cGMP-stimulated cyclic nucleotide phosphodiesterase regulates the basal calcium current in human atrial myocytes.

EHNA (Erythro-9-[2-hydroxy-3-nonyl]adenine) is a wellknown inhibitor of adenosine deaminase. Recently, EHNA was shown to block the activity of purified soluble cGMPstimulated phosphodiesterase (PDE2) from frog, human, and porcine heart with an apparent Ki value of approximately 1 microM and with negligible effects on Ca2+/calmodulin PDE (PDE1), cGMP-inhibited PDE (PDE3), and low Km cAMP-specific PDE (PDE4) (Méry, P.F., C. Pavoine, F. Pecker, and R. Fischmeister. 1995. Mol. Pharmacol. 48:121-130; Podzuweit, T., P. Nennstiel, and A. Muller. 1995. Cell. Signalling. 7:733- 738). To investigate the role of PDE2 in the regulation of cardiac L-type Ca2+ current (ICa), we have examined the effect of EHNA on ICa in freshly isolated human atrial myocytes. Extracellular application of 0.1-10 microM EHNA induced an increase in the amplitude of basal ICa ( approximately 80% at 1 microM) without modification of the current-voltage or inactivation curves. The maximal stimulatory effect of EHNA on ICa was comparable in amplitude with the maximal effect of isoprenaline (1 microM), and the two effects were not additive. The effect of EHNA was not a result of adenosine deaminase inhibition, since 2'-deoxycoformycin (1-30 microM), another adenosine deaminase inhibitor with no effect on PDE2, or adenosine (1-10 microM) did not increase ICa. In the absence of intracellular GTP, the substrate of guanylyl cyclase, EHNA did not increase ICa. However, under similar conditions, intracellular perfusion with 0.5 microM cGMP produced an 80% increase in ICa. As opposed to human cardiomyocytes, EHNA (1-10 microM) did not modify ICa in isolated rat ventricular and atrial myocytes. We conclude that basal ICa is controlled by PDE2 activity in human atrial myocytes. Both PDE2 and PDE3 may contribute to keep the cyclic nucleotides concentrations at minimum in the absence of adenylyl and/or guanylyl cyclase stimulation.

Nitric oxide regulates the calcium current in isolated human atrial myocytes.

Cardiac Ca2+ current (ICa) was shown to be regulated by cGMP in a number of different species. Recently, we found that the NO-donor SIN-1 (3-morpholino-sydnonimine) exerts a dual regulation of ICa in frog ventricular myocytes via an accumulation of cGMP. To examine whether NO also regulates Ca2+ channels in human heart, we investigated the effects of SIN-1 on ICa in isolated human atrial myocytes. An extracellular application of SIN-1 produced a profound stimulatory effect on basal ICa at concentrations > 1 pM. Indeed, 10 pM SIN-1 induced a approximately 35% increase in ICa. The stimulatory effect of SIN-1 was maximal at 1 nM (approximately 2-fold increase in ICa) and was comparable with the effect of a saturating concentration (1 microM) of isoprenaline, a beta-adrenergic agonist. Increasing the concentration of SIN-1 to 1-100 microM reduced the stimulatory effect in two thirds of the cells. The stimulatory effect of SIN-1 was not mimicked by SIN-1C, the cleavage product of SIN-1 produced after liberation of NO. This suggests that NO mediates the effects of SIN-1 on ICa. Because, in frog heart, the stimulatory effect of SIN-1 on ICa was found to be due to cGMP-induced inhibition of cGMP-inhibited phosphodiesterase (cGI-PDE), we compared the effects of SIN-1 and milrinone, a cGI-PDE selective inhibitor, on ICa in human. Milrinone (10 microM) induced a strong stimulation of ICa (approximately 150%), demonstrating that cGI-PDE controls the amplitude of basal ICa in this tissue. In the presence of milrinone, SIN-1 (0.1-1 nM) had no stimulatory effect on ICa, suggesting that the effects of SIN-1 and MIL were not additive. We conclude that NO may stimulate ICa in human atrial myocytes via inhibition of the cGI-PDE.

Intracellular calcium transients induced by different kinds of stimulus during myogenesis of rat skeletal muscle cells studied by laser cytofluorimetry with Indo-1.

Resting intracellular calcium levels and intracellular calcium transients induced by three types of stimulus (acetylcholine, high potassium and caffeine) were recorded, during in vitro myogenesis, by means of a ratiometric fluorescence method using the calcium probe Indo-1 under laser illumination. Resting levels seemed to decrease with the age of cultured cells and the depolarization-induced transients, through 100 mM K+ or Ach application, were progressively faster and larger as the muscle cells developed. An additive mechanism, likely due to calcium entry into the cell through nicotinic acetylcholine receptors, could explain the differences observed in Ach-induced responses as compared with the 100 mM K(+)-induced ones. In myoballs (the older cells) the calcium transients exhibited progressively a biphasic shape. From data obtained in different conditions (tetrodotoxin, nifedipine, strontium and free Ca EGTA) and those indicating the appearance of caffeine-releasable intracellular calcium stores only at 2-3 days stage, and from the previously reported developmental appearance of calcium currents and contraction, it was proposed that, in young myotubes, the calcium transients were more dependent on extracellular calcium than in older cells. These developmental data are discussed in the light of a known model of the in situ biogenesis of the structures involved in excitation-contraction coupling (ECC) like transverse tubules and triads.

