Morag J. Young

Mechanisms of Mineralocorticoid Action

Sodium transport in epithelial tissues is regulated by the physiological mineralocorticoid aldosterone. The response to aldosterone is mediated by the mineralocorticoid receptor (MR), for which the crystal structure of the ligand-binding domain has recently been established. The classical mode of action for this receptor involves the regulation of gene transcription. Several genes have now been shown to be regulated by aldosterone in epithelial tissues. Of these, the best characterized is serum- and glucocorticoid-regulated kinase, which increases sodium influx through the epithelial sodium channel. Turnover of these channels in the cell membrane is mediated by Nedd4–2, a ubiquitin protein ligase; serum- and glucocorticoid-regulated kinase interacts with and phosphorylates Nedd4–2, thereby rendering it unable to bind the sodium channels. In nonepithelial tissues, particularly the cardiovascular system, aldosterone also has direct effects, activating an inflammatory cascade, leading to cardiac fibrosis. A critical role for the MR in cardiovascular disease has now been demonstrated by the beneficial response to MR blockade in 2 large clinical trials in patients with cardiac failure. It is these nonepithelial actions of MR activation that need to be exploited for the development of antagonists that target the cardiovascular system while avoiding the undesirable side effects of renal MR blockade.

Deletion of Mineralocorticoid Receptors From Macrophages Protects Against Deoxycorticosterone/Salt-Induced Cardiac Fibrosis and Increased Blood Pressure

Increased mineralocorticoid levels plus high salt promote vascular inflammation and cardiac tissue remodeling. Mineralocorticoid receptors are expressed in many cell types of the cardiovascular system, including monocytes/macrophages and other inflammatory cell types. Although mineralocorticoid receptors are expressed in monocytes/macrophages, their role in regulating macrophage function to date has not been investigated. We, thus, used the Cre/LoxP-recombination system to selectively delete mineralocorticoid receptors from monocytes/macrophages with the lysozyme M promoter used to drive Cre expression (MRflox/flox/LysMCre/− mice). Male mice from each genotype (MRflox/flox or wild-type and MRflox/flox/LysMCre/− mice) were uninephrectomized, given 0.9% NaCl solution to drink, and treated for 8 days or 8 weeks with either vehicle (n=10) or deoxycorticosterone (n=10). Equivalent tissue macrophage numbers were seen for deoxycorticosterone treatment of each genotype at 8 days; in contrast, plasminogen activator inhibitor type 1 and NAD(P)H oxidase subunit 2 levels were increased in wild-type but not in MRflox/flox/LysMCre/− mice given deoxycorticosterone. Baseline expression of other inflammatory genes was reduced in MRflox/flox/LysMCre/− mice compared with wild-type mice. At 8 weeks, deoxycorticosterone-induced macrophage recruitment and connective tissue growth factor and plasminogen activator inhibitor type 1 mRNA levels were similar for each genotype; in contrast, MRflox/flox/LysMCre/− mice showed no increase in cardiac fibrosis or blood pressure, as was seen in wild-type mice at 8 weeks. These data demonstrate the following points: (1) mineralocorticoid receptor signaling regulates basal monocyte/macrophage function; (2) macrophage recruitment is not altered by loss of mineralocorticoid receptor signaling in these cells; and (3) a novel and significant role is seen for macrophage signaling in the regulation of cardiac remodeling and systolic blood pressure in the deoxycorticosterone/salt model.

Myocardial autophagy activation and suppressed survival signaling is associated with insulin resistance in fructose-fed mice.

Fructose intake is linked with the increasing prevalence of insulin resistance and there is now evidence for a specific insulin-resistant cardiomyopathy. The aim of this study was to determine the cardiac-specific myocardial remodeling effects of high fructose dietary intake. Given the links between insulin signaling, reactive oxygen species generation and autophagy induction, we hypothesized that autophagy contributes to pathologic remodeling in the insulin-resistant heart, and in particular may be a feature of high fructose diet-induced cardiac phenotype. Male C57Bl/6 mice were fed a high fructose (60%) diet or nutrient-matched control diet for 12 weeks. Systemic and myocardial insulin-resistant status was characterized. Superoxide production (lucigenin) and cellular growth and death signaling pathways were examined in myocardial tissue. Myocardial structural remodeling was evaluated by measurement of heart weight indices and histological analysis of collagen deposition (picrosirius red). Fructose-fed mice exhibited hyperglycemia and glucose intolerance, but plasma insulin and blood pressure were unchanged. High fructose intake suppressed the myocardial Akt cell survival signaling coincident with increased cardiac superoxide generation (21% increase, p<0.05). Fructose feeding induced elevated autophagy (LC3B-II: LC3B-I ratio: 46% increase, p<0.05) but not apoptosis signaling (unchanged Bax-1:Bcl-2 ratio). Despite a 28% increase in interstitial fibrosis, no difference in heart weight was observed in fructose-fed mice. We provide the first evidence that myocardial autophagy activation is associated with systemic insulin resistance, and that high level fructose intake inflicts direct cardiac damage. Upregulated autophagy is associated with elevated cardiac superoxide production, suppressed cell survival signaling and fibrotic infiltration in fructose-fed mice. The novel finding that autophagy contributes to cardiac pathology in insulin resistance identifies a new therapeutic target for diabetic cardiomyopathy.

