Endogenous DUX4 expression in FSHD myotubes is sufficient to cause cell death and disrupts RNA splicing and cell migration pathways.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by chromatin relaxation that results in aberrant expression of the transcription factor Double Homeobox 4 (DUX4). DUX4 protein is present in a small subset of FSHD muscle cells, making its detection and analysis of its effects historically difficult. Using a DUX4-activated reporter, we demonstrate the burst expression pattern of endogenous DUX4, its method of signal amplification in the unique shared cytoplasm of the myotube, and FSHD cell death that depends on its activation. Transcriptome analysis of DUX4-expressing cells revealed that DUX4 activation disrupts RNA metabolism including RNA splicing, surveillance and transport pathways. Cell signaling, polarity and migration pathways were also disrupted. Thus, DUX4 expression is sufficient for myocyte death, and these findings suggest mechanistic links between DUX4 expression and cell migration, supporting recent descriptions of phenotypic similarities between FSHD and an FSHD-like condition caused by FAT1 mutations.

Discovery of a highly potent, selective, orally bioavailable inhibitor of KAT6A/B histone acetyltransferases with efficacy against KAT6A-high ER+ breast cancer.

KAT6A, and its paralog KAT6B, are histone lysine acetyltransferases (HAT) that acetylate histone H3K23 and exert an oncogenic role in several tumor types including breast cancer where KAT6A is frequently amplified/overexpressed. However, pharmacologic targeting of KAT6A to achieve therapeutic benefit has been a challenge. Here we describe identification of a highly potent, selective, and orally bioavailable KAT6A/KAT6B inhibitor CTx-648 (PF-9363), derived from a benzisoxazole series, which demonstrates anti-tumor activity in correlation with H3K23Ac inhibition in KAT6A over-expressing breast cancer. Transcriptional and epigenetic profiling studies show reduced RNA Pol II binding and downregulation of genes involved in estrogen signaling, cell cycle, Myc and stem cell pathways associated with CTx-648 anti-tumor activity in ER-positive (ER+) breast cancer. CTx-648 treatment leads to potent tumor growth inhibition in ER+ breast cancer in vivo models, including models refractory to endocrine therapy, highlighting the potential for targeting KAT6A in ER+ breast cancer.

iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease modeling.

Skeletal muscle myoblasts (iMyoblasts) were generated from human induced pluripotent stem cells (iPSCs) using an efficient and reliable transgene-free induction and stem cell selection protocol. Immunofluorescence, flow cytometry, qPCR, digital RNA expression profiling, and scRNA-Seq studies identify iMyoblasts as a PAX3+/MYOD1+ skeletal myogenic lineage with a fetal-like transcriptome signature, distinct from adult muscle biopsy myoblasts (bMyoblasts) and iPSC-induced muscle progenitors. iMyoblasts can be stably propagated for >12 passages or 30 population doublings while retaining their dual commitment for myotube differentiation and regeneration of reserve cells. iMyoblasts also efficiently xenoengrafted into irradiated and injured mouse muscle where they undergo differentiation and fetal-adult MYH isoform switching, demonstrating their regulatory plasticity for adult muscle maturation in response to signals in the host muscle. Xenograft muscle retains PAX3+ muscle progenitors and can regenerate human muscle in response to secondary injury. As models of disease, iMyoblasts from individuals with Facioscapulohumeral Muscular Dystrophy revealed a previously unknown epigenetic regulatory mechanism controlling developmental expression of the pathological DUX4 gene. iMyoblasts from Limb-Girdle Muscular Dystrophy R7 and R9 and Walker Warburg Syndrome patients modeled their molecular disease pathologies and were responsive to small molecule and gene editing therapeutics. These findings establish the utility of iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease pathogenesis and for the development of muscle stem cell therapeutics.

