Aldosterone increases T-type calcium channel expression and in vitro beating frequency in neonatal rat cardiomyocytes

Objective: Although aldosterone has been implicated in the pathogenesis of cardiac hypertrophy and heart failure, its cellular mechanism of action on cardiomyocyte function is not yet completely elucidated. This study was designed to investigate the effect of aldosterone on calcium channel expression and cardiomyocyte contraction frequency. Methods: Cultured neonatal rat ventricular cardiomyocytes were stimulated in vitro with 1 μmol/L aldosterone for 24 h. Calcium currents were then measured with the patch clamp technique, while calcium channel expression was assessed by real-time RT-PCR. Results: In the present study, we show that aldosterone increases Ca2+ currents by inducing channel expression. Indeed, aldosterone led to a substantial increase of L- and T-type Ca2+ current amplitudes, and we found a concomitant 55% increase of the mRNA coding for α1C and β2 subunits of cardiac L channels. Although T-type currents were relatively small under control conditions, they increased 4-fold and T channel α1H isoform expression rose in the same proportion after aldosterone treatment. Because T channels have been implicated in the modulation of membrane electrical activity, we investigated whether aldosterone affects the beating frequency of isolated cardiomyocytes. In fact, aldosterone dose-dependently increased the spontaneous beating frequency more than 4-fold. This effect of aldosterone was prevented by actinomycin D and spironolactone and reduced by RU486, suggesting a mixed mineralocorticoid/glucocorticoid receptor-dependent transcriptional mechanism. Moreover, inhibition of T currents with Ni2+ or mibefradil significantly reduced beating frequency towards control values, while conditions affecting L-type currents completely blocked contractions. Conclusion: Aldosterone modulates the expression of cardiac voltage-operated Ca2+ channels and accelerates beating in cultured neonatal rat ventricular myocytes. This chronotropic action of aldosterone appears to be linked to increased T channel activity and could contribute to the deleterious effect of an excess of this steroid in vivo on cardiac functio

Aldosterone Upregulates Ca 2+

International audienceAbstract —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 Ca 2+ 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 of I Ca significantly. This “long-term” aldosterone treatment had no significant effects on the kinetics and voltage dependence of I Ca inactivation. Moreover, no demonstrable influence of aldosterone on I Ca could 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 in I Ca density. 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 in I Ca . Therefore, the long latency I Ca stimulation effect of aldosterone might result from an increased channel expression. We suggest that this genomic action contributes to the increased I Ca observed during cardiac remodeling

Ca2+ Controls Functional Expression of the Cardiac K+ Transient Outward Current via the Calcineurin Pathway

International audienceThe transient outward K+ current (Ito) modulates transmembrane Ca2+ influx into cardiomyocytes, which, in turn, might act on Ito. Here, we investigated whether Ca2+ modifies functional expression of Ito. Whole-cell Ito were recorded using the patch clamp technique in single right ventricular myocytes isolated from adult rats and incubated for 24 h at 37 degrees C in a serum-free medium containing various Ca2+ concentrations ([Ca2+]o). Increasing the [Ca2+]o from 0.5 to 1.0 and 2.5 mM produced a gradual decrease in Ito density without change in current kinetics. Quantitativereverse transcriptase-PCR showed that a decrease of the Kv4.2 mRNA could account for this decrease. In the acetoxymethyl ester form of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM)-loaded myocytes (a permeant Ca2+ chelator), Ito density increased significantly when cells were exposed for 24 h to either 1 or 2.5 mM [Ca2+]o. Moreover, 24-h exposure to the Ca2+ channel agonist, Bay K8644, in 1 mM [Ca2+]o induced a decrease in Ito density, whereas the Ca2+ channel antagonist, nifedipine, blunted Ito decrease in 2.5 mM [Ca2+]o. The decrease of Ito in 2.5 mM [Ca2+]o was also prevented by co-incubation with either the calmodulin inhibitor W7 or the calcineurin inhibitors FK506 or cyclosporin A. Furthermore, in myocytes incubated for 24 h with 2.5 mM [Ca2+]o, calcineurin activity was significantly increased compared with 1 mM [Ca2+]o. Our data suggest that modulation of [Ca2+]i via L-type Ca2+ channels, which appears to involve the Ca2+/calmodulin-regulated protein phosphatase calcineurin, down-regulates the functional expression of Ito. This effect might be involved in many physiological and pathological modulations of Ito channel expression in cardiac cells, as well other cell types

Molecular Dynamics of the Sodium Channel Pore Vary with Gating: Interactions between P-Segment Motions and Inactivation

