Cardiac Mineralocorticoid Receptor Research Articles

Aldosterone Effect on Cardiac Structure and Function.

Cardiac remodelling could be a key mechanism in aldosteronemediated cardiovascular morbidity and mortality. Experimental and clinical evidence has demonstrated that aldosterone causes cardiac structural remodelling and dysfunction by its profibrotic and pro-hypertrophic effects, which result mainly from the direct effects on myocardial collagen deposition, inflammation, and oxidative stress. Clinical studies have investigated the aldosterone effects on the heart in different clinical conditions, including general population, essential hypertension, primary aldosteronism, heart failure, and atrial fibrillation. Robust findings indicate that aldosterone or the activation of the cardiac mineralocorticoid receptor can cause damage to myocardial tissue by mechanisms independent of the blood pressure, leading to tissue hypertrophy, fibrosis, and dysfunction. Aldosterone-mediated cardiovascular morbidity and mortality mainly result from cardiac structural and functional alterations. In different clinical settings, aldosterone can induce cardiac structural remodelling and dysfunction via several pathological mechanisms, including cardiac fibrosis, inflammation, and oxidative stress. Aldosterone antagonists could effectively decrease or reverse the detrimental aldosterone-mediated changes in the heart.

GRK5 is an essential co-repressor of the cardiac mineralocorticoid receptor and is selectively induced by finerenone.

BACKGROUNDIn the heart, aldosterone (Aldo) binds the mineralocorticoid receptor (MR) to exert damaging, adverse remodeling-promoting effects. We recently showed that G protein-coupled receptor-kinase (GRK)-5 blocks the cardiac MR by directly phosphorylating it, thereby repressing its transcriptional activity. MR antagonist (MRA) drugs block the cardiac MR reducing morbidity and mortality of advanced human heart failure. Non-steroidal MRAs, such as finerenone, may provide better cardio-protection against Aldo than classic, steroidal MRAs, like spironolactone and eplerenone. AIMTo investigate potential differences between finerenone and eplerenone at engaging GRK5-dependent cardiac MR phosphorylation and subsequent blockade. METHODSWe used H9c2 cardiomyocytes, which endogenously express the MR and GRK5.RESULTSGRK5 phosphorylates the MR in H9c2 cardiomyocytes in response to finerenone but not to eplerenone. Unlike eplerenone, finerenone alone potently and efficiently suppresses cardiac MR transcriptional activity, thus displaying inverse agonism. GRK5 is necessary for finerenone’s inverse agonism, since GRK5 genetic deletion renders finerenone incapable of blocking cardiac MR transcriptional activity. Eplerenone alone does not fully suppress cardiac MR basal activity regardless of GRK5 expression levels. Finally, GRK5 is necessary for the anti-apoptotic, anti-oxidative, and anti-fibrotic effects of both finerenone and eplerenone against Aldo, as well as for the higher efficacy and potency of finerenone at blocking Aldo-induced apoptosis, oxidative stress, and fibrosis.CONCLUSIONFinerenone, but not eplerenone, induces GRK5-dependent cardiac MR inhibition, which underlies, at least in part, its higher potency and efficacy, compared to eplerenone, as an MRA in the heart. GRK5 acts as a co-repressor of the cardiac MR and is essential for efficient MR antagonism in the myocardium

The Cardiac Mineralocorticoid Receptor (MR): A Therapeutic Target Against Ventricular Arrhythmias.

Mineralocorticoid antagonists have been shown to be useful in the treatment of severe heart failure and may even save lives in this context. However, the reason for the beneficial action of these drugs, as well as the physiological role played by the cardiac mineralocorticoid receptor (MR), are still poorly understood. While the proinflammatory action of aldosterone on the heart and the resulting fibrosis partly explain the improvement due to the anti-mineralocorticoid therapy, the reduction in sudden death is probably related to a lower occurrence of ventricular arrhythmias. In this review, the author explains the physiological mechanism linking the positive chronotropic response induced by aldosterone observed in vitro with isolated ventricular cardiomyocytes and the increased risk of ventricular arrhythmias reported in vivo in hyperaldosteronism. He describes the molecular steps involved between MR activation and acceleration of spontaneous myocyte contractions, including expression of a specific micro RNA (miR204), down-regulation of a silencing transcription factor (NRSF), and re-expression of a fetal gene encoding a low threshold voltage-gated calcium channel (CaV3.2). Finally, he provides evidence suggesting aldosterone-independent and redox-sensitive mechanisms of MR activation in cardiac myocytes. Taken together, this information suggests that the use of anti-mineralocorticoid therapy could benefit the heart by preventing ventricular arrhythmias, not only in established hyperaldosteronism, but also in various pathological situations such as Cushing’s disease, oxidative stress, or even diabetes mellitus.

Cardiac Mineralocorticoid Receptor and the Na+/H+ Exchanger: Spilling the Beans.

