Regulation of Ca2+ signaling in transgenic mouse cardiac myocytes overexpressing calsequestrin.

To probe the physiological role of calsequestrin in excitation-contraction coupling, transgenic mice overexpressing cardiac calsequestrin were developed. Transgenic mice exhibited 10-fold higher levels of calsequestrin in myocardium and survived into adulthood, but had severe cardiac hypertrophy, with a twofold increase in heart mass and cell size. In whole cell-clamped transgenic myocytes, Ca2+ channel- gated Ca2+ release from the sarcoplasmic reticulum was strongly suppressed, the frequency of occurrence of spontaneous or Ca2+ current-triggered "Ca2+ sparks" was reduced, and the spark perimeter was less defined.

Molecular determinants of L-type Ca2+ channel inactivation. Segment exchange analysis of the carboxyl-terminal cytoplasmic motif encoded by exons 40-42 of the human alpha1C subunit gene.

Recently we have described a splice variant of the L-type Ca2+ channel (alpha1C,86) in which 80 amino acids (1572-1651) of the conventional alpha1C,77 were substituted by another 81 amino acids due to alternative splicing of exons 40-42. Ba2+ current (IBa) through alpha1C,86 exhibited faster inactivation kinetics, was strongly voltage-dependent, and had no Ca2+-dependent inactivation. An oligonucleotide-directed segment substitution and expression of the mutated channels in Xenopus oocytes were used to study the molecular determinants for gating of the channel within the 80-amino acid domain.

Glutathione is a cofactor for H2O2-mediated stimulation of Ca2+-induced Ca2+ release in cardiac myocytes.

Reactive oxygen species are known to cause attenuation of cardiac muscle contraction. This attenuation is usually preceded by transient augmentation of twitch amplitude as well as cytosolic Ca2+. The present study examines the role of an endogenous antioxidant, glutathione in the mechanism of H2O2-mediated augmentation of Ca2+ release from the sarcoplasmic reticulum.

Comparative effects of loratadine and terfenadine on cardiac K+ channels.

Nonsedating H1-receptor antagonists appear to have wide and variable effects on the QT interval, mediated through modulation of cardiac K+ channels. By using the whole-cell patch-clamp technique, we examined the effects of terfenadine, loratadine, and descarboethoxyloratadine on a large family of K+ channels in ventricular myocytes and in Xenopus oocytes expressing the HERG delayed rectifier. The channels studied included the inward rectifier (I(Kl)) of rat and guinea pig, the transient outward K+ current (I(to)) of rat, the maintained K+ current (I(ped)) of rat, and the delayed rectifier K+ channels (I(Ks) and I(Kr)) of guinea pig myocytes.

Calcium signaling in transgenic mice overexpressing cardiac Na(+)-Ca2+ exchanger.

We have produced transgenic mice which overexpress cardiac Na(+)-Ca2+ exchange activity. Overexpression has been assessed by Western blot, Northern blot, and immunofluorescence. Functional overexpression was analyzed using membrane vesicles and isolated ventricular myocytes.

A caffeine-sensitive Ca2+ store modulates K+-evoked secretion in chromaffin cells.

Catecholamine release from bovine adrenal medulla chromaffin cells superfused with a Krebs-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid solution was monitored on-line with an electrochemical detector. Caffeine (10 mM) progressively depressed the magnitude of secretory responses to depolarizing pulses of 70 mM K+ and 2 mM Ca2+ (70 K+/2 Ca2+) in cells superfused with a Krebs-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid solution containing 0 mM Ca2+ + 0.5 mM EGTA; blockade reached 80% at the third 70 K+/2 Ca2+ challenge given in the presence of caffeine.

Suppression of mammalian K+ channel family by ebastine.

Nonsedating H1 receptor (H1-R) antagonists exert variable effects on QT interval, most likely mediated through modulation of cardiac K+ channels. We examined the effects of a novel H1-R antagonist, ebastine, on a family of K+ currents in isolated rat and guinea pig ventricular cardiomyocytes as well as on HERG-induced rapidly delayed rectifier K+ current (I(Kr)) in Xenopus laevis oocytes. The effect of ebastine was compared with that of two other H1-R antagonists, terfenadine and loratadine, with and without reported cardiotoxicity, respectively.

Ca2+ sparks within 200 nm of the sarcolemma of rat ventricular cells: evidence from total internal reflection fluorescence microscopy.

