Recurrent "outsider" intronic variation in the SLC5A6 gene causes severe mixed axonal and demyelinating neuropathy, cyclic vomiting and optic atrophy in 3 families from Maghreb.

Sodium dependent multivitamin transporter (SMVT) deficiency is a very rare autosomal recessive disorder characterized by multisystemic clinical manifestations due to combined biotin, panthotenic acid and lipoic acid deficiency. About 10 families have been described so far. Accurate diagnosis is crucial because of the possibility of a supplementation treatment with proven efficacy. Here we describe 4 new patients (3 additional families) originating from the same world region (Algeria, Maghreb). All patients, born form consanguineous parents, were homozygous carriers of the same intronic variation, outside of canonical sites, in the SLC5A6 gene encoding SMVT. RNA study in one family allowed confirming the pathogenic effect of the variation and re-classifying this variant of uncertain significance as pathogenic, opening the possibility of genetic counseling and treatment. The identification of the same variation in three distinct and apparently unrelated families is suggestive of a founder effect. The phenotype of all patients was very similar, with systematic optic atrophy (initially considered as a very rare sign), severe cyclic vomiting, and rapidly progressive mixed axonal and demyelinating sensory motor neuropathy.

Cardiac expression of neutrophil gelatinase-associated lipocalin in a model of cancer cachexia-induced cardiomyopathy.

AIMS: Cachexia is a severe consequence of cancer. Although cancer-induced heart atrophy leads to cardiac dysfunction and heart failure (HF), biomarkers for their diagnosis have not been identified. Neutrophil gelatinase-associated lipocalin (NGAL) is an aldosterone-responsive gene increased in HF. We studied NGAL and its association with aldosterone levels in a model of cancer cachexia-induced cardiomyopathy. METHODS AND RESULTS: Rats were injected with Yoshida 108 AH-130 hepatoma cells to induce tumour. Cachectic rats were treated daily, for 16 days, with placebo or with 5 or 50 mg/kg/day of spironolactone. Cardiac function was analysed by echocardiography at baseline and at Day 11. Weight loss and atrophy of lean body and fat mass of cachectic rats were significantly attenuated by spironolactone. Cardiac dysfunction of tumour-bearing rats was improved by spironolactone. Plasma aldosterone was up-regulated from 337 ± 7 pg/mL in sham animals to 591 ± 31 pg/mL in the cachectic rats (P < 0.001 vs. sham). Treatment with 50 or 5 mg/kg/day of spironolactone reduced plasma aldosterone to 396 ± 22 and 391 ± 25 pg/mL (P < 0.01 vs. placebo). Plasma levels of NGAL were also increased in cachectic rats (1.462 ± 0.3603 µg/mL) than in controls (0.0936 ± 6 µg/mL, P < 0.001). Spironolactone treatment (50 mg/kg/day) significantly reduced cardiac mRNA and protein NGAL levels (P < 0.05 and P < 0.001 vs. placebo, respectively). NGAL mRNA and protein levels were overexpressed in cachectic animal hearts treated with placebo, compared with control (P < 0.05 and P < 0.01 vs. sham). Spironolactone treatment at 50 mg/kg/day reduced significantly cardiac NGAL (P < 0.05 and P < 0.001 vs. placebo). CONCLUSIONS: Cancer cachexia induced increased levels of aldosterone and NGAL, contributing to worsening cardiac damage in cancer cachexia-induced cardiomyopathy. Spironolactone treatment may greatly attenuate cardiac dysfunction and lean mass atrophy associated with cancer cachexia.

Short in-Frame Insertions/Deletions in the Coding Sequence of the α-Globin Gene. Consequences of the 3D Structure and Resulting Phenotypes: Hb Choisy as an Example.

