Zinc modulation of basal and ß-adrenergically stimulated L-type Ca2+ current in rat ventricular cardiomyocytes: consequences in cardiac diseases.

Zinc exists in biological systems as bound and histochemically reactive free Zn(2+) in the nanomolar range. Zinc is required as either structural or catalytic component for a large number of enzymes. It also modulates current passage through many ion channels. Here, we reinvestigated the effects of extracellular and intracellular Zn(2+) on the L-type Ca(2+) current (I (CaL)) and its modulation by ß-adrenergic stimulation in rat ventricular cardiomyocytes. In the absence of Ca(2+) ions, Zn(2+) could permeate through the L-type channel at much lower concentrations and at a more positive voltage range, but with a lower permeability than Ca(2+). In the presence of Ca(2+), extracellular Zn(2+) demonstrated strong bimodal inhibitory effects on the I (CaL), with half-inhibition occurring around 30 nM, i.e., in the range of concentrations found in the plasma. Intracellular Zn(2+) also significantly inhibited the I (CaL) with a half-inhibitory effect at 12.7 nM. Moreover, ß-adrenergic stimulation was markedly reduced by intracellular Zn(2+) at even lower concentrations (<1 nM) as a consequence of Zn(2+)-induced inhibition of the adenylyl cyclase. All these effects appeared independent of redox variations and were not affected by dithiothreitol. Thus, both basal intracellular and extracellular Zn(2+) modulate transmembrane Ca(2+) movements and their regulation by ß-adrenergic stimulation. Considering that, in many pathological situations, including diabetes, the extracellular Zn(2+) concentration is reduced and the intracellular one is increased, our results help to explain both Ca(2+) overload and marked reduction in the ß-adrenergic stimulation in these diseases.

Effects of high-altitude exercise training on contractile function of rat skinned cardiomyocyte.

OBJECTIVE: Previous studies have questioned whether there is an improved cardiac function after high-altitude training. Accordingly, the present study was designed specifically to test whether this apparent blunted response of the whole heart to training can be accounted for by altered mechanical properties at the cellular level. METHODS: Adult rats were trained for 5 weeks under normoxic (N, NT for sedentary and trained animals, respectively) or hypobaric hypoxic (H, HT) conditions. Cardiac morphology and function were evaluated by echocardiography. Calcium Ca2+ sensitivity of the contractile machinery was estimated in skinned cardiomyocytes isolated from the left ventricular (LV) sub-epicardium (Epi) and sub-endocardium (Endo) at short and long sarcomere lengths (SL). RESULTS: Cardiac remodelling was harmonious (increase in wall thickness with chamber dilatation) in NT rats and disharmonious (hypertrophy without chamber dilatation) in HT rats. Contrary to NT rats, HT rats did not exhibit enhancement in global cardiac performance evaluated by echocardiography. Stretch- dependent Ca2+ sensitization of the myofilaments (cellular index of the Frank-Starling mechanism) increased from Epi to Endo in N rats. Training in normoxic conditions further increased this stretch-dependent Ca2+ sensitization. Chronic hypoxia did not significantly affect myofibrilar Ca2+ sensitivity. In contrast, high-altitude training decreased Ca2+ sensitivity of the myofilaments at both SL, mostly in Endo cells, resulting in a loss of the transmural gradient of the stretch-dependent Ca2+ sensitization. Expression of myosin heavy chain isoforms was affected both by training and chronic hypoxia but did not correlate with mechanical data. CONCLUSIONS: Training at sea level increased the transmural gradient of stretch-dependent Ca2+ sensitization of the myofilaments, accounting for an improved Frank-Starling mechanism. High-altitude training depressed myofilament response to Ca2+, especially in the Endo layer. This led to a reduction in this transmural gradient that may contribute to the lack of improvement in LV function via the Frank-Starling mechanism.

Defects in ryanodine receptor calcium release in skeletal muscle from post-myocardial infarct rats.

