Sylvain Richard

High Frequency–Induced Upregulation of Human Cardiac Calcium Currents

BACKGROUNDnIn mammalian heart cells, Ca2+ influx through voltage-gated L-type Ca2+ channels can be upregulated by high rates of stimulation. We have investigated this important adaptive regulation in human cardiomyocytes.nnnMETHODS AND RESULTSnUsing the whole-cell patch-clamp technique, we found a high frequency-induced upregulation (HFIUR) of the dihydropyridine-sensitive L-type Ca2+ current (ICa) in human cardiomyocytes. ICa was potentiated in a graded manner with increasing rates of stimulation between 0.3 and 5 Hz. Both moderate increase of ICa peak amplitude and marked slowing of current decay contributed to large increases of Ca2+ influx (up to 80%). The maximal potentiation of ICa was reached rapidly after the change in the rate of stimulation (no more than a few seconds). Beta-Adrenergic stimulation of the cells by isoproterenol (1 micromol/L), which is well known to induce a slow (approximately 1 minute) cAMP-mediated potentiation of ICa, could enhance (when present) or promote (when absent) the HFIUR of ICa. As a consequence, the increasing effect of isoproterenol on Ca2+ influx through Ca2+ channels was dependent on the rate of stimulation. HFIUR of ICa was altered in patients with ejection fraction lower than 40% and in patients pretreated with Ca2+ antagonists or beta-blockers.nnnCONCLUSIONSnUpregulation of Ca2+ entry through voltage-gated Ca2+ channels by high rates of beating may be involved in the frequency-dependent regulation of contractility (Bowditch staircase) of the human heart. This process, which is highly sensitive to beta-adrenergic stimulation, may be crucial in adaptation to exercise and stress.

Mineralocorticoid Receptor Antagonism Prevents the Electrical Remodeling That Precedes Cellular Hypertrophy After Myocardial Infarction

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 Ca2+ current (ICa) and a decrease in K+ transient outward current (Ito) underlay this effect. Consistently, the relative expression of mRNA coding for the Ca2+ channel &agr;1C subunit (Cav1.2) increased, and that of the K+ channel Kv4.2 subunit decreased. Three weeks after MI, AP prolongation endured, whereas cellular hypertrophy developed. ICa density, Cav1.2, and Kv4.2 mRNA levels regained control levels, but Ito 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.

Increased Ca2+ Sensitivity of the Ryanodine Receptor Mutant RyR2R4496C Underlies Catecholaminergic Polymorphic Ventricular Tachycardia

Cardiac ryanodine receptor (RyR2) mutations are associated with autosomal dominant catecholaminergic polymorphic ventricular tachycardia, suggesting that alterations in Ca2+ handling underlie this disease. Here we analyze the underlying Ca2+ release defect that leads to arrhythmia in cardiomyocytes isolated from heterozygous knock-in mice carrying the RyR2R4496C mutation. RyR2R4496C−/− littermates (wild type) were used as controls. [Ca2+]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), [Ca2+]i transients and sarcoplasmic reticulum Ca2+ load were similar in wild-type and RyR2R4496C cells. However, paced RyR2R4496C ventricular myocytes presented abnormal Ca2+ release during the diastolic period, viewed as Ca2+ waves, consistent with the occurrence of delayed afterdepolarizations. The occurrence of this abnormal Ca2+ release was enhanced at faster stimulation rates and by &bgr;-adrenergic stimulation, which also induced triggered activity. Spontaneous Ca2+ sparks were more frequent in RyR2R4496C myocytes, indicating increased RyR2R4496C activity. When permeabilized cells were exposed to different cytosolic [Ca2+]i, RyR2R4496C showed a dramatic increase in Ca2+ sensitivity. Isoproterenol increased [Ca2+]i transient amplitude and Ca2+ spark frequency to the same extent in wild-type and RyR2R4496C cells, indicating that the &bgr;-adrenergic sensitivity of RyR2R4496C 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 RyR2R4496C mutation is attributable to the increased Ca2+ sensitivity of RyR2R4496C, which induces diastolic Ca2+ release and lowers the threshold for triggered activity.

Effects of Amiodarone and Dronedarone on Voltage‐Dependent Sodium Current in Human Cardiomyocytes

Introduction: Amiodarone (AM) is a highly effective antiarrhythmic agent used in the management of both atrial and ventricular arrhythmias. Its noniodinated analogue dronedarone (SR) may have fewer side effects than AM. In this study, we compared the effects of AM and SR on the sodium current INa in human atrial myocytes.

