Actions of pyrethroid insecticides on sodium currents, action potentials, and contractile rhythm in isolated mammalian ventricular myocytes and perfused hearts.

Pyrethroid insecticides are known to modify neuronal sodium channels, inducing persistent, steady-state sodium current at depolarized membrane potentials. Cardiac myocytes are also rich in sodium channels but comparatively little is known about the effect of pyrethroids on the heart, or on the cardiac sodium channel isoform. In the present study therefore, we determined the actions of type I and type II pyrethroids against rat and guinea pig ventricular myocytes under current and voltage clamp, and on isolated perfused rat hearts. In myocytes, tefluthrin (type I) and fenpropathrin and alpha-cypermethrin (type II) prolonged action potentials and evoked afterdepolarizations. The time course of sodium current (I(Na)) was also prolonged by these compounds. Pyrethroids delayed I(Na) inactivation, when measured under selective conditions as current sensitive to 30 microM tetrodotoxin, by increasing the proportion of slowly inactivating current at the expense of fast inactivating current. Further experiments, focusing on fenpropathrin, revealed that its effects on I(Na) inactivation time course were dose-dependent, and the Na(+) "window-current" was increased in its presence. In unstimulated, isolated hearts perfused with the same pyrethroids, the variability in contraction amplitude increased due to variations in the intervals between heartbeats. These potentially arrhythmogenic changes are consistent with the effects observed at the cellular level. The type I pyrethroid tetramethrin had little effect in any of the preparations. These findings suggest that some pyrethroids possess considerable mammalian cardiac arrhythmogenic potential, the manifestation of which in vivo may depend on the route of exposure.

Loading of calcium and strontium into the sarcoplasmic reticulum in rat ventricular muscle.

Previous work suggests that strontium ions (Sr(2+)) are less effective than calcium ions (Ca(2+)) at supporting excitation-contraction (EC) coupling in cardiac muscle. We therefore tested whether this was due to differences in the uptake and release of Ca(2+)and Sr(2+)by the sarcoplasmic reticulum (SR) of rat ventricular trabeculae and myocytes at 22-24 degrees C. In permeabilized trabeculae, isometric contractions activated by exposure to Ca(2+)- and Sr(2+)-containing solutions produced similar maximal force, but were four times more sensitive to Ca(2+)than to Sr(2+). The rate of loading and maximal SR capacity for caffeine-releasable Ca(2+)and Sr(2+)were similar. In isolated, voltage-clamped ventricular myocytes, the SR content was measured as Na(+)-Ca(2+)exchange current during caffeine-induced SR cation releases. The SR Ca(2+)load reached a steady maximum during a train of voltage clamp depolarizations. A similar maximal Sr(2+)load was not observed, suggesting that the SR capacity for Sr(2+)exceeds that for Ca(2+). Therefore, the relative inability of Sr(2+)to support cardiac EC coupling appears not to be due to failure of the SR to sequester Sr(2+). Instead, increases in cytosolic [Sr(2+)] seem to poorly activate Sr(2+)release from the SR.

A novel anionic conductance affects action potential duration in isolated rat ventricular myocytes.

Effects of extracellular anions were studied in electrophysiological experiments on freshly isolated rat ventricular myocytes. Under current-clamp, action potential duration (APD) was prolonged by reducing the extracellular Cl(-) concentration and shortened by replacement of extracellular Cl(-) with I(-). Under voltage-clamp, membrane potential steps or ramps evoked an anionic background current (I(AB)) carried by either Cl(-), Br(-), I(-) or NO(3)(-). Activation of I(AB) was Ca(2+)- and cyclic AMP-independent, and was unaffected by cell shrinkage. I(AB) was insensitive to stilbene and fenamate anion transport blockers at concentrations that inhibit Ca(2+)-, cyclic AMP- and swelling-activated Cl(-) currents in ventricular cells of other mammals. These results suggest that I(AB) may be carried by a novel class of Cl(-) channel. Correlation of anion substitution experiments on membrane current and action potentials revealed that I(AB) could play a major role in controlling rat ventricular APD. These findings have important implications for those studying cardiac Cl(-) channels as potential targets for novel antiarrythmic agents.