Defective calcium (Ca2+) signaling and impaired contractile function have been observed in skeletal muscle secondary to impaired myocardial function. However, the molecular basis for these muscle defects have not been identified. In this study, we evaluated the alterations of the ryanodine-sensitive Ca2+ release channels (RyR1) by analyzing global and local Ca2+ signaling in a rat postmyocardial infarction (PMI) model of myocardial overload. Ca2+ transients, measured with multiphoton imaging in individual fibers within a whole extensor digitorum longus (EDL) muscle, exhibited significantly reduced amplitude and a prolonged time course in PMI. Spatio-temporal properties of spontaneous Ca2+ sparks in fibers isolated from PMI EDL muscles were also significantly altered. In addition, RyR1 from PMI skeletal muscles were PKA-hyperphosphorylated and depleted of the FK506 binding protein (FKBP12). These data show that PMI skeletal muscles exhibit altered local Ca2+ signaling, associated with hyperphosphorylation of RyR1. The observed changes in Ca2+ signaling may contribute to defective excitation-contraction coupling in muscle that can contribute to the reduced exercise capacity in PMI, out of proportion to the degree of cardiac dysfunction.

The p42/44mitogen-activated protein kinase inhibitor PD 98059, but not U 0126, increases a K+ current in cardiomyocytes.

1. The effects of the mitogen-activated protein kinase (MAPK) inhibitors PD 98059 and U 0126, useful tools to investigate MAPK involvement in intracellular signal transduction pathways, were assessed on cardiomyocytes. 2. In rat freshly isolated ventricular myocytes, under current-clamp conditions, PD 98059 (40 micro mol/L) shortened the action potential. Under whole-cell patch-clamp, this compound slowly induced a fast activating sustained outward K+ current that was sensitive to 1 mmol/L Ba2+, 100 micro mol/L Gd3+, 3 mmol/L 4-aminopyridine and 100 micro mol/L tetracain. The PD 98059-induced current was prevented by 40 micro mol/L AACOCF3, a cytosolic phospholipase A2 inhibitor. 3. U 0126 (1 micro mol/L), a recently developed highly potent p42/44 MAPK inhibitor, did not alter K+ currents. 4. PD 98059, but not U 0126, increased arachidonic acid content, probably as a consequence of its reported cyclo-oxygenase inhibitory effect. 5. These observations indicate that PD 98059 activates a TREK-1 like current. Thus, this MAPK inhibitor has to be used with caution because alterations in cell metabolism can be secondary to changes in electrophysiological behaviour.

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

The L-type Ca2+ current (I(Ca-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 I(Ca-L) in the development of chronic atrial fibrillation (AF). Alterations of single channel activity and regulation of macroscopic I(Ca-L) have also been found in heart failure (HF), ugh some of the data are divergent and puzzling. The T-type Ca2+ current (I(Ca-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.

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

1. 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 (I(to1)). 2. Exposure to 100 nM aldosterone for 48 h at 37 degrees C produced a 1.6-fold decrease in the I(to1) 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 microM), a specific mineralocorticoid receptor antagonist, prevented the aldosterone-induced decrease in I(to1) density. 3. When myocytes were incubated for 24 h with aldosterone, concentrations up to 1 microM did not change I(to1) density, whereas L-type Ca(2+) current (I(Ca,L)) density increased. After 48 h, aldosterone caused a further increase in I(Ca,L). The delay in the I(to1) response to aldosterone might indicate that it occurs secondary to an increase in I(Ca,L). 4. After 24 h of aldosterone pretreatment, further co-incubation for 24 h either with an I(Ca,L) antagonist (100 nM nifedipine) or with a permeant Ca(2+) chelator (10 microM BAPTA-AM) prevented a decrease in I(to1) density. 5. After 48 h of aldosterone treatment, we observed a 2.5-fold increase in the occurrence of spontaneous Ca(2+) sparks, which was blunted by co-treatment with nifedipine. 6. We conclude that aldosterone decreases I(to1) density. We suggest that this decrease is secondary to the modulation of intracellular Ca(2+) signalling, which probably arises from the aldosterone-induced increase in I(Ca,L). These results provide new insights into how cardiac ionic currents are modulated by hormones.

Heart failure after myocardial infarction: altered excitation-contraction coupling.