A small group of hemoglobin (Hb) variants result from 'in-frame' deletion/insertion (del/ins). We describe a new variant of this group (Hb Choisy), found on the α1 gene, which is the exact counterpart of a previously published deletional variant, Hb J-Biskra [codons 51-58 (or codons 52-59) (-24 bp) (-TCTGCCCAGGTTAAGGGCCACGGC); HBA1: c.157_180del (or HBA2)]. In Hb J-Biskra, the sequence Ser-Ala-Gln-Val-Lys-Gly-His-Gly located from positions α52(E1) to α59(E8) is deleted, while in Hb Choisy the same sequence (Ser-Ala-Gln-Val-Lys-Gly-His-Gly) is inserted at position α52(E1). The variant carrying the insertion appears to be less damaging than the one with the deletion. A possible explanation could be that the additional sequence is located in the C to E interhelical region, and is less disturbing to the general structure of the globin chain. This insertion/deletion (ins/del) is likely favored by the repetition, at an interval of 16 nucleotides, of an eight nucleotide sequence. Comparison of variants of this group, found in the HbVar database, shows that structural modifications resulting from insertions are frequently less damaging than that caused by deletions.

Protective Effect of Inflammasome Activation by Hydrogen Peroxide in a Mouse Model of Septic Shock.

OBJECTIVES: To study the effect of a lack of antioxidant defenses during lethal pneumonia induced by Klebsiella pneumonia, compared to wild-type mice. SETTING: Laboratory experiments. SUBJECTS: C57Bl6 and glutathione peroxidase 1 knockout mice. INTERVENTION: Murine acute pneumonia model induced by Klebsiella pneumonia. MEASUREMENTS AND MAIN RESULTS: We show here that despite a lack of one of the major antioxidant defense enzymes, glutathione peroxidase 1 knockout mice are protected during lethal pneumonia induced by Klebsiella pneumonia, compared to wild-type mice. Furthermore, this protective effect was suppressed when antioxidant defenses were restored. Infected glutathione peroxidase 1 mice showed an early and significant, albeit transient, increase in the activity of the NOD-like receptor family, pyrin domain containing 3 inflammasome when compared with wild-type mice. The key role of the NOD-like receptor family, pyrin domain containing 3 inflammasome during acute pneumonia was confirmed in vivo when the protective effect was suppressed by treating glutathione peroxidase 1 mice with an interleukin-1 receptor antagonist. Additionally we report, in vitro, that increased concentrations of active caspase-1 and interleukin-1ß are related to an increased concentration of hydrogen peroxide in bacterially infected glutathione peroxidase 1 macrophages and that restoring hydrogen peroxide antioxidant defenses suppressed this effect. CONCLUSIONS: Our findings demonstrate that, contrary to current thinking, an early intervention targeting NOD-like receptor family, pyrin domain containing 3 inflammasome activity induces a timely and efficient activation of the innate immune response during acute infection. Our findings also demonstrate a role for hydrogen peroxide in the mechanisms tightly regulating NOD-like receptor family, pyrin domain containing 3 activation.

Frequent interruptions of sedentary time modulates contraction- and insulin-stimulated glucose uptake pathways in muscle: Ancillary analysis from randomized clinical trials.

Epidemiological studies have observed associations between frequent interruptions of sitting time with physical activity bouts and beneficial metabolic outcomes, even in individuals who regularly exercise. Frequent interruptions to prolonged sitting reduce postprandial plasma glucose. Here we studied potential skeletal muscle mechanisms accounting for this improved control of glycemia in overweight adults under conditions of one day uninterrupted sitting and sitting interrupted with light-intensity or moderate-intensity walking every 20-min (n = 8); and, after three days of either uninterrupted sitting or light-intensity walking interruptions (n = 5). Contraction- and insulin-mediated glucose uptake signaling pathways as well as changes in oxidative phosphorylation proteins were examined. We showed that 1) both interventions reduce postprandial glucose concentration, 2) acute interruptions to sitting over one day stimulate the contraction-mediated glucose uptake pathway, 3) both acute interruptions to sitting with moderate-intensity activity over one day and light-intensity activity over three days induce a transition to modulation of the insulin-signaling pathway, in association with increased capacity for glucose transport. Only the moderate-intensity interruptions resulted in greater capacity for glycogen synthesis and likely for ATP production. These observations contribute to a mechanistic explanation of improved postprandial glucose metabolism with regular interruptions to sitting time, a promising preventive strategy for metabolic diseases.