Current evidence reveals that cardiac mineralocorticoid receptor (MR) activation following myocardial stretch plays an important physiological role in adapting developed force to sudden changes in hemodynamic conditions. Its underlying mechanism involves a previously unknown nongenomic effect of the MR that triggers redox-mediated Na+/H+ exchanger (NHE1) activation, intracellular Na+ accumulation, and a consequent increase in Ca2+ transient amplitude through reverse Na+/Ca2+ exchange. However, clinical evidence assigns a detrimental role to MR activation in the pathogenesis of severe cardiac diseases such as congestive heart failure. This mini review is meant to present and briefly discuss some recent discoveries about locally triggered cardiac MR signals with the objective of shedding some light on its physiological but potentially pathological consequences in the heart.

Antagonistic Roles of GRK2 and GRK5 in Cardiac Aldosterone Signaling Reveal GRK5-Mediated Cardioprotection via Mineralocorticoid Receptor Inhibition

Aldosterone (Aldo), when overproduced, is a cardiotoxic hormone underlying heart failure and hypertension. Aldo exerts damaging effects via the mineralocorticoid receptor (MR) but also activates the antiapoptotic G protein-coupled estrogen receptor (GPER) in the heart. G protein-coupled receptor (GPCR)-kinase (GRK)-2 and -5 are the most abundant cardiac GRKs and phosphorylate GPCRs as well as non-GPCR substrates. Herein, we investigated whether they phosphorylate and regulate cardiac MR and GPER. To this end, we used the cardiomyocyte cell line H9c2 and adult rat ventricular myocytes (ARVMs), in which we manipulated GRK5 protein levels via clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and GRK2 activity via pharmacological inhibition. We report that GRK5 phosphorylates and inhibits the cardiac MR whereas GRK2 phosphorylates and desensitizes GPER. In H9c2 cardiomyocytes, GRK5 interacts with and phosphorylates the MR upon β2-adrenergic receptor (AR) activation. In contrast, GRK2 opposes agonist-activated GPER signaling. Importantly, GRK5-dependent MR phosphorylation of the MR inhibits transcriptional activity, since aldosterone-induced gene transcription is markedly suppressed in GRK5-overexpressing cardiomyocytes. Conversely, GRK5 gene deletion augments cardiac MR transcriptional activity. β2AR-stimulated GRK5 phosphorylates and inhibits the MR also in ARVMs. Additionally, GRK5 is necessary for the protective effects of the MR antagonist drug eplerenone against Aldo-induced apoptosis and oxidative stress in ARVMs. In conclusion, GRK5 blocks the cardiotoxic MR-dependent effects of Aldo in the heart, whereas GRK2 may hinder beneficial effects of Aldo through GPER. Thus, cardiac GRK5 stimulation (e.g., via β2AR activation) might be of therapeutic value for heart disease treatment via boosting the efficacy of MR antagonists against Aldo-mediated cardiac injury.

Aldosterone And Osteopontin Promote Cardiac Fibrosis Via GRK2‐dependent β2‐Adrenergic Receptor Dysfunction

BackgroundCardiac β2‐adrenergic receptors (ARs) are known to inhibit collagen production and fibrosis in cardiac fibroblasts and myocytes. β2AR is a Gs protein‐coupled receptor (GPCR) and, upon its activation, stimulates generation of cyclic 3′, 5′‐adenosine monophosphate (cAMP). cAMP has two effectors: protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac1/2). Epac1 has been shown to inhibit cardiac fibroblast activation and fibrosis. Osteopontin (OPN) is a ubiquitous pro‐inflammatory and pro‐fibrotic cytokine, including in the heart. OPN expression is known to be induced by the cardiotoxic hormone aldosterone in various tissues and OPN has been reported to dysregulate β2AR signaling and function in bone cells. Thus, we hypothesized here that aldosterone upregulates OPN in the heart in order to perturb β2AR anti‐fibrotic function.MethodsWe used the rat cardiomyoblast cell line H9c2 and human cardiac fibroblasts (HCFs). We measured OPN levels and fibrosis markers via real time PCR and β2AR function via cAMP accumulation studies and co‐immunoprecipitation/western blotting for several downstream signaling mediators. We also deleted OPN via CRISPR/Cas9 to examine its role in cardiac β2AR function and fibrosis.ResultsAldosterone upregulates OPN via the mineralocorticoid receptor (MR) in H9c2 cardiomyocytes. This is prevented by β2AR activation with salbutamol (albuterol), which stimulates GPCR‐kinase (GRK)‐5 to phosphorylate the cardiac MR and inhibit its transcriptional activity. Importantly, CRISPR/Cas9‐mediated OPN gene deletion enhances β2AR‐dependent cAMP generation in H9c2 cardiomyocytes and upregulates Epac1 protein levels. The functional result of this is that β2AR’s inhibition of transforming growth factor (TGF)‐β‐dependent fibrosis in H9c2 cardiomyocytes is robustly augmented. In addition, pharmacological inhibition of GRK2, the major GRK that phosphorylates and desensitizes the cardiac β2AR, with Cmpd101 or OPN CRIPSR/Cas9‐mediated knockout completely abrogates isoproterenol (agonist)‐induced β2AR functional desensitization in HCFs (Isoproterenol’s ΔEC50: 94±16 μM in control HCFs, p<0.05, n=4; 6±7.2 μM in Cmpd101‐treated HCFs, Not significant at p=0.05, n=4; 5.3+6.1 μM in OPN knockout HCFs, Not significant at p=0.05, n=4). Mechanistically, OPN dysregulates the β2AR via enhancement of GRK2‐mediated functional desensitization, since OPN forms a multi‐protein complex with GRK2 and GRK2‐interacting protein (GIT)‐1 in HCFs, thereby facilitating GRK2 plasma membrane recruitment. This reduces β2AR’s anti‐fibrotic cAMP signaling in the heart.ConclusionsWe have uncovered a direct inhibitory effect of OPN on cardiac β2AR’s anti‐fibrotic signaling: facilitation of GRK2‐mediated receptor desensitization. Since aldosterone/MR upregulates OPN, a combination of an MR blocker with OPN or GRK2 blockade could be of value in cardiac fibrosis treatment.Support or Funding Information1) American Heart Association (A.L.)2) NSU President’s Faculty Research & Development Grant (A.L.)

