Macromolecular Complex Including MLL3, Carabin and Calcineurin Regulates Cardiac Remodeling.

BACKGROUND: Cardiac hypertrophy is an intermediate stage in the development of heart failure. The structural and functional processes occurring in cardiac hypertrophy include extensive gene reprogramming, which is dependent on epigenetic regulation and chromatin remodeling. However, the chromatin remodelers and their regulatory functions involved in the pathogenesis of cardiac hypertrophy are not well characterized. METHODS: Protein interaction was determined by immunoprecipitation assay in primary cardiomyocytes and mouse cardiac samples subjected or not to transverse aortic constriction for 1 week. Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) experiments were performed on the chromatin of adult mouse cardiomyocytes. RESULTS: We report that the calcium-activated protein phosphatase CaN (calcineurin), its endogenous inhibitory protein carabin, the STK24 (STE20-like protein kinase 3), and the histone monomethyltransferase, MLL3 (mixed lineage leukemia 3) form altogether a macromolecular complex at the chromatin of cardiomyocytes. Under basal conditions, carabin prevents CaN activation while the serine/threonine kinase STK24 maintains MLL3 inactive via phosphorylation. After 1 week of transverse aortic constriction, both carabin and STK24 are released from the CaN-MLL3 complex leading to the activation of CaN, dephosphorylation of MLL3, and in turn, histone H3 lysine 4 monomethylation. Selective cardiac MLL3 knockdown mitigates hypertrophy, and chromatin immunoprecipitation and DNA sequencing analysis demonstrates that MLL3 is de novo recruited at the transcriptional start site of genes implicated in cardiomyopathy in stress conditions. We also show that CaN and MLL3 colocalize at chromatin and that CaN activates MLL3 histone methyl transferase activity at distal intergenic regions under hypertrophic conditions. CONCLUSIONS: Our study reveals an unsuspected epigenetic mechanism of CaN that directly regulates MLL3 histone methyl transferase activity to promote cardiac remodeling.

Age-Related Shift in Cardiac and Metabolic Phenotyping Linked to Inflammatory Cytokines and Antioxidant Status in Mice.

Age-related alterations in cardiac function, metabolic, inflammatory and antioxidant profiles are associated with an increased risk of cardiovascular mortality and morbidity. Here, we examined cardiac and metabolic phenotypes in relation to inflammatory status and antioxidant capacity in young, middle-aged and old mice. Real-time reverse transcription-polymerase chain reactions were performed on myocardium and immunoassays on plasma. Left ventricular (LV) structure and function were assessed by echocardiography using high-frequency ultrasound. Middle-aged mice exhibited an altered metabolic profile and antioxidant capacity compared to young mice, whereas myocardial expression of inflammatory factors (TNFα, IL1ß, IL6 and IL10) remained unchanged. In contrast, old mice exhibited increased expression of inflammatory cytokines and plasma levels of resistin compared to young and middle-aged mice (p < 0.05). The pro-inflammatory signature of aged hearts was associated with alterations in glutathione redox homeostasis and elevated contents of 4-hydroxynonenal (4-HNE), a marker of lipid peroxidation and oxidative stress. Furthermore, echocardiographic parameters of LV systolic and diastolic functions were significantly altered in old mice compared to young mice. Taken together, these findings suggest age-related shifts in cardiac phenotype encompass the spectrum of metabo-inflammatory abnormalities and altered redox homeostasis.

Inhibition of EPAC1 signaling pathway alters atrial electrophysiology and prevents atrial fibrillation.

