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ABSTRACT: The properties of left ventricular cardiac myocytes vary transmurally. This may be related to the gradients of stress and strain experienced in vivo across the ventricular wall. We tested the hypothesis that within the rat left ventricle there are transmural differences in the expression of genes for proteins that are involved in mechanosensitive pathways and in associated physiological responses. Real time reverse transcription polymerase chain reaction was used to measure messenger RNA (mRNA) levels of selected targets in sub-epicardial (EPI) and sub-endocardial (ENDO) myocardium. Carbon fibres were attached to single myocytes to stretch them and to record contractility. We observed that the slow positive inotropic response to stretch was not different between EPI and ENDO myocytes and consistent with this, that the mRNA expression of two proteins implicated in the slow response, non-specific cationic mechanosensitive channels (TRPC-1) and Na/H exchanger, were not different. However, mRNA levels of other targets, e.g. the mechanosensitive K(+) channel TREK-1, Brain Natriuretic Peptide and Endothelin-1 receptor B, were significantly greater in ENDO than EPI. No targets had significantly greater mRNA levels in EPI than ENDO. On the basis of these findings, we suggest that the response of the ventricle to stretch will depend upon both the regional differences in stimuli and the relative expression of the mechanosensitive targets and that generally, stretch sensitivity is predicted to be greater in ENDO.
Pflügers Archiv - European Journal of Physiology 08/2007; 454(4):545-9. · 4.46 Impact Factor
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ABSTRACT: Serum levels of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) increase during an inflammatory response and have been reported to induce a negative inotropic effect on the myocardium. Alfentanil, an opioid analgesic often used in the critical care of patients with sepsis, has been shown to enhance ventricular contractility. This study characterised the effects of TNF-alpha and IL-1beta on contraction and the Ca(2+) transient and investigated whether depressed ventricular function was ameliorated by alfentanil.
Isolated rat ventricular myocytes were loaded with fura-2 and electrically stimulated at 1 Hz. Contraction and Ca(2+) transients were measured after 60, 120 and 180 min incubations in TNF-alpha (0.05 ng ml(-1)) and IL-1beta (2 ng ml(-1)). The effects of 10 microM alfentanil on contractility and Ca(2+) transients of TNF-alpha and IL-1beta treated cells were determined. Key results:After 180 min of TNF-alpha and IL-1beta treatment, the amplitude of contraction, the Ca(2+) transient and sarcoplasmic reticulum (SR) Ca(2+) content were significantly reduced. Alfentanil significantly increased contraction of TNF-alpha and IL-1beta treated cells via a small increase in the Ca(2+) transient and a larger increase in myofilament Ca(2+) sensitivity, effects that were not blocked by 10 microM naloxone, a broad spectrum opioid receptor antagonist.
TNF-alpha and IL-1beta induce a significant negative inotropic effect on ventricular myocytes in a time dependent manner through disruption of SR Ca(2+) handling and the Ca(2+) transient. This negative inotropic effect was ameliorated by alfentanil, but this response may not be mediated via opioid receptors.
British Journal of Pharmacology 04/2007; 150(6):720-6. · 4.41 Impact Factor
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ABSTRACT: The electrogenic Na+/Ca2+ exchanger (NCX) represents the main extrusion pathway for Ca2+ in ventricular muscle and therefore plays an important role in the regulation of cytosolic Ca2+ and contraction. Halothane and sevoflurane modulate cytosolic Ca2+ regulation and at steady state are negatively inotropic, however, the involvement of anaesthetic-induced changes in NCX activity in these effects requires further study.
Ventricular myocytes were isolated using a standard collagenase/protease dispersion technique and superfused with a physiological salt solution at 30 degrees C. Whole-cell patch-clamp technique was used to control membrane voltage. I(NCX) (identified as Ni2+ sensitive current) was recorded using a ramp clamp protocol under conditions to inhibit contaminating currents.
With 0.6 mM sevoflurane, outward I(NCX) at positive voltages (> or = 0 mV) and inward I(NCX) at voltages negative to -60 mV was significantly reduced (P<0.05, n=13; I(NCX) reduced by 48% at +50 and 65% of control at -120 mV). Halothane (0.6 mM) inhibited outward I(NCX) at voltages positive to -10 mV and inward I(NCX) at voltages negative to -80 mV (P<0.05, n=10; I(NCX) reduced by 64% at +50 and 65% of control at -120 mV). Anaesthetic-induced inhibition of both inward and outward current was not voltage-dependent.