Vascular Effects of Calcium Channel Antagonists: New Evidence

Calcium channel antagonists have a well-established role in the management of cardiovascular diseases. L-type calcium channels in vascular cells are a key therapeutic target in hypertension and are the preferred molecular target of the initial calcium channel antagonists. However, third-generation dihydropyridine (DHP) calcium channel antagonists, including manidipine, nilvadipine, benidipine and efonidipine, appear to have effects in addition to blockade of the L-type calcium channel. Voltage-gated calcium channels are widely expressed throughout the cardiovascular system. They constitute the main route for calcium entry, essential for the maintenance of contraction. Cardiac and vascular cells predominantly express L-type calcium channels. More recently, T-type channels have been discovered, and there is emerging evidence of their significance in the regulation of arterial resistance. A lack of functional expression of L-type channels in renal efferent arterioles may be consistent with an important role of T-type channels in the regulation of efferent arteriolar tone. Although the exact role of T-type calcium channels in vascular beds remains to be determined, they could be associated with gene-activated cell replication and growth during pathology. The three major classes of calcium channel antagonists are chemically distinct, and exhibit different functional effects depending on their biophysical, conformation-dependent interactions with the L-type calcium channel. The DHPs are more potent vasodilators, and generally have less cardiode-pressant activity than representatives of other classes of calcium channel antagonist such as diltiazem (a phenylalkylamine) and verapamil (a benzothiazepine). In contrast to older calcium channel antagonists, the newer DHPs, manidipine, nilvadipine, benidipine and efonidipine, dilate not only afferent but also efferent renal arterioles, a potentially beneficial effect that may improve glomerular hypertension and provide renoprotection. The underlying mechanisms for the heterogenous effects of calcium channel antagonists in the renal microvasculature are unclear. A credible hypothesis suggests a contribution of T-type calcium channels to efferent arteriolar tone, and that manidipine, nilvadipine and efonidipine inhibit both L and T-type channels. However, other mechanisms, including an effect on neuronal P/Q-type calcium channels (recently detected in arterioles), the microheterogeneity of vascular beds, and other types of calcium influx may also play a role. This article presents recent data about the expression and physiological role of calcium channels in arteries and the molecular targets of the calcium channel antagonists, particularly those exhibiting distinct renovascular effects.

Regulation of the frequency‐dependent facilitation of L‐type Ca2+ currents in rat ventricular myocytes.

1. An increase in the rate of stimulation induces an augmentation of L‐type Ca2+ currents (ICa) and concomitant slowing of current decay in rat ventricular cells. This facilitation is quasi immediate (1‐3 s), graded with the rate of stimulation, and occurs only from negative holding potentials. We investigated this effect using trains of stimulation at 1 Hz and the whole‐cell patch‐clamp technique (18‐22 degrees C). 2. The decay of ICa is normally bi‐exponential and comprises fast and slow current components (ICa,fc and ICa,sc, respectively). Facilitation of ICa was observed only when ICa,fc was predominant. 3. Facilitation developed during the run‐up of ICa with the interconversion of ICa,sc into ICa,fc, and vanished during the run‐down of ICa with the loss of ICa,fc.Ni2+ (300 microM) and nifedipine (1 microM) suppressed facilitation owing to the preferential inhibition of ICa,fc. 4. Facilitation of ICa was not altered (when present) or favoured (when absent) by the cAMP‐dependent phosphorylation of Ca2+ channels promoted by isoprenaline or by intracellular application of cAMP or of the catalytic subunit of protein kinase A (C‐sub). A similar effect was observed when the dihydropyridine agonist Bay K 8644 was applied. In both cases, facilitation was linked to a preferential increase of ICa,fc. 5. Following intracellular application of inhibitors of protein kinase A in combination with a non‐hydrolysable ATP analogue, ICa consisted predominantly of ICa,sc and no facilitation was observed. The calmodulin antagonist naphthalenesulphonamide had no effect on facilitation. 6. When Bay K 8644 was applied in combination with isoprenaline, cAMP or C‐sub, the decay of ICa was slowed with the predominant development of ICa,sc, and facilitation of ICa was nearly abolished. Facilitation also depended on extracellular Ca2+, and was suppressed when Ba2+ replaced Ca2+ as the permeating ion. 7. When no EGTA was included in the patch pipette, facilitation was not further enhanced but a use‐dependent decrease of ICa frequently occurred. When BAPTA was used in place of EGTA, the rate of inactivation of ICa was reduced and facilitation was abolished. 8. In conclusion, the facilitation of ICa that reflects a voltage‐driven interconversion of ICa,fc into ICa,sc is also regulated by Ca2+ and by cAMP‐dependent phosphorylation. The presence of the gating pattern typified by ICa,fc is required. Ca2+ may exert its effect near the inner pore of the Ca2+ channel protein and control the distribution between the closed states of the two gating pathways.