BACKGROUND: Heart failure (HF) frequently follows the occurrence of myocardial infarction (MI). Questions about how HF develops and what cellular defects contribute to this dysfunction led to this study. Methods and Results-- MI was induced in rats by coronary artery ligation. Clinical examination of the post-MI (PMI) surviving animals indicated that they were in overt HF by all measures. Cellular examination of the cardiomyocytes by patch-clamp and confocal [Ca(2+)](i) imaging methods indicated that cellular function was significantly compromised. At the single-cell level, [Ca(2+)](i) transient amplitudes were reduced and contractions were decreased and slowed, although Ca(2+) current (I(Ca)) remained unchanged. The excitation-contraction coupling (ECC) gain function measured as Delta[Ca(2+)](i)/I(Ca) was significantly decreased. Ouabain, a cardiotonic steroid that blocks the Na(+),K(+)-ATPase and activates Ca(2+) entry via cardiac Na(+) channels, largely alleviated this defect. CONCLUSIONS: After MI, I(Ca) becomes less able to trigger release of Ca(2+) from the sarcoplasmic reticulum. This failure of ECC is a major factor contributing to the development of contractile dysfunction and HF in PMI animals. The improved ECC gain, enhanced Ca(2+) entry, and augmented Ca(2+) signaling due to cardiotonic steroids contribute to the beneficial effects of these agents.

Microtubule disruption by colchicine reversibly enhances calcium signaling in intact rat cardiac myocytes.

Using the whole-cell patch-clamp configuration in rat ventricular myocytes, we recently reported that microtubule disruption increases calcium current (I(Ca)) and [Ca(2+)](i) transient and accelerates their kinetics by adenylyl cyclase activation. In the present report, we further analyzed the effects of microtubule disruption by 1 micromol/L colchicine on Ca(2+) signaling in cardiac myocytes with intact sarcolemma. In quiescent intact cells, it is possible to investigate ryanodine receptor (RyR) activity by analyzing the characteristics of spontaneous Ca(2+) sparks. Colchicine treatment decreased Ca(2+) spark amplitude (F/F(0): 1.78+/-0.01, n=983, versus 1.64+/-0.01, n=1660, recorded in control versus colchicine-treated cells; P<0.0001) without modifying the sarcoplasmic reticulum Ca(2+) load and enhanced their time to peak (in ms: 6.85+/-0.09, n=1185, versus 7.33+/-0.13, n=1647; P<0.0001). Microtubule disruption also induced the appearance of Ca(2+) sparks in doublets. These alterations may reflect RyR phosphorylation. To further investigate Ca(2+) signaling in cardiac myocytes with intact sarcolemma, we analyzed [Ca(2+)](i) transient evoked by field stimulation. Cells were loaded with the fluorescence Ca(2+) indicator, Fluo-3 cell permeant, and stimulated at 1 HZ: [Ca(2+)](i) transient amplitude was greater and its decay was accelerated in colchicine-treated, field-stimulated myocytes. This effect is reversible. When colchicine-treated myocytes were placed in a colchicine-free solution for 30 minutes, tubulin was repolymerized into microtubules, as shown by immunofluorescence, and the increase in [Ca(2+)](i) transient was reversed. In summary, we demonstrate that microtubule disruption by colchicine reversibly modulates Ca(2+) signaling in cardiac cells with intact sarcolemma.

Adenosine 5'-triphosphate: a P2-purinergic agonist in the myocardium.

ATP, besides an intracellular energy source, is an agonist when applied to a variety of different cells including cardiomyocytes. Sources of ATP in the extracellular milieu are multiple. Extracellular ATP is rapidly degraded by ectonucleotidases. Today ionotropic P2X(1--7) receptors and metabotropic P2Y(1,2,4,6,11) receptors have been cloned and their mRNA found in cardiomyocytes. On a single cardiomyocyte, micromolar ATP induces nonspecific cationic and Cl(-) currents that depolarize the cells. ATP both increases directly via a G(s) protein and decreases Ca(2+) current. ATP activates the inward-rectifying currents (ACh- and ATP-activated K(+) currents) and outward K(+) currents. P2-purinergic stimulation increases cAMP by activating adenylyl cyclase isoform V. It also involves tyrosine kinases to activate phospholipase C-gamma to produce inositol 1,4,5-trisphosphate and Cl(-)/HCO(3)(-) exchange to induce a large transient acidosis. No clear correlation is presently possible between an effect and the activation of a given P2-receptor subtype in cardiomyocytes. ATP itself is generally a positive inotropic agent. Upon rapid application to cells, ATP induces various forms of arrhythmia. At the tissue level, arrhythmia could be due to slowing of electrical spread after both Na(+) current decrease and cell-to-cell uncoupling as well as cell depolarization and Ca(2+) current increase. In as much as the information is available, this review also reports analog effects of UTP and diadenosine polyphosphates.