Mechanisms of mineralocorticoid receptor-mediated cardiac fibrosis and vascular inflammation

Purpose of reviewThis review addresses the growing area of cardiovascular mineralocorticoid responses and highlights recent work investigating the underlying mechanisms that regulate mineralocorticoid receptor activation and translate mineralocorticoid receptor signaling into cardiac inflammation and fibrosis. Recent findingsMineralocorticoid receptor activation has been shown to regulate numerous pathways, including the plasminogen activation system and angiotensin II signaling pathways, which encompass both genomic and nongenomic responses. Mineralocorticoid receptor activation has been demonstrated to result in increased tissue oxidative stress and vascular inflammation, while recent studies provide evidence for changes in tissue oxidative stress, in turn regulating mineralocorticoid receptor activation by alternate ligands. These studies suggest possible mechanisms for the protection afforded by mineralocorticoid receptor blockade in the RALES and EPHESUS trials where plasma aldosterone levels were low or normal. SummaryExperimental models of cardiac fibrosis and clinical observations have established mineralocorticoid receptor activation as a key player in the initiation and progression of cardiovascular disease. Studies over the last 12 months address the mechanisms underlying mineralocorticoid receptor-mediated vascular inflammation and cardiac fibrosis and focus on oxidative stress, inflammation and early tissue remodeling, and describe an increasing range of tissue signaling pathways and novel mechanisms of mineralocorticoid receptor activation that contribute to the pathology of cardiac fibrosis.

Aldosterone and the Heart

Classically, aldosterone is a steroid hormone secreted from the adrenal cortex, which acts on kidney, colon and sweat/salivary glands to promote unidirectional sodium transport. Currently, there is excellent experimental evidence for aldosterone acting directly on the central nervous system to raise blood pressure, and on the heart to cause cardiac hypertrophy and fibrosis. In addition, there is emerging evidence for aldosterone synthesis in the heart, and for as yet unexplained benefits of aldosterone antagonism in the treatment of cardiac failure.

Corticosteroid receptors, macrophages and cardiovascular disease

The mineralocorticoid receptor (MR) and glucocorticoid receptor are ligand-activated transcription factors that have important physiological and pathophysiological actions in a broad range of cell types including monocytes and macrophages. While the glucocorticoids cortisol and corticosterone have well-described anti-inflammatory actions on both recruited and tissue resident macrophages, a role for the mineralocorticoid aldosterone in these cells is largely undefined. Emerging evidence, however, suggests that MR signalling may promote pro-inflammatory effects. This review will discuss the current understanding of the role of corticosteroid receptors in macrophages and their effect on diseases involving inflammation, with a particular focus on cardiovascular disease.

Mineralocorticoid receptors and pathophysiological roles for aldosterone in the cardiovascular system.

For almost 40 years since its discovery in 1953, the mineralocorticoid hormone, aldosterone, was considered to affect blood volume, and thus blood pressure, by its action to retain sodium at epithelial tissues. Over the past decade, direct effects of aldosterone on the heart and blood vessels, and on the cerebral control of blood pressure, have been established in experimental animals. Simultaneously, the incidence of primary aldosteronism in essential hypertension is now acknowledged to be 10-20%, rather than <or= 1%, underscoring a previously unrecognized role for aldosterone in hypertension. The 30% improvement in mortality (and 35% in morbidity) seen in the RALES trial with the addition of low-dose spironolactone to best practice therapy in moderate to severe heart failure, similarly points to an unrecognized role for aldosterone in the pathophysiology of heart failure. Currently, both experimental and clinical studies are directed towards establishing the mechanisms involved in these pathophysiological effects of aldosterone in the cardiovascular system, and of the role of mineralocorticoid receptor antagonists in offsetting or blocking such effects. A brief account of the current state of these mechanisms in at a cellular and tissue level forms the basis of this review.

The mineralocorticoid receptor and its coregulators.

The mineralocorticoid receptor (MR) is a member of the nuclear receptor superfamily and is essential for controlling sodium transport in epithelial tissues such as the kidney and colon. Moreover, it is also present in other non-epithelial tissues and is capable of activation by both mineralocorticoids and glucocorticoids. A challenge in understanding transcriptional regulation by the MR and other nuclear receptors is to determine how tissue- and ligand-specificity is achieved. Over the past decade, it has become clear that a heterogeneous group of non-receptor proteins termed as coregulators are required to either enhance or repress nuclear receptor-mediated transactivation of target genes. A subset of these coregulators may be expected to confer specificity to MR-mediated responses by virtue of their variable tissue expression and selectivity for different ligands. Specific coregulator-MR interactions may be a suitable target in the rational design of tissue-specific MR modulators as has been described for other steroid receptors. However, the number of coregulators identified to date for the MR is very limited compared with other nuclear receptors. Understanding the full complement of MR coregulators is essential for unraveling the complexity of MR signaling pathways and will facilitate the development of selective MR modulators.

