Using simulated noise to define optimal QT intervals for computer analysis of ambulatory ECG.

The ambulatory electrocardiogram (ECG) is an important medical tool, not only for diagnosis of adverse cardiac events, but also to predict the risk of such events occurring. The 24-hour ambulatory ECG has certain problems and drawbacks because the signal is corrupted by noise from various sources and also several other conditions which may alter the ECG morphology. We have developed a Windows based program for the computer analysis of ambulatory ECG which attempts to address these problems. The software includes options for importing ECG data, different methods of waveform analysis, data-viewing, and exporting the extracted time series. In addition, the modular structure allows for flexible maintenance and expansion of the software. The ECG was recorded using a Holter device and oversampled to enhance the fidelity of the low sampling rate of the ambulatory ECG. The influence of different sampling rates on the interval variability were studied. The noise sensitivity of the implemented algorithm was tested with several types of simulated noise and the precision of the interval measurement was reported with SD values. Our simulations showed that, in most of the cases, defining the end of QT interval at the maximum of the T wave gave the most precise measurement. The definition of the onset of the ventricular repolarization duration is most precisely made on the maximum or descending maximal slope of the R wave. We also analyzed some examples of time series from patients using power spectrum estimates in order to validate the low level QT interval variability.

Effect of 3,4-diaminopyridine on rat extensor digitorum longus muscle paralyzed by local injection of botulinum neurotoxin.

The actions of the K+ channel blocker, 3,4-diaminopyridine (3,4-DAP), were studied in the rat extensor digitorum longus (EDL) muscle following local inhibition of neuromuscular transmission by botulinum neurotoxin (BoNT). Local paralysis of the EDL muscle was induced by s.c. injections of BoNT serotypes A, B, E or F over the anterior tibialis muscle. One to 14 days later, the rats were anesthetized with urethane, and isometric twitch tensions following stimulation of the peroneal nerve were measured in situ. Muscles were paralyzed within 24 hr of administration of 5 mouse LD50 units (U) of BoNT/A and remained inhibited for the entire 14-day period of observation. Similar levels of inhibition, but of shorter duration, were observed after local injection of 20 U of BoNT/E, 10(4) U of BoNT/B or 20 U of BoNT/F. 3,4-DAP (4 mg/kg, i.v.) potentiated twitch tensions markedly in BoNT/A intoxicated muscle. The increase in tension developed rapidly (halftime = 5.81 +/- 0.6 min), persisted for approximately 1 hr, then decayed slowly with a halftime of 25.2 +/- 4.6 min. Subsequent administration of 3,4-DAP restored tensions to the original maxima, and this procedure could be repeated up to eight times with no decrement. The action of 3,4-DAP was comparable when given 1, 2, 3 or 7 days after BoNT/A and enhanced when administered 14 days after toxin injection. 3,4-DAP was less effective in reversing BoNT/E-induced muscle paralysis and nearly ineffective in antagonizing the paralytic actions of BoNT/B or BoNT/F. The results indicate that 3,4-DAP is of benefit in BoNT/A and BoNT/E intoxication, but is of marginal value after exposure to serotypes B and F.

Conformational analysis of a toxic peptide from Trimeresurus wagleri which blocks the nicotinic acetylcholine receptor.

The 22-residue toxic peptide (WTX1) from the venom of the Southeast Asian snake Trimeresurus wagleri has multiple sites of action, but its lethal effect has been attributed to blocking the postsynaptic acetylcholine receptor at the neuromuscular junction. The 3-dimensional structure of WTX1 was studied using 2-dimensional nuclear magnetic resonance spectroscopy, circular dichroism, and computer simulations. In aqueous solution, WTX1 was shown to have extended and flexible "tails" defined by a short, rigid disulfide-bonded loop. The flexible regions can undergo structural rearrangement when moved from an aqueous to a less polar environment and may contribute to its effectiveness at different receptor sites. By substituting Gly or Phe for His at position 10, significant effects on the disulfide bond formation and, thereby, the activity of the peptide were observed. These results suggest that even subtle differences in single residues can have profound effects on the dynamics of folding, disulfide bond formation, and activity of this toxic peptide.

On the possible origin of giant or slow-rising miniature end-plate potentials at the neuromuscular junction.