Simultaneous activation of p38 MAPK and p42/44 MAPK by ATP stimulates the K+ current ITREK in cardiomyocytes.

Living cells exhibit multiple K(+) channel proteins; among these is the recently reported atypical two-pore domain K(+) channel protein TREK-1. Most K(+) currents are modulated by neurohormones and under various pathological conditions. Here, in rat ventricular cardiomyocytes using the whole-cell patch-clamp technique, we characterize for the first time a native TREK-1-like current (I(TREK)) that is activated by ATP, a purine agonist applied at a micromolar range. This current is sensitive to arachidonic acid, intracellular acidosis, and various K(+) current inhibitors. Reverse transcription-polymerase chain reaction reveals the presence of a TREK-1-like mRNA in rat cardiomyocytes that shows 93% identity with mouse TREK-1. ATP effects are greatly attenuated in the presence of arachidonic acid or HCO(-)(3)-induced intracellular acidosis. Using a series of inhibitors, we further demonstrate that the ATP-induced stimulation of I(TREK) implies the activation of cytosolic phospholipase A(2) and the release of arachidonic acid. These events require the simultaneous involvement of p38 MAPK and p42/44 MAPK, respectively, via a cAMP-dependent protein kinase and a tyrosine kinase pathway, whereas the two MAPKs conjugate to activate a mitogen- and stress-activated protein kinase (MSK-1). Our results thus demonstrate the occurrence of a TREK-1-like current in cardiac cells whose activation by purine agonists implies a dual-MAPK cytosolic pathway.

Is titin the length sensor in cardiac muscle? Physiological and physiopathological perspectives.

One of the most salient physiological characteristics of cardiac muscle is that a dilated heart pumps more vigorously, a phenomenon known as the Frank-Starling relationship (see Allen and Kentish, 1985). At least two cellular mechanisms participate in this phenomenon: the reduction of the interfilament lattice spacing which favors the formation of cross-bridges (Wang and Fuchs, 1995) and the increased affinity of troponin C (TnC) for calcium (Ca2+) (Babu et al., 1988). In the latter case, it has been established that TnC itself is not the length sensor (Moss et al., 1991). The intracellular structure(s) able to sense changes in cell length has always been challenged and is still not known. We previously observed on intact isolated cardiac cells that active tension is more closely related to passive tension than to sarcomere length per se (Cazorla et al., 1997). This might have some physiological implications in the working heart since we found that sub-epicardial cells are more supple than sub-endocardial cells. In the present work on skinned cells, we studied the relationship between different levels of passive tension (modulated by a mild trypsin digestion) and the shift in pCa50 of tension-pCa relations induced by a stretch of cells from 1.9 to 2.3 microns sarcomere length. A significant correlation was obtained between passive tension and the stretch-induced shift in pCa50, or stretch-sensitivity of the active force. These observations led us to assume that titin might play a role in sensing cell length to modulate the contractile activity. Besides, it is known that myocardial infarcted cells are less sensitive to stretch. We propose that, in such a rat model, alterations of titin might participate in heart failure.

gamma-aminobutyric acid type B receptors are expressed and functional in mammalian cardiomyocytes.