MicroRNA-194 Modulates Glucose Metabolism and Its Skeletal Muscle Expression Is Reduced in Diabetes.

BACKGROUND: The regulation of microRNAs (miRNAs) at different stages of the progression of type 2 diabetes mellitus (T2DM) and their role in glucose homeostasis was investigated. METHODS: Microarrays were used to assess miRNA expression in skeletal muscle biopsies taken from healthy individuals and patients with pre-diabetes or T2DM, and insulin resistant offspring of rat dams fed a high fat diet during pregnancy. RESULTS: Twenty-three miRNAs were differentially expressed in patients with T2DM, and 7 in the insulin resistant rat offspring compared to their controls. Among these, only one miRNA was similarly regulated: miR-194 expression was significantly reduced by 25 to 50% in both the rat model and in human with pre-diabetes and established diabetes. Knockdown of miR-194 in L6 skeletal muscle cells induced an increase in basal and insulin-stimulated glucose uptake and glycogen synthesis. This occurred in conjunction with an increased glycolysis, indicated by elevated lactate production. Moreover, oxidative capacity was also increased as we found an enhanced glucose oxidation in presence of the mitochondrial uncoupler FCCP. When miR-194 was down-regulated in vitro, western blot analysis showed an increased phosphorylation of AKT and GSK3ß in response to insulin, and an increase in expression of proteins controlling mitochondrial oxidative phosphorylation. CONCLUSIONS: Type 2 diabetes mellitus is associated with regulation of several miRNAs in skeletal muscle. Interestingly, miR-194 was a unique miRNA that appeared regulated across different stages of the disease progression, from the early stages of insulin resistance to the development of T2DM. We have shown miR-194 is involved in multiple aspects of skeletal muscle glucose metabolism from uptake, through to glycolysis, glycogenesis and glucose oxidation, potentially via mechanisms involving AKT, GSK3 and oxidative phosphorylation. MiR-194 could be down-regulated in patients with early features of diabetes as an adaptive response to facilitate tissue glucose uptake and metabolism in the face of insulin resistance.

Neutrophil Gelatinase-Associated Lipocalin, a Novel Mineralocorticoid Biotarget, Mediates Vascular Profibrotic Effects of Mineralocorticoids.

Activation of the mineralocorticoid receptor has been shown to be deleterious in cardiovascular diseases (CVDs). We have recently shown that lipocalin 2 (Lcn2), or neutrophil gelatinase-associated lipocalin (NGAL), is a primary target of aldosterone/mineralocorticoid receptor in the cardiovascular system. Lcn2 is a circulating protein, which binds matrix metalloproteinase 9 and modulates its stability. We hypothesized that Lcn2 could be a mediator of aldosterone/mineralocorticoid receptor profibrotic effects in the cardiovascular system. Correlations between aldosterone and profibrotic markers, such as procollagen type I N-terminal peptide, were investigated in healthy subjects and subjects with abdominal obesity. The implication of Lcn2 in the mineralocorticoid pathway was studied using Lcn2 knockout mice subjected to a nephrectomy/aldosterone/salt (NAS) challenge for 4 weeks. In human subjects, NGAL/matrix metalloproteinase 9 was positively correlated with plasma aldosterone and fibrosis biomarkers. In mice, loss of Lcn2 prevented the NAS-induced increase of plasma procollagen type I N-terminal peptide, as well as the increase of collagen fibers deposition and collagen I expression in the coronary vessels and the aorta. The lack of Lcn2 also blunted the NAS-induced increase in systolic blood pressure. Ex vivo, treatment of human fibroblasts with recombinant Lcn2 induced the expression of collagen I and the profibrotic galectin-3 and cardiotrophin-1 molecules. Our results showed that Lcn2 plays a key role in aldosterone/mineralocorticoid receptor-mediated vascular fibrosis. The clinical data indicate that this may translate in human patients. Lcn2 is, therefore, a new biotarget in cardiovascular fibrosis induced by mineralocorticoid activation.