Signature currents: a patch-clamp method for determining the selectivity of ion-channel blockers in isolated cardiac myocytes.

BACKGROUND: We describe a simple method using membrane potential ramps for rapidly determining the ion-channel selectivity of drugs that affect action-potential duration in isolated cardiac myocytes. The method allows the simultaneous assay of compounds on a number of ionic currents in a single cardiac cell. METHODS: Trains of membrane potential ramps were applied from -90 to +70 mV at 0.33 Hz to obtain a consistent "signature current," in which the major individual currents involved in the cardiac action potential could be easily identified. Confirmatory experiments were performed using known inhibitors of these currents. RESULTS: The identities of the currents in the signature were established by varying the concentrations of extracellular cations and by adding known ion channel blockers to superfusion solutions. Inhibition of each current had a characteristic and reproducible effect on the overall signature current. CONCLUSIONS: The consistent current signature in the presence and absence of blockers suggests that this method could be used for tertiary electrophysiological evaluation of compounds, eg, in a drug discovery program focusing on antiarrhythmic agents. The ability to assay for secondary effects of novel compounds against multiple currents in the target cell type is convenient and avoids the artefacts associated with using artificial expression systems.

Calcium-induced release of strontium ions from the sarcoplasmic reticulum of rat cardiac ventricular myocytes.

1. The effects of strontium ions, Sr2+, on Ca(2+)-dependent feedback mechanisms during excitation-contraction coupling were examined in voltage-clamped rat ventricular myocytes in which intracellular [Ca2+] and [Sr2+] were monitored with the fluorescent indicator, indo-1. 2. Voltage clamp depolarizations and caffeine applications during superfusion in Ca(2+)-free, Sr(2+)-containing solutions were employed to exchange intracellular Ca2+ with Sr2+. Myocytes were loaded with Sr2+ by applying voltage clamp depolarizations during superfusion in Na(+)-free, Sr(2+)-containing solutions. 3. Caffeine applications produced large fluorescence transients in Sr(2+)-loaded cells. Thus, Sr2+ could be sequestered and released from the sarcoplasmic reticulum. 4. Ca2+ influx, but not Sr2+ influx, via sarcolemmal Ca2+ channels evoked ryanodine-sensitive fluorescence transients in Sr(2+)-loaded cells. These results demonstrated that Ca2+ influx-induced Sr2+ release (CISR) from the sarcoplasmic reticulum occurred in these experiments, even though Sr2+ influx-induced Sr2+ release was not observed. 5. The amplitude of the Ca2+ influx-induced fluorescence transient was 17 +/- 1% of the caffeine-induced transient (n = 5 cells), an indication that fractional utilization of Sr2+ sequestered in the sarcoplasmic reticulum during CISR was low. 6. With increased Sr2+ loading, the amplitude of Ca2+ influx- and caffeine-induced fluorescence transients increased, but fractional utilization of sarcoplasmic reticulum divalent cation stores was independent of the degree of Sr2+ loading. These data suggest that Ca2+ influx directly activated the release of divalent cations from the sarcoplasmic reticulum, but mechanisms promoting positive feedback of Sr2+ release were minimal during CISR. 7. By comparison, in Ca(2+)-loaded myocytes, Ca2+ influx-induced Ca2+ release (CICR) utilized a greater fraction of caffeine-releasable stores than CISR. Fractional utilization of Ca2+ stores during CICR increased with the degree of Ca2+ loading. 8. Taken together, these results suggest that Ca(2+)-dependent feedback mechanisms play a major role in determining the extent of sarcoplasmic reticulum Ca2+ release during cardiac excitation-contraction coupling under a wide range of conditions.

Control of sarcoplasmic reticulum calcium release during calcium loading in isolated rat ventricular myocytes.