Macrophage Mineralocorticoid Receptor Signaling Plays a Key Role in Aldosterone-Independent Cardiac Fibrosis

Mineralocorticoid receptor (MR) activation promotes the development of cardiac fibrosis and heart failure. Clinical evidence demonstrates that MR antagonism is protective even when plasma aldosterone levels are not increased. We hypothesize that MR activation in macrophages drives the profibrotic phenotype in the heart even when aldosterone levels are not elevated. The aim of the present study was to establish the role of macrophage MR signaling in mediating cardiac tissue remodeling caused by nitric oxide (NO) deficiency, a mineralocorticoid-independent insult. Male wild-type (MRflox/flox) and macrophage MR-knockout (MRflox/flox/LysMCre/+; mac-MRKO) mice were uninephrectomized, maintained on 0.9% NaCl drinking solution, with either vehicle (control) or the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (L-NAME; 150 mg/kg/d) for 8 wk. NO deficiency increased systolic blood pressure at 4 wk in wild-type L-NAME/salt-treated mice compared with all other groups. At 8 wk, systolic blood pressure was increased above control in both L-NAME/salt treated wild-type and mac-MRKO mice by approximately 28 mm Hg by L-NAME/salt. Recruitment of macrophages was increased 2- to 3-fold in both L-NAME/salt treated wild-type and mac-MRKO. Inducible NOS positive macrophage infiltration and TNFα mRNA expression was greater in wild-type L-NAME/salt-treated mice compared with mac-MRKO, demonstrating that loss of MR reduces M1 phenotype. mRNA levels for markers of vascular inflammation and oxidative stress (NADPH oxidase 2, p22phox, intercellular adhesion molecule-1, G protein-coupled chemokine receptor 5) were similar in treated wild-type and mac-MRKO mice compared with control groups. In contrast, L-NAME/salt treatment increased interstitial collagen deposition in wild-type by about 33% but not in mac-MRKO mice. mRNA levels for connective tissue growth factor and collagen III were also increased above control treatment in wild-type (1.931 ± 0.215 vs. 1 ± 0.073) but not mac-MRKO mice (1.403 ± 0.150 vs. 1.286 ± 0.255). These data demonstrate that macrophage MR are necessary for the translation of inflammation and oxidative stress into interstitial and perivascular fibrosis after NO deficiency, even when plasma aldosterone is not elevated.

Mineralocorticoid receptor antagonism induces browning of white adipose tissue through impairment of autophagy and prevents adipocyte dysfunction in high-fat-diet-fed mice

The mineralocorticoid receptor (MR) controls adipocyte function, but its role in the conversion of white adipose tissue (WAT) into thermogenic fat has not been elucidated. We investigated responses to the MR antagonists spironolactone (spiro; 20 mg/kg/d) and drospirenone (DRSP; 6 mg/kg/d) in C57BL/6 mice fed a high‐fat (HF) diet for 90 d. DRSP and spiro curbed HF diet‐induced impairment in glucose tolerance, and prevented body weight gain and white fat expansion. Notably, either MR antagonist induced up‐regulation of brown adipocyte‐specific transcripts and markedly increased protein levels of uncoupling protein 1 (UCP1) in visceral and inguinal fat depots when compared with the HF diet group. Positron emission tomography and magnetic resonance spectroscopy confirmed acquisition of brown fat features in WAT. Interestingly, MR antagonists markedly reduced the autophagic rate both in murine preadipocytes in vitro (10‐5 M) and in WAT depots in vivo, with a concomitant increase in UCP1 protein expression. Moreover, the autophagy repressor bafilomycin A1 (10‐8 M) mimicked the effect of MR antagonists, increasing UCP1 protein expression in primary preadipocytes. Hence, we showed that adipocyte MR regulates brown remodeling of WAT through a modulation of autophagy. These results provide a rationale for the use of MR antagonists to prevent the adverse metabolic consequences of adipocyte dysfunction.—Armani, A., Cinti, F., Marzolla, V., Morgan, J., Cranston, G. A., Antelmi, A., Carpinelli, G., Canese, R., Pagotto, U., Quarta, C., Malorni, W., Matarrese, P., Marconi, M., Fabbri, A., Rosano, G., Cinti, S., Young, M. J., Caprio, M. Mineralocorticoid receptor antagonism induces browning of white adipose tissue through impairment of autophagy and prevents adipocyte dysfunction in high‐fat‐diet‐fed mice. FASEB J. 28, 3745–3757 (2014). www.fasebj.org