Giant or slow-rising miniature end-plate potentials (GMEPPs) caused by vesicular release of acetylcholine (ACh) occur at any time in about 50% of mouse diaphragm neuro muscular junctions, but generally at frequencies less than 0.03 s-1. Their frequency is, unlike that of miniature end-plate potentials (MEPPs), not affected by nerve terminal depolarization. Unlike MEPPs and stimulus-evoked end-plate potentials, GMEPPs have a prolonged time-to-peak and show an increase in time-to-peak with amplitude. By using these differences in amplitude and time course, GMEPPs can be separated from MEPPs. In contrast to MEPPs, GMEPPs are not blocked by botulinum neurotoxin type A. GMEPPs have a greater temperature sensitivity than MEPPs, disappearing at temperatures below 15 degrees C. Long-term paralysis by botulinum toxin and certain drugs which inhibit protein kinase C or affect actin filament polymerization (cytochalasins) enhance the frequency of GMEPPs. End-plate current recordings show that similar postsynaptic ACh receptors are activated by MEPPs and GMEPPs. It is suggested that GMEPPs are not caused by mechanisms involved in regulated neurotransmitter release but are generated by constitutive secretion.

Multiple effects of salicylaldoxime on rat cardiac action potentials.

The effect of salicylaldoxime, 2-(OH)C6H4CH = NOH, on the resting membrane potential and action potential characteristics was studied using isolated right ventricular strips from rat heart. Salicylaldoxime (1-3 mM) reversibly hyperpolarized the cells, increased action potential amplitude, decreased the maximal rate of rise (Vmax) and prolonged duration. The prolongation of the action potential produced by 1 mM salicyaldoxime could not be reversed with isoprenaline (10 microM). Salicyalaldoxime (0.3-1 mM) had no effect on the Ca(2+)-dependent slow action potential for periods up to 60 min. Initial exposure to 3 mM salicylaldoxime produced no changes in the slow action potential, but after 30 min. there was a gradual reduction in amplitude. This effect was completely reversible within 10-15 min. of washout. These data suggest that salicyaladoxime can block Na+, K+ and Ca2+ currents in rat cardiac muscle. Furthermore, it appears that the slow inward Ca2+ current, as measured by the slow action potential, may be sensitive to a dephoshorylating action of this oxime.

Salicylaldoxime blocks K+ and Ca2+ currents in rat cardiac myocytes.

The effects of salicylaldoxime, 2-(OH)C6H4CH = NOH, on the action potential duration, transient outward K+ current and slow inward Ca2+ current were studied in isolated rat ventricular myocytes. The application of salicylaldoxime (0.1-2.0 mM) reversibly increased the action potential duration and reduced in a dose-dependent manner both the transient outward K+ and the slow inward Ca2+ currents. The effect of salicylaldoxime on these two ionic currents was similar to that of 2,3-butanedione monoxime, but was about ten times more potent. Compounds which block both K+ and Ca2+ currents may represent a new type of Class III antiarrhythmic agent which counteracts arrhythmias initiated by re-entry with reduced proarrhythmic risk via triggered activity.

2,3-Butanedione monoxime (BDM) increases initial yields and improves long-term survival of isolated cardiac myocytes.

When BDM is added to the perfusion solutions used during the preparation of single, enzymatically dispersed mammalian cardiomyocytes, higher yields of calcium-tolerant cells are obtained. As the principal component of a storage solution, BDM also improves the survival of myocytes maintained in cold storage. These data support the hypothesis that BDM can act as a cardioprotective agent under certain conditions.

Multiple effects of 2,3-butanedione monoxime.

2,3-Butanedione monoxime, also known as diacetyl monoxime, is a nucleophilic agent which dephosphorylates acetylcholinesterase poisoned with organophosphates. This "chemical phosphatase" activity stimulated studies of the effect of 2,3-butanedione monoxime on phosphorylation-dependent cellular processes. As a result of these studies, we know that the drug affects a number of mechanisms including muscle contraction, ionic current flow and synaptic transmission. Furthermore, it may be used as a component of cardioplegic solutions since it protects cardiac tissue exposed to certain ischaemic conditions. While this MiniReview reveals the diversity of its cellular actions, there continues to be unresolved questions regarding its molecular mechanism.

A novel peptide toxin from Trimeresurus wagleri acts pre- and post-synaptically to block transmission at the rat neuromuscular junction.

The neuromuscular effects of a peptide toxin (peptide I) from venom of Trimeresurus wagleri were investigated using the rat extensor digitorum longus muscle/peroneal nerve preparation. Sub-micromolar concentrations depressed endplate currents (EPCs) produced in response to nerve stimulation. Since quantal content of EPCs was not altered, it appears that the site of action is post-synaptic. However, higher concentrations (1.4-2.9 microM) also inhibited spontaneous release of transmitter. Nerve stimulation in the presence of peptide I caused 'rundown' of EPC amplitude, evidence that the peptide acts pre-synaptically to interfere with transmitter release. Recovery from this effect occurred within 3-5 min. of washing, but EPC amplitude took 20-30 min. to recover. The dual action of this peptide makes it unusual amongst naturally-occurring toxins, and these data suggest that further investigation of the peptide (and its analogues) could yield new information about neurotransmitter release.