1. Isolated rat ventricular myocytes were whole-cell voltage clamped using electrodes containing fluorescent Ca2+ indicators. Cytosolic [Ca2+] ([Ca2+]i) was estimated with calcium green-2 in combination with carboxy SNARF-1 to remove movement artifacts, or with indo-1. 2. Sarcoplasmic reticulum (SR) Ca2+ was depleted using 20 mM caffeine in Na(+)-containing superfusion solution, and cells were Ca2+ loaded by voltage clamp depolarizations applied during superfusion with Na(+)-free 2 mM Ca2+ solution. Ca2+ currents (ICa) and fluorescence transients elicited by these depolarizations were measured, and the releasable Ca2+ content of the Sr was estimated from the amplitude of fluorescence transients elicited by the rapid application of 20 mM caffeine. 3. Depolarization-induced [Ca2+]i transients increased in amplitude and duration during superfusion with Na(+)-free 2 mM Ca2+ solution, independent of changes in peak ICa. Caffeine application confirmed that the SR Ca2+ content increased during this manoeuvre. 4. With increased Ca2+ loading, the fraction of releasable SR Ca2+ involved in depolarization-induced transients increased, and the gradation in [Ca2+]i transient amplitude produced by beat-to-beat variation of voltage clamp pulse duration (10-100 ms) was progressively lost. This duration dependence of [Ca2+]i transients was maintained during Ca2+ loading when the Ca2+ buffering capacity of the electrode solution was increased with 100 microM BAPTA, 150 microM EGTA, or 60 microM indo-1. 5. These data suggest that Ca2+ released from the SR during a stimulated [Ca2+]i transient promotes further SR Ca2+ release to a degree which is smoothly graded with SR Ca2+ content. The effects of exogenous Ca2+ buffers suggest that this positive feedback is mediated, at least in part, by [Ca2+]i.

A method for recording intracellular [Ca2+] transients in cardiac myocytes using calcium green-2.

Calcium green-2 (Ca green) is a non-ratiometric fluorescent Ca2+ indicator with an affinity for Ca2+ (dissociation constant Kd = 3 microM) that is lower than more commonly used indicators such as fura-2 and fluo-3. This low Ca2+ affinity, coupled with a high quantum yield, allows cells to be loaded with low concentrations of Ca green, avoiding problems of cytosolic Ca2+ buffering and a low signal-to-noise ratio. This communication presents a method for monitoring intracellular [Ca2+] changes in isolated rat ventricular myocytes loaded with Ca green and the fluorescent pH indicator carboxy SNARF-1 (SNARF). SNARF provides a Ca(2+)-insensitive signal with which Ca green fluorescence can be corrected for cell motion and dye-loading artifacts.

Effects of gadolinium on length-dependent force in guinea-pig papillary muscle.

The degree to which stretch-activated channels operate during physiological length changes in multicellular heart preparations, or how much the channels could contribute to length-dependent activation, is not known. We studied the relationship between muscle length and contractile force in guinea-pig papillary muscles superfused with gadolinium chloride (10 microM), a stretch-activated channel blocker, and compared the effects to those with nifedipine (0.25 microM), a calcium channel blocker. Gadolinium reduced contractile force statistically significantly more at the longer muscle lengths than at the short muscle lengths. This did not apply with nifedipine, although a marginally greater effect at longer lengths was perceptible. The results can only partly be explained by gadolinium having a non-specific action via the calcium channel, or Na(+)-Ca2+ exchange, and are consistent with the possibility that stretch-activated channels contribute to length-dependent activation in cardiac muscle, and thus to 'Starling's Law of the Heart'.

Influences of stimulation frequency and temperature on interval-force relationships in guinea-pig papillary muscles.