Calcium current-dependent staircase in rat myotubes and myoballs developing in culture.

Calcium current and contraction were simultaneously recorded in whole-cell patch-clamped rat skeletal muscle cells grown in primary culture. Repetitive depolarizations at low frequency, which elicited calcium currents, led to a staircase response, characterized by the progressive increase of both twitch amplitude and activation rate. It was sometimes possible to elicit a staircase response in 2 or 3 day old postfusion myotubes which did not or weakly contract initially. The staircase response was dependent on calcium entry through calcium channels, since it was reversed when calcium current was depressed by means of inorganic calcium blockers or depolarization to large positive potential. The entry of calcium was also necessary to allow the development of a staircase response following caffeine-induced contractures which partly emptied the intracellular stores of calcium. These features are consistent with the idea that calcium currents allow the initial loading of intracellular calcium stores and, in later stages, serve to replenish and maintain them constant.

Changes in cytosolic resting ionized calcium level and in calcium transients during in vitro development of normal and Duchenne muscular dystrophy cultured skeletal muscle measured by laser cytofluorimetry using indo-1.

Intracellular calcium activity was recorded during in vitro myogenesis of human normal and DMD muscle, using the calcium probe Indo-1 under laser illumination, at rest and during different kinds of stimulation (acetylcholine, high K+, caffeine). In myoblasts, the resting intracellular calcium level was significantly larger in DMD cells (89 +/- 9 nM; n = 40 vs 37 +/- 5 nM; n = 22) but there was no significant difference in myotubes, after fusion (44 +/- 4 nM; n = 34 vs 36 +/- 4 nM; n = 52). A similar evolution was observed in cells cultured from FSH biopsies. The amplitude of ACh- and high K(+)-induced calcium transients was significantly halved in DMD myotubes as compared to control ones and non-significantly decreased for caffeine responses. Some alterations in the kinetics of responses were observed in DMD muscle: the rising phases of ACh- and high K(+)-elicited transients and the decaying phase of the ACh-responses were significantly slowed down. It is concluded that: (i) in aneurally cultured human muscle, an increase in the basal level of internal calcium can occur at early stages of myogenesis before the expression of the dystrophin gene; and (ii) the changes in calcium transients induced by depolarization or direct stimulation of sarcoplasmic reticulum are not susceptible of inducing a calcium overload in DMD cells.

Progressive predominance of 'skeletal' versus 'cardiac' types of excitation-contraction coupling during in vitro skeletal myogenesis.

Simultaneous recordings, in the presence or absence of cadmium, of current and contraction of skeletal muscle cells in primary culture (myoballs) showed that the relative part of contraction depending on calcium current progressively decreases with the age of cells whereas the cadmium-insensitive component becomes predominant. The coexistence of "cardiac" and "skeletal" excitation-contraction coupling mechanisms in developing muscle and its developmental regulation are consistent with recent molecular data showing the expression, in newborn animals, of an abbreviated isoform of the alpha 1 subunit of the DHP-receptor of muscle cells.

Activation of a slow outward current by the calcium released during contraction of cultured rat skeletal muscle cells.

A slow outward current, activated during depolarization, which induced contraction in whole-cell patch-clamped rat skeletal muscle cells in primary culture [10], was extensively characterized in the present study. This current, Io, was simultaneously recorded with the contraction as a slow outward current during the test pulse, and a slow outward bell-shaped tail after repolarization. Io never appeared below the threshold potential for contraction, and the tail amplitude displayed a similar evolution with peak contraction amplitude as a function of membrane potential. This feature is consistent with the fact that Io was suppressed when contraction was blocked by 5 microM nifedipine [10], and it suggests that Io was dependent on calcium released during contraction. This was confirmed by the fact that the presence of 10 mM EGTA in the patch pipette prevented the development of both contraction and Io, and that Io could be activated during caffeine-induced contractures without applying depolarizations. Io could be carried by K+ or Cs+ ions, but not by Na+. The pharmacology of Io was different from that of Ca(2+)-dependent BK and SK channels, since it was resistant to tetraethylammonium (135 mM), charybdotoxin (25 nM) and apamin (50 nM). Io was also insensitive to 4-aminopyridine (1 mM) but blocked by 5 mM Ba2+ without change to contraction. It was concluded that rat cultured myoballs exhibit a Cs+ permeation through an atypical K+ channel type, which is activated by the calcium released during contraction.