Inhibition of Ca2+ efflux via NCX (i.e. inward I(NCX)) during an exposure to halothane or sevoflurane would be expected to limit the negative inotropic effects of these agents and help maintain SR Ca2+ content.
BJA British Journal of Anaesthesia 09/2005; 95(3):305-9. · 4.24 Impact Factor
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ABSTRACT: The volatile anaesthetics isoflurane and sevoflurane induce both negative and positive inotropic effects in ventricular myocytes, the mechanisms of which are not fully understood. Previous data suggest that changes in myofilament Ca(2+) sensitivity contribute to their sustained negative inotropic effects. In this study, the role of changes in myofilament Ca(2+) sensitivity in both positive and negative inotropic effects of these agents was examined in intact ventricular myocytes.
Contractility and cytosolic Ca(2+) (fura-2) were recorded optically in ventricular myocytes stimulated electrically (1 Hz) at 30 degrees C. Myofilament Ca(2+) sensitivity was assessed from plots of cell length against fura-2 fluorescence ratio (Fr) from individual twitches at various points before, during and after a 1 or 4 min exposure to 0.6 mM anaesthetic.
Isoflurane reduced mean (sd) myofilament Ca(2+) sensitivity from 10.3 (1.9) to 5.9 (1.6) microm Fr(-1) (P<0.001) throughout a 1 min exposure, which returned to control on removal. In contrast, on initial exposure to sevoflurane, Ca(2+) sensitivity was reduced from 10.8 (1.3) to 4.3 (0.9) microm Fr(-1) (P<0.001) but this recovered partially towards control over 3 min. On removal, sensitivity was increased above control (to 17.7 (2.2) microm Fr(-1); P<0.001) before preanaesthetic levels were restored.
These data show that both isoflurane and sevoflurane reduce apparent myofilament Ca(2+) sensitivity at steady state. However, sevoflurane (but not isoflurane) induced transient changes in apparent myofilament Ca(2+) sensitivity, which would contribute to its inotropic profile.
BJA British Journal of Anaesthesia 04/2005; 94(3):279-86. · 4.24 Impact Factor
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ABSTRACT: Some of the cellular targets affected by volatile anaesthetics (e.g. halothane) which contribute to the negative inotropic effects of these agents are also affected during the progression of diabetic cardiomyopathy. A previous report suggested that halothane inhibited contraction to a lesser extent in papillary muscle from diabetic animals and so the aim of this study was to investigate possible mechanisms underlying this effect.
Contractility and cytosolic calcium ion (Ca(2+)) transients were measured (fura-2) in ventricular myocytes isolated from control and streptozotocin (STZ)-induced diabetic rats in the absence and presence of halothane 0.6 mmol litre(-1) at 1 Hz stimulation. Sarcoplasmic reticulum (SR) Ca(2+) content was assessed by rapid application of caffeine. All experiments were carried out at 36-37 degrees C.
The amplitude of shortening, the electrically evoked Ca(2+) transient, SR Ca(2+) content and myofilament Ca(2+) sensitivity, though not altered by STZ treatment, were significantly reduced by halothane to a similar extent in control and STZ myocytes. The time course of contraction and Ca(2+) transient were prolonged in myocytes from STZ-treated rats compared with controls but this was not altered further by halothane. STZ treatment appeared to reduce Ca(2+) efflux from the cell, an effect reversed by halothane.
In contrast to a previous report, we could find no evidence of amelioration of the negative inotropic effect of halothane in myocytes from the STZ-induced diabetic rat. Contractility, the cytosolic Ca(2+) transient, SR Ca(2+) content and myofilament Ca(2+) sensitivity were qualitatively similar in control and STZ myocytes and were all depressed to the same extent by halothane.
BJA British Journal of Anaesthesia 03/2004; 92(2):246-53. · 4.24 Impact Factor
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ABSTRACT: Halothane shortens ventricular action potential duration (APD), as a consequence of its inhibitory effects on a variety of membrane currents, an effect that is greater in sub-endocardial than sub-epicardial myocytes. In hypertrophied ventricle, APD is prolonged as a consequence of electrical remodelling. In this study, we compared the effects of halothane on transmural APD in myocytes from normal and hypertrophied ventricle.
Myocytes were isolated from the sub-endocardium and sub-epicardium of the left ventricle of spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. Action potentials were recorded before, during, and after a 1-min exposure to 0.6 mM halothane and APD measured from the peak of the action potential to repolarization at -50 mV (APD(-50 mV)). Data are presented as mean (SEM).