Total internal reflection fluorescence microscopy (TIRFM) was used to measure local calcium releases in resting cardiac myocytes stained with fluo-3AM. The measured fluorescence originated from regions where cells were close to, and develop adhesions to, a totally reflecting glass surface. The excitation of the fluorescent Ca2+ indicator dye by the exponentially attenuated evanescent wave penetrated approximately 200 nm into the fluid phase.

Ca(2+)-signaling in cardiac myocytes: evidence from evolutionary and transgenic models.

Cardiac contraction is regulated by a number of Ca(2+)-mediated processes. Here we consider the effects of modification imposed on the Ca(2+)-signalling mechanism by evolutionary developments and transgenic manipulations. Ca(2+)-signalling appears to be mediated via influx of Ca2+ through the DHP receptor in preference to the Na(+)-Ca2+ exchange protein, and activates the ryanodine receptor and the Ca2+ release from the SR.

Lack of cystic fibrosis transmembrane regulator-type chloride current in pediatric human atrial myocytes.

Mature cardiomyocytes have been shown to possess a cyclic AMP-mediated chloride channel (I(Cl)) which is the product of the cystic fibrosis transmembrane regulator (CFTR) gene. Species variability has been demonstrated for other ion channels. This study was designed to evaluate human I(Cl) regulation using the whole-cell patch-clamp bioassay.

Cross-signaling between L-type Ca2+ channels and ryanodine receptors in rat ventricular myocytes.

Calcium-mediated cross-signaling between the dihydropyridine (DHP) receptor, ryanodine receptor, and Na(+)-Ca2+ exchanger was examined in single rat ventricular myocytes where the diffusion distance of Ca2+ was limited to < 50 nm by dialysis with high concentrations of Ca2+ buffers. Dialysis of the cell with 2 mM Ca(2+)- indicator dye, Fura-2, or 2 mM Fura-2 plus 14 mM EGTA decreased the magnitude of ICa-triggered intracellular Ca2+ transients (Cai-transients) from 500 to 20-100 nM and completely abolished contraction, even though the amount of Ca2+ released from the sarcoplasmic reticulum remained constant (approximately 140 microM). Inactivation kinetics of ICa in highly Ca(2+)-buffered cells was retarded when Ca2+ stores of the sarcoplasmic reticulum (SR) were depleted by caffeine applied 500 ms before activation of ICa, while inactivation was accelerated if caffeine-induced release coincided with the activation of ICa.

Glass-funnel technique for the recording of membrane currents and intracellular perfusion of Xenopus oocytes.

In this report we present a description of a modified version of the "glass-funnel" technique for the recording of membrane currents and intracellular perfusion of Xenopus laevis oocytes. The technique is based on the ability of the devitellinated oocyte to form a high-resistance seal with the glass, permitting separation of the oocyte into two, i.e.

Regulation of cardiac sodium-calcium exchanger by beta-adrenergic agonists.

Na+-Ca2+ exchanger and Ca2+ channel are two major sarcolemmal Ca2+-transporting proteins of cardiac myocytes. Although the Ca2+ channel is effectively regulated by protein kinase A-dependent phosphorylation, no enzymatic regulation of the exchanger protein has been identified as yet. Here we report that in frog ventricular myocytes, isoproterenol down-regulates the Na+-Ca2+ exchanger, independent of intracellular Ca2+ and membrane potential, by activation of the beta-receptor/adenylate-cyclase/cAMP-dependent cascade, resulting in suppression of transmembrane Ca2+ transport via the exchanger and providing for the well-documented contracture-suppressant effect of the hormone on frog heart.

Species differences in the activity of the Na(+)-Ca2+ exchanger in mammalian cardiac myocytes.

1. Species differences in the activity of the exchanger were evaluated in isolated myocytes from rat, guinea-pig, hamster ventricles and human atria. Fluorescence measurements using fura-2 were carried out in conjunction with the whole-cell patch-clamp technique for simultaneous recording of membrane currents and intracellular Ca2+ concentration.

Evaluation of T- and L-type Ca2+ currents in shark ventricular myocytes.

Two types of Ca2+ currents with characteristics of T- and L-type Ca2+ currents were recorded in ventricular myocytes of dogfish (Squalus acanthias). The T-type Ca2+ current activated near -70 mV and had a peak current density of 9.8 pA/pF at -34 mV.

Regulation of potassium channels by nonsedating antihistamines.

Background: Terfenadine and astemizole are widely prescribed nonsedating antihistamines that have been associated with QT-interval prolongation and ventricular arrhythmias. Since potassium channels are intrinsically involved in repolarization, this study was designed to evaluate the effect of the nonsedating antihistamines on potassium channel modulation.

