, but protected cultured cells from insulin-mediated oxidative stress, DNA damage, and mutation. Treatment of the rats with metformin protected their kidneys from oxidative stress and genomic damage induced by hyperinsulinemia. Metformin may protect patients from genomic damage induced by elevated insulin levels. This may support efforts to reduce the elevated cancer risk that is associated with hyperinsulinemia.

Eya4 Induces Hypertrophy via Regulation of p27kip1.

BACKGROUND: E193, a heterozygous truncating mutation in the human transcription cofactor Eyes absent 4 (Eya4), causes hearing impairment followed by dilative cardiomyopathy. METHODS AND RESULTS: In this study, we first show Eya4 and E193 alter the expression of p27(kip1) in vitro, suggesting Eya4 is a negative regulator of p27. Next, we generated transgenic mice with cardiac-specific overexpression of Eya4 or E193. Luciferase and chromatin immunoprecipitation assays confirmed Eya4 and E193 bind and regulate p27 expression in a contradictory manner. Activity and phosphorylation status of the downstream molecules casein kinase-2α and histone deacetylase 2 were significantly elevated in Eya4- but significantly reduced in E193-overexpressing animals compared with wild-type littermates. Magnetic resonance imaging and hemodynamic analysis indicate Eya4-overexpression results in an age-dependent development of hypertrophy already under baseline conditions with no obvious functional effects, whereas E193 animals develop onset of dilative cardiomyopathy as seen in human E193 patients. Both cardiac phenotypes were aggravated on pressure overload. Finally, we identified a new heterozygous truncating Eya4 mutation, E215, which leads to similar clinical features of disease and a stable myocardial expression of the mutant protein as seen with E193. CONCLUSIONS: Our results implicate Eya4/Six1 regulates normal cardiac function via p27/casein kinase-2α/histone deacetylase 2 and indicate that mutations within this transcriptional complex and signaling cascade lead to the development of cardiomyopathy.

Monitoring of monocyte recruitment in reperfused myocardial infarction with intramyocardial hemorrhage and microvascular obstruction by combined fluorine 19 and proton cardiac magnetic resonance imaging.

BACKGROUND: Monocytes and macrophages are indispensable in the healing process after myocardial infarction (MI); however, the spatiotemporal distribution of monocyte infiltration and its correlation to prognostic indicators of reperfused MI have not been well described. METHODS AND RESULTS: With combined fluorine 19/proton ((1)H) magnetic resonance imaging, we noninvasively visualized the spatiotemporal recruitment of monocytes in vivo in a rat model of reperfused MI. Blood monocytes were labeled by intravenous injection of (19)F-perfluorocarbon emulsion 1 day after MI. The distribution patterns of monocyte infiltration were correlated to the presence of microvascular obstruction (MVO) and intramyocardial hemorrhage. In vivo, (19)F/(1)H magnetic resonance imaging performed in series revealed that monocyte infiltration was spatially inhomogeneous in reperfused MI areas. In the absence of MVO, monocyte infiltration was more intense in MI regions with serious ischemia-reperfusion injuries, indicated by severe intramyocardial hemorrhage; however, monocyte recruitment was significantly impaired in MVO areas accompanied by severe intramyocardial hemorrhage. Compared with MI with isolated intramyocardial hemorrhage, MI with MVO resulted in significantly worse pump function of the left ventricle 28 days after MI. CONCLUSIONS: Monocyte recruitment was inhomogeneous in reperfused MI tissue. It was highly reduced in MVO areas defined by magnetic resonance imaging. The impaired monocyte infiltration in MVO regions could be related to delayed healing and worse functional outcomes in the long term. Therefore, monocyte recruitment in MI with MVO could be a potential diagnostic and therapeutic target that could be monitored noninvasively and longitudinally by (19)F/(1)H magnetic resonance imaging in vivo.

Insulin-mediated oxidative stress and DNA damage in LLC-PK1 pig kidney cell line, female rat primary kidney cells, and male ZDF rat kidneys in vivo.