Muscular dystrophies are a group of inherited genetic diseases characterised by progressive muscle weakness. They lead to disability or even death, and no cure exists against these conditions. Advances in genome sequencing have identified many mutations that underly muscular dystrophies, opening the door to new therapies that could repair incorrect genes or rebuild damaged muscles. However, testing these ideas requires better ways to recreate human muscular dystrophy in the laboratory. One strategy for modelling muscular dystrophy involves coaxing skin or other cells from an individual into becoming 'induced pluripotent stem cells'; these can then mature to form almost any adult cell in the body, including muscles. However, this approach does not usually create myoblasts, the 'precursor' cells that specifically mature into muscle during development. This limits investigations into how disease-causing mutations impact muscle formation early on. As a response, Guo et al. developed a two-step protocol of muscle maturation followed by stem cell growth selection to isolate and grow 'induced myoblasts' from induced pluripotent stem cells taken from healthy volunteers and muscular dystrophy patients. These induced myoblasts can both make more of themselves and become muscle, allowing Guo et al. to model three different types of muscular dystrophy. These myoblasts also behave as stem cells when grafted inside adult mouse muscles: some formed human muscle tissue while others remained as precursor cells, which could then respond to muscle injury and start repair. The induced myoblasts developed by Guo et al. will enable scientists to investigate the impacts of different mutations on muscle tissue and to better test treatments. They could also be used as part of regenerative medicine therapies, to restore muscle cells in patients.

A non-viral genome editing platform for site-specific insertion of large transgenes.

BACKGROUND: The precise, functional and safe insertion of large DNA payloads into host genomes offers versatility in downstream genetic engineering-associated applications, spanning cell and gene therapies, therapeutic protein production, high-throughput cell-based drug screening and reporter cell lines amongst others. Employing viral- and non-viral-based genome engineering tools to achieve specific insertion of large DNA-despite being successful in E. coli and animal models-still pose challenges in the human system. In this study, we demonstrate the applicability of our lambda integrase-based genome insertion tool for human cell and gene therapy applications that require insertions of large functional genes, as exemplified by the integration of a functional copy of the F8 gene and a Double Homeobox Protein 4 (DUX4)-based reporter cassette for potential hemophilia A gene therapy and facioscapulohumeral muscular dystrophy (FSHD)-based high-throughput drug screening purposes, respectively. Thus, we present a non-viral genome insertion tool for safe and functional delivery of large seamless DNA cargo into the human genome that can enable novel designer cell-based therapies. METHODS: Previously, we have demonstrated the utility of our phage λ-integrase platform to generate seamless vectors and subsequently achieve functional integration of large-sized DNA payloads at defined loci in the human genome. To further explore this tool for therapeutic applications, we used pluripotent human embryonic stem cells (hESCs) to integrate large seamless vectors comprising a 'gene of interest'. Clonal cell populations were screened for the correct integration events and further characterized by southern blotting, gene expression and protein activity assays. In the case of our hemophilia A-related study, clones were differentiated to confirm that the targeted locus is active after differentiation and actively express and secrete Factor VIII. RESULTS: The two independent approaches demonstrated specific and functional insertions of a full-length blood clotting F8 expression cassette of ~ 10 kb and of a DUX4 reporter cassette of ~ 7 kb in hESCs. CONCLUSION: We present a versatile tool for site-specific human genome engineering with large transgenes for cell/gene therapies and other synthetic biology and biomedical applications.

Retinoic acid receptor α as a novel contributor to adrenal cortex structure and function through interactions with Wnt and Vegfa signalling.

Primary aldosteronism (PA) is the most frequent form of secondary arterial hypertension. Mutations in different genes increase aldosterone production in PA, but additional mechanisms may contribute to increased cell proliferation and aldosterone producing adenoma (APA) development. We performed transcriptome analysis in APA and identified retinoic acid receptor alpha (RARα) signaling as a central molecular network involved in nodule formation. To understand how RARα modulates adrenal structure and function, we explored the adrenal phenotype of male and female Rarα knockout mice. Inactivation of Rarα in mice led to significant structural disorganization of the adrenal cortex in both sexes, with increased adrenal cortex size in female mice and increased cell proliferation in males. Abnormalities of vessel architecture and extracellular matrix were due to decreased Vegfa expression and modifications in extracellular matrix components. On the molecular level, Rarα inactivation leads to inhibition of non-canonical Wnt signaling, without affecting the canonical Wnt pathway nor PKA signaling. Our study suggests that Rarα contributes to the maintenance of normal adrenal cortex structure and cell proliferation, by modulating Wnt signaling. Dysregulation of this interaction may contribute to abnormal cell proliferation, creating a propitious environment for the emergence of specific driver mutations in PA.