gamma-Hydroxybutyrate (GHB), an anesthetic adjuvant analog of gamma-aminobutyrate (GABA), depresses cell excitability in hippocampal neurons by inducing hyperpolarization through the activation of a prominent inwardly rectifying K(+) (Kir3) conductance. These GABA type B (GABA(B))-like effects are clearly shown at high concentrations of GHB corresponding to blood levels usually reached during anesthesia and are mimicked by the GABA(B) agonist baclofen. Recent studies of native GABA(B) receptors (GABA(B)Rs) have favored the concept that GHB is also a selective agonist. Furthermore, cloning has demonstrated that GABA(B)Rs assemble heteromeric complexes from the GABA(B)R1 and GABA(B)R2 subtypes and that these assemblies are activated by GHB. The surprisingly high tissue content, together with anti-ischemic and protective effects of GHB in the heart, raises the question of a possible influence of GABA(B) agonists on excitable cardiac cells. In the present study, we provide electrophysiological evidence that GHB activates an inwardly rectifying K(+) current in rat ventricular myocytes. This effect is mimicked by baclofen, reversibly inhibited by GABA(B) antagonists, and prevented by pertussis toxin pretreatment. Both GABA(B)R1 and GABA(B)R2 are detected in cardiomyocytes by Western blotting and are shown to coimmunoprecipitate. Laser scanning confocal microscopy discloses an even distribution of the two receptors in the sarcolemma and along the transverse tubular system. Hence, we conclude that GABA(B)Rs are distributed not only in neuronal tissues but also in the heart, where they can be activated and induce electrophysiological alterations through G-protein-coupled inward rectifier potassium channels.

Late post-myocardial infarction induces a tetrodotoxin-resistant Na(+)Current in rat cardiomyocytes.

Left ventricular remodeling after myocardial infarction is accompanied by electrical abnormalities that might predispose to rhythm disturbances. To get insight into the ionic mechanisms involved, we studied myocytes isolated from four different regions of the rat ventricles, 4-6 months after ligation of the left coronary artery. Using the whole-cell patch-clamp technique, we never observed T-type Ca(2+)current in both diseased and control hearts. In contrast, in 41 out of 78 cells isolated from 16 post-myocardial infarcted rats, analysed in the presence of 30 m m Na(+)ions, we found a tetrodotoxin (TTX)-resistant Na(+)current with quite variable amplitude in every investigated region. Albeit being resistant to 100 microM TTX, this Na(+)-dependent current was highly sensitive to lidocaine since 3 microM lidocaine induced about 65% tonic block. It was also inhibited by 5 microM nifedipine and 2 m m Co(2+), but was insensitive to 100 microM Ni(2+). The TTX-resistant Na(+)channel availability was shifted rightward by 25-30 mV with respect to TTX-sensitive Na(+)current; therefore, a large "window current" might flow in the voltage range from -70 to -20 mV. In conclusion, in late post-myocardial infarction, a Na(+)current with specific kinetics and pharmacology may provide inward charges in a critical range of membrane voltages that are able to alter action potential time course and trigger ventricular arrhythmia. These apparent new characteristics of the Na(+)channel might result in part from environmental changes during heart remodeling.

Inositol 1,4,5-trisphosphate directs Ca(2+) flow between mitochondria and the Endoplasmic/Sarcoplasmic reticulum: a role in regulating cardiac autonomic Ca(2+) spiking.

The signaling role of the Ca(2+) releaser inositol 1,4, 5-trisphosphate (IP(3)) has been associated with diverse cell functions. Yet, the physiological significance of IP(3) in tissues that feature a ryanodine-sensitive sarcoplasmic reticulum has remained elusive. IP(3) generated by photolysis of caged IP(3) or by purinergic activation of phospholipase Cgamma slowed down or abolished autonomic Ca(2+) spiking in neonatal rat cardiomyocytes. Microinjection of heparin, blocking dominant-negative fusion protein, or anti-phospholipase Cgamma antibody prevented the IP(3)-mediated purinergic effect. IP(3) triggered a ryanodine- and caffeine-insensitive Ca(2+) release restricted to the perinuclear region. In cells loaded with Rhod2 or expressing a mitochondria-targeted cameleon and TMRM to monitor mitochondrial Ca(2+) and potential, IP(3) induced transient Ca(2+) loading and depolarization of the organelles. These mitochondrial changes were associated with Ca(2+) depletion of the sarcoplasmic reticulum and preceded the arrest of cellular Ca(2+) spiking. Thus, IP(3) acting within a restricted cellular region regulates the dynamic of calcium flow between mitochondria and the endoplasmic/sarcoplasmic reticulum. We have thus uncovered a novel role for IP(3) in excitable cells, the regulation of cardiac autonomic activity.

Effects of preconditioning on myocardial interstitial levels of ATP and its catabolites during regional ischemia and reperfusion in the rat.

