New perspectives on the key role of calcium in the progression of heart disease.

The heart continuously adapts to adjust its output to a continuum of pathophysiological situations ensuring adequate blood distribution. These situations range from high performance in well-trained athletes to failure in a variety of cardiac syndromes. Changes in the concentration of intracellular Ca2+ ([Ca2+]i) are crucial. They have immediate and late effects that can be oversimplified as follows. Immediate effects result from abrupt and large variations in [Ca2+]i triggering contraction after binding to the contractile proteins. These variations are involved in the process known to as excitation-contraction (EC) coupling. In contrast, the late effects involve a process that is, by analogy, referred to as excitation-transcription (ET) coupling. This process involves activation of gene expression by Ca2+. In this scheme, specific and localised elevations of Ca2+ can be converted into changes in gene expression with long-term effects on the adaptation of the heart to a sustained stimulus. There is emerging evidence of an extraordinary diversity of responses, depending on the location, intensity, and duration of Ca2+ signals that can be activated during pathology. Whereas alterations of cellular and molecular mechanisms underlying chronic pathology are relatively well defined, the initial changes and their hierarchy are unknown. However, the actual picture suggests promising perspectives for new therapeutic interventions on old targets or new strategies. Some of these aspects are reviewed here.

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.

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.

Cluster organization and pore structure of ion channels formed by beticolin 3, a nonpeptidic fungal toxin.

Beticolin 3 (B3) belongs to a family of nonpeptidic phytotoxins produced by the fungus Cercospora beticola, which present a broad spectrum of cytotoxic effects. We report here that, at cytotoxic concentration (10 microM), B3 formed voltage-independent, weakly selective ion channels with multiple conductance levels in planar lipid bilayers. In symmetrical standard solutions, conductance values of the first levels were, respectively, 16 +/- 1 pS, 32 +/- 2 pS, and 57 +/- 2 pS (n = 4) and so on, any conductance level being roughly twice the lower one. Whether a cluster organization of elementary channels or different channel structures underlies this particular property was addressed by investigating the ionic selectivity and the pore size corresponding to the first three conductance levels. Both selectivity and pore size were found to be almost independent of the conductance level. This indicated that multiple conductance behavior resulted from a cluster organization of "B3 elementary channels." According to the estimated pore size and analyses of x-ray diffraction of B3 microcrystals, a structural model for "B3 elementary channels" is proposed. The ability to form channels is likely to be involved in the biological activity of beticolins.

Molecular dynamics of the sodium channel pore vary with gating: interactions between P-segment motions and inactivation.

Disulfide 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 micro1 sodium channels engineered to contain two cysteines within the P segments. Rates of catalyzed internal disulfide formation (kSS) were measured in K1237C+W1531C mutant channels expressed in oocytes. During repetitive voltage-clamp depolarizations, increasing the pulse duration had biphasic effects on the kSS, 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 (IM) facilitates pore motions. Consistent with the known antagonism between alkali metals and a component of slow inactivation, kSS 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.

Extracellular K+ dependence of inward rectification kinetics in human left ventricular cardiomyocytes.

BACKGROUND: In human ventricular cells, the inwardly rectifying K+ current (IK1) 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 IK1 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 IK1 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.

Molecular motions within the pore of voltage-dependent sodium channels.

The pores of ion channel proteins are often modeled as static structures. In this view, selectivity reflects rigidly constrained backbone orientations. Such a picture is at variance with the generalization that biological proteins are flexible, capable of major internal motions on biologically relevant time scales. We tested for motions in the sodium channel pore by systematically introducing pairs of cysteine residues throughout the pore-lining segments. Two distinct pairs of residues spontaneously formed disulfide bonds bridging domains I and II. Nine other permutations, involving all four domains, were capable of disulfide bonding in the presence of a redox catalyst. The results are inconsistent with a single fixed backbone structure for the pore; instead, the segments that line the permeation pathway appear capable of sizable motions.

Regional alteration of the transient outward current in human left ventricular septum during compensated hypertrophy.

BACKGROUND: 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 Ca2+ 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.

A chloride current component induced by hypertrophy in rat ventricular myocytes.

The 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.

Modulation of electrical heterogeneity by compensated hypertrophy in rat left ventricle.

Modulation of the regional distribution of the action potential by left ventricular hypertrophy and the role of the L-type Ca2+ current (I(Ca)) and transient outward current (I(to)) in the action potential duration (APD) were investigated in normal and hypertrophied rat ventricular myocytes from the apex (A), septum (S) and left ventricular free wall (FW) by using whole cell current- and voltage-clamp techniques. Hypertrophy was induced by abdominal aortic constriction. In control cells, the APD measured at 20% repolarization (APD20) assumed the shortest values in the A and the longest in the S, whereas FW cells showed intermediate values. Hypertrophy significantly prolonged the APD20 and increased APD variability within the A and FW regions but did not modify the APD in S cells. Analysis of the APD, I(Ca), and I(to) at the instant of 20% repolarization in the same cell showed that in control cells the shortest APD20 was associated with a prominent I(to) in the A and FW, whereas the long APD20 was identified with a lower I(to) in S myocytes. Hypertrophy-induced prolongation ofAPD20 was associated with a reduction in the I(to) in the A and FW. Significant correlations could be established between the APD20 and the "net current," defined as the algebraic addition of I(to) and I(Ca) in the A and FW control groups but not in the control S or hypertrophied cells whatever their origin. Our results indicate that interregional APD heterogeneity is lost while intraregional APD variability is increased in the A and FW during the hypertrophic process. These effects are largely due to a change in the balance between the I(Ca) and I(to), which is a major contributing factor to the heterogeneity of the initial phase of repolarization in the normal rat ventricle.

Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis.

The pores of voltage-gated ion channels are lined by protein loops that determine selectivity and conductance. The relative orientations of these "P" loops remain uncertain, as do the distances between them. Using site-directed mutagenesis, we introduced pairs of cysteines into the P loops of micro1 rat skeletal muscle sodium channels and sought functional evidence of proximity between the substituted residues. Only cysteinyl residues that are in close proximity can form disulfide bonds or metal-chelating sites. The mutant Y401C (domain I) spontaneously formed a disulfide bond when paired with E758C in the P loop of domain II; the same residue, when coupled with G1530C in domain IV, created a high-affinity binding site for Cd2+ ions. The results provide the first specific constraints for intramolecular dimensions of the sodium channel pore.

Heterogeneity of the early outward current in ventricular cells isolated from normal and hypertrophied rat hearts.

1. The nature, magnitude and kinetics of the 4-aminopyridine-sensitive early outward current (Ito) were analysed in isolated ventricular myocytes from the septum, the apex and the left ventricular free wall of rat ventricles using the whole-cell voltage clamp method. The modulatory effect of pressure overload-induced cardiac hypertrophy on the regional variations of Ito was assessed in each topographical class of cells. 2. Voltage clamp experiments were performed at room temperature (20-25 degrees C) in the absence of Na+ on both sides of the membrane and in the presence of 3 mM CoCl2. Ito was studied from a holding potential of -80 mV and determined by subtraction of total outward currents elicited by the same protocols in the presence of 3 mM 4-aminopyridine (4-AP) from those obtained in its absence. 3. In normal hearts, membrane passive properties were very similar in each topographical class of cells. Our results confirmed that the predominant early outward current in rat ventricular cells was 4-AP-sensitive, time and voltage dependent, and demonstrated that the magnitude of the current varied on a regional basis: current density of Ito in left ventricular free wall cells (30.1 +/- 9.2 pA/pF at +60 mV) was larger than in apex cells (20.2 +/- 1.7 pA/pF) or in septum cells (11.9 +/- 3.3 pA/pF). We noticed a larger variability in data from left ventricular free wall compared with other regions. 4. No shift in steady-state voltage dependence of Ito activation and inactivation was found. However, the maximal computed chord conductances were (in microS/pF): 0.18 +/- 0.07 for left ventricular free wall cells, 0.13 +/- 0.02 for apex cells, and 0.08 +/- 0.02 for septum cells. These findings might reflect a differential distribution in functional channel densities. 5. No difference in voltage-dependent Ito activation kinetics was present with respect to topography. However, inactivation time constants in septum were longer than those of both other groups. 6. Left ventricular hypertrophy was induced by abdominal aortic constriction and its effects compared to the findings from normal rats. Hypertrophied cells had similar resting potentials but higher capacitance values than normal cells. Although Ito magnitude appeared not to be modified, the current density-voltage curves were slightly shifted to more positive potentials and significantly decreased as compared to normal cells (in pA/pF, at +60 mV): 8.4 +/- 5.0 in the left free wall group, 11.6 +/- 2.0 in the apex group, and 3.8 +/- 1.5 in the septum group.(ABSTRACT TRUNCATED AT 400 WORDS)

Pro-arrhythmic effect of nicorandil in isolated rabbit atria and its suppression by tolbutamide and quinidine.

Nicorandil, a potent vasodilator substance which exerts its effects through complex mechanisms including KATP channel activation, has so far been reported to exert antiarrhythmic but not pro-arrhythmic cardiac activity. We now examined the effects of 10(-4) M nicorandil on spontaneously active or electrically driven isolated rabbit atria. Nicorandil (a) significantly reduced the action potential duration at both 50% (by approximately 45%) and 80% (by approximately 30%) repolarization and the effective refractory period (by approximately 25%) and (b) reproducibly induced short periods of tachycardia either in normal Tyrode solution after a single extra-stimulus or in low-potassium media in the absence of extra-stimulation. Quinidine (10(-5) M) or the KATP channel inhibitor, tolbutamide (10(-5) M), suppressed the nicorandil-induced arrhythmias. It is suggested that the pro-arrhythmic effect of nicorandil results from its KATP channel opener activity and occurs essentially when the underlying conditions facilitate re-entry.

Slow inward current in single cells isolated from adult human ventricles.

Characteristics 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.