Methods And Results: The whole-cell patch-clamp technique was used to study K+ currents in enzymatically isolated rat and guinea pig ventricular myocytes.

Evidence for presence of Ca2+ channel-gated Ca2+ stores in neonatal human atrial myocytes.

The characteristics of Ca2+ signaling in fura 2-loaded whole cell-clamped myocytes obtained from samples of human atrial appendages of 3-day to 4-yr-old patients were examined. In isolated myocytes, activation of Ca2+ current (ICa) (2.47 +/- 0.

Functional coupling of Ca2+ channels and ryanodine receptors in cardiac myocytes.

In skeletal muscle, dihydropyridine receptors are functionally coupled to ryanodine receptors of the sarcoplasmic reticulum in triadic or diadic junctional complexes. In cardiac muscle direct physical or functional couplings have not been demonstrated. We have tested the hypothesis of functional coupling of L-type Ca2+ channels and ryanodine receptors in rat cardiac myocytes by comparing the efficacies of Ca2+ in triggering Ca2+ release when the ion enters the cell via the Ca2+ channels or the Na+/Ca2+ exchanger.

Signaling of Ca2+ release and contraction in cardiac myocytes.

Cross signaling between Ca2+ channel and ryanodine receptor was explored in whole cell clamped rat ventricular myocyte under conditions where global myoplasmic Ca2+ concentrations were strongly buffered by dialyzing the myocytes with high concentrations of Fura 2 and EGTA. Ca2+ channel and ryanodine receptor were respectively activated by a depolarizing pulse to -10 mV and rapid (< 50 ms) application of 5 mM caffeine. Temporal analysis of kinetics of inactivation of Ca2+ channel with respect to the time of application of caffeine pulse provided experimental evidence that signalling between the ryanodine and Ca2+ channel is mediated exclusively through the Ca2+ microdomains surrounding the DHP/ryanodine receptor complex independent of global myoplasmic Ca2+ concentrations.

Enhanced Na(+)-Ca2+ exchange activity in cardiomyopathic Syrian hamster.

The signaling of contraction by Ca2+ in the Syrian hamster (BIO 14.6) heart in the late stage of the cardiomyopathy (220 to 300 days old) was compared with that in age-matched healthy hamster hearts. Membrane current and cell shortening or intracellular Ca2+ transients were measured simultaneously in isolated whole-cell-clamped myocytes.

ATP modulation of calcium channels in chromaffin cells.

1. The effects of externally applied micromolar concentrations of adenosine 5'-triphosphate (ATP) on Ca2+ currents (ICa) were studied in whole-cell clamped adrenaline-secreting chromaffin cells. 2.

Ca2+ channel modulating effects of heparin in mammalian cardiac myocytes.

1. The effect of heparin on L-type Ca2+ channels in rabbit, rat and guinea-pig cardiac myocytes was studied using the whole-cell patch clamp method. 2.

Modulation of contraction by intracellular Na+ via Na(+)-Ca2+ exchange in single shark (Squalus acanthias) ventricular myocytes.

1. The effect of direct alteration of intracellular Na+ concentration on contractile properties of whole-cell clamped shark ventricular myocytes was studied using an array of 256 photodiodes to monitor the length of the isolated myocytes. 2.

Characteristics of calcium currents in rabbit portal vein myocytes.

The properties of voltage-dependent Ca2+ channels were studied in isolated portal vein myocytes using the whole cell voltage-clamp method. Ca2+ currents (ICa) were identified based on their activation and inactivation potential, their dependence on external Ca2+ ([Ca2+]o), their suppression by organic or inorganic Ca2+ channel blockers, their augmentation by BAY K 8644, and their insensitivity to tetrodotoxin or alterations in external Na+ ([Na+]o). Changing the holding potential from -90 to -40 mV decreased ICa from 4.

Gating of the cardiac Ca2+ release channel: the role of Na+ current and Na(+)-Ca2+ exchange.

In cardiac myocytes, calcium influx through the calcium channel is the primary pathway for triggering calcium release. Recently it has been suggested that the calcium-induced calcium release mechanism can also be activated indirectly by the sodium current, which elevates the sodium concentration under the cell membrane, thereby favoring the entry of "trigger" calcium via the sodium-calcium exchanger. To test this hypothesis, sodium current was suppressed by reducing the external sodium concentration or applying tetrodotoxin.