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 Ca2+ currents (ICa) correlates with plasma aldosterone levels. Larger values of ICa are associated with high aldosterone concentrations in SL models, whereas smaller values of ICa were observed in the SR model. Neither the time‐ nor the voltage‐dependent properties of ICa varied measurably. In parallel, we determined whether modulation of ICa by blood concentration of aldosterone has a major physiological impact on the excitation–contraction coupling of the cardiomyocytes. Action potential duration, [Ca2+]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 ICa in mouse cardiomyocytes. This regulation has important consequences for excitation–contraction coupling and, potentially, for other Ca2+‐regulated functions in cardiomyocytes.

Voltage-gated Ca2+ currents in the human pathophysiologic heart: a review.

Abstract The L-type Ca2+ current (ICa-L) plays a key role in the cardiac excitation-contraction (E-C) coupling. Thus, it is a major target for many transmitters and hormones modulating cardiac function and, therefore, for pharmacological drugs to regulate inotropy. Ca2+ (and other) ion currents are commonly studied in animal tissues for practical reasons. Investigations in human cardiomyocytes started extensively only ten years ago with the development of patch-clamp techniques, enzymatic cell dissociation procedures, and surgical techniques. These studies have already provided valuable information concerning the nature, biophysics, pharmacology and regulation of human cardiac ionic currents in normal and diseased tissues. Interesting advances have been made to understand the role of ICa-L in the development of chronic atrial fibrillation (AF). Alterations of single channel activity and regulation of macroscopic ICa-L have also been found in heart failure (HF), although some of the data are divergent and puzzling. The T-type Ca2+ current (ICa-T) has never been recorded in human cardiomyocytes. After a rapid overview of the basic properties of human cardiac Ca2+ currents, we focus on selected aspects of pathophysiology that are still unsolved.

Inhibition of T-type calcium currents by dihydropyridines in mouse embryonic dorsal root ganglion neurons.

The effects of dihydropyridines (DHPs) normally considered to be specific for L-type calcium channels were studied on the T-type Ca channel current of acutely isolated dorsal root ganglion (DRG) neurons taken from 13-day-old (E13) mouse embryos. Potent but reversible inhibitory effects of the DHP nicardipine were found in the micromolar range. For example, 5 microM nicardipine suppressed 93 +/- 5% of T-type currents. In comparison, other classical DHPs such as nifedipine, PN 200-110 and nitrendipine had only weak effects (less than 20% inhibition) at the same concentration. The inhibition by nicardipine was found slightly to be voltage dependent and the drug induced a leftward shift in the steady-state inactivation. The DHP agonist (-)-Bay K 8644, which dramatically increased the L-type current, weakly decreased T-type Ca currents (17 +/- 8% at 5 microM). In conclusion, neuronal T-type Ca channels may be potential targets for some dihydropyridines. This property is not only a feature of the central nervous system (J. Physiol., 412 (1989) 181-195) and can be extended to peripheral neurons.

Coexpression of theβ2 subunit does not induce voltage-dependent facilitation of the class C L-type Ca channel

Voltage-dependent facilitation of L-type Ca2+ channels is an important regulatory mechanism by which excitable cells modulate Ca2+ entry during a train of action potentials. Expression of theαl andβ subunits of theα1C Ca2+ channel is necessary and sufficient to reproduce this kind of facilitation inXenopus oocytes. Here we show that, by expressing theα1C together with differentβ subunits in oocytes, theβ1,β3 andβ4, but not theβ2 subunits are permissive for Ca2+ channel facilitation. The poor facilitation observed in rat ventricular cells, together with the presence of theβ2 subunit mRNA, suggest thatβ2 may be theβ subunit associated with functional cardiac L-type Ca2+ channels.