Does glucocorticoid receptor blockade exacerbate tissue damage after mineralocorticoid/salt administration?

Mineralocorticoid receptor (MR) antagonism reverses established inflammation, oxidative stress, and cardiac fibrosis in the mineralocorticoid/salt-treated rat, whereas withdrawal of the mineralocorticoid deoxycorticosterone (DOC) alone does not. Glucocorticoid receptors (GRs) play a central role in regulating inflammatory responses but are also involved in cardiovascular homeostasis. Physiological glucocorticoids bind MR with high affinity, equivalent to that for aldosterone, but are normally prevented from activating MR by pre-receptor metabolism by 11beta-hydroxysteroid dehydrogenase 2. We have previously shown a continuing fibrotic and hypertrophic effect after DOC withdrawal, putatively mediated by activation of glucocorticoid/MR complexes; the present study investigates whether this effect is moderated by antiinflammatory effects mediated via GR. Uninephrectomized rats, drinking 0.9% saline solution, were treated as follows: control; DOC (20 mg/wk) for 4 wk; DOC for 4 wk and no steroid wk 5-8; DOC for 4 wk plus the MR antagonist eplerenone (50 mg/kg.d) wk 5-8; DOC for 4 wk plus the GR antagonist RU486 (2 mg/d) wk 5-8; and DOC for 4 wk plus RU486 and eplerenone for wk 5-8. After steroid withdrawal, mineralocorticoid/salt-induced cardiac hypertrophy is sustained, but not hypertension. Inflammation and fibrosis persist after DOC withdrawal, and GR blockade with RU486 has no effect on these responses. Rats receiving RU486 for wk 5-8 after DOC withdrawal showed marginal blood pressure elevation, whereas eplerenone alone or coadministered with RU486 reversed all DOC/salt-induced circulatory and cardiac pathology. Thus, sustained responses after mineralocorticoid withdrawal appear to be independent of GR signaling, in that blockade of endogenous antiinflammatory effects via GR does not lead to an increase in the severity of responses in the mineralocorticoid/salt-treated rat after steroid withdrawal.

Mineralocorticoid receptor activation and cardiac fibrosis.

MR (mineralocorticoid receptor) activation by either administration of exogenous mineralocorticoids or by allowing endogenous glucocorticoids to activate the MR has been shown to produce oxidative stress and vascular inflammation at the earliest stages of the development of cardiac fibrosis in experimental animals. These studies suggest potential mechanisms for the benefits observed in recent large scale clinical trials investigating the cardioprotective effects of MR antagonists given in conjunction with current best practice therapy for moderate-to-severe heart failure and heart failure post-myocardial infarction. Given that few patients had elevated plasma aldosterone, novel mechanisms involved in activating the MR in the failing heart are now being investigated.

Deoxycorticosterone/Salt-Mediated Cardiac Inflammation and Fibrosis Are Dependent on Functional CLOCK Signaling in Male Mice.