GRK5‐mediated inhibitory phosphorylation is essential for inverse agonism at the cardiac mineralocorticoid receptor

BackgroundIn the heart, aldosterone binds the mineralocorticoid receptor (MR) to exert damaging effects, e.g. fibrosis, apoptosis, oxidative stress, etc. GRK2 and GRK5 are the most abundant cardiac G protein‐coupled receptor (GPCR)‐kinases (GRKs). Both phosphorylate GPCRs but also non‐GPCR substrates. We recently showed that GRK5 blocks the cardio‐toxic MR‐dependent effects of aldosterone in the heart by directly phosphorylating the cardiac MR and inhibiting its transcriptional activity. MR antagonist (MRA) drugs are beneifical in human CHF by blocking the cardiac MR. Novel non‐steroidal MRAs, such as finerenone, may provide better cardio‐protection against aldosterone's cardio‐toxic actions than the classic steroidal MRAs, such as sprironolactone and eplerenone. Indeed, finerenone was very recently shown to be a more potent and efficacious inverse agonist at the MR than eplerenone in terms of cardiac fibrosis/adverse remodeling attenuation. Thus, in the present study, we sought to investigate potential differences between finerenone and eplerenone at engaging GRK5‐dependent cardiac MR phosphorylation and subsequent blockade.MethodsWe used the cardiomyocyte cell line H9c2 and neonatal rat ventricular myocytes (NRVMs). We measured MR phosphorylation via phospho‐serine immunoblotting & transcriptional activity via the luciferase reporter assay.ResultsGRK5 phosphorylates the MR in H9c2 cardiomyocytes in response to finerenone but not to eplerenone. Contrary to eplerenone, finerenone alone potently and efficiently suppresses MR transcriptional activity in H9c2 cardiac myocytes. GRK5‐dependent phosphorylation is necessary for the robust inverse agonism displayed by finerenone at the cardiac MR, since CRISPR‐mediated GRK5 gene deletion renders finerenone incapable of blocking cardiac MR transcriptional activity. Conversely, this non‐steroidal MRA fully suppresses cardiac MR transcriptional activity upon GRK5 overexpression in H9c2 cardiomyocytes. Eplerenone alone cannot suppress cardiac MR basal activity upon GRK5 overexpression or knockout. Finally, finerenone blocks aldosterone's effects more efficiently than eplerenone in control and GRK5‐overexpressing NRVMs.ConclusionsFinerenone appears a more efficacious cardiac MR blocker than eplerenone, partly via GRK5‐dependent cardiac MR inhibition, which eplerenone does not induce. This non‐canonical effect of GRK5 on the MR is essential for the efficient blockade of aldosterone's deleterious actions in the heart.Support or Funding Information1) American Heart Association 2) NSU President's Faculty Research & Development GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract WP549: GRK5-Mediated Inhibitory Phosphorylation of the Mineralocorticoid Receptor Protects Against Aldosterone`s Stroke-Promoting Vascular Effects