Introduction: Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and is associated with increased mortality and morbidity. The Exchange Protein directly Activated by cAMP (EPAC), has been implicated in pro-arrhythmic signaling pathways in the atria, but the underlying mechanisms remain unknown. Methods: In this study, we investigated the involvement of EPAC1 and EPAC2 isoforms in the genesis of AF in wild type (WT) mice and knockout (KO) mice for EPAC1 or EPAC2. We also employed EPAC pharmacological modulators to selectively activate EPAC proteins (8-CPT-AM; 10 µM), or inhibit either EPAC1 (AM-001; 20 µM) or EPAC2 (ESI-05; 25 µM). Transesophageal stimulation was used to characterize the induction of AF in vivo in mice. Optical mapping experiments were performed on isolated mouse atria and cellular electrophysiology was examined by whole-cell patch-clamp technique. Results: In wild type mice, we found 8-CPT-AM slightly increased AF susceptibility and that this was blocked by the EPAC1 inhibitor AM-001 but not the EPAC2 inhibitor ESI-05. Consistent with this, in EPAC1 KO mice, occurrence of AF was observed in 3/12 (vs. 4/10 WT littermates) and 4/10 in EPAC2 KO (vs. 5/10 WT littermates). In wild type animals, optical mapping experiments revealed that 8-CPT-AM perfusion increased action potential duration even in the presence of AM-001 or ESI-05. Interestingly, 8-CPT-AM perfusion decreased conduction velocity, an effect blunted by AM-001 but not ESI-05. Patch-clamp experiments demonstrated action potential prolongation after 8-CPT-AM perfusion in both wild type and EPAC1 KO mice and this effect was partially prevented by AM-001 in WT. Conclusion: Together, these results indicate that EPAC1 and EPAC2 signaling pathways differentially alter atrial electrophysiology but only the EPAC1 isoform is involved in the genesis of AF. Selective blockade of EPAC1 with AM-001 prevents AF in mice.

Cyclic AMP-binding protein Epac1 acts as a metabolic sensor to promote cardiomyocyte lipotoxicity.

Cyclic adenosine monophosphate (cAMP) is a master regulator of mitochondrial metabolism but its precise mechanism of action yet remains unclear. Here, we found that a dietary saturated fatty acid (FA), palmitate increased intracellular cAMP synthesis through the palmitoylation of soluble adenylyl cyclase in cardiomyocytes. cAMP further induced exchange protein directly activated by cyclic AMP 1 (Epac1) activation, which was upregulated in the myocardium of obese patients. Epac1 enhanced the activity of a key enzyme regulating mitochondrial FA uptake, carnitine palmitoyltransferase 1. Consistently, pharmacological or genetic Epac1 inhibition prevented lipid overload, increased FA oxidation (FAO), and protected against mitochondrial dysfunction in cardiomyocytes. In addition, analysis of Epac1 phosphoproteome led us to identify two key mitochondrial enzymes of the the ß-oxidation cycle as targets of Epac1, the long-chain FA acyl-CoA dehydrogenase (ACADL) and the 3-ketoacyl-CoA thiolase (3-KAT). Epac1 formed molecular complexes with the Ca2+/calmodulin-dependent protein kinase II (CaMKII), which phosphorylated ACADL and 3-KAT at specific amino acid residues to decrease lipid oxidation. The Epac1-CaMKII axis also interacted with the α subunit of ATP synthase, thereby further impairing mitochondrial energetics. Altogether, these findings indicate that Epac1 disrupts the balance between mitochondrial FA uptake and oxidation leading to lipid accumulation and mitochondrial dysfunction, and ultimately cardiomyocyte death.

Atrial Natriuretic Peptide Orchestrates a Coordinated Physiological Response to Fuel Non-shivering Thermogenesis.

Atrial natriuretic peptide (ANP) is a cardiac hormone controlling blood volume and pressure in mammals. It is still unclear whether ANP controls cold-induced thermogenesis in vivo. Here, we show that acute cold exposure induces cardiac ANP secretion in mice and humans. Genetic inactivation of ANP promotes cold intolerance and suppresses half of cold-induced brown adipose tissue (BAT) activation in mice. While white adipocytes are resistant to ANP-mediated lipolysis at thermoneutral temperature in mice, cold exposure renders white adipocytes fully responsive to ANP to activate lipolysis and a thermogenic program, a physiological response that is dramatically suppressed in ANP null mice. ANP deficiency also blunts liver triglycerides and glycogen metabolism, thus impairing fuel availability for BAT thermogenesis. ANP directly increases mitochondrial uncoupling and thermogenic gene expression in human white and brown adipocytes. Together, these results indicate that ANP is a major physiological trigger of BAT thermogenesis upon cold exposure in mammals.

Mitochondrial 4-HNE derived from MAO-A promotes mitoCa2+ overload in chronic postischemic cardiac remodeling.

