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.

Cardiac ryanodine receptor (RyR2) mutations are associated with autosomal dominant catecholaminergic polymorphic ventricular tachycardia, suggesting that alterations in Ca ²⁺ handling underlie this disease. Here we analyze the underlying Ca ²⁺ release defect that leads to arrhythmia in cardiomyocytes isolated from heterozygous knock-in mice carrying the RyR2 ^R4496C mutation. RyR2 ^{R4496C−/−} littermates (wild type) were used as controls. [Ca ²⁺ ] _i transients were obtained by field stimulation in fluo-3–loaded cardiomyocytes and viewed using confocal microscopy. In our basal recording conditions (2-Hz stimulation rate), [Ca ²⁺ ] _i transients and sarcoplasmic reticulum Ca ²⁺ load were similar in wild-type and RyR2 ^R4496C cells. However, paced RyR2 ^R4496C ventricular myocytes presented abnormal Ca ²⁺ release during the diastolic period, viewed as Ca ²⁺ waves, consistent with the occurrence of delayed afterdepolarizations. The occurrence of this abnormal Ca ²⁺ release was enhanced at faster stimulation rates and by β-adrenergic stimulation, which also induced triggered activity. Spontaneous Ca ²⁺ sparks were more frequent in RyR2 ^R4496C myocytes, indicating increased RyR2 ^R4496C activity. When permeabilized cells were exposed to different cytosolic [Ca ²⁺ ] _i , RyR2 ^R4496C showed a dramatic increase in Ca ²⁺ sensitivity. Isoproterenol increased [Ca ²⁺ ] _i transient amplitude and Ca ²⁺ spark frequency to the same extent in wild-type and RyR2 ^R4496C cells, indicating that the β-adrenergic sensitivity of RyR2 ^R4496C cells remained unaltered. This effect was independent of protein expression variations because no difference was found in the total or phosphorylated RyR2 expression levels. In conclusion, the arrhythmogenic potential of the RyR2 ^R4496C mutation is attributable to the increased Ca ²⁺ sensitivity of RyR2 ^R4496C , which induces diastolic Ca ²⁺ release and lowers the threshold for triggered activity.

—Aldosterone is associated with the pathogenesis and progression of left ventricular hypertrophy and heart failure, independent of its relation with arterial blood pressure. However, little information exists about the possible influence of this mineralocorticoisteroid on cardiomyocyte electrical activity. The present study was designed to determine the role of aldosterone on whole-cell Ca²⁺current (I_Ca) in isolated adult rat ventricular myocytes using the patch-clamp technique. We found that incubation of cells with 1 μmol/L aldosterone for 24 hours increases the density ofI_Casignificantly. This “long-term” aldosterone treatment had no significant effects on the kinetics and voltage dependence ofI_Cainactivation. Moreover, no demonstrable influence of aldosterone onI_Cacould be detected during short-term exposure (up to 6 hours), under our experimental conditions. The classical aldosterone intracellular receptor antagonist spironolactone (250-fold excess) was able to blunt the aldosterone-induced increase inI_Cadensity. These effects were also observed with lower concentrations of aldosterone (10 and 100 nmol/L). Moreover, inhibitors of transcription (actinomycin D, 5 μg/mL) and protein synthesis (cycloheximide, 20 μg/mL) prevented the aldosterone-dependent increase inI_Ca. Therefore, the long latencyI_Castimulation effect of aldosterone might result from an increased channel expression. We suggest that this genomic action contributes to the increasedI_Caobserved during cardiac remodeling.

Background— Cardiac hypertrophy underlies arrhythmias and sudden death, for which mineralocorticoid receptor (MR) activity has recently been implicated. We sought to establish the sequence of ionic events that link the initiating insult and MR to hypertrophy development. Methods and Results— Using whole-cell, patch-clamp and quantitative reverse transcription–polymerase chain reaction techniques on right ventricular myocytes of a myocardial infarction (MI) rat model, we examined the cellular response over time. One week after MI, no sign of cellular hypertrophy was found, but action potential duration (APD) was lengthened. Both an increase in Ca ²⁺ current ( I _Ca ) and a decrease in K ⁺ transient outward current ( I _to ) underlay this effect. Consistently, the relative expression of mRNA coding for the Ca ²⁺ channel α1C subunit (Ca _v 1.2) increased, and that of the K ⁺ channel K _v 4.2 subunit decreased. Three weeks after MI, AP prolongation endured, whereas cellular hypertrophy developed. I _Ca density, Ca _v 1.2, and K _v 4.2 mRNA levels regained control levels, but I _to density remained reduced. Long-term treatment with RU28318, an MR antagonist, prevented this electrical remodeling. In a different etiologic model of abdominal aortic constriction, we confirmed that APD prolongation and modifications of ionic currents precede cellular hypertrophy. Conclusions— Electrical remodeling, which is triggered at least in part by MR activation, is an initial, early cellular response to hypertrophic insults.