Tetrahydroaminoacridine (tacrine) stimulates neurosecretion at mammalian motor endplates.

1. Tacrine (20 microM) induced, like 4-aminoquinoline (4-AQ, 200 microM), the appearance of a population of miniature endplate potentials (m.e.p.ps) with more than twice the normal amplitude or time-to-peak. The times-to-peak of nerve impulse-evoked endplate potentials were not similarly affected. 2. Cholinesterase inhibition by edrophonium (25 microM) did not prevent tacrine or 4-AQ from inducing this population of m.e.p.ps. 3. Nerve-muscle preparations in which the normal calcium-sensitive quantal release of acetylcholine had been blocked by botulinum neurotoxin type A also responded to tacrine by an increase in the frequency of giant or slow m.e.p.ps. 4. Reduction of the temperature from 30 degrees to 14 degrees C reduced the frequency of giant or slow m.e.p.ps induced either by tacrine or by 4-AQ. A similar effect was obtained by colchicine (5 mM). This supports the idea that proximo-distal axonal transport is required for the secretory activity. 5. The neurosecretion evoked by tacrine could explain the therapeutic effects of the drug claimed in the treatment of Alzheimer's type of dementia.

Rapid screening of experimental cardiac drugs using atria and a microcomputer.

The authors investigated the use of a programmable digitizer, multiplexer, and laboratory microcomputer as a rapid and accurate means of evaluating potential inotropic or antiarrhythmic drugs using mammalian atrial preparations.

Inhibition of cardiac phosphodiesterase III by the novel cardiotonic agent 6-[4-(4'-pyridyl)aminophenyl]-4,5-dihydro-3(2H)-pyridazinone hydrochloride.

The novel cardiotonic agent 6[4-(4'-pyridyl)aminophenyl]-4,5-dihydro-3(2H)-pyridazinone hydrochloride (MCI-154) was investigated for its cardiovascular effects and its mechanism of action. In the anaesthetized rat MCI-154 (0.01-0.3 mumol/kg i.v., bolus injection) produced a dose-dependent increase in left ventricular dP/dt, and a decrease in mean arterial pressure. A relatively small increase in heart rate was observed. The drug inhibited selectively canine cardiac phosphodiesterase III (IC50 2.5 +/- 0.6 mumol/l). In skinned porcine trabeculae, MCI-154 produced only a small increase in the Ca2+-sensitivity of the contractile proteins. The results suggest that MCI-154 is a potent cardiotonic agent, and that inhibition of phosphodiesterase III may be a important component of this effect.

Cardiovascular effects of the novel cardiotonic agent DPI 201-106 in the anaesthetized rat.

DPI 201-106 (4-[3-(4-diphenylmethyl-1-piperazinyl)-2-hydroxypropoxy]-1H-indole -2- carbonitrile) was given intravenously to anaesthetized male rats. DPI caused an increase in left ventricular dP/dt (LV dP/dt), giving a significant increase at 0.03 mumol/kg. At this dose DPI had no effect on either mean arterial pressure (MAP) or heart rate (HR). At higher doses, MAP decreased transiently. At 0.3 and 1 mumol/kg, HR was decreased. The results indicate that DPI produces positive inotropic and negative chronotropic effects in the anaesthetized rat.

Effect of ethanol on motor performance and hippocampal population spikes in some standard and selectively outbred rat strains.

Ethanol sensitivity of Wistar and Long-Evans rats was compared in vivo and in vitro. Ethanol was more effective in reducing motor performance in Long-Evans than in Wistar rats, as determined by the tilting plane test. In addition, ethanol produced a greater reduction in the population spikes recorded from hippocampal slices (in vitro) of Long-Evans rats compared to Wistar rats. When rats from the Wistar, Long-Evans, and Sprague-Dawley strains were crossbred and then selectively outbred for high (ANT) and low (AT) sensitivity to ethanol-induced impairment of motor performance, no differences were observed in the ethanol sensitivity of the hippocampal population spike between these two strains. These data suggest that differences in ethanol sensitivity may exist among standard laboratory rodent strains. Selective outbreeding may reduce or eliminate the differences in ethanol sensitivity of brain regions or neurons other than those directly involved in producing the selected behavior. Therefore, it may be incorrect to assume a general difference in ethanol sensitivity when these traits are not coselected during outbreeding, thus indicating different neuronal pools in terms of sensitivity to ethanol.

Antagonism of ethanol-induced depressant effects by 4-aminopyridine in the central nervous system of the rat.