Potential vascular mechanisms of ramipril induced increases in walking ability in patients with intermittent claudication.

RATIONALE: We recently reported that ramipril more than doubled maximum walking times in patients with peripheral artery disease with intermittent claudication. OBJECTIVE: Our aim was to conduct exploratory analyses of the effects of ramipril therapy on circulating biomarkers of angiogenesis/arteriogenesis, thrombosis, inflammation, and leukocyte adhesion in patients with intermittent claudication. METHODS AND RESULTS: One hundred sixty-five patients with intermittent claudication (mean, 65.3 [SD, 6.7] years) were administered ramipril 10 mg per day (n=82) or matching placebo (n=83) for 24 weeks in a randomized, double-blind study. Plasma biomarkers of angiogenesis/arteriogenesis (vascular endothelial growth factor-A, fibroblast growth factor-2), thrombosis (D-dimer, von Willebrand factor, thrombin-antithrombin III), inflammation (high-sensitivity C-reactive protein, osteopontin), and leukocyte adhesion (soluble vascular cell adhesion molecule-1, soluble intracellular adhesion molecule-1) were measured at baseline and 24 weeks. Relative to placebo, ramipril was associated with increases in vascular endothelial growth factor-A by 38% (95% confidence interval [CI], 34%-42%) and fibroblast growth factor-2 by 64% (95% CI, 44-85%; P<0.001 for both), and reductions in D-dimer by 24% (95% CI, -30% to -18%), von Willebrand factor by 22% (95% CI, -35% to -9%), thrombin-antithrombin III by 16% (95% CI, -19% to -13%), high-sensitivity C-reactive protein by 13% (95% CI, -14% to -9%), osteopontin by 12% (95% CI, -14% to -10%), soluble vascular cell adhesion molecule-1 by 14% (95% CI, -18% to -10%), and soluble intracellular adhesion molecule-1 by 15% (95% CI, -17% to -13%; all P<0.001). With the exception of von Willebrand factor, all the above changes correlated significantly with the change in maximum walking time (P=0.02-0.001) in the group treated with ramipril. CONCLUSIONS: Ramipril is associated with an increase in the biomarkers of angiogenesis/arteriogenesis and reduction in the markers of thrombosis, inflammation, and leukocyte adhesion. This study informs strategies to improve mobility in patients with intermittent claudication. CLINICAL TRIAL REGISTRATION INFORMATION URL: http://clinicaltrials.gov. Unique identifier: NCT00681226.

Maternal overnutrition programs changes in the expression of skeletal muscle genes that are associated with insulin resistance and defects of oxidative phosphorylation in adult male rat offspring.

Children of obese mothers have increased risk of metabolic syndrome as adults. Here we report the effects of a high-fat diet in the absence of maternal obesity at conception on skeletal muscle metabolic and transcriptional profiles of adult male offspring. Female Sprague Dawley rats were fed a diet rich in saturated fat and sucrose [high-fat diet (HFD): 23.5% total fat, 9.83% saturated fat, 20% sucrose wt:wt] or a normal control diet [(CD) 7% total fat, 0.5% saturated fat, 10% sucrose wt:wt] for the 3 wk prior to mating and throughout pregnancy and lactation. Maternal weights were not different at conception; however, HFD-fed dams were 22% heavier than controls during pregnancy. On a normal diet, the male offspring of HFD-fed dams were not heavier than controls but demonstrated features of insulin resistance, including elevated plasma insulin concentration [40.1 ± 2.5 (CD) vs 56.2 ± 6.1 (HFD) mU/L; P = 0.023]. Next-generation mRNA sequencing was used to identify differentially expressed genes in the offspring soleus muscle, and gene set enrichment analysis (GSEA) was used to detect coordinated changes that are characteristic of a biological function. GSEA identified 15 upregulated pathways, including cytokine signaling (P < 0.005), starch and sucrose metabolism (P < 0.017), inflammatory response (P < 0.024), and cytokine-cytokine receptor interaction (P < 0.037). A further 8 pathways were downregulated, including oxidative phosphorylation (P < 0.004), mitochondrial matrix (P < 0.006), and electron transport/uncoupling (P < 0.022). Phosphorylation of the insulin signaling protein kinase B was reduced [2.86 ± 0.63 (CD) vs 1.02 ± 0.27 (HFD); P = 0.027] and mitochondrial complexes I, II, and V protein were downregulated by 50-68% (P < 0.005). On a normal diet, the male offspring of HFD-fed dams did not become obese adults but developed insulin resistance, with transcriptional evidence of muscle cytokine activation, inflammation, and mitochondrial dysfunction. These data indicate that maternal overnutrition, even in the absence of prepregnancy obesity, can promote metabolic dysregulation and predispose offspring to type 2 diabetes.