Chronic remodeling postmyocardial infarction consists in various maladaptive changes including interstitial fibrosis, cardiomyocyte death and mitochondrial dysfunction that lead to heart failure (HF). Reactive aldehydes such as 4-hydroxynonenal (4-HNE) are critical mediators of mitochondrial dysfunction but the sources of mitochondrial 4-HNE in cardiac diseases together with its mechanisms of action remain poorly understood. Here, we evaluated whether the mitochondrial enzyme monoamine oxidase-A (MAO-A), which generates H2O2 as a by-product of catecholamine metabolism, is a source of deleterious 4-HNE in HF. We found that MAO-A activation increased mitochondrial ROS and promoted local 4-HNE production inside the mitochondria through cardiolipin peroxidation in primary cardiomyocytes. Deleterious effects of MAO-A/4-HNE on cardiac dysfunction were prevented by activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2), the main enzyme for 4-HNE metabolism. Mechanistically, MAO-A-derived 4-HNE bound to newly identified targets VDAC and MCU to promote ER-mitochondria contact sites and MCU higher-order complex formation. The resulting mitochondrial Ca2+ accumulation participated in mitochondrial respiratory dysfunction and loss of membrane potential, as shown with the protective effects of the MCU inhibitor, RU360. Most interestingly, these findings were recapitulated in a chronic model of ischemic remodeling where pharmacological or genetic inhibition of MAO-A protected the mice from 4-HNE accumulation, MCU oligomer formation and Ca2+ overload, thus mitigating ventricular dysfunction. To our knowledge, these are the first evidences linking MAO-A activation to mitoCa2+ mishandling through local 4-HNE production, contributing to energetic failure and postischemic remodeling.

Identification of a pharmacological inhibitor of Epac1 that protects the heart against acute and chronic models of cardiac stress.

AIMS: Recent studies reported that cAMP-binding protein Epac1-deficient mice were protected against various forms of cardiac stress, suggesting that pharmacological inhibition of Epac1 could be beneficial for the treatment of cardiac diseases. To test this assumption, we characterized an Epac1-selective inhibitory compound and investigated its potential cardioprotective properties. METHODS AND RESULTS: We used the Epac1-BRET (bioluminescence resonance energy transfer) for searching for non-cyclic nucleotide Epac1 modulators. A thieno[2,3-b]pyridine derivative, designated as AM-001 was identified as a non-competitive inhibitor of Epac1. AM-001 has no antagonist effect on Epac2 or protein kinase A activity. This small molecule prevents the activation of the Epac1 downstream effector Rap1 in cultured cells, in response to the Epac1 preferential agonist, 8-CPT-AM. In addition, we found that AM-001 inhibited Epac1-dependent deleterious effects such as cardiomyocyte hypertrophy and death. Importantly, AM-001-mediated inhibition of Epac1 reduces infarct size after mouse myocardial ischaemia/reperfusion injury. Finally, AM-001 attenuates cardiac hypertrophy, inflammation and fibrosis, and improves cardiac function during chronic ß-adrenergic receptor activation with isoprenaline (ISO) in mice. At the molecular level, ISO increased Epac1-G protein-coupled receptor kinase 5 (GRK5) interaction and induced GRK5 nuclear import and histone deacetylase type 5 (HDAC5) nuclear export to promote the activity of the prohypertrophic transcription factor, myocyte enhancer factor 2 (MEF2). Inversely, AM-001 prevented the non-canonical action of GRK5 on HDAC5 cytoplasmic shuttle to down-regulate MEF2 transcriptional activity. CONCLUSION: Our study represents a 'proof-of-concept' for the therapeutic effectiveness of inhibiting Epac1 activity in cardiac disease using small-molecule pharmacotherapy.

Multifunctional Mitochondrial Epac1 Controls Myocardial Cell Death.