In WKY myocytes, halothane reduced APD(-50 mV) from 21 (2) to 18 (2) ms (P<0.001, n=15) in sub-epicardial myocytes but abbreviated APD(-50 mV) to a greater extent in sub-endocardial myocytes (37 (4) to 28 (3) ms; P<0.001, n=14). In SHR myocytes, APD(-50 mV) values were prolonged compared with WKY and APD(-50 mV) was reduced by halothane from 36 (6) to 27 (4) ms (P<0.016) and from 77 (10) to 38 (4) ms (P<0.001) in sub-epicardial and sub-endocardial myocytes, respectively.
In the SHR, hypertrophic remodelling was not homogeneous; APD(-50 mV) was prolonged to a greater extent in sub-endocardial than sub-epicardial cells. Halothane reduced APD to a greater extent in sub-endocardium than sub-epicardium in both WKY and SHR but this effect was larger proportionately in SHR myocytes. The transmural gradient of repolarization was reduced in WKY and effectively abolished in SHR by halothane, which might disturb normal ventricular repolarization.
BJA British Journal of Anaesthesia 04/2003; 90(4):501-3. · 4.24 Impact Factor
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ABSTRACT: Short-term (6 weeks) voluntary wheel running exercise in young female rats that were in an active growth phase resulted in whole-heart hypertrophy and myocyte concentric hypertrophy, when compared to sedentary controls. The cross-sectional area of ventricular myocytes from trained rats was significantly greater than for those isolated from sedentary rats, with the greatest change in morphology seen in sub-endocardial cells. There was no statistically significant effect of training on cell shortening in the absence of external mechanical loading, in [Ca2+](i) transients, or in myofilament Ca2+ sensitivity (assessed during re-lengthening following tetanic stimulation). Under the external mechanical load of carbon fibres, absolute force developed in myocytes from trained rats was significantly greater than in those from sedentary rats. This suggests that increased myocyte cross-sectional area is a major contractile adaptation to exercise in this model. Training did not alter the passive mechanical properties of myocytes or the relative distribution of titin isomers, which was exclusively of the short, N2B form. However, training did increase the steepness of the active tension-sarcomere length relationship, suggesting an exercise-induced modulation of the Frank-Starling mechanism. This effect would be expected to enhance cardiac contractility. Training lengthened the action potential duration of sub-epicardial myocytes, reducing the transmural gradient in action potential duration. This observation may be important in understanding the cellular causes of T-wave abnormalities found in the electrocardiograms of some athletes. Our study shows that voluntary exercise modulates the morphological, mechanical and electrical properties of cardiac myocytes, and that this modulation is dependent upon the regional origin of the myocytes.
The Journal of Physiology 07/2002; 541(Pt 3):863-75. · 4.72 Impact Factor
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ABSTRACT: Short-term (6 weeks) voluntary wheel running exercise in young female rats that were in an active growth phase resulted in whole-heart hypertrophy and myocyte concentric hypertrophy, when compared to sedentary controls. The cross-sectional area of ventricular myocytes from trained rats was significantly greater than for those isolated from sedentary rats, with the greatest change in morphology seen in sub-endocardial cells. There was no statistically significant effect of training on cell shortening in the absence of external mechanical loading, in [Ca2+]i transients, or in myofilament Ca2+ sensitivity (assessed during re-lengthening following tetanic stimulation). Under the external mechanical load of carbon fibres, absolute force developed in myocytes from trained rats was significantly greater than in those from sedentary rats. This suggests that increased myocyte cross-sectional area is a major contractile adaptation to exercise in this model. Training did not alter the passive mechanical properties of myocytes or the relative distribution of titin isomers, which was exclusively of the short, N2B form. However, training did increase the steepness of the active tension-sarcomere length relationship, suggesting an exercise-induced modulation of the Frank-Starling mechanism. This effect would be expected to enhance cardiac contractility. Training lengthened the action potential duration of sub-epicardial myocytes, reducing the transmural gradient in action potential duration. This observation may be important in understanding the cellular causes of T-wave abnormalities found in the electrocardiograms of some athletes. Our study shows that voluntary exercise modulates the morphological, mechanical and electrical properties of cardiac myocytes, and that this modulation is dependent upon the regional origin of the myocytes.
The Journal of Physiology 05/2002; 541(3):863 - 875. · 4.72 Impact Factor
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ABSTRACT: Halothane inhibits the 4-aminopyridine-sensitive transient outward K(+) current (I(to)) which in many species, including humans, plays an important role in determining action potential duration. As I(to) is greater in the ventricular subepicardium than subendocardium, halothane may have differential effects on action potential duration and, therefore, contraction in cells isolated from these two regions.