International audienceDisulfide trapping studies have revealed that the pore-lining (P) segments of voltage-dependent sodium channels undergo sizable motions on a subsecond time scale. Such motions of the pore may be necessary for selective ion translocation. Although traditionally viewed as separable properties, gating and permeation are now known to interact extensively in various classes of channels. We have investigated the interaction of pore motions and voltage-dependent gating in μ1 sodium channels engineered to contain two cysteines within the P segments. Rates of catalyzed internal disulfide formation ( k SS ) were measured in K1237C+W1531C mutant channels expressed in oocytes. During repetitive voltage-clamp depolarizations, increasing the pulse duration had biphasic effects on the k SS , which first increased to a maximum at 200 msec and then decreased with longer depolarizations. This result suggested that occupancy of an intermediate inactivation state ( I M ) facilitates pore motions. Consistent with the known antagonism between alkali metals and a component of slow inactivation, k SS varied inversely with external [Na + ] o . We examined the converse relationship, namely the effect of pore flexibility on gating, by measuring recovery from inactivation in Y401C+E758C (YC/EC) channels. Under oxidative conditions, recovery from inactivation was slower than in a reduced environment in which the spontaneous YC/EC cross-link is disrupted. The most prominent effects were slowing of a component with intermediate recovery kinetics, with diminution of its relative amplitude. We conclude that occupancy of an intermediate inactivation state facilitates motions of the P segments; conversely, flexibility of the P segments alters an intermediate component of inactivation

Regional Alteration of the Transient Outward Current in Human Left Ventricular Septum During Compensated Hypertrophy

International audienceBackground A large calcium-insensitive transient outward current ( I to ) has been recorded in atria, left ventricular (LV) free wall, and right ventricular septal subendocardium of the human heart. Recent studies suggested a major contribution of this current to the electrical heterogeneity of the heart. However, no data have been reported on the distribution of I to density within the LV septal wall from compensated human LV hypertrophy. Methods and Results Microelectrode and patch-clamp techniques were used to record action potentials and I to in myocytes isolated from superficial (<3 mm deep) and deep (3 to 6 mm deep) layers of LV septum from patients with aortic stenosis and compensated LV hypertrophy. Subendocardial specimens were also obtained from undiseased donor hearts. In none of the superficial subendocardial cells from diseased hearts was a macroscopic I to recorded (n=42), whereas in cells from the same location from donor hearts, a typical I to was clearly present, with a peak density of 5.88±0.78 pA/pF at +60 mV (n=4). However, in deep layers from patients with compensated LV hypertrophy, macroscopic I to was present, with a peak density of 10.50±2.58 pA/pF at +60 mV (n=4). The absence of I to in superficial septal cells from hypertrophied hearts was not due to a divalent cation–related shift of the current kinetics. Instead, extracellular Ca 2+ removal induced an I to -like current, possibly carried by K + ions, with a peak density of 30.7±2.6 pA/pF at +60 mV (n=29). However, its magnitude, kinetics, and pharmacological characteristics did not allow identification of this current as the usual I to . Conclusions Both topography and pathology can be major modulating factors of the regional distribution of I to density in human LV septum. Therefore, they may play a prominent role in determining electrical gradients within this region from which the early depolarization vectors start and the left-to-right activation sequence of the interventricular septum proceeds

Extracellular K +

International audienceBackground —In human ventricular cells, the inwardly rectifying K + current ( I K1 ) is very similar to that of other mammalian species, but detailed knowledge about the K + -dependent distribution of open and blocked states during rectification and about the K + -dependent modulation of inactivation on hyperpolarization is currently lacking. Methods and Results —We used the whole-cell patch-clamp technique to record I K1 in myocytes isolated from subendocardial layers of left ventricular septum from patients with nonfailing hearts with aortic stenosis and cardiac hypertrophy who were undergoing open-heart surgery. Outward currents were very small at voltages positive to the reversal potential but increased at high external [K + ]. Chord conductance measurements and kinetic analyses allowed us to estimate the proportion of channels in the open state and of those showing either slow unblock or instantaneous unblock (the so-called slow or instantaneous “activation”) on hyperpolarization: the distribution in the individual states was dependent on external [K + ]. The proportion of channels unblocking slowly was greater than that of channels unblocking instantaneously on hyperpolarization from the plateau voltage range. Hence, because of the previously reported link between the presence of highly protonated blocking molecules and slow unblock kinetics, it is suggested that high cellular concentrations of spermine may account for the low outward current density recorded in these cells. The current decrease observed on extended hyperpolarization was significantly relieved by an increase in external [K + ]. Conclusions —The pattern of I K1 current alterations observed in the present model of human ventricular hypertrophy might favor enhanced excitability and underlie ventricular arrhythmias, possibly via increased intracellular polyamine levels