RATIONALE: Although the second messenger cyclic AMP (cAMP) is physiologically beneficial in the heart, it largely contributes to cardiac disease progression when dysregulated. Current evidence suggests that cAMP is produced within mitochondria. However, mitochondrial cAMP signaling and its involvement in cardiac pathophysiology are far from being understood. OBJECTIVE: To investigate the role of MitEpac1 (mitochondrial exchange protein directly activated by cAMP 1) in ischemia/reperfusion injury. METHODS AND RESULTS: We show that Epac1 (exchange protein directly activated by cAMP 1) genetic ablation (Epac1-/-) protects against experimental myocardial ischemia/reperfusion injury with reduced infarct size and cardiomyocyte apoptosis. As observed in vivo, Epac1 inhibition prevents hypoxia/reoxygenation-induced adult cardiomyocyte apoptosis. Interestingly, a deleted form of Epac1 in its mitochondrial-targeting sequence protects against hypoxia/reoxygenation-induced cell death. Mechanistically, Epac1 favors Ca2+ exchange between the endoplasmic reticulum and the mitochondrion, by increasing interaction with a macromolecular complex composed of the VDAC1 (voltage-dependent anion channel 1), the GRP75 (chaperone glucose-regulated protein 75), and the IP3R1 (inositol-1,4,5-triphosphate receptor 1), leading to mitochondrial Ca2+ overload and opening of the mitochondrial permeability transition pore. In addition, our findings demonstrate that MitEpac1 inhibits isocitrate dehydrogenase 2 via the mitochondrial recruitment of CaMKII (Ca2+/calmodulin-dependent protein kinase II), which decreases nicotinamide adenine dinucleotide phosphate hydrogen synthesis, thereby, reducing the antioxidant capabilities of the cardiomyocyte. CONCLUSIONS: Our results reveal the existence, within mitochondria, of different cAMP-Epac1 microdomains that control myocardial cell death. In addition, our findings suggest Epac1 as a promising target for the treatment of ischemia-induced myocardial damage.

[Molecular determinants of pathological cardiac remodeling: the examples of Epac and Carabin]. / Les acteurs moléculaires du remodelage cardiaque pathologique - Exemple des protéines Epac et carabin.

Physical exercise or hypertension requires that the heart increases its hemodynamic work. However, this adaptation is based on distinct cardiac remodelling according to the physiological or pathological origin of the stress. As shown here with two examples, understanding the molecular events leading to cardiac remodeling may offer new opportunities for the development of therapies for heart failure. The recently described Epac1 protein is an effector of the second messenger cAMP. Following a pathological stress, the cAMP-binding protein Epac1 induces cardiac hypertrophy and fibrosis as well as alteration of calcium cycling suggesting that Epac1 pharmacological inhibition may be of therapeutic value. Furthermore, the protein carabin is an important regulator of several effectors of pathological cardiac remodelling. Experimental manipulation of carabin expression profoundly alters the development of heart failure.

Exchange protein directly activated by cAMP 1 promotes autophagy during cardiomyocyte hypertrophy.

AIMS: Stimulation of ß-adrenergic receptors (ß-AR) increases cAMP production and contributes to the pathogenesis of cardiac hypertrophy and failure through poorly understood mechanisms. We previously demonstrated that Exchange protein directly activated by cAMP 1 (Epac1)-induced hypertrophy in primary cardiomyocytes. Among the mechanisms triggered by cardiac stress, autophagy has been highlighted as a protective or harmful response. Here, we investigate whether Epac1 promotes cardiac autophagy and how altered autophagy has an impact on Epac1-induced cardiomyocyte hypertrophy. METHODS AND RESULTS: We reported that direct stimulation of Epac1 with the agonist, Sp-8-(4-chlorophenylthio)-2'-O-methyl-cAMP (Sp-8-pCPT) promoted autophagy activation in neonatal cardiomyocytes. Stimulation of ß-AR with isoprenaline (ISO) mimicked the effect of Epac1 on autophagy markers. Conversely, the induction of autophagy flux following ISO treatment was prevented in cardiomyocytes pre-treated with a selective inhibitor of Epac1, CE3F4. Importantly, we found that Epac1 deletion in mice protected against ß-AR-induced cardiac remodelling and prevented the induction of autophagy. The signalling mechanisms underlying Epac1-induced autophagy involved a Ca(2+)/calmodulin-dependent kinase kinase ß (CaMKKß)/AMP-dependent protein kinase (AMPK) pathway. Finally, we provided evidence that pharmacological inhibition of autophagy using 3-methyladenine (3-MA) or down-regulation of autophagy-related protein 5 (Atg5) significantly potentiated Epac1-promoted cardiomyocyte hypertrophy. CONCLUSION: Altogether, these findings demonstrate that autophagy is an adaptive response to antagonize Epac1-promoted cardiomyocyte hypertrophy.

Abnormal plasma microparticles impair vasoconstrictor responses in patients with cirrhosis.