Activation of the mineralocorticoid receptor (MR) promotes inflammation, fibrosis, and hypertension. Clinical and experimental studies show that MR antagonists have significant therapeutic benefit for all-cause heart failure; however, blockade of renal MRs limits their widespread use. Identification of key downstream signaling mechanisms for the MR in the cardiovascular system may enable development of targeted MR antagonists with selectivity for pathological MR signaling and lower impact on physiological renal electrolyte handling. One candidate pathway is the circadian clock, the dysregulation of which is associated with cardiovascular diseases. We have previously shown that the circadian gene Per2 is dysregulated in hearts with selective deletion of cardiomyocyte MR. We therefore investigated MR-mediated cardiac inflammation and fibrosis in mice that lack normal regulation and oscillation of the circadian clock in peripheral tissues, that is, CLOCKΔ19 mutant mice. The characteristic cardiac inflammatory/fibrotic response to a deoxycorticosterone (DOC)/salt for 8 weeks was significantly blunted in CLOCKΔ19 mice when compared with wild-type mice, despite a modest increase at "baseline" for fibrosis and macrophage number in CLOCKΔ19 mice. In contrast, cardiac hypertrophy in response to DOC/salt was significantly greater in CLOCKΔ19 vs wild-type mice. Markers for renal inflammation and fibrosis were similarly attenuated in the CLOCKΔ19 mice given DOC/salt. Moreover, increased CLOCK expression in H9c2 cardiac cells enhanced MR-mediated transactivation of Per1, suggesting cooperative signaling between these transcription factors. This study demonstrates that the full development of MR-mediated cardiac inflammation and fibrosis is dependent on intact signaling by the circadian protein CLOCK.

The role of the glucocorticoid receptor in mineralocorticoid/salt-mediated cardiac fibrosis.

The pathophysiological consequences of excess mineralocorticoid for salt status include hypertension, vascular inflammation, and cardiac fibrosis. Mineralocorticoid receptor (MR) blockade can both prevent and reverse established inflammation and fibrosis due to exogenous mineralocorticoids or endogenous glucocorticoid activation of the MR. Glucocorticoids also exert potent antiinflammatory effects via glucocorticoid receptors (GR) in the vascular wall. We propose that GR signaling may ameliorate mineralocorticoid/salt-induced vascular inflammation and fibrosis in the mineralocorticoid/salt model. In the present study, the role of GR in the mineralocorticoid/salt model was explored in uninephrectomized rats that were maintained on 0.9% saline solution to drink and treated as follows: control (CON), no further treatment; deoxycorticosterone (DOC; 20 mg/wk) for 4 wk (DOC4); DOC for 8 wk (DOC8); DOC for 8 wk plus the GR antagonist RU486 (2 mg/d) wk 5-8 (DOC8/RU486); and DOC for 8 wk plus RU486 and the MR antagonist eplerenone (EPL; 50 mg/kg.d) for wk 5-8 (DOC8/RU486+EPL). DOC treatment significantly increased systolic blood pressure, cardiac fibrosis, inflammation (ED-1-positive macrophages and osteopontin), and mRNA for markers of oxidative stress (p22phox, gp91phox, and NAD(P)H-4). GR blockade reduced the DOC-mediated increase in systolic blood pressure and the number of infiltrating ED-1-positive macrophages but had no effect on fibrosis, oxidative stress, or osteopontin mRNA levels. EPL reversed DOC-induced pathology in the absence or presence of GR blockade. Thus, blocking agonist activity at the GR neither enhances nor attenuates the fibrotic response, although it may modulate systolic blood pressure and macrophage recruitment in the mineralocorticoid/salt model.

Cardiac Tissue Injury and Remodeling Is Dependent Upon MR Regulation of Activation Pathways in Cardiac Tissue Macrophages.

Macrophage mineralocorticoid receptor (MR) signaling is an important mediator of cardiac tissue inflammation and fibrosis. The goal of the present study was to determine the cellular mechanisms of MR signaling in macrophages that promote cardiac tissue injury and remodeling. We sought to identify specific markers of MR signaling in isolated tissue macrophages (cardiac, aortic) vs splenic mononuclear cells from wild-type and myeloid MR-null mice given vehicle/salt or deoxycorticosterone (DOC)/salt for 8 weeks. Cardiac tissue fibrosis in response to 8 weeks of DOC/salt treatment was found in the hearts from wild-type but not myeloid MR-null mice. This was associated with an increased expression of the profibrotic markers TGF-ß1 and matrix metalloproteinase-12 and type 1 inflammatory markers TNFα and chemokine (C-X-C motif) ligand-9 in cardiac macrophages. Differential expression of immunomodulatory M2-like markers (eg, arginase-1, macrophage scavenger receptor 1) was dependent on the tissue location of wild-type and MR-null macrophages. Finally, intact MR signaling is required for the phosphorylation of c-Jun NH2-terminal kinase in response to a proinflammatory stimulus in bone marrow monocytes/macrophages in culture. These data suggest that the activation of the c-Jun NH2-terminal kinase pathway in macrophages after a tissue injury and inflammatory stimuli in the DOC/salt model is MR dependent and regulates the transcription of downstream profibrotic factors, which may represent potential therapeutic targets in heart failure patients.