The interstitial accumulation of adenine nucleotide breakdown products (ANBP) in the myocardium during ischemia has been shown to provide a useful index of the ischemic injury, whereas reperfusion ANBP washout rate has been regarded as an index of reperfusion damage. The purpose of this study was, using cardiac microdialysis, to examine in the rat model of regional ischemia/reperfusion the relationship between the duration of ischemia and these indices and to assess the profile of interstitial ATP concentrations and the beneficial effects of ischemic preconditioning (IP). The rats underwent 10, 20, 30 or 40 min of coronary artery occlusion and 50 min of reperfusion. Regional ischemia, with its duration, provoked a progressive increase in dialysate ANBP in the ischemic zone. The rate of purine washout during reperfusion exponentially declined with an increase in duration of the ischemic period. IP, induced by three 5-min episodes of ischemia, each separated by 5 min of reperfusion, significantly reduced the accumulation of ANBP during the 30-min period of sustained ischemia and resulted in a marked acceleration of reperfusion ANBP washout, indicating the improvement of postischemic microcirculation. These effects were suggested to be, at least in part, responsible for the infarct size limitation observed. Using the relationship between the duration of ischemia and ANBP washout rate, it could be demonstrated that IP produced similar facilitation of purine washout as shortening of the ischemic period in nonpreconditioned rats from 30 to approximately 7 min. Regional 20-min ischemia induced an early peak increase in interstitial fluid ATP which correlated with the maximal incidence of ventricular arrhythmias, whereas IP abolished both ATP release and arrhythmias during the sustained ischemia. These findings suggest that ATP may be an important mediator of ischemia-induced ventricular arrhythmias.

Microtubule disruption modulates Ca(2+) signaling in rat cardiac myocytes.

Microtubules have been shown to alter contraction in cardiac myocytes through changes in cellular stiffness. However, an effect on excitation-contraction coupling has not been examined. Here we analyze the effects of microtubule disruption by 1 micromol/L colchicine on calcium currents (I(Ca)) and [Ca(2+)](i) transients in rat ventricular myocytes. I(Ca) was studied using the whole-cell patch-clamp technique. Colchicine treatment increased I(Ca) density (peak values, -4.6+/-0.4 and -9.1+/-1.3 pA/pF in 11 control and 12 colchicine-treated myocytes, respectively; P<0.05). I(Ca) inactivation was well fitted by a biexponential function. The slow component of inactivation was unchanged, whereas the fast component was accelerated after colchicine treatment (at -10 mV, 11.8+/-1.0 versus 6.7+/-1.0 ms in control versus colchicine-treated cells; P<0.005). [Ca(2+)](i) transients were analyzed by fluo-3 epifluorescence simultaneously with I(Ca). Peak [Ca(2+)](i) transients were significantly increased in cardiac myocytes treated with colchicine. The values of F/F(0) at 0 mV were 1.1+/-0.02 in 9 control cells and 1.4+/-0.1 in 11 colchicine-treated cells (P<0.05). beta-Adrenergic stimulation with 1 micromol/L isoproterenol increased both I(Ca) and [Ca(2+)](i) transient in control cells. However, no significant change was induced by isoproterenol on colchicine-treated cells. Colchicine and isoproterenol effects were similar and not additive. Inhibition of adenylyl cyclase by 200 micromol/L 2'-deoxyadenosine 3'-monophosphate blunted the colchicine effect. We suggest that beta-adrenergic stimulation and microtubule disruption share a common pathway to enhance I(Ca) and [Ca(2+)](i) transient.

Modulation of voltage-dependent facilitation of the T-type calcium current by sodium ion in isolated frog atrial cells.

