1Department of Anesthesiology, CHU Bicêtre, Université Paris Sud, Hôpital de Bicêtre, Assistance Publique, Hôpitaux de Paris, F-94275 Le Kremlin-Bicêtre, France. 2Laboratoire dOptique Appliquée, Ecole PolytechniqueENSTAINSERM U451, Centre de lYvette, F-91761 Palaiseau, France. 3Service de Physiologie, CHU Bicêtre, Université Paris Sud, Hôpital de Bicêtre, F-94275 Le Kremlin-Bicêtre, France*Corresponding author
Accepted for publication: August 9, 2000
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Abstract |
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Br J Anaesth 2001; 86: 1039
Key words: anaesthetics i.v., thiopental; heart, myocardium; rat
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Introduction |
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Numerous cellular processes regulating the homeostasis of contractile performance in rat myocardium differ from those observed in other species. Thus, in rat, Ca2+-induced Ca2+-release from the SR is more strongly developed than in other species.11 In mammalian species, SR Ca2+-ATPase activity is more prominent than Na+/Ca2+ exchanger activity, but in the rat this dominance is much more marked (13-fold vs 2.5-fold).12 Myosin ATPase activity and the percentage of the rapid V1 myosin isoform are higher in rat than in guinea-pig myocardium.13 The plateau duration of the action potential (AP) in the rat is extremely short compared with that in the guinea pig14 and most other mammals, including humans. Moreover, changes in AP duration and in the magnitude of contraction occurring during variations in heart rate are opposite in rat and in guinea pig.14 Because of the special characteristics of rat heart muscle, as well as the various species-related effects of thiopental, the direction of the inotropic effect of thiopental on rat myocardium cannot be predicted. The aims of the present study were to test the effects of thiopental on contractile and electrical activities in rat cardiac left ventricular (LV) papillary muscle and to address the cellular mechanisms that might be involved in the positive inotropic effect induced by thiopental in rat myocardium.
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Materials and methods |
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Mechanical study
The heart was removed rapidly under ether anaesthesia. LV papillary muscles were carefully excised and suspended vertically in 40 ml of KrebsHenseleit solution containing (in mM) 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.1 KH2PO4, 25 NaHCO3, 2.5 CaCl2, 4.5 glucose, bubbled with 95% oxygen/5% carbon dioxide (pH 7.4) and maintained at 29°C. The preparations were field-stimulated at 0.12 Hz by two platinum electrodes with rectangular wave pulses (5 ms duration) just above threshold. After a 1 h stabilization period at Lmax (the initial muscle length at the apex of the length-active isometric tension curve), papillary muscles recovered their optimal performance. Suitable preparations were selected on the basis of the following criteria: (i) well individualized cylindrical shape, with cross-sectional area not exceeding 1.3 mm2 and length at Lmax of 3.5 mm, (ii) ratio of resting force to total isometric force <0.25 and (iii) ratio of maximum shortening and lengthening velocities <0.85 at load equal to preload at Lmax. Adherent or bifid muscles were excluded from the study.
The electromagnetic lever system used to record mechanical data has been described elsewhere.15 Signals of force and length were recorded and digitized; the sampling rate was 1 kHz. All analyses were performed on the basis of digital data without filtering. Mechanical parameters characterizing the contraction phase (inotropic state) were calculated from three twitches with different levels of preload and afterload (Figure 1). The first twitch was isotonic and was loaded with preload only. The second twitch was loaded with preload then abruptly clamped to zero-load immediately after the electrical stimulus, using the zero-load clamp technique. The muscle was released from preload to zero load with critical damping in order to slow the first rapid shortening overshoot resulting from the recoil of the series passive elastic component. This technique allows measurement of the maximum shortening velocity of the unloaded muscle, which is a good parameter of inotropy.16 The third twitch was fully isometric.
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Control values of each mechanical parameter were first recorded using each muscle as its own control. Thiopental sodium (Specia) was added to the bath solution at three concentrations: 3.8x106 M (1 mg litre1) in group 1 (n=9); 3.8x105 M (10 mg litre1) in group 2 (n=13) and 1.1x104 M (30 mg litre1) in group 3 (n=9). Taking into account the protein binding of thiopental in blood, the concentration of 3.8x105 M corresponds to the free drug concentration, reached immediately after a bolus intravenous injection of 6 mg kg1 thiopental in humans.17 This concentration is higher than the concentration compatible with tracheal intubation in the rat.18 In group 4 (n=15), the effect of 3.8x105 M thiopental was studied after adding 1x106 M atenolol (Zeneca Pharma), a selective ß1 adrenergic antagonist. In group 5 (n=9), 3.8x105 M thiopental was tested after adding 1 mM 4-aminopyridine (4-AP, Merck) to inhibit19 the transient outward K+ current (Ito). In group 6 (n=9), 1 mM 4-AP was applied after treatment with 3.8x105 M thiopental. Changes in mechanical parameters were studied 30 min after adding atenolol and 4-AP and 45 min after adding thiopental.
