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.

CAMP is a powerful second messenger whose known general effector is protein kinase A (PKA). The identification of a cAMP binding protein, Epac, raises the question of its role in Ca²⁺ signalling in cardiac myocytes. In this study, we analysed the effects of Epac activation on Ca²⁺ handling by using confocal microscopy in isolated adult rat cardiomyocytes. [Ca²⁺]_i transients were evoked by electrical stimulation and Ca²⁺ sparks were measured in quiescent myocytes. Epac was selectively activated by the cAMP analogue 8‐(4‐chlorophenylthio)‐2′‐O‐methyladenosine‐3′,5′‐cyclic monophosphate (8‐CPT). Patch‐clamp was used to record the L‐type calcium current (I_Ca), and Western blot to evaluate phosphorylated ryanodine receptor (RyR). [Ca²⁺]_i transients were slightly reduced by 10 μm 8‐CPT (F/F₀: decreased from 4.7 ± 0.5 to 3.8 ± 0.4, P < 0.05), an effect that was boosted when cells were previously infected with an adenovirus encoding human Epac. I_Ca was unaltered by Epac activation, so this cannot explain the decreased [Ca²⁺]_i transients. Instead, a decrease in the sarcoplasmic reticulum (SR) Ca²⁺ load underlies the decrease in the [Ca²⁺]_i transients. This decrease in the SR Ca²⁺ load was provoked by the increase in the SR Ca²⁺ leak induced by Epac activation. 8‐CPT significantly increased Ca²⁺ spark frequency (Ca²⁺ sparks s⁻¹ (100 μm)⁻¹: from 2.4 ± 0.6 to 6.9 ± 1.5, P < 0.01) while reducing their amplitude (F/F₀: 1.8 ± 0.02 versus 1.6 ± 0.01, P < 0.001) in a Ca²⁺/calmodulin kinase II (CaMKII)‐dependent and PKA‐independent manner. Accordingly, we found that Epac increased RyR phosphorylation at the CaMKII site. Altogether, our data reveal a new signalling pathway by which cAMP governs Ca²⁺ release and signalling in cardiac myocytes.

Uromodulin is the most abundant protein in the urine. It is exclusively produced by renal epithelial cells and it plays key roles in kidney function and disease. Uromodulin mainly exerts its function as an extracellular matrix whose assembly depends on a conserved, specific proteolytic cleavage leading to conformational activation of a Zona Pellucida (ZP) polymerisation domain. Through a comprehensive approach, including extensive characterisation of uromodulin processing in cellular models and in specific knock-out mice, we demonstrate that the membrane-bound serine protease hepsin is the enzyme responsible for the physiological cleavage of uromodulin. Our findings define a key aspect of uromodulin biology and identify the first in vivo substrate of hepsin. The identification of hepsin as the first protease involved in the release of a ZP domain protein is likely relevant for other members of this protein family, including several extracellular proteins, as egg coat proteins and inner ear tectorins.

Mutations in α, β, or γ subunits of the epithelial sodium channel (ENaC) can downregulate ENaC activity and cause a severe salt-losing syndrome with hyperkalemia and metabolic acidosis, designated pseudohypoaldosteronism type 1 in humans. In contrast, mice with selective inactivation of αENaC in the collecting duct (CD) maintain sodium and potassium balance, suggesting that the late distal convoluted tubule (DCT2) and/or the connecting tubule (CNT) participates in sodium homeostasis. To investigate the relative importance of ENaC-mediated sodium absorption in the CNT, we used Cre-lox technology to generate mice lacking αENaC in the aquaporin 2–expressing CNT and CD. Western blot analysis of microdissected cortical CD (CCD) and CNT revealed absence of αENaC in the CCD and weak αENaC expression in the CNT. These mice exhibited a significantly higher urinary sodium excretion, a lower urine osmolality, and an increased urine volume compared with control mice. Furthermore, serum sodium was lower and potassium levels were higher in the genetically modified mice. With dietary sodium restriction, these mice experienced significant weight loss, increased urinary sodium excretion, and hyperkalemia. Plasma aldosterone levels were significantly elevated under both standard and sodium-restricted diets. In summary, αENaC expression within the CNT/CD is crucial for sodium and potassium homeostasis and causes signs and symptoms of pseudohypoaldosteronism type 1 if missing.

The E3 ubiquitin ligase NEDD4-2 (encoded by the Nedd4L gene) regulates the amiloride-sensitive epithelial Na⁺ channel (ENaC/SCNN1) to mediate Na⁺ homeostasis. Mutations in the human β/γENaC subunits that block NEDD4-2 binding or constitutive ablation of exons 6–8 of Nedd4L in mice both result in salt-sensitive hypertension and elevated ENaC activity (Liddle syndrome). To determine the role of renal tubular NEDD4-2 in adult mice, we generated tetracycline-inducible, nephron-specific Nedd4L KO mice. Under standard and high-Na⁺ diets, conditional KO mice displayed decreased plasma aldosterone but normal Na⁺/K⁺ balance. Under a high-Na⁺ diet, KO mice exhibited hypercalciuria and increased blood pressure, which were reversed by thiazide treatment. Protein expression of βENaC, γENaC, the renal outer medullary K⁺ channel (ROMK), and total and phosphorylated thiazide-sensitive Na⁺Cl^– cotransporter (NCC) levels were increased in KO kidneys. Unexpectedly, Scnn1a mRNA, which encodes the αENaC subunit, was reduced and proteolytic cleavage of αENaC decreased. Taken together, these results demonstrate that loss of NEDD4-2 in adult renal tubules causes a new form of mild, salt-sensitive hypertension without hyperkalemia that is characterized by upregulation of NCC, elevation of β/γENaC, but not αENaC, and a normal Na⁺/K⁺ balance maintained by downregulation of ENaC activity and upregulation of ROMK.

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.