Hyperinsulinemia, a condition with excessively high insulin blood levels, is related to an increased cancer incidence. Diabetes mellitus is the most common of several diseases accompanied by hyperinsulinemia. Because an elevated kidney cancer risk was reported for diabetic patients, we investigated the induction of genomic damage by insulin in LLC-PK1 pig kidney cells, rat primary kidney cells, and ZDF rat kidneys. Insulin at a concentration of 5nM caused a significant increase in DNA damage in vitro. This was associated with the formation of reactive oxygen species (ROS). In the presence of antioxidants, blockers of the insulin, and IGF-I receptors, and a phosphatidylinositol 3-kinase inhibitor, the insulin-mediated DNA damage was reduced. Phosphorylation of protein kinase B (PKB or AKT) was increased and p53 accumulated. Inhibition of the mitochondrial and nicotinamide adenine dinucleotide phosphatase oxidase-related ROS production reduced the insulin-mediated damage. In primary rat cells, insulin also induced genomic damage. In kidneys from healthy, lean ZDF rats, which were infused with insulin to yield normal or high blood insulin levels, while keeping blood glucose levels constant, the amounts of ROS and the tumor protein (p53) were elevated in the high-insulin group compared with the control level group. ROS and p53 were also elevated in diabetic obese ZDF rats. Overall, insulin-induced oxidative stress resulted in genomic damage. If the same mechanisms are active in patients, hyperinsulinemia might cause genomic damage through the induction of ROS contributing to the increased cancer risk, against which the use of antioxidants and/or ROS production inhibitors might exert protective effects.

Estrogen and estrogen receptors in cardiovascular oxidative stress.

The cardiovascular system of a premenopausal woman is prepared to adapt to the challenges of increased cardiac output and work load that accompany pregnancy. Thus, it is tempting to speculate whether enhanced adaptability of the female cardiovascular system might be advantageous under conditions that promote cardiovascular disease. In support of this concept, 17ß-estradiol as the major female sex hormone has been shown to confer protective cardiovascular effects in experimental studies. Mechanistically, these have been partially linked to the prevention and protection against oxidative stress. Current evidence indicates that estrogens attenuate oxidative stress at two levels: first, by preventing generation of reactive oxygen species (ROS) and, second, by scavenging ROS in the myocardium and in the vasculature. The purpose of this review is to give an overview on current concepts on conditions and mechanisms by which estrogens protect the cardiovascular system against ROS-mediated cellular injury.

The estrogen receptor-α is required and sufficient to maintain physiological glucose uptake in the mouse heart.

Estrogens attenuate cardiac hypertrophy and increase cardiac contractility via their cognate estrogen receptors (ERs) ERα and ERß. Because female sex hormones enhance global glucose use and because myocardial function and mass are tightly linked to cardiac glucose metabolism, we tested the hypothesis that expression and activation of the ERα might be required and sufficient to maintain physiological cardiac glucose uptake in the murine heart. Cardiac glucose uptake quantified in vivo by 18F-fluorodeoxyglucose positron emission tomography was strongly impaired in ovariectomized compared with gonadal intact female C57BL/6JO mice. The selective ERα agonist 16α-LE2 and the nonselective ERα and ERß agonist 17ß-estradiol completely restored cardiac glucose uptake in ovariectomized mice. Cardiac 18F-fluorodeoxyglucose uptake was strongly decreased in female ERα knockout mice compared with wild-type littermates. Analysis of cardiac mRNA accumulation by quantitative RT-PCR revealed an upregulation of genes involved in glycolisis and tricarboxylic acid cycle by ERα treatment. In conclusion, systemic activation of ERα is sufficient, and its expression is required to maintain physiological glucose uptake in the murine heart, which is likely to contribute to known cardioprotective estrogen effects.

Both estrogen receptor subtypes, ERα and ERß, prevent aldosterone-induced oxidative stress in VSMC via increased NADPH bioavailability.