Intraperitoneal injection of ethanol (2 g/kg) produced significant motor impairment in rats, as measured by performance on the tilting plane. Administration of 3 mg/kg 4-aminopyridine (4-AP) antagonized the depressant effect of ethanol on motor performance. Using slices of hippocampus, in vitro, 4-aminopyridine (10-100 microM) also antagonized the ethanol-induced depressant effect on orthodromically-elicited population spikes in the CA1 pyramidal cell layer. This antagonism appears to result from the ability of 4-aminopyridine to enhance release of transmitter in both excitatory and inhibitory neurones. Due to a number of unwanted side effects, further evaluation of 4-aminopyridine and its analogues needs to be done before it can be considered useful in the management of acute intoxication with ethanol.

Real-time acquisition and analysis of cardiac action potentials and twitches using a programmable digitizer and a microcomputer.

Action potentials and single twitches from papillary muscles were acquired and analyzed in real-time using a programmable digitizer and a microcomputer. This combination provides greater control and flexibility during data acquisition than standard analog-to-digital converters. The present system was designed to facilitate evaluation of experimental drugs. However, the combination of a programmable digitizer and a laboratory microcomputer can be applied to a variety of applications in biomedical research.

Effect of 3,4-diaminopyridine on the survival of mice injected with botulinum neurotoxin type A, B, E, or F.

To determine the efficacy of 3,4-diaminopyridine (3,4-DAP) as a potential treatment for botulism, its effect on the survival times of mice injected with type A, B, E, or F botulinum toxin (Bo Tx) was examined. Mice were injected ip with 10, 20, or 40 LD50 of Bo Tx. Three hours later, when the mice displayed signs of botulism, half of each group of mice was treated with 3,4-DAP, an agent which increases nerve-evoked transmitter release. At each dose of type A Bo Tx tested, 3,4-DAP definitely prolonged survival. In contrast, treatment with the drug did not significantly increase the survival time of mice injected with type B, E, or F Bo Tx. The differences in efficacy of 3,4-DAP against the four serotypes of Bo Tx together with previously reported variations in specific toxicity and duration of paralysis may reflect differences in the pharmacological activity of these neurotoxins.

Comparison of the action of types A and F botulinum toxin at the rat neuromuscular junction.

Blockade of neuromuscular transmission was produced in the lower hind limb of the rat by local injection of either type A or type F botulinum toxin (BoTx). At 1, 3, 7, and 10 days after injection, the extensor digitorum longus (edl) nerve-muscle preparation was excised and analyzed for alterations in muscle mechanical properties or spontaneous and nerve stimulus-evoked quantal transmitter release. Muscles receiving type A toxin were paralyzed up to and including 7 days after injection. Muscles treated with type F toxin, although completely paralyzed at 1 and 3 days after injection, twitched in response to nerve stimulation by 7 days. Both toxins induced a marked decrease in the frequency of miniature end-plate potentials, but type A did so to a greater extent. Between 1 and 3 days after toxin injection nerve impulse-evoked transmitter release was reduced in both type A- and type F-treated muscles. Evoked release was temperature sensitive in type A-treated muscles but not in those treated with type F. 3,4-Diaminopyridine (3,4-DAP), a compound which increases nerve-evoked transmitter release by increasing Ca2+ influx, was more effective in reversing the paralysis in type A than in type F-treated muscles. 3,4-DAP induced asynchronous end-plate potentials in response to nerve stimulation in type F-paralyzed muscles, but not in muscles treated with type A. Amidination of the amino groups (presumably lysine) on the toxin by treatment with ethylacetimidate increased the potency and efficacy of only type F BoTx. The results show that type F BoTx differs from type A, mainly by its lower potency, efficacy, shorter duration of action, and by being less effectively antagonized by 3,4-DAP.

Comparison of the effects of botulinum neurotoxin types A and E at the rat neuromuscular junction.

Local blockade of transmitter release was produced by s.c. injection of purified botulinum neurotoxin (NT) types A or E above the tibialis anterior muscle of adult male rats. Extensor digitorum longus nerve-muscle preparation was examined for toxin-induced alterations in single twitch and tetanic tension (in situ) or transmitter release (in vitro). For both single twitch and tetanic tension, muscles treated with type E NT recovered from an initial partial paralysis (induced with 56 mouse LD50) or full paralysis (induced with 565 mouse LD50) by 7 days after NT injection, while those treated with only 5 mouse LD50 of type A remained either fully or partially paralysed through 10 days. Also, miniature end-plate potential frequency and mean quantal content were reduced for a longer period of time and/or to a greater extent for muscles treated with type A NT than for those treated with type E. The present results are consistent with the observed higher specific toxicity (i.p. injections in mice) for type A NT than for type E, although these differences may be exaggerated after s.c. injections. The differences in the paralytic effect between types A and E may be determined by differences in amino acid sequence, which causes type E to dissociate more easily from its site of action and/or be detoxified more rapidly. The clinical implications of these findings are discussed.