Prevention of liver cancer cachexia-induced cardiac wasting and heart failure.

AIMS: Symptoms of cancer cachexia (CC) include fatigue, shortness of breath, and impaired exercise capacity, which are also hallmark symptoms of heart failure (HF). Herein, we evaluate the effects of drugs commonly used to treat HF (bisoprolol, imidapril, spironolactone) on development of cardiac wasting, HF, and death in the rat hepatoma CC model (AH-130). METHODS AND RESULTS: Tumour-bearing rats showed a progressive loss of body weight and left-ventricular (LV) mass that was associated with a progressive deterioration in cardiac function. Strikingly, bisoprolol and spironolactone significantly reduced wasting of LV mass, attenuated cardiac dysfunction, and improved survival. In contrast, imidapril had no beneficial effect. Several key anabolic and catabolic pathways were dysregulated in the cachectic hearts and, in addition, we found enhanced fibrosis that was corrected by treatment with spironolactone. Finally, we found cardiac wasting and fibrotic remodelling in patients who died as a result of CC. In living cancer patients, with and without cachexia, serum levels of brain natriuretic peptide and aldosterone were elevated. CONCLUSION: Systemic effects of tumours lead not only to CC but also to cardiac wasting, associated with LV-dysfunction, fibrotic remodelling, and increased mortality. These adverse effects of the tumour on the heart and on survival can be mitigated by treatment with either the ß-blocker bisoprolol or the aldosterone antagonist spironolactone. We suggest that clinical trials employing these agents be considered to attempt to limit this devastating complication of cancer.

Effects of breaking up prolonged sitting on skeletal muscle gene expression.

Breaking up prolonged sitting has been beneficially associated with cardiometabolic risk markers in both observational and intervention studies. We aimed to define the acute transcriptional events induced in skeletal muscle by breaks in sedentary time. Overweight/obese adults participated in a randomized three-period, three-treatment crossover trial in an acute setting. The three 5-h interventions were performed in the postprandial state after a standardized test drink and included seated position with no activity and seated with 2-min bouts of light- or moderate-intensity treadmill walking every 20 min. Vastus lateralis biopsies were obtained in eight participants after each treatment, and gene expression was examined using microarrays validated with real-time quantitative PCR. There were 75 differentially expressed genes between the three conditions. Pathway analysis indicated the main biological functions affected were related to small-molecule biochemistry, cellular development, growth and proliferation, and carbohydrate metabolism. Interestingly, differentially expressed genes were also linked to cardiovascular disease. For example, relative to prolonged sitting, activity bouts increased expression of nicotamide N-methyltransferase, which modulates anti-inflammatory and anti-oxidative pathways and triglyceride metabolism. Activity bouts also altered expression of 10 genes involved in carbohydrate metabolism, including increased expression of dynein light chain, which may regulate translocation of the GLUT-4 glucose transporter. In addition, breaking up sedentary time reversed the effects of chronic inactivity on expression of some specific genes. This study provides insight into the muscle regulatory systems and molecular processes underlying the physiological benefits induced by interrupting prolonged sitting.