Sodium ions have been reported to alter the permeation properties of L- and N-type Ca2+ channels. Here in frog atrial cardiomyocytes under whole-cell patch-clamp conditions, we have examined the effects of lowering the external Na+ concentration on the amplitude of T-type Ca2+ current, ICaT, and on the relief of its steady-state inactivation by large depolarizing prepulses, ICaT facilitation. A partial reduction in Na+ ion concentration did not significantly alter ICaT amplitude elicited at -50 mV. However, after a large depolarization, low- Na+ solutions enhanced the relief of inactivation and induced ICaT facilitation. This facilitation occurred independently of the divalent charge carrier, high intracellular Ca2+ buffering or the intracellular Na+ content. Its effects were additional to the beta-adrenergic effects mediated by a decrease of Gi/o-protein inhibitory tone. In Ca2+-free solution the very large T-type current, then carried by Na+ ions, showed only a weak relief of inactivation. In conclusion, ICaT facilitation--which, as previously reported, is modulated by the transient voltage-dependent relief of Gi-protein inhibitory tone--is further enhanced in a low-Na+ solution. In Ca2+-free solution, relief of inactivation due to re-openings dependent on the divalent charge carrier is improbable. It thus appears that for a short while after a large depolarization, external Na+ compete with Ca2+ ions on permeation-controlling sites, so as to modulate channel re-openings and thus the amplitude of voltage-facilitated ICaT independently of the control exerted by the inhibitory G-protein.

Cellular and in vivo electrophysiological effects of dronedarone in normal and postmyocardial infarcted rats.

We studied the effects of dronedarone (SR 33589) on the action potentials, membrane ionic currents, and arrhythmic activity in control rats and in rats after myocardial infarction, a model known to develop anomalous electrical activity. Dronedarone increased action potential duration in normal hearts. It had little effect on the action potentials that were already prolonged in the postmyocardial infarcted (PMI) rats. Particularly, dronedarone reduced the late sustained K(+) current, I(K) (or Isus) by 69%. Dronedarone induced only a tonic block of I(K). Similar relative inhibitions of I(K) by dronedarone were obtained in young, sham, and PMI rats, even if I(K) was less in sham than in young and further reduced in PMI rats. The EC(50) values were 0.78 and 0.85 microM in sham and PMI rats. Dronedarone induced a weak increase in the fast transient outward current, I(to). Time-to-peak and inactivation time constant of I(to) were decreased by dronedarone that also induced a marked slowing of I(to) recovery from inactivation. Similar effects were observed on the reduced I(to) recorded in PMI rats. Holter monitoring study in control, unthetered animals showed that dronedarone had no proarrhythmic effect. On rats, which after myocardial infarction exhibited ventricular premature beats, dronedarone significantly decreased beat occurrence during the 7-day treatment; this effect was sustained for two more weeks. Thus, dronedarone exerts antiarrhythmic effects on PMI rat heart. Its effects are attributable for the most part to the inhibition of outward K(+) currents and the increase in effective refractory period.

Aldosterone upregulates Ca(2+) current in adult rat cardiomyocytes.

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 micromol/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 microg/mL) and protein synthesis (cycloheximide, 20 microg/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.

Frequency-dependent increase in cardiac Ca2+ current is due to reduced Ca2+ release by the sarcoplasmic reticulum.

"Ca(2+)-current facilitation" describes several features of increase in current amplitude often associated with a reduction in inactivation rate. The aim of this study was to investigate the mechanism of frequency-dependent increase in L-type Ca2+ current, I(Ca) taking advantage of recent knowledge on the control of Ca2+ current inactivation in cardiac cells. The frequency-dependent increase in I(Ca) was studied in adult rat ventricular myocytes using the whole-cell patch-clamp technique. I(Ca) was elicited by a train of 200-ms depolarizing pulses to +20 mV applied at various frequencies (0.2 up to 1.3 Hz). The increase in frequency induced a rate-dependent enhancement of I(Ca), or facilitation phenomena. In most cells, that showed two inactivation phases of I(Ca), facilitation was mainly related to slowing of the fast I(Ca) inactivation phase that occurred besides increase in peak I(Ca) amplitude. Both the decrease and slowing of the fast component of inactivation phase were attenuated on beta -adrenergic-stimulated current. Frequency-dependent I(Ca) facilitation paralleled a reduction in Ca2+ transient measured with fluo-3. After blocking sarcoplasmic reticulum-Ca2+ release by thapsigargin, the fast I(Ca) inactivation phase was reduced and facilitation was eliminated. Facilitation could not then be restored by 1 microM isoprenaline. Thus in rat ventricular myocytes, frequency-dependent facilitation of I(Ca)reflects a reduced Ca(2+)-dependent inactivation consecutive, in most part, to reduced Ca2+ load and Ca2+ release by the sarcoplasmic reticulum rather than being an intrinsic characteristic of the L-type Ca2+ channel.