BACKGROUND & AIMS: Circulating membrane-shed microparticles (MPs) participate in regulation of vascular tone. We investigated the cellular origins of MPs in plasma from patients with cirrhosis and assessed the contribution of MPs to arterial vasodilation, a mechanism that contributes to portal hypertension. METHODS: We analyzed MPs from blood samples of 91 patients with cirrhosis and 30 healthy individuals (controls) using flow cytometry; their effects on the vascular response to vasoconstrictors were examined in vitro and in vivo. RESULTS: Circulating levels of leuko-endothelial (CD31(+)/41(-)), pan-leukocyte (CD11a(+)), lymphocyte (CD4(+)), and erythrocyte (CD235a(+)) MPs were higher in patients with cirrhosis than in controls. Plasma of patients with cirrhosis contained hepatocyte-derived MPs (cytokeratin-18(+)), whereas plasma from controls did not. The severity of cirrhosis and systemic inflammation were major determinants of the levels of leuko-endothelial and hepatocyte MPs. MPs from patients with advanced cirrhosis significantly impaired contraction of vessels in response to phenylephrine, whereas MPs from healthy controls or from patients of Child-Pugh class A did not. This effect depended on cyclooxygenase type 1 and required phosphatidylserine on the surface of MPs. Intravenous injection of MPs from patients with cirrhosis into BALB/C mice decreased mean arterial blood pressure. CONCLUSIONS: Cirrhosis is associated with increases in circulating subpopulations of MPs, likely resulting from systemic inflammation and liver cell damage. The overall pool of circulating MPs from patients with advanced cirrhosis impairs vasoconstrictor responses and decreases blood pressure, contributing to the arterial vasodilation associated with portal hypertension.

Molecular signature of mineralocorticoid receptor signaling in cardiomyocytes: from cultured cells to mouse heart.

Excess mineralocorticoid signaling is deleterious for cardiovascular functions, as demonstrated by the beneficial effects of mineralocorticoid receptor (MR) antagonism on morbidity and mortality in patients with heart failure. However, the understanding of signaling pathways after MR activation in the heart remains limited. We performed transcriptomic analyses in the heart of double-transgenic mice with conditional, cardiomyocyte-specific, overexpression of the MR (MRcardio mice) or the glucocorticoid receptor (GR; GRcardio mice). Some of the genes induced in MRcardio mice were selected for comparative evaluation (real time PCR) in vivo in the heart of mice and ex vivo in the MR-expressing cardiomyocyte H9C2 cell line after aldosterone or corticosterone treatment. We demonstrate that chronic MR overexpression in the heart results in a limited number of induced (n = 24) and repressed (n = 22) genes compared with their control littermates. These genes are specifically modulated by MR because there is limited overlap (three induced, four repressed) with the genes that are regulated in the heart of GRcardio mice (compared with control mice: 70 induced, 73 repressed). Interestingly, some MR-induced genes that are up-regulated in vivo in mice are also induced by 24-h aldosterone treatment in H9C2 cells, such as plasminogen activator inhibitor 1 and Serpina-3 (alpha1-antichymotrypsin). The signaling pathways that are affected by long-term activation of MR may be of particular interest to design novel therapeutic targets in cardiac diseases.

Mineralocorticoid modulation of cardiac ryanodine receptor activity is associated with downregulation of FK506-binding proteins.

BACKGROUND: The mineralocorticoid pathway is involved in cardiac arrhythmias associated with heart failure through mechanisms that are incompletely understood. Defective regulation of the cardiac ryanodine receptor (RyR) is an important cause of the initiation of arrhythmias. Here, we examined whether the aldosterone pathway might modulate RyR function. METHODS AND RESULTS: Using the whole-cell patch clamp method, we observed an increase in the occurrence of delayed afterdepolarizations during action potential recordings in isolated adult rat ventricular myocytes exposed for 48 hours to aldosterone 100 nmol/L, in freshly isolated myocytes from transgenic mice with human mineralocorticoid receptor expression in the heart, and in wild-type littermates treated with aldosterone. Sarcoplasmic reticulum Ca(2+) load and RyR expression were not altered; however, RyR activity, visualized in situ by confocal microscopy, was increased in all cells, as evidenced by an increased occurrence and redistribution to long-lasting and broader populations of spontaneous Ca(2+) sparks. These changes were associated with downregulation of FK506-binding proteins (FKBP12 and 12.6), regulatory proteins of the RyR macromolecular complex. CONCLUSIONS: We suggest that in addition to modulation of Ca(2+) influx, overstimulation of the cardiac mineralocorticoid pathway in the heart might be a major upstream factor for aberrant Ca(2+) release during diastole, which contributes to cardiac arrhythmia in heart failure.