Neutrophil gelatinase-associated lipocalin is a novel mineralocorticoid target in the cardiovascular system.

Mineralocorticoid receptor (MR) activation may be deleterious to the cardiovascular system, and MR antagonists improve morbidity and mortality of patients with heart failure. However, mineralocorticoid signaling in the heart remains largely unknown. Using a pan-genomic transcriptomic analysis, we identified neutrophil gelatinase-associated lipocalin (NGAL or lipocalin 2) as a strongly induced gene in the heart of mice with conditional and targeted MR overexpression in cardiomyocytes (whereas induction was low in glucocorticoid receptor-overexpressing mice). NGAL mRNA levels were enhanced after hormonal stimulation by the MR ligand aldosterone in cultured cardiac cells and in the heart of wild-type mice. Mineralocorticoid pathological challenge induced by nephrectomy/aldosterone/salt treatment upregulated NGAL expression in the heart and aorta and its plasma levels. We show evidence for MR binding to an NGAL promoter, providing a mechanism for NGAL regulation. We propose that NGAL may be a marker of mineralocorticoid-dependent injury in the cardiovascular system in mice.

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.

[Contribution of transgenesis models in discovery of new pharmacological targets in heart failure: aldosterone receptor as an example]. / Apport des modèles transgéniques dans la recherche de nouvelles cibles pharmacologiques dans l'insuffisance cardiaque: l'exemple du récepteur de l'aldostérone.

Heart failure is a complex illness with multiple etiologies. Therapeutic targets are numerous such as neurohormonal blockade (adrenergic and renin-angiotensin-aldosterone systems). Progress in therapeutic strategy and efficacy could be expected from individual treatment taking into account biomarkers so called "theragnostic". Animal models, classical or genetically modified, have conducted to a better identification and validation of signal pathways in heart failure. Furthermore these models allow testing of new hypothesis and therapeutic approaches, although cardiovascular physiology and physiopathological patterns are far different in animals than in humans (haemodynamic conditions, kinetic development of the illness, old age, ...). Last, identification of putative theragnostic biomarkers is easier (i.e. free access, homogeneity of experimental cohorts). Genetically modified models by classical additive transgenesis techniques or gene targeting have an important role in discovery of new pharmacological targets in heart failure. Study of the role of aldosterone and of its receptor (mineralocorticosteroid receptor) in the physiopathology of heart failure and as a new therapeutic target will be used as an Ariane's clew to demonstrate that translational bidirectional research (from integrated experimental model to bedside and from clinical results to bench) has allowed from these 15 last years: i) to propose changes in therapeutic strategies and ii) to stimulate research of new targets and new mineralocorticosteroid receptor antagonists.

Cnksr3 is a direct mineralocorticoid receptor target gene and plays a key role in the regulation of the epithelial sodium channel.

Aldosterone is the principal hormonal regulator of sodium homeostasis in vertebrates. It exerts its actions through the mineralocorticoid receptor (MR) that regulates the transcription of specific target genes. In recent years, a number of MR target genes have been identified that are involved in the regulation of the epithelial sodium channel (ENaC), a key modulator of renal sodium absorption. Here we report the identification of cnksr3 as a direct MR target gene that is up-regulated in response to physiological concentrations of aldosterone. The cnksr3 promoter exhibits two functional aldosterone-responsive regions, which were bound by the MR as assessed by chromatin immunoprecipitation (ChIP). In vivo, CNKSR3 was highly expressed in the renal cortical collecting duct (CCD), the prime target segment of aldosterone-regulated sodium retention in the kidney. CCD cell lines stably overexpressing or silencing CNKSR3 were electrophysiologically analyzed and show that CNKSR3 expression correlated with and is required for ENaC-mediated transepithelial sodium transport. In parallel, CNKSR3 expression led to decreased MEK phosphorylation. We conclude that CNKSR3, a homologue of scaffold proteins involved in MAPK pathway regulation, is a direct target of MR and is required for the maintenance of transepithelial sodium transport in the kidney.

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.