A cross sectional study of two independent cohorts identifies serum biomarkers for facioscapulohumeral muscular dystrophy (FSHD).

Measuring the severity and progression of facioscapulohumeral muscular dystrophy (FSHD) is particularly challenging because muscle weakness progresses over long periods of time and can be sporadic. Biomarkers are essential for measuring disease burden and testing treatment strategies. We utilized the sensitive, specific, high-throughput SomaLogic proteomics platform of 1129 proteins to identify proteins with levels that correlate with FSHD severity in a cross-sectional study of two independent cohorts. We discovered biomarkers that correlate with clinical severity and disease burden measured by magnetic resonance imaging. Sixty-eight proteins in the Rochester cohort (n = 48) and 51 proteins in the Seattle cohort (n = 30) had significantly different levels in FSHD-affected individuals when compared with controls (p-value ≤ .005). A subset of these varied by at least 1.5 fold and four biomarkers were significantly elevated in both cohorts. Levels of creatine kinase MM and MB isoforms, carbonic anhydrase III, and troponin I type 2 reliably predicted the disease state and correlated with disease severity. Other novel biomarkers were also discovered that may reveal mechanisms of disease pathology. Assessing the levels of these biomarkers during clinical trials may add significance to other measures of quantifying disease progression or regression.

Mineralocorticoid receptors in the heart: lessons from cell-selective transgenic animals.

The clinical impact of cardiovascular disease cannot be underestimated. Equally, the importance of cost-effective management of cardiac failure is a pressing issue in the face of an ageing population and the increasing incidence of metabolic disorders worldwide. Targeting the mineralocorticoid receptor (MR) offers one approach for the treatment of heart failure with current strategies for novel MR therapeutics focusing on harnessing their cardio-protective benefits, but limiting the side effects of existing agents. It is now well accepted that activation of the MR in the cardiovascular system promotes tissue inflammation and fibrosis and has negative consequences for cardiac function and patient outcomes following cardiac events. Indeed, blockade of the MR using one of the two available antagonists (spironolactone and eplerenone) provides significant cardio-protective effects in the clinical and experimental setting. Although the pathways downstream of MR that translate receptor activation into tissue inflammation, fibrosis and dysfunction are still being elucidated, a series of recent studies using cell-selective MR (NR3C2)-null or MR-overexpressing mice have offered many new insights into the role of MR in cardiovascular disease and the control of blood pressure. Dissecting the cell-specific roles of MR signalling in the heart and vasculature to identify those pathways that are critical for MR-dependent responses is an important step towards achieving cardiac-selective therapeutics. The goal of this review is to discuss recent advances in this area that have emerged from the study of tissue-selective MR-null mice, and other targeted transgenic models and their relevance to clinical disease.

Cardiomyocyte Mineralocorticoid Receptor Activation Impairs Acute Cardiac Functional Recovery After Ischemic Insult.