Myocytes were isolated from the subendocardium and subepicardium of the rat left ventricle. Myocytes from each region were electrically stimulated at 1 Hz to measure contractions and action potentials and exposed to 0.6 mm halothane (approximately 2 x minimum alveolar concentration(50) for the rat) for 1 min. The time from the peak of the action potential to repolarization at 0 and -50 mV was measured to assess the effects of halothane on action potential duration.
Halothane inhibited contraction to a significantly (P = 0.002) greater extent in subendocardial myocytes than in subepicardial myocytes: the amplitude of contraction during control conditions was 3.6 +/- 0.4 microm and 3.2 +/- 0.7 microm in subendocardial and subepicardial cells, respectively, and this was reduced to 1.1 +/- 0.2 microm (29 +/- 2% of control, P < 0.0001, n = 10) and 1.4 +/- 0.3 microm (46 +/- 3% of control, P = 0.007, n = 7), respectively, after a 1-min exposure to 0.6 mm halothane. Control action potential duration (at -50 mV) was 67 +/- 10 and 28 +/- 4 ms in subendocardial and subepicardial myocytes, respectively, and these values were reduced to 39 +/- 6 ms (58 +/- 3% of control, P < 0.001) and 20 +/- 3 ms (73 +/- 5% of control, P = 0.009) by halothane, respectively.
Action potential duration was reduced to a greater extent in subendocardial than subepicardial myocytes, which would contribute to the greater negative inotropic effect of halothane in the subendocardium. Furthermore, the transmural difference in action potential duration was reduced by halothane, which could contribute to its arrhythmogenic properties.
Anesthesiology 11/2001; 95(5):1213-9. · 5.36 Impact Factor
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ABSTRACT: A model of voluntary exercise, in which rats are given free access to a running wheel over a 14-week period, led to left ventricular hypertrophy. To test whether the hypertrophic response to exercise was uniformly distributed across the ventricular wall, single ventricular myocytes were isolated from the sub-epicardium (EPI) and sub-endocardium (ENDO) of exercised rats and from sedentary rats for comparison. Cellular hypertrophy (approximately 20 % greater cell volume) was seen in ENDO cells from exercised animals, but no significant changes were observed in EPI cells when compared with sedentary controls. This regional effect of exercise may be a response to transmural changes in ventricular wall stress and/or strain. Cell contraction was measured as cell shortening in ENDO and EPI cells at stimulation frequencies between 1 and 9 Hz at 37 degrees C. Exercise training had no effect on cell shortening. Positive and negative contraction-frequency relationships (CFRs) were found in both EPI and ENDO cells between 1 and 5 Hz; at higher frequencies (5-9 Hz), all myocytes displayed a negative CFR. The CFR of a myocyte was, therefore, independent of regional origin and unaffected by exercise. These results suggest that, in vivo, the rat heart displays a negative CFR. We conclude that increased cell size may be a more important adaptive response to exercise than a modification of excitation-contraction coupling.
Journal of Experimental Biology 04/2001; 204(Pt 6):1191-9. · 3.00 Impact Factor
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ABSTRACT: The aim of this study was to describe and compare the effects of isoflurane, sevoflurane, and halothane at selected concentrations (i.e., concentrations that led to equivalent depression of the electrically evoked Ca2+ transient) on myofilament Ca2+ sensitivity, sarcoplasmic reticulum (SR) Ca2+ content, and the fraction of SR Ca2+ released during electrical stimulation (fractional release) in rat ventricular myocytes.
Single rat ventricular myocytes loaded with fura-2 were electrically stimulated at 1 Hz, and the Ca2+ transients and contractions were recorded optically. Cells were exposed to each anesthetic for 1 min. Changes in myofilament Ca2+ sensitivity were assessed by comparing the changes in the Ca2+ transient and contraction during exposure to anesthetic and low Ca2+. SR Ca2+ content was assessed by exposure to 20 mm caffeine.
Isoflurane and halothane caused a depression of myofilament Ca2+ sensitivity, unlike sevoflurane, which had no effect on myofilament Ca2+ sensitivity. All three anesthetics decreased the electrically stimulated Ca2+ transient. SR Ca2+ content was reduced by both isoflurane and halothane but was unchanged by sevoflurane. Fractional release was reduced by both isoflurane and sevoflurane, but was unchanged by halothane.
Depressed myofilament Ca2+ sensitivity contributes to the negative inotropic effects of isoflurane and halothane but not sevoflurane. The decrease in the Ca2+ transient is either responsible for or contributory to the negative inotropic effects of all three anesthetics and is either primarily the result of a decrease in fractional release (isoflurane and sevoflurane) or primarily the result of a decrease in SR Ca2+ content (halothane).