Relationships between contractile force and the preceding and pre-preceding stimulation intervals were studied in papillary muscles by interposing variable test intervals during steady-state pacing. The strength of test contractions increased exponentially to a maximum as the preceding (test) interval was lengthened. Contractility decreased as an exponential function of pre-preceding interval. At 37 degrees C, the half times for these processes were unaffected by increasing the steady-state frequency from 1 to 3 Hz. At 27 degrees C, the force increase with preceding interval was accelerated and the decay with pre-preceding interval was retarded as the stimulation frequency was increased from 0.33 to 2 Hz. The time-courses of force increase and decay were similar to each other during stimulation at an optimum frequency characteristic for the temperature. Cooling from 37 to 27 degrees C prolonged the half times for force increase and decay by factors of 4.5 and 3 respectively. The slope of the linear relationship between the force of the contraction pre-preceded by the test interval and the immediately subsequent contraction (recirculation fraction) was also halved. These results suggest that high stimulation frequency and low temperature uncouples cellular processes underlying the interval dependence of cardiac contractility. The temperature sensitivities are consistent with these processes being enzymatic. The reduced recirculation fraction provides a mechanism for the lowered threshold frequency for sustained mechanical alternans at 27 degrees C.

Effects of nifedipine and low [Ca2+] on mechanical restitution during hypothermia in guinea pig papillary muscles.

Increasing the frequency of steady state stimulation increases the rate of mechanical restitution in hypothermic (27 degrees C) guinea pig papillary muscles. In this paper we have investigated the influences of the calcium antagonist nifedipine and of reduced extracellular calcium concentration on this phenomenon. We found that nifedipine abolished the frequency dependent increase in the restitution rate, which was also sensitive to extracellular [Ca2+]. These findings suggest that the level of intracellular [Ca2+] can influence the rate of restitution. It is implied that this effect is mediated via ICa, the inward calcium current, which makes a larger than normal contribution to direct contractile activation in hypothermic myocardium.

Mechanical restitution during alternans in guinea pig papillary muscles.

OBJECTIVE: The aim was to investigate alternate acceleration and retardation of mechanical restitution as a possible mechanism for mechanical alternans in isolated myocardium. METHODS: Mechanical alternans was induced in papillary muscles from the right ventricles of 11 guinea pigs (200-300 g) by rapid pacing under hypothermic conditions (T = 27 degrees C). Mechanical restitution curves were constructed by measuring the force responses to stimuli applied following variable test intervals during steady state pacing. Curves were obtained under control conditions (steady state stimulation interval 3 s), and for the beats following the large and small contractions during mechanical alternans. Monoexponentials were fitted to the restitution curves. RESULTS: The mean rate constant for restitution following the large beat in alternans was found to be slightly but significantly smaller than that following the small. Both rate constants obtained during alternans were significantly larger than the control rate constant (restitution was faster in alternans). In addition, as the alternation widened, the restitution curve of the beat following the small contraction developed a higher plateau than that following the large. CONCLUSIONS: The results confirm that the small beat in alternans is followed by faster restitution than the large. This alone is insufficient to explain the observed extent of alternans. The restitution curve for the beat following the small contraction must also rise to a higher plateau. Both the amount of calcium available for intracellular release and the rate at which it is made available vary from beat to beat.

The elementary steps of the actomyosin ATPase in muscle fibres studied with caged-ATP.

Caged-ATP (P3-1(2-nitrophenyl) ethyladenosine 5'-triphosphate) has been used to introduce millimolar concentrations of ATP rapidly into glycerinated muscle fibres, thus removing the limit imposed by diffusion to the time resolution of kinetic measurements. We have combined this technique with a rapid freezing method to arrest ATP hydrolysis abruptly. The work of Ferenczi et al. describes the time course of hydrolysis by fibres in the presence and absence of calcium, and for fibres stretched to a length where there is no myofilament overlap. Ferenczi measured the rate of the phosphate burst in muscle fibres. Here we show how these measurements can be extended to measure the equilibrium constant for the hydrolysis step and the rate of ATP release from the active site. Our initial results indicate that in chemically skinned psoas fibres of the rabbit at 12 degrees C, pH 7.1 and ionic strength of 200 mM, the equilibrium constant for the hydrolysis step is 6, and the rate of release of ATP from the active site is 13 s-1.