Electrophysiological study
LV papillary muscles were pinned to the bottom of the tissue chamber maintained at 29°C and superfused with KrebsHenseleit solution at a rate of 15 ml min1. Muscles were electrically stimulated, at a frequency of 0.2 Hz, by means of square pulses (5 ms duration) delivered through bipolar earth-isolated platinum electrodes, using an optoelectric coupling device. Transmembrane resting (RP) and action (AP) potentials were recorded with conventional glass microelectrodes (resistance 2530 M; tip potential less than ±3 mV) filled with 3 M KCl and connected to a differential voltage follower via AgAgCl platinum black electrodes (20). Stimulated AP were recorded in Krebs Henseleit control solution after a stabilization period of 30 min before and 10 min after the addition of thiopental (3.8x106 M) and (3.8x105 M), 1 mM 4-AP or both.
The following AP parameters were measured: resting membrane potential (RP, in mV), overshoot (in mV), duration of the plateau (APD0, in ms) measured at 0 mV, and duration of the slow repolarizing phase of the AP (APD10, in ms) measured at +10 mV from the RP. In the figures, m corresponds to the number of impalements recorded from n muscles tested. Transmembrane potentials were displayed on a Nicolet 310 oscilloscope, digitized with a sampling rate of 1 kHz without filtering via a Labmaster acquisition card (DMA 100 OEM) driven by the software program Acquis1, linked to the mass storage of a personal computer.
Statistical analysis
Data are expressed as mean (SD). Comparisons with control values were made using Students paired t test. Comparisons between groups were made using Students t-test with the Bonferroni correction when appropriate. All P values were two-tailed; a P value of <0.05 was necessary to reject the null hypothesis.
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Results |
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Discussion |
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Rat cardiac AP consists of a spike induced by the fast inward Na+ current, followed by a short plateau generated by the inward Ca2+ current, which is modulated by the transient outward K+ current (Ito), and a slow repolarization phase thought to be due to the development of the delayed outward K+ current and the electrogenicity (3 Na+/1 Ca2+) of the Na+/Ca2+ exchanger.14 16 27 The importance of Ito28 in determining early repolarization during the plateau has been shown in normal29 and pathological30 mammalian cardiac muscles. Inhibition of Ito by 4-AP in rat heart muscle increases the duration of the AP plateau.19
In our study, the lengthening of the plateau induced by thiopental suggests that the anaesthetic exerted a 4-AP-like effect. Lengthening of the AP of rat cardiac ventricular myocytes, after application of a control solution containing 4-AP, leads to a more sustained Ca2+ current associated with an increased contractile response.25 4-AP had a marked positive inotropic effect on rat left ventricular papillary muscle. Thus, the extended plateau induced by thiopental which occurred in a range of membrane potentials within which a large proportion of Ca2+ channels remains activated, may partly account for the increase in contractile force.
Thiopental also lengthened the slow repolarizing phase of the AP. This lengthening occurred in the presence or absence of 4-AP and may be a result of a decrease in both the delayed outward K+ current and IK1.9 23 Lengthening of AP duration has been associated with an increase of inotropy because greater Ca2+ influx is expected during this period. Given that, in rat myocardium, Ca2+-induced Ca2+ release from the SR is highly sensitive to Ca2+ influx into the cells,31 greater Ca2+ influx during the AP may contribute to increased Ca2+ release from the SR and thus to a positive inotropic effect. Such a mechanism is suggested to explain the positive inotropic effect of an 1-adrenergic agent32 and of the immunosuppressant agent FK 506.33 However, to maintain a positive effect on the systolic Ca2+ transient, a drug may also modify processes other than Ca2+-induced Ca2+ release.21 For example, in the case of FK 506 it has been suggested that the sustained positive inotropic effect could be due to inhibition of the Na+/Ca2+ exchange, resulting in an increase in Ca2+ availability for SR uptake. In the case of thiopental, further studies are necessary to investigate such a mechanism.
One of the limitations of our study is the use of a low stimulation frequency. In the rat, a low stimulation frequency induces an increase in inotropy because a prolonged diastole leads to an increase in SR Ca2+ uptake. Each contraction takes place after an 8 s diastolic pause, allowing a return to baseline diastolic Ca2+ level, thus, a drug-induced anomaly of this uptake can be hidden. However, most mechanical studies use such a low frequency to avoid the many rhythm anomalies induced by high frequencies. Moreover, it has been shown that thiopental does not alter SR uptake, at least in the rabbit.10
In conclusion, this study shows that thiopental increases inotropy in rat cardiac papillary muscle. This positive inotropic effect can be linked to the lengthening of both the plateau and the slow repolarizing phase of the AP. This leads to an increase in the Ca2+ influx into the cardiac cells, which improves mechanical performance.
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Acknowledgements |
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References |
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