Introduction: Stroke is one of the leading causes of death in the Western world. Mineralocorticoid receptor (MR) antagonism is a potential therapy for ischemic and hemorrhagic stroke patients, since aldosterone exerts deleterious effects in the cerebral vasculature via the MR. Prominent among these is activation of epidermal growth factor (EGF)-dependent vessel wall remodeling. GRK2 and GRK5 are the most abundant G protein-coupled receptor (GPCR)-kinases (GRKs) in the heart and vessels and both phosphorylate GPCRs but also non-GPCR substrates at Ser/Thr residues. The human MR gets phosphorylated at various Ser residues, including Ser-843 in the ligand binding domain (LBD) and Ser-601 in the N-terminal domain (NTD). These Ser phosphorylations occur in the cytoplasm and result in MR inhibition via direct transcriptional activity repression or cytosolic retention, respectively. Hypothesis: GRK5 phosphorylates and inhibits the MR in the heart and vessels. Methods: We used rat cardiomyocytes (H9c2) and aortic smooth muscle cells (ASMCs). We checked for GRK interactions with the MR via co-immunoprecipitation. We measured MR phosphorylation via phospho-Ser immunoblotting and MR transcriptional activity via the luciferase reporter assay. We also measured EGF and EGF receptor (EGFR) expression via real time-PCR. Results: GRK5, but not GRK2, phosphorylates the MR in H9c2 cardiomyocytes. Beta2-adrenoceptor activation stimulates this non-canonical effect of GRK5. The GRK5-phosphorylated MR is incapable of activating gene transcription, since aldosterone-induced MR transcriptional activity is markedly suppressed upon GRK5 overexpression in both cardiomyocytes and ASMCs. Conversely, CRISPR-mediated GRK5 gene deletion augments cardiac and ASMC aldosterone-dependent MR transcriptional activity. Finally, beta2-adrenoceptor-stimulated GRK5 phosphorylates and inhibits the MR in ASMCs, thereby suppressing aldosterone-induced EGF and EGFR mRNA expression. Conclusions: GRK5 blocks the toxic MR-dependent effects of aldosterone in the heart and vascular smooth muscles. Thus, GRK5 stimulation (e.g. via beta2-adrenoceptor activation) may protect against the stroke-promoting effects of aldosterone in the cerebral vasculature.

Novel Insights into the Crosstalk between Mineralocorticoid Receptor and G Protein-Coupled Receptors in Heart Adverse Remodeling and Disease

The mineralocorticoid hormone aldosterone regulates sodium and potassium homeostasis but also adversely modulates the maladaptive process of cardiac adverse remodeling post-myocardial infarction. Through activation of its mineralocorticoid receptor (MR), a classic steroid hormone receptor/transcription factor, aldosterone promotes inflammation and fibrosis of the heart, the vasculature, and the kidneys. This is why MR antagonists reduce morbidity and mortality of heart disease patients and are part of the mainstay pharmacotherapy of advanced human heart failure. A plethora of animal studies using cell type–specific targeting of the MR gene have established the importance of MR signaling and function in cardiac myocytes, vascular endothelial and smooth muscle cells, renal cells, and macrophages. In terms of its signaling properties, the MR is distinct from nuclear receptors in that it has, in reality, two physiological hormonal agonists: not only aldosterone but also cortisol. In fact, in several tissues, including in the myocardium, cortisol is the primary hormone activating the MR. There is a considerable amount of evidence indicating that the effects of the MR in each tissue expressing it depend on tissue- and ligand-specific engagement of molecular co-regulators that either activate or suppress its transcriptional activity. Identification of these co-regulators for every ligand that interacts with the MR in the heart (and in other tissues) is of utmost importance therapeutically, since it can not only help elucidate fully the pathophysiological ramifications of the cardiac MR’s actions, but also help design and develop novel better MR antagonist drugs for heart disease therapy. Among the various proteins the MR interacts with are molecules involved in cardiac G protein-coupled receptor (GPCR) signaling. This results in a significant amount of crosstalk between GPCRs and the MR, which can affect the latter’s activity dramatically in the heart and in other cardiovascular tissues. This review summarizes the current experimental evidence for this GPCR-MR crosstalk in the heart and discusses its pathophysiological implications for cardiac adverse remodeling as well as for heart disease therapy. Novel findings revealing non-conventional roles of GPCR signaling molecules, specifically of GPCR-kinase (GRK)-5, in cardiac MR regulation are also highlighted.

Abstract 056: Differential Roles of GRK2 and GRK5 in Cardiac Aldosterone Signaling Suggest GRK5-Mediated Cardio-protection Against Mineralocorticoids