Activation of vascular mineralocorticoid (MR) or estrogen receptors (ER) exerts opposing effects on vascular remodeling. As we have previously shown, activation of either estrogen receptor subtype, ERα or ERß, is fully sufficient to attenuate vascular remodeling in aldosterone salt-treated rats. To further elucidate the underlying mechanism(s) we tested the hypothesis that ER and MR activation might differentially modulate vascular reactive oxygen species (ROS) generation. In support of this concept, aldosterone increased ROS generation in vascular smooth muscle cells as determined by quantitative dihydroethidium fluorescence microscopy. Co-treatment with the selective ERα agonist 16α-LE2, the selective ERß agonist 8ß-VE2 or the non-selective ER agonist 17ß-estradiol (E2) significantly reduced aldosterone-induced ROS generation. The pure ER antagonist ICI 182,780 completely blocked these salutary effects of E2, 16α-LE2 and 8ß-VE2. Activation of ERα or ERß fully blocked the reduction of intracellular nicotinamide adenine dinucleotide phosphate (NADPH) levels observed in aldosterone treated vascular smooth muscle cells. Intracellular NADPH levels were closely associated with expression and activity of the NADPH generating enzyme glucose-6-phosphate dehydrogenase. In conclusion, estrogens attenuate the detrimental vascular effects of excessive MR activation at least in part by preventing the depletion of intracellular NADPH levels.

Medroxyprogesterone acetate aggravates oxidative stress and left ventricular dysfunction in rats with chronic myocardial infarction.

The role of estrogens during myocardial ischemia has been extensively studied. However, effects of a standard hormone replacement therapy including 17ß-estradiol (E2) combined with medroxyprogesterone acetate (MPA) have not been assessed, and this combination could have contributed to the negative outcomes of the clinical studies on hormone replacement. We hypothesized that adding MPA to an E2 treatment would aggravate chronic heart failure after experimental myocardial infarction (MI). To address this issue, we evaluated clinical signs of heart failure as well as left ventricular (LV) dysfunction and remodeling in ovariectomized rats subjected to chronic MI receiving E2 or E2 plus MPA. After eight weeks MI E2 showed no effects. Adding MPA to E2 aggravated LV remodeling and dysfunction as judged by increased heart weight, elevated myocyte cross-sectional areas, increased elevated left ventricle end diastolic pressure, and decreased LV fractional shortening. Impaired LV function in rats receiving MPA plus E2 was associated with increased cardiac reactive oxygen species generation and myocardial expression levels of NADPH oxidase subunits. These results support the interpretation that adding MPA to an E2 treatment complicates cardiovascular injury damage post-MI and therefore contributes to explain the adverse outcome of prospective clinical studies.

Differential effects of 17beta-estradiol and of synthetic progestins on aldosterone-salt-induced kidney disease.

UNLABELLED: Elevated mineralocorticoid levels and female sex hormones have been shown to confer opposing effects on renal injury, but their combined effects are still unknown. OBJECTIVE: Identify the function of estrogens and of different synthetic progestins on aldosterone salt-mediated renal disease. METHODS: The role of 17beta-estradiol, medroxyprogesterone acetate (MPA), and drospirenone during renal injury was studied in Wistar rats subjected to uni-nephrectomy plus aldosterone salt treatment. RESULTS: Aldo-salt treatment of intact, ovariectomized, and estradiol-treated female rats resulted in remnant kidney hypertrophy without structural damage. Co-treatment with MPA, but not with drospirenone, increased kidney hypertrophy, fluid turnover, sodium retention, and potassium excretion. Medroxyprogesterone acetate also caused glomerular, vascular, tubular, and interstitial lesions that were accompanied by increased blood pressure and enhanced NADPH oxidase (p67phox) and sodium channel (alpha-ENaC) expression. Drospirenone, a progestin with anti-mineralocorticoid function, and spironolactone prevented kidney hypertrophy, hypertension, and sodium retention. Drospirenone and spironolactone also increased renal angiotensin II type 2 receptor expression and relieved aldosterone-induced suppression of serum angiotensin II levels. CONCLUSION: The choice of specific synthetic progestins has profound implications on the development of kidney injury and renal gene expression under conditions of elevated aldosterone serum levels and salt intake.