Cardiomyocyte overexpression of neuronal nitric oxide synthase delays transition toward heart failure in response to pressure overload by preserving calcium cycling.

BACKGROUND: Defects in cardiomyocyte Ca(2+) cycling are a signature feature of heart failure (HF) that occurs in response to sustained hemodynamic overload, and they largely account for contractile dysfunction. Neuronal nitric oxide synthase (NOS1) influences myocyte excitation-contraction coupling through modulation of Ca(2+) cycling, but the potential relevance of this in HF is unknown. METHODS AND RESULTS: We generated a transgenic mouse with conditional, cardiomyocyte-specific NOS1 overexpression (double-transgenic [DT]) and studied cardiac remodeling, myocardial Ca(2+) handling, and contractility in DT and control mice subjected to transverse aortic constriction (TAC). After TAC, control mice developed eccentric hypertrophy with evolution toward HF as revealed by a significantly reduced fractional shortening. In contrast, DT mice developed a greater increase in wall thickness (P<0.0001 versus control+TAC) and less left ventricular dilatation than control+TAC mice (P<0.0001 for both end-systolic and end-diastolic dimensions). Thus, DT mice displayed concentric hypertrophy with fully preserved fractional shortening (43.7+/-0.6% versus 30.3+/-2.6% in control+TAC mice, P<0.05). Isolated cardiomyocytes from DT+TAC mice had greater shortening, intracellular Ca(2+) transients, and sarcoplasmic reticulum Ca(2+) load (P<0.05 versus control+TAC for all parameters). These effects could be explained, at least in part, through modulation of phospholamban phosphorylation status. CONCLUSIONS: Cardiomyocyte NOS1 may be a useful target against cardiac deterioration during chronic pressure-overload-induced HF through modulation of calcium cycling.

Targeted skin overexpression of the mineralocorticoid receptor in mice causes epidermal atrophy, premature skin barrier formation, eye abnormalities, and alopecia.

The mineralocorticoid receptor (MR) is a transcription factor of the nuclear receptor family, activation of which by aldosterone enhances salt reabsorption in the kidney. The MR is also expressed in nonclassical aldosterone target cells (brain, heart, and skin), in which its functions are incompletely understood. To explore the functional importance of MR in mammalian skin, we have generated a conditional doxycycline-inducible model of MR overexpression, resulting in double-transgenic (DT) mice [keratin 5-tTa/tetO-human MR (hMR)], targeting the human MR specifically to keratinocytes of the epidermis and hair follicle (HF). Expression of hMR throughout gestation resulted in early postnatal death that could be prevented by antagonizing MR signaling. DT mice exhibited premature epidermal barrier formation at embryonic day 16.5, reduced HF density and epidermal atrophy, increased keratinocyte apoptosis at embryonic day 18.5, and premature eye opening. When hMR expression was initiated after birth to overcome mortality, DT mice developed progressive alopecia and HF cysts, starting 4 months after hMR induction, preceded by dystrophy and cycling abnormalities of pelage HF. In contrast, interfollicular epidermis, vibrissae, and footpad sweat glands in DT mice were normal. This new mouse model reveals novel biological roles of MR signaling and offers an instructive tool for dissecting nonclassical functions of MR signaling in epidermal, hair follicle, and ocular physiology.

Conditional glucocorticoid receptor expression in the heart induces atrio-ventricular block.

Corticosteroid hormones (aldosterone and glucocorticoids) and their receptors are now recognized as major modulators of cardiovascular pathophysiology, but their specific roles remain elusive. Glucocorticoid hormones (GCs), which are widely used to treat acute and chronic diseases, often have adverse cardiovascular effects such as heart failure, hypertension, atherosclerosis, or metabolic alterations. The direct effects of GC on the heart are difficult to evaluate, as changes in plasma GC concentrations have multiple consequences due to the ubiquitous expression of the glucocorticoid receptor (GR), resulting in secondary effects on cardiac function. We evaluated the effects of GR on the heart in a conditional mouse model in which the GR was overexpressed solely in cardiomyocytes. The transgenic mice displayed electrocardiogram (ECG) abnormalities: a long PQ interval, increased QRS and QTc duration as well as chronic atrio-ventricular block, without cardiac hypertrophy or fibrosis. The ECG alterations were reversible on GR expression shutoff. Isolated ventricular cardiomyocytes showed major ion channel remodeling, with decreases in I(Na), I(to), and I(Kslow) activity and changes in cell calcium homeostasis (increase in C(al), in Ca2+ transients and in sarcoplasmic reticulum Ca2+ load). This phenotype differs from that observed in mice overexpressing the mineralocorticoid receptor in the heart, which displayed ventricular arrhythmia. Our mouse model highlights novel effects of GR activation in the heart indicating that GR has direct and specific cardiac effects in the mouse.