Background: Aldosterone (Aldo) contributes significantly to the morbidity & mortality of heart failure (HF). In the heart, Aldo binds the mineralocorticoid receptor (MR) to exert damaging effects, e.g. fibrosis, apoptosis, oxidative stress, etc. Additionally, Aldo can activate the G protein-coupled estrogen receptor (GPER), which may have beneficial, anti-apoptotic effects for cardiomyocytes, e.g. in ischemia/reperfusion injury. GRK2 and GRK5 are the most abundant G protein-coupled receptor (GPCR)-kinases (GRKs) in the heart and both phosphorylate GPCRs but also non-GPCR substrates. The human MR is known to undergo inhibitory phosphorylation at Ser-843, inside its ligand binding domain, which blocks its transcriptional activity. Hypothesis: In the heart, GRK5 phosphorylates and inhibits the MR, whereas GRK2 phosphorylates and desensitizes GPER. Methods: We used the cardiomyocyte cell line H9c2 and isolated adult rat ventricular myocytes (ARVMs). We performed co-immunoprecipitation experiments for GRK interactions with MR or GPER. We measured MR phosphorylation via immunoblotting and MR transcriptional activity via the luciferase reporter assay. We also measured apoptosis and oxidative stress in ARVMs. Results: GRK5, but not GRK2, phosphorylates the MR in H9c2 cardiomyocytes. Beta 2 -adrenoceptor activation stimulates this non-canonical effect of GRK5. In contrast, GRK2, but not GRK5, phosphorylates and desensitizes agonist-activated GPER. The GRK5-phosphorylated MR is incapable of activating gene transcription, since MR transcriptional activity is markedly suppressed upon GRK5 overexpression. Conversely, CRISPR-mediated GRK5 gene deletion augments cardiac MR transcriptional activity. Importantly, GRK5 is necessary for the protective effects of the MR antagonist drug eplerenone against Aldo-induced apoptosis/oxidative stress in ARVMs. Finally, beta 2 -adrenoceptor-stimulated GRK5 phosphorylates and inhibits the MR in ARVMs. Conclusions: GRK5 blocks the cardio-toxic MR-dependent effects of Aldo in the heart, whereas GRK2 may hinder GPER`s cardio-protective effects. Thus, cardiac GRK5 stimulation (e.g. via beta 2 -adrenoceptor activation) enhances the cardio-protection exerted by Aldo inhibitors in HF.

The Aldosterone Receptor Antagonist Eplerenone Inhibits Isoproterenol-Induced Collagen-I and 11β-HSD1 Expression in Rat Cardiac Fibroblasts and the Left Ventricle

β-Adrenergic receptor (β-AR)-induction of collagen-I synthesis is partially mediated by the cardiac mineralocorticoid receptor (MR) system. However, it remains unclear whether the selective MR antagonist, eplerenone, inhibits collagen-I synthesis induced by β-AR stimulation. We investigated the effects of eplerenone on the responses to a non-selective β-AR agonist, isoproterenol, which induced collagen-I synthesis in primary cardiac fibroblasts (CFs) and the left ventricle. mRNAs encoding the MR and 11β-hydroxysteroid dehydrogenase type I (11β-HSD1) were evident in the left ventricle and primary CFs. mRNAs encoding the CYP family 11 subfamily B member 2 (CYP11-B2) were not detected, even after isoproterenol treatment. In vivo, isoproterenol induced collagenous fiber accumulation in the left ventricle. The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), 11β-HSD1 levels, and mRNA/protein levels of collagen-I increased upon exposure to isoproterenol, but these increases were inhibited by eplerenone co-treatment. In primary CFs, isoproterenol increased the phosphorylation of ERK1/2 and the expression levels of both 11β-HSD1 and collagen-I; these isoproterenol-attributable effects were inhibited by co-treatment with eplerenone and PD98059, a specific inhibitor of mitogen-activated protein kinase/ERK kinase activity. The results suggest that 11β-HSD1 but not CYP11-B2 is expressed in primary CFs. Eplerenone inhibited isoproterenol-induced ERK1/2 phosphorylation and expression of 11β-HSD1 and collagen-I in primary CFs, as well as the progression of cardiac fibrosis in the left ventricle. Therefore, eplerenone inhibited the isoproterenol-induced increases in 11β-HSD1 and collagen-I expression in primary CFs, and progression of cardiac fibrosis in the left ventricle.

Abstract P295: Endothelial Sodium Channel Activation Promotes Cardiac Stiffness in Obese Female Mice

Cardiac diastolic dysfunction (DD) and diastolic heart failure is increasing in concert with obesity and aging population in the United States. In obese and diabetic women, DD is more common than in their male counterparts. This disproportionate increase in DD in obese females may partly explain their loss of sex-related cardiovascular (CV) disease protection. Recent studies have suggested a role for endothelial sodium channel (ENaC) activation in promotion of endothelial stiffness and suppression of flow- (nitric oxide) mediated vasodilation. Moreover, increased mineralocorticoid receptor (MR) activation mediated endothelial stiffness is promoted, in part, by ENaC activation. In this regard, we have recently reported increased plasma aldosterone levels, aortic and cardiac stiffness, and cardiac and vascular MR expression in female mice fed a high fat and high fructose diet (western diet [WD]). This increase in CV stiffness was prevented by very low dose MR antagonism. Accordingly, we hypothesized that inhibition of MR-mediated ENaC activation by using a very low dose of the ENaC inhibitor, amiloride would prevent cardiac stiffening (DD) in WD-fed female mice. Four week old C57BL6/J mice were fed a WD containing high fat (46%), sucrose (17.5%), and high fructose corn syrup (17.5%) with or without a very low dose of amiloride (1mg/kg/day) for 16 weeks. Amiloride significantly attenuated WD-induced impairment of cardiac relaxation in vivo as measured by high resolution magnetic resonance imaging (MRI) as well as cardiac interstitial fibrosis as measured by immunohistochemistry by picrosirius red staining. Moreover, amiloride prevented the development of DD in obese female mice without having effects on blood pressure. These observations support a role for ENaC activation in diet-induced cardiac stiffening (DD) in obese females.