Loss of mineralocorticoid receptor signaling selectively in cardiomyocytes can ameliorate cardiac fibrotic and inflammatory responses caused by excess mineralocorticoids. The aim of this study was to characterize the role of cardiomyocyte mineralocorticoid receptor signaling in ischemia-reperfusion injury and recovery and to identify a role of mineralocorticoid receptor modulation of cardiac function. Wild-type and cardiomyocyte mineralocorticoid receptor knockout mice (8 weeks) were uninephrectomized and maintained on (1) high salt (0.9% NaCl, 0.4% KCl) or (2) high salt plus deoxycorticosterone pellet (0.3 mg/d, 0.9% NaCl, 0.4% KCl). After 8 weeks of treatment, hearts were isolated and subjected to 20 minutes of global ischemia plus 45 minutes of reperfusion. Mineralocorticoid excess increased peak contracture during ischemia regardless of genotype. Recovery of left ventricular developed pressure and rates of contraction and relaxation post ischemia-reperfusion were greater in knockout versus wild-type hearts. The incidence of arrhythmic activity during early reperfusion was significantly higher in wild-type than in knockout hearts. Levels of autophosphorylated Ca(2+)/calmodulin protein kinase II (Thr287) were elevated in hearts from wild-type versus knockout mice and associated with increased sodium hydrogen exchanger-1 expression. These findings demonstrate that cardiomyocyte-specific mineralocorticoid receptor-dependent signaling contributes to electromechanical vulnerability in acute ischemia-reperfusion via a mechanism involving Ca(2+)/calmodulin protein kinase II activation in association with upstream alteration in expression regulation of the sodium hydrogen exchanger-1.

Endothelial cell mineralocorticoid receptors regulate deoxycorticosterone/salt-mediated cardiac remodeling and vascular reactivity but not blood pressure.

Recent studies have identified novel pathological roles for mineralocorticoid receptors (MR) in specific cell types in cardiovascular disease. The mechanisms by which MR promotes inflammation and fibrosis involve multiple cell-specific events. To identify the role of MR in endothelial cells (EC-MR), the current study explored the vascular responses to aldosterone in wild-type (WT) and EC-null mice (EC-MRKO). Nitric oxide function was impaired in the thoracic aorta and mesenteric arteries of aldosterone-treated WT mice. Although endothelial nitric oxide function was equivalently impaired in the mesenteric arteries of aldosterone-treated EC-MRKO mice, endothelial function was unaffected in the aorta, suggesting a differential role for EC-MR depending on the vascular bed. Second, the contribution of EC-MR to cardiovascular inflammation, fibrosis, and hypertension was determined in WT and EC-MRKO treated with deoxycorticosterone/salt for 8 days or 8 weeks. At 8 days, loss of EC-MR prevented macrophage infiltration and the expression of proinflammatory genes in the myocardium. Increased cardiac fibrosis was not detected in either genotype at this time, mRNA levels of profibrotic genes were significantly lower in EC-MRKO mice versus WT. At 8 weeks, deoxycorticosterone/salt treatment increased macrophage recruitment and proinflammatory gene expression in WT but not in EC-MRKO. Collagen deposition and connective tissue growth factor expression were significantly reduced in EC-MRKO versus WT. Interestingly, systolic blood pressure was equivalently elevated in deoxycorticosterone/salt treated WT and EC-MRKO. Our data demonstrate that (1) EC-MR signaling contributes to vascular nitric oxide function in large conduit arteries but not in resistance vessels and (2) an independent role for EC-MR in the inflammatory and profibrotic response to deoxycorticosterone/salt.

Genetics of mineralocorticoid excess: an update for clinicians.

Aldosterone plays a major role in the regulation of sodium and potassium homeostasis and blood pressure. More recently, aldosterone has emerged as a key hormone mediating end organ damage. In extreme cases, dysregulated aldosterone production leads to primary aldosteronism (PA), the most common form of secondary hypertension. However, even within the physiological range, high levels of aldosterone are associated with an increased risk of developing hypertension over time. PA represents the most common and curable form of hypertension, with a prevalence that increases with the severity of hypertension. Although genetic causes underlying glucocorticoid-remediable aldosteronism, one of the three Mendelian forms of PA, were established some time ago, somatic and inherited mutations in the potassium channel GIRK4 have only recently been implicated in the formation of aldosterone-producing adenoma (APA) and in familial hyperaldosteronism type 3. Moreover, recent findings have shown somatic mutations in two additional genes, involved in maintaining intracellular ionic homeostasis and cell membrane potential, in a subset of APAs. This review summarizes our current knowledge on the genetic determinants that contribute to variations in plasma aldosterone and renin levels in the general population and the genetics of familial and sporadic PA. Various animal models that have significantly improved our understanding of the pathophysiology of excess aldosterone production are also discussed. Finally, we outline the cardiovascular, renal, and metabolic consequences of mineralocorticoid excess beyond blood pressure regulation.