Genetic and pharmacologic strategies to determine the function of estrogen receptor alpha and estrogen receptor beta in cardiovascular system.

BACKGROUND: The biological functions of estrogens extend beyond the female and male reproductive tract, affecting the cardiovascular and renal systems. Traditional views on the role of postmenopausal hormone therapy (HT) in protecting against heart disease, which were challenged by clinical end point studies that found adverse effects of combined HT, are now being replaced by more differentiated concepts suggesting a beneficial role of early and unopposed HT that does not include a progestin. OBJECTIVE: We reviewed recent insights, concepts, and research results on the biology of both estrogen receptor (ER) subtypes, ERalpha and ERbeta, in cardiac and vascular tissues. Knowledge of these ER subtypes is crucial to understanding gender and estrogen effects and to developing novel, exciting strategies that may have a profound clinical impact. METHODS: This review focuses on in vivo studies and includes data presented at the August 2007 meeting of the American Physiological Society as well as data from a search of the MEDLINE and Ovid databases from January 1986 to November 2007. Search results were restricted to English-language publications, using the following search terms: estrogen, estrogen receptor alpha, estrogen receptor beta, estrogen receptor alpha agonist, estrogen receptor alpha antagonist, estrogen receptor beta agonist, estrogen receptor beta antagonist, PPT, DPN, heart, vasculature, ERKO mice, BERKO mice, transgenic mice, and knockout mice. RESULTS: Genetic mouse models and pharmacologic studies that employed selective as well as nonselective ER agonists support the concept that both ER subtypes confer protective effects in experimental models of human heart disease, including hypertension, cardiac hypertrophy, and chronic heart failure. CONCLUSIONS: Genetic models and novel ligands hold the promise of further improving our understanding of estrogen action in multiple tissues and organs. These efforts will ultimately enhance the safety and efficacy of HT and may also result in new applications for synthetic female sex hormone analogues.

Ligand-dependent activation of ER{beta} lowers blood pressure and attenuates cardiac hypertrophy in ovariectomized spontaneously hypertensive rats.

AIMS: The biological effects of oestrogens are mediated by two different oestrogen receptor (ER) subtypes, ERalpha and ERbeta, which might play different, redundant, or opposing roles in cardiovascular disease. Previously, we have shown that the selective ERalpha agonist 16alpha-LE2 improves vascular relaxation, attenuates cardiac hypertrophy, and increases cardiac output without lowering elevated blood pressure in spontaneously hypertensive rats (SHR). Because ERbeta-deficient mice exhibit elevated blood pressure and since the ERbeta agonist 8beta-VE2 attenuated hypertension in aldosterone-salt-treated rats, we have now tested the hypothesis that the isotype-selective ERbeta agonist 8beta-VE2 might be capable of lowering elevated blood pressure in ovariectomized SHR. METHODS AND RESULTS: Treatment of ovariectomized SHR with 8beta-VE2 for 12 weeks conferred no uterotrophic effects but lowered elevated systolic blood pressure (-38 +/- 5 mmHg, n = 31, P < 0.001 vs. placebo) as well as peripheral vascular resistance (-31.3 +/- 4.6%, P < 0.001 vs. placebo). 8beta-VE2 enhanced aortic ERbeta expression (+75.7 +/- 7.1%, P < 0.01 vs. placebo), improved NO-dependent vasorelaxation, augmented phosphorylation of the vasodilator-stimulated phosphoprotein in isolated aortic rings (P < 0.05 vs. placebo), increased cardiac output (+20.4 +/- 2.5%, P < 0.01 vs. placebo), and attenuated cardiac hypertrophy (-22.2 +/- 3.2%, p < 0.01 vs. placebo). 8beta-VE2, in contrast to oestradiol, did not enhance cardiac alpha-myosin heavy chain expression. CONCLUSION: Ligand-dependent activation of ERbeta confers blood pressure lowering effects in SHR that are superior to those of 17beta-estradiol or the ERalpha agonist 16alpha-LE2 and attenuates cardiac hypertrophy primarily by a reduction of cardiac afterload without promoting uterine growth.