PS 14-14 24-HOUR URINARY SODIUM PREDICTS LEFT VENTRICULAR HYPERTROPHY REGRESSION TO SPIRONOLACTONE IN PATIENTS WITH RESISTANT HYPERTENSION

Objective: Patients with resistant hypertension (RHTN) commonly have primary aldosteronism (PA), which is associated with left ventricular hypertrophy (LVH). Aldosterone activates mineralocorticoid receptors (MR) and induces hypertrophy. Experimental studies indicate a paradoxical activation of the MR in sodium-loaded rats despite adequate suppression of aldosterone. MR antagonists slow down cardiac hypertrophy. We hypothesized that the MR antagonist spironolactone (SPL) would cause greater LVH reduction in patients on high sodium (Na) diet independent of aldosterone. Design and Method: Overall 34 patients with RHTN, defined as BP ≥ 140/90 mmHg despite ≥3 different medications, including a diuretic, were treated with SPL. Cardiac magnetic resonance imaging and biochemical evaluation was performed at baseline, 3 and 6 months in patients with PA and non-PA. PA was defined as renin activity (PRA) <1 ng/ml/h and urinary aldosterone ≥12 ug /24h. We dichotomized patients according to UNa level (UNa ≥200 mEq/24h: high Na diet) and PA status. LVH reduction was indexed by left ventricular mass (LVM) and interventricular septum thickness (IVS) regression. Results: LVM and IVS regression after treatment with SPL at 3 and 6 months was greater in patients with PA on a normal sodium diet and less pronounced in patients on a high sodium diet suggesting that Na blunts the effects of cardiac MR when treated with SPL. However, in patients with non-PA high Na intake did not blunt the effects of SPL. Conclusions: Contrary to our hypothesis, high dietary Na blunted LVH regression in patients with PA treated with SPL. Further studies are needed to elucidate mechanisms for sodium dependent MR activation in patients with PA and non-PA.

High dietary sodium blunts effects of mineralocorticoid receptor antagonism on left ventricular hypertrophy regression in patients with resistant hypertension

Purpose of Study: Patients with resistant hypertension (RHTN) commonly have primary aldosteronism (PA), which is associated with left ventricular hypertrophy (LVH). Aldosterone activates mineralocorticoid receptors (MR) and induces hypertrophy. Experimental studies indicate a paradoxical activation of the MR in sodium-loaded rats despite adequate suppression of aldosterone. MR antagonists slow down cardiac hypertrophy. We hypothesized that the MR anatagonist spironolactone (SPL) would cause greater LVH reduction in patients on high sodium (Na) diet independent of aldosterone. Methods Used: Overall 34 patients with RHTN, defined as BP 140/90 mmHg despite 3 different medications, including a diuretic, were treated with SPL. Cardiac magnetic resonance imaging and biochemical evaluation was performed at baseline, 3 and 6 months in patients with PA and non-PA. PA was defined as renin activity (PRA)<1 ng/ml/h and urinary aldosterone 12 ug /24h. We dichotomized patients according to UNa level (UNa 200 mEq/24h: high Na diet) and PA status. LVH reduction was indexed by left ventricular mass (LVM) and interventricular septum thickness (IVS) regression. Summary of Results: LVM and IVS regression after treatment with SPL at 3 and 6 months was greater in patients with PA on a normal sodium diet and less pronounced in patients on a high sodium diet suggesting that Na blunts the effects of cardiac MR when treated with SPL. However, in patients with non-PA high Na intake did not blunt the effects of SPL. Conclusions: Contrary to our hypothesis, high dietary Na blunted LVH regression in patients with PA treated with SPL. Further studies are needed to elucidate mechanisms for sodium dependent MR activation in patients with PA and non-PA.

Cooperative Role of Mineralocorticoid Receptor and Caveolin-1 in Regulating the Vascular Response to Low Nitric Oxide-High Angiotensin II-Induced Cardiovascular Injury.