KCNJ5 mutations in aldosterone producing adenoma and relationship with adrenal cortex remodeling.

Somatic mutations of KCNJ5, coding for the potassium channel GIRK4, have recently been implicated in the formation of aldosterone producing adenoma (APA). While a causal link between KCNJ5 mutations, membrane depolarization and aldosterone production has been established, the precise mechanism by which these mutations promote cell proliferation and APA formation remains unclear. The aim of our study was to correlate KCNJ5 mutation status with morphological and functional characteristics of the adrenal cortex adjacent to APA. While GIRK4 was expressed in APA and in the zona glomerulosa of the adjacent cortex, significantly lower levels were detected in APA harboring a KCNJ5 mutation. There was no correlation between KCNJ5 mutation status and the morphological measures of adrenal cortex remodeling, including nodulation, vascularization and expression of CYP11B2. The cell composition of APA was not significantly different between groups. These results indicate that KCNJ5 mutations are not correlated with adrenal cortex remodeling in APA.

Mechanisms of mineralocorticoid salt-induced hypertension and cardiac fibrosis.

For 50 years aldosterone has been thought to act primarily on epithelia to regulate fluid and electrolyte homeostasis. Mineralocorticoid receptors (MR), however, are also expressed in nonepithelial tissues such as the heart and vascular smooth muscle. Recently pathophysiologic effects of nonepithelial MR activation by aldosterone have been demonstrated, in the context of inappropriate mineralocorticoid for salt status, including coronary vascular inflammation and cardiac fibrosis. Consistent with experimental studies, clinical trials (RALES, EPHESUS), have demonstrated a reduced mortality and morbidity when MR antagonists are included in the treatment of moderate-severe heart failure. The pathogenesis of MR-mediated cardiovascular disease is a complex, multifactorial process that involves loss of vascular reactivity, hypertension, inflammation of the vasculature and end organs (heart and kidney), oxidative stress and tissue fibrosis (cardiac and renal). This review will discuss the mechanisms by which MR, located in the various cell types that comprise the heart, plays a central role in the development of cardiomyocyte failure, tissue inflammation, remodelling and hypertension.

Cardiomyocyte mineralocorticoid receptors are essential for deoxycorticosterone/salt-mediated inflammation and cardiac fibrosis.

Because the role of mineralocorticoid receptors in specific cell types in cardiac remodeling remains unknown, we have compared cardiac responses with deoxycorticosterone/salt in cardiomyocyte mineralocorticoid receptor-null (MyoMRKO) and wild-type (WT) mice at 8 days and 8 weeks. No differences in cardiac function between untreated WT and MyoMRKO mice were found, whereas profibrotic markers were reduced in MyoMRKO hearts at baseline. At 8 days, MyoMRKO showed monocyte/macrophage recruitment equivalent to WT mice in response to deoxycorticosterone/salt but a suppression of markers of fibrosis compared with WT. At 8 weeks, MyoMRKO mice showed no deoxycorticosterone/salt-induced increase in inflammatory cell infiltration and collagen deposition or in proinflammatory gene expression. Although some profibrotic markers were equivalently increased in both genotypes, MyoMRKO mice also showed increased baseline levels of mRNA and protein for the transforming growth factor-ß/connective tissue growth factor inhibitor decorin compared with WT that was accompanied by higher levels of matrix metalloproteinase 2/matrix metalloproteinase 9 activity. These data point to a direct role for cardiomyocyte mineralocorticoid receptor in both deoxycorticosterone/salt-induced tissue inflammation and remodeling and suggest potential mechanisms for the cardioprotective effects of selective mineralocorticoid receptor blockade in cardiomyocytes that may involve regulation of matrix metalloproteinase 2/matrix metalloproteinase 9 activity and the transforming growth factor-ß-connective tissue growth factor profibrotic pathway.

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.

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.