Slow inward current in single cells isolated from adult human ventricles

International audienceCharacteristics of the slow inward current (Isi) in human ventricular myocytes isolated from septal specimens obtained in patients undergoing corrective cardiac surgery were studied using the whole-cell clamp method. A first series of experiments was performed under normal standard superfusion. Clamping from -60 mV evoked an inward current with a threshold at about -35 mV, a maximum around +10 mV and an apparent reversal potential at about +55 mV. No overlapping transient or background outward currents were detected in the -60 to +30 mV potential range, but time-dependent and steady-state outward currents were elicited at potentials above +30 mV. An overlap of steady-state activation and inactivation curves was present between -30 and +10 mV and a slight relief from inactivation was observed for voltages positive to +10 mV. The time course of inactivation consisted of fast and slow phases with time constants differing by a factor of eight. Slow time constants of inactivation were shorter at potentials that elicited larger Isi, and longer at potentials inducing smaller Isi. Recovery from inactivation evolved slowly with 100% reactivation occurring in about 4000 ms. Switching the holding potential from -60 to -40 mV led to a reversible decline of Isi without any change of the decay time constants. Isi was significantly increased by 0.1 microM isoproterenol. Total or partial inhibition by inorganic (2 mM Mn2+, 3 mM Co2+, 1 mM Cd2+) and organic (1 microM methoxyverapamil, 5 microM diltiazem) calcium antagonists did not unmask any transient outward current. However, a consistent increase of Isi was reversibly observed with 3 mM 4-aminopyridine while using standard solutions. A second series of experiments carried out with K(+)- and Na(+)-free solutions did not demonstrate any significant change from data observed with standard solutions except a reduction of outward currents at steps above +30 mV and alteration of inactivation kinetics. In this experimental setting, 4-aminopyridine also increased Isi but to a lesser degree. We conclude that Isi, as compared to the outward currents, is dominant in the diseased human ventricular cells we have studied

A chloride current component induced by hypertrophy in rat ventricular myocytes

International audienceThe effect of hypertrophy on membrane currents of rat left ventricular myocytes was studied with the whole cell voltage-clamp method. We found that the slope of the total time-independent current density-voltage relationship was increased in hypertrophied cells. No change in the zero-current potential was observed. Surprisingly, the dominant time-independent current, the inward rectifier K+ current (measured as the Ba(2+)-sensitive current density) was unchanged. We therefore investigated the identity of the outwardly rectifying Ba(2+)-resistant current seen in the hypertrophied rat ventricular myocytes but not present in control cells. We found that this current 1) was not carried by monovalent cations, 2) was partially blocked by anthracene-9-carboxylic acid (9-AC), and 3) was sensitive to variations in extracellular Cl concentration. These findings are consistent with the current being carried at least partially by Cl-. The presence of an additional Cl(-)-dependent component in hypertrophied cells is supported by the actions of 9-AC on the measured action potentials (APs). 9-AC had no effect on control cells APs but prolonged hypertrophied cell APs. We conclude that a Cl- current component develops in hypertrophied rat heart cells. This component appears to shorten the AP duration and might thus provide protection from cardiac arrhythmias

Effects of aldosterone on transient outward K+ current density in rat ventricular myocytes

Aldosterone, a major ionic homeostasis regulator, might also regulate cardiac ion currents. Using the whole-cell patch-clamp technique, we investigated whether aldosterone affects the 4-aminopyridine-sensitive transient outward K+ current (Ito1).Exposure to 100 nm aldosterone for 48 h at 37 °C produced a 1.6-fold decrease in the Ito1 density compared to control myocytes incubated without aldosterone. Neither the time- nor voltage-dependent properties of the current were significantly altered after aldosterone treatment. RU28318 (1 μm), a specific mineralocorticoid receptor antagonist, prevented the aldosterone-induced decrease in Ito1 density.When myocytes were incubated for 24 h with aldosterone, concentrations up to 1 μm did not change Ito1 density, whereas L-type Ca2+ current (ICa,L) density increased. After 48 h, aldosterone caused a further increase in ICa,L. The delay in the Ito1 response to aldosterone might indicate that it occurs secondary to an increase in ICa,L.After 24 h of aldosterone pretreatment, further co-incubation for 24 h either with an ICa,L antagonist (100 nm nifedipine) or with a permeant Ca2+ chelator (10 μm BAPTA-AM) prevented a decrease in Ito1 density.After 48 h of aldosterone treatment, we observed a 2.5-fold increase in the occurrence of spontaneous Ca2+ sparks, which was blunted by co-treatment with nifedipine.We conclude that aldosterone decreases Ito1 density. We suggest that this decrease is secondary to the modulation of intracellular Ca2+ signalling, which probably arises from the aldosterone-induced increase in ICa,L. These results provide new insights into how cardiac ionic currents are modulated by hormones