Aldosterone interacts with mineralocorticoid receptor (MR) to stimulate sodium reabsorption in renal tubules and may also affect the vasculature. Caveolin-1 (cav-1), an anchoring protein in plasmalemmal caveolae, binds steroid receptors and also endothelial nitric oxide synthase, thus limiting its translocation and activation. To test for potential MR/cav-1 interaction in the vasculature, we investigated if MR blockade in cav-1-replete or -deficient states would alter vascular function in a mouse model of low nitric oxide (NO)-high angiotensin II (AngII)-induced cardiovascular injury. Wild-type (WT) and cav-1 knockout mice (cav-1(-/-)) consuming a high salt diet (4% NaCl) received Nω-nitro-l-arginine methyl ester (L-NAME) (0.1-0.2 mg/ml in drinking water at days 1-11) plus AngII (0.7-2.8 mg/kg per day via an osmotic minipump at days 8-11) ± MR antagonist eplerenone (EPL) 100 mg/kg per day in food. In both genotypes, blood pressure increased with L-NAME + AngII. EPL minimally changed blood pressure, although its dose was sufficient to block MR and reverse cardiac expression of the injury markers cluster of differentiation 68 and plasminogen activator inhibitor-1 in L-NAME+AngII treated mice. In aortic rings, phenylephrine and KCl contraction was enhanced with EPL in L-NAME+AngII treated WT mice, but not cav-1(-/-) mice. AngII-induced contraction was not different, and angiotensin type 1 receptor expression was reduced in L-NAME + AngII treated WT and cav-1(-/-) mice. In WT mice, acetylcholine-induced relaxation was enhanced with L-NAME + AngII treatment and reversed with EPL. Acetylcholine relaxation in cav-1(-/-) mice was greater than in WT mice, not modified by L-NAME + AngII or EPL, and blocked by ex vivo L-NAME, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), or endothelium removal, suggesting the role of NO-cGMP. Cardiac endothelial NO synthase was increased in cav-1(-/-) versus WT mice, further increased with L-NAME + AngII, and not affected by EPL. Vascular relaxation to the NO donor sodium nitroprusside was increased with L-NAME + AngII in WT mice but not in cav-1(-/-) mice. Plasma aldosterone levels increased and cardiac MR expression decreased in L-NAME + AngII treated WT and cav-1(-/-) mice and did not change with EPL. Thus, during L-NAME + AngII induced hypertension, MR blockade increases contraction and alters vascular relaxation via NO-cGMP, and these changes are absent in cav-1 deficiency states. The data suggest a cooperative role of MR and cav-1 in regulating vascular contraction and NO-cGMP-mediated relaxation during low NO-high AngII-dependent cardiovascular injury.

Cardiac macrophages and apoptosis after myocardial infarction: effects of central MR blockade.

After myocardial infarction (post-MI), inflammation and apoptosis contribute to progressive cardiac remodeling and dysfunction. Cardiac mineralocorticoid receptor (MR) and β-adrenergic signaling promote apoptosis and inflammation. Post-MI, MR activation in the brain contributes to sympathetic hyperactivity and an increase in cardiac aldosterone. In the present study, we assessed the time course of macrophage infiltration and apoptosis in the heart as detected by both terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and active caspase-3 immunostaining in both myocytes and nonmyocytes, as well as the effects of central MR blockade by intracerebroventricular infusion of eplerenone at 5 μg/day on peak changes in macrophage infiltration and apoptosis post-MI. Macrophage numbers were markedly increased in the infarct and peri-infarct zones and to a minor extent in the noninfarct part of the left ventricle at 10 days post-MI and decreased over the 3-mo study period. Apoptosis of both myocytes and nonmyocytes was clearly apparent in the infarct and peri-infarct areas at 10 days post-MI. For TUNEL, the increases persisted at 4 and 12 wk, but the number of active caspase-3-positive cells markedly decreased. Central MR blockade significantly decreased CD80-positive proinflammatory M1 macrophages and increased CD163-positive anti-inflammatory M2 macrophages in the infarct. Central MR blockade also reduced apoptosis of myocytes by 40-50% in the peri-infarct and to a lesser extent of nonmyocytes in the peri-infarct and infarct zones. These findings indicate that MR activation in the brain enhances apoptosis both in myocytes and nonmyocytes in the peri-infarct and infarct area post-MI and contributes to the inflammatory response.

Mineralocorticoid receptor and cardiac arrhythmia

Mineralocorticoid receptor (MR) activation has been shown to play a deleterious role in the development of heart disease in studies using specific MR antagonists (spironolactone, eplerenone) in both experimental models and patients. Pharmacological MR blockade attenuates the transition to heart failure (HF) in models of systolic left ventricular dysfunction and myocardial infarction, as well as diastolic dysfunction, in rats and mice. In humans, MR antagonism is highly beneficial in patients with mild or advanced HF and postinfarct HF. The consequences of aldosterone and MR activation for cardiac arrhythmia and its prevention and/or correction by MR antagonists are often underestimated. Activation of MR modulates cardiac electrical activity, causing atrial and ventricular arrhythmias. A pro-arrhythmogenic effect of aldosterone (possibly partly dependent on fibrosis) has been suggested by several studies. Cardiac MR activation has important consequences for the control of cellular calcium homeostasis, action potential lengthening, modulation of calcium transients and sarcoplasmic reticulum diastolic leaks, resulting in the promotion of rhythm disorders. Aldosterone and/or MR activation (in both cardiomyocytes and coronary vessels) result in vascular dysfunction and also contribute to pro-arrhythmogenic conditions. Together, the pro-arrhythmic effects of aldosterone and/or MR may explain the highly beneficial effect of MR antagonism, namely a decrease in the incidence of sudden death, observed in the Randomized Aldactone Evaluation Study (RALES) and Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (EPHESUS) studies.