A direct relationship between plasma aldosterone and cardiac L-type Ca2+ current in mice.

Aldosterone is involved in a variety of pathophysiological processes that ultimately cause cardiovascular diseases. Despite this, the physiological role of aldosterone in heart function remains elusive. We took advantage of transgenic mouse models characterized by a renal salt-losing (SL) or salt-retaining (SR) phenotype, thus exhibiting chronically high or low plasma aldosterone levels, respectively, to investigate the chronic effects of aldosterone in cardiomyocytes devoid of pathology. On a diet containing normal levels of salt, these animals do not develop any evidence of cardiovascular disease. Using the whole cell patch-clamp technique on freshly isolated adult ventricular cardiomyocytes, we observed that the amplitude of L-type Ca(2)(+) currents (I(Ca)) correlates with plasma aldosterone levels. Larger values of I(Ca) are associated with high aldosterone concentrations in SL models, whereas smaller values of I(Ca) were observed in the SR model. Neither the time- nor the voltage-dependent properties of I(Ca) varied measurably. In parallel, we determined whether modulation of I(Ca) by blood concentration of aldosterone has a major physiological impact on the excitation-contraction coupling of the cardiomyocytes. Action potential duration, [Ca(2)(+)](i) transient amplitude and contraction are increased in the SL model and decreased in the SR model. In conclusion, we demonstrate that the blood concentration of aldosterone exerts chronic regulation of I(Ca) in mouse cardiomyocytes. This regulation has important consequences for excitation-contraction coupling and, potentially, for other Ca(2)(+)-regulated functions in cardiomyocytes.

Conditional mineralocorticoid receptor expression in the heart leads to life-threatening arrhythmias.

BACKGROUND: Life-threatening cardiac arrhythmia is a major source of mortality worldwide. Besides rare inherited monogenic diseases such as long-QT or Brugada syndromes, which reflect abnormalities in ion fluxes across cardiac ion channels as a final common pathway, arrhythmias are most frequently acquired and associated with heart disease. The mineralocorticoid hormone aldosterone is an important contributor to morbidity and mortality in heart failure, but its mechanisms of action are incompletely understood. METHODS AND RESULTS: To specifically assess the role of the mineralocorticoid receptor (MR) in the heart, in the absence of changes in aldosteronemia, we generated a transgenic mouse model with conditional cardiac-specific overexpression of the human MR. Mice exhibit a high rate of death prevented by spironolactone, an MR antagonist used in human therapy. Cardiac MR overexpression led to ion channel remodeling, resulting in prolonged ventricular repolarization at both the cellular and integrated levels and in severe ventricular arrhythmias. CONCLUSIONS: Our results indicate that cardiac MR triggers cardiac arrhythmias, suggesting novel opportunities for prevention of arrhythmia-related sudden death.

Conditional gene expression in renal collecting duct epithelial cells: use of the inducible Cre-lox system.

The renal collecting duct plays a key role in control of ion and fluid homeostasis. Genes encoding for ion transporters, hormone receptors, or regulatory proteins specifically expressed in the collecting duct are mutated in several genetic diseases with altered blood pressure. Suitable cellular models expressing genes in a conditional way should represent attractive systems for structure-function analyses and generation of appropriate physiopathological models of related diseases. However, generation of such systems remains laborious and quite inefficient. We adapted and improved a conditional Cre-lox-inducible system in the highly differentiated aldosterone-sensitive rat cortical collecting duct (RCCD2) cell line. The inducible MerCreMer recombinase allowed tight control and high levels of transgene expression, whereas flanking a selection marker with two loxP sites strongly improved the selection procedure. We have used this system to conditionally express an enhanced green fluorescent protein-tagged human mineralocorticoid receptor. In the future, this will allow structure-function analyses as well as mineralocorticoid receptor trafficking studies in these epithelial cells, which retain the features of the native collecting duct. Improvements in the conditional Cre-lox expression system have potentially wide applications in other epithelial or nonepithelial cell lines.