Role of the cytoskeleton in the regulation of Cl- channels in human embryonic skeletal muscle cells.

The effects of volume change and cytoskeleton manipulation on the Cl- channels in human embryonic skeletal muscle cells were studied. Trypsination, used for production of myoballs, changes the channel properties only a little. When the external osmolarity was reduced from 300 to 270 mosmol/l, the specific Cl- conductance, gCl, (at -80 mV) of myoballs increased from 5.1 +/- 1.9 to 30.4 +/- 12.2 microS/cm2 (SD; n = 6) within 15 min. Concomitantly, the kinetics of Cl- currents, elicited by clamping the membrane potential from a negative to positive values, changed from activation and subsequent slow inactivation to instantaneous activation with fast inactivation. G protein activation, protein kinase action or [Ca2+]i elevation seemed not to be involved in these effects. Similar changes were produced in the absence of a transmembrane osmotic gradient by 500 nM intracellular cytochalasin D (gCl = 34.3 +/- 10.3 microS/cm2; n = 6) or 12.5 microM colchicine (gCl = 15.4 +/- 1.4 microS/cm2; n = 5). When the external osmolarity was increased to 418 mosmol/l, 1 microM cytochalasin D did not affect gCl. In four of six cell-attached patches the open probability of the intermediate Cl- channel was increased after reduction of the bath osmolarity. In inside-out patches, the drugs increased the open probability of the channels. It is concluded that the Cl- channels are under control of the cytoskeleton.

Appearance and evolution of calcium currents and contraction during the early post-fusional stages of rat skeletal muscle cells developing in primary culture.

Primary cultures from enzymatically dissociated satellite cells of newborn rat skeletal muscles enabled developmental in vitro studies of mechanical and electrical properties during the first steps of myogenesis. The present work focused on the appearance, evolution and roles of two types of calcium currents (ICa,T and ICa,L) and of depolarization-induced contractile activity during the early stages of muscle cell development in primary culture. Prefusional mononucleated cells (myoblasts), young myotubes of 1 day (with less than 10 nuclei) or 2-3 days (more than 9 nuclei) and myoballs from 4-6, 7-9, 10-12 and 13-16 days cultures were patch-clamped (whole-cell configuration), and calcium currents and contraction simultaneously recorded. Sodium but not calcium currents could be recorded at the myoblast stage. In young myotubes (1 day), ICa,L was present with high incidence as compared to ICa,T, which was poorly expressed. Contractile responses appeared at the next stage (2-3 days) while the occurrence of ICa,T progressively increased. This developmental evolution of the calcium currents and contraction expression was accompanied by some changes in their characteristics: the ICa,T/ICa,L amplitudes ratio progressively increased and the time-to-peak of contraction progressively decreased with the age of myoballs. Physiological functions for calcium currents in developing muscle are suggested and discussed: ICa,T, which is transiently expressed, could be involved in the pacemaker-like activity while ICa,L could serve as an early contraction triggering mechanism and/or initially to fill and then to maintain the intracellular calcium stores.

Human sodium channel myotonia: slowed channel inactivation due to substitutions for a glycine within the III-IV linker.

1. Three families with a form of myotonia (muscle stiffness due to membrane hyperexcitability) clinically distinct from previously classified myotonias were examined. The severity of the disease greatly differed among the families. 2. Three dominant point mutations were discovered at the same nucleotide position of the SCN4A gene encoding the adult skeletal muscle Na+ channel alpha-subunit. They predict the substitution of either glutamic acid, valine or alanine for glycine1306, a highly conserved residue within the supposed inactivation gate. Additional SCN4A mutations were excluded. 3. Electrophysiological studies were performed on biopsied muscle specimens obtained for each mutation. Patch clamp recordings on sarcolemmal blebs revealed an increase in the time constant of fast Na+ channel inactivation, tau h, and in late channel openings as compared to normal controls. tau h was increased from 1.2 to 1.6-2.1 ms and the average late currents from 0.4 to 1-6% of the peak early current. 4. Intracellular recordings on resealed fibre segments revealed an abnormal tetrodotoxin-sensitive steady-state inward current, and repetitive action potentials. Since K+ and Cl- conductances were normal, only the increase in the number of non-inactivating Na+ channels has to be responsible for the membrane hyperexcitability. 5. Length, ramification and charge of the side-chains of the substitutions correlated well with the Na+ channel dysfunction and the severity of myotonia, with alanine as the most benign and glutamic acid as the substitution with a major steric effect. 6. Our electrophysiological and molecular genetic studies strongly suggest that these Na+ channel mutations cause myotonia. The naturally occurring mutants allowed us to gain further insight into the mechanism of Na+ channel inactivation.