Myocardial Mineralocorticoid Receptor Activation by Stretching and Its Functional Consequences

Myocardial stretch triggers an angiotensin II-dependent autocrine/paracrine loop of intracellular signals, leading to reactive oxygen species-mediated activation of redox-sensitive kinases. Based on pharmacological strategies, we previously proposed that mineralocorticoid receptor (MR) is necessary for this stretch-triggered mechanism. Now, we aimed to test the role of MR after stretch by using a molecular approach to avoid secondary effects of pharmacological MR blockers. Small hairpin interference RNA capable of specifically knocking down the MR was incorporated into a lentiviral vector (l-shMR) and injected into the left ventricular wall of Wistar rats. The same vector but expressing a nonsilencing sequence (scramble) was used as control. Lentivirus propagation through the left ventricle was evidenced by confocal microscopy. Myocardial MR expression, stretch-triggered activation of redox-sensitive kinases (ERK1/2-p90(RSK)), the consequent Na(+)/H(+) exchanger-mediated changes in pHi (HEPES-buffer), and its mechanical counterpart, the slow force response, were evaluated. Furthermore, reactive oxygen species production in response to a low concentration of angiotensin II (1.0 nmol/L) or an equipotent concentration of epidermal growth factor (0.1 μg/mL) was compared in myocardial tissue slices from both groups. Compared with scramble, animals transduced with l-shMR showed (1) reduced cardiac MR expression, (2) cancellation of angiotensin II-induced reactive oxygen species production but preservation of epidermal growth factor-induced reactive oxygen species production, (3) cancellation of stretch-triggered increase in ERK1/2-p90(RSK) phosphorylation, (4) lack of stretch-induced Na(+)/H(+) exchanger activation, and (5) abolishment of the slow force response. Our results provide strong evidence that MR activation occurs after myocardial stretch and is a key factor to promote redox-sensitive kinase activation and their downstream consequences.

Mineralocorticoid receptor antagonists: emerging roles in cardiovascular medicine

Spironolactone was first developed over 50 years ago as a potent mineralocorticoid receptor (MR) antagonist with undesirable side effects; it was followed a decade ago by eplerenone, which is less potent but much more MR-specific. From a marginal role as a potassium-sparing diuretic, spironolactone was shown to be an extraordinarily effective adjunctive agent in the treatment of progressive heart failure, as was eplerenone in subsequent heart failure trials. Neither acts as an aldosterone antagonist in the heart as the cardiac MR are occupied by cortisol, which becomes an aldosterone mimic in conditions of tissue damage. The accepted term “MR antagonist”, (as opposed to “aldosterone antagonist” or, worse, “aldosterone blocker”), should be retained, despite the demonstration that they act not to deny agonist access but as inverse agonists. The prevalence of primary aldosteronism is now recognized as accounting for about 10% of hypertension, with recent evidence suggesting that this figure may be considerably higher: in over two thirds of cases of primary aldosteronism therapy including MR antagonists is standard of care. MR antagonists are safe and vasoprotective in uncomplicated essential hypertension, even in diabetics, and at low doses they also specifically lower blood pressure in patients with so-called resistant hypertension. Nowhere are more than 1% of patients with primary aldosteronism ever diagnosed and specifically treated. Given the higher risk profile in patients with primary aldosteronism than that of age, sex, and blood pressure matched essential hypertension, on public health grounds alone the guidelines for first-line treatment of all hypertension should mandate inclusion of a low-dose MR antagonist.

Mineralocorticoid receptors and the heart, multiple cell types and multiple mechanisms: a focus on the cardiomyocyte

MR (mineralocorticoid receptor) activation in the heart plays a central role in the development of cardiovascular disease, including heart failure. The MR is present in many cell types within the myocardium, including cardiomyocytes, macrophages and the coronary vasculature. The specific role of the MR in each of these cell types in the initiation and progression of cardiac pathophysiology is not fully understood. Cardiomyocyte MRs are increasingly recognized to play a role in regulating cardiac function, electrical conduction and fibrosis, through direct signal mediation and through paracrine MR-dependent activity. Although MR blockade in the heart is an attractive therapeutic option for the treatment of heart failure and other forms of heart disease, current antagonists are limited by side effects owing to MR inactivation in other tissues (including renal targets). This has led to increased efforts to develop therapeutics that are more selective for cardiac MRs and which may have reduced the occurrence of side effects in non-cardiac tissues. A major clinical consideration in the treatment of cardiovascular disease is of the differences between males and females in the incidence and outcomes of cardiac events. There is clinical evidence that female sensitivity to endogenous MRs is more pronounced, and experimentally that MR-targeted interventions may be more efficacious in females. Given that sex differences have been described in MR signalling in a range of experimental settings and that the MR and oestrogen receptor pathways share some common signalling intermediates, it is becoming increasingly apparent that the mechanisms of MRs need to be evaluated in a sex-selective manner. Further research targeted to identify sex differences in cardiomyocyte MR activation and signalling processes has the potential to provide the basis for the development of cardiac-specific MR therapies that may also be sex-specific.