Both estrogen receptor subtypes, alpha and beta, attenuate cardiovascular remodeling in aldosterone salt-treated rats.

Experimental and population-based studies indicate that female gender and estrogens protect the cardiovascular system against aldosterone-induced injury. Understanding the function of estrogens in heart disease requires more precise information on the role of both estrogen receptor (ER) subtypes, ERalpha and ERbeta. Therefore, we determined whether selective activation of ERalpha or of ERbeta would confer redundant, specific, or opposing effects on cardiovascular remodeling in aldosterone salt-treated rats. The ERalpha agonist 16alpha-LE2, the ERbeta agonist 8beta-VE2, and the nonselective estrogen receptor agonist 17beta-estradiol lowered elevated blood pressure, cardiac mass, and cardiac myocyte cross-sectional areas, as well as increased perivascular collagen accumulation and vascular osteopontin expression in ovariectomized rats receiving chronic aldosterone infusion plus a high-salt diet for 8 weeks. Uterus atrophy was prevented by 16alpha-LE2 and 17beta-estradiol but not by 8beta-VE2. Cardiac proteome analyses by 2D gel electrophoresis, mass spectrometry, and peptide sequencing identified specific subsets of proteins involved in cardiac contractility, energy metabolism, cellular stress response and extracellular matrix formation that were regulated in opposite directions by aldosterone salt treatment and by different estrogen receptor agonists. We conclude that activation of either ERalpha or ERbeta protects the cardiovascular system against the detrimental effects of aldosterone salt treatment and confers redundant, as well as specific, effects on cardiac protein expression. Nonfeminizing ERbeta agonists such as 8beta-VE2 have a therapeutic potential in the treatment of hypertensive heart disease.

Medroxyprogesterone acetate but not drospirenone ablates the protective function of 17 beta-estradiol in aldosterone salt-treated rats.

Controversial results obtained from human and animal studies on the prevention of heart disease by estrogens and progestins warrant a better understanding of nuclear hormone receptor function and interaction. To address this issue and taking into account that effects of synthetic progestins are not only referable to action through the progesterone receptor but may also be mediated by other steroid receptors, we characterized cardiovascular function and inflammatory gene expression in aldosterone salt-treated rats on long-term administration of 17beta-estradiol, medroxyprogesterone acetate, and drospirenone, a new progestogen exhibiting antimineralocorticoid activity. The complex pattern of cardiovascular injury in ovariectomized Wistar rats induced by chronic aldosterone infusion plus a high-salt diet was significantly attenuated in sham-ovariectomized rats and by coadministration of 17beta-estradiol in ovariectomized animals after 8 weeks of continuous treatment. The beneficial role of 17beta-estradiol on blood pressure, cardiac hypertrophy, vascular osteopontin expression, perivascular fibrosis, and impaired NO-dependent relaxation of isolated aortic rings was completely abrogated by coadministration of medroxyprogesterone acetate. In contrast, drospirenone was either neutral or additive to 17beta-estradiol in protecting against aldosterone salt-induced cardiovascular injury and inflammation. The current results support the hypothesis of complex interactions among estrogen, progesterone, glucocorticoid, androgen, and mineralocorticoid receptor signaling in cardiovascular injury and inflammation. Novel progestins, such as drospirenone, confer superior effects compared with medroxyprogesterone acetate in a model of aldosterone-induced heart disease because of its antimineralocorticoid properties.