Anesthesiology 11/2000; 93(4):1034-44. · 5.36 Impact Factor
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ABSTRACT: Whether propofol contributes a direct negative inotropic effect is controversial. Our principal aim in this study was to determine whether negative inotropic effects of propofol occur at clinically relevant concentrations. We constructed the concentration-response relationship for the negative inotropic effects on intact, isolated, stimulated rat ventricular myocytes. Contraction was measured as cell shortening by using an optical system. Propofol was applied as dilutions of the commercial preparation in physiological saline solution. The drug vehicle had a minimal effect on myocyte contractility. Propofol produced a concentration-dependent reduction in evoked contraction at concentrations greater than 5 microM. The maximum effect was observed at >100 microM, with the K(0.5) calculated to be 34.5 microM (95% CI, 21.8-54.7 microM). In further experiments, we investigated the relationship between changes in contractility and changes in Ca(2+) transient (measured by using fura-2 fluorescence) after the application of propofol. By using the shift in the relationship of the cell length to fura-2 fluorescence ratio in the relaxation phase of a contraction as an index of Ca(2+) response of the myofilaments, we demonstrated that some of the negative inotropic effect of propofol may be caused by a reduction in myofilament Ca(2+) sensitivity. We confirmed this by comparing the reduction in contractility in the presence of propofol with that caused by reducing the extracellular Ca(2+) concentration. We observed that, for a decrease in the fura-2 fluorescence ratio of 21%, propofol caused a 12% (95% CI, 2% to 22%) greater reduction in contractility than predicted from reducing the extracellular Ca(2+) concentration. However, the K(0.5) for the negative inotropic effect of propofol we observed is more than 80 times the 50% effective concentration value for anesthesia. The potential relevance of these findings for clinical use of propofol in humans is discussed. Implications: By using intact, isolated rat heart ventricle cells, we investigated the mechanisms and concentration dependence of the depressant effect of propofol on contractility of the heart. We conclude that direct effects of propofol on the heart are unlikely to be of significance at the clinical dosage usually given.
Anesthesia & Analgesia 09/2000; 91(2):276-82. · 3.29 Impact Factor
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ABSTRACT: In previous studies, regional variations in the expression of the Na+-Ca2+ exchanger (NCX) have been examined qualitatively in human heart using the C2C12 monoclonal antibody [Wang, J., Schwinger, R.H., Frank, K., Muller-Ehmsen, J., Martin-Vasallo, P., Pressley, T.A., Xiang, A., Erdmann, E. & McDonough, A.A. (1996) J. Clin. Invest. 98, 1650-1658]. Although NCX expression was found to be significantly lower in the atria compared to the septum, no significant differences were found between atrial and ventricular tissue. NCX has been located in the general sarcolemma and t-tubules of ventricular muscle and as t-tubules are sparse in atrial tissue compared to ventricular tissue, it is surprising that NCX expression was found to be similar in both atria and ventricles [Wang et al. (1996)]. To reinvestigate this, we have used SDS/PAGE and a quantitative Western blotting technique to determine the pattern of expression of NCX in guinea-pig heart in tissue samples from left atrium, right atrium, septum, left ventricle and right ventricle. NCX protein expression was 17.5 +/- 3.9 pmol.mg-1 of protein in the left atrium and 29.2 +/- 6.1 pmol.mg-1 of protein in the right atrium, which were both significantly lower (P < 0.05) than NCX expression in the septum, left ventricle and right ventricle (64.7 +/- 15.2, 76.8 +/- 19.5 and 69.4 +/- 14.1 pmol.mg-1 of protein, respectively, n = 7). These differences in NCX expression may reflect variations in the cellular location of NCX protein in these regions. To study this, we used confocal immunofluorescence of single isolated myocytes to examine differences in the proportion of fluorescent staining on the general surface membrane compared with the interior of the cell (which presumably reflects a t-tubular location). We found that the general membrane staining was 79.0 +/- 1.2% in cells from the atria which was significantly higher (P < 0. 001) than that seen in cells from the septum, left ventricle and right ventricle, with 48.1 +/- 1.1%, 48.2 +/- 1.8% and 45.6 +/- 1.3%, respectively (n = 20). These results illustrate a similar pattern of NCX expression in guinea-pig and human, with expression in atrial tissue significantly lower than in ventricular tissue. However, the cellular location of NCX differs regionally; in atrial tissue, the majority of the NCX protein is located in the general sarcolemma whereas in ventricular and septal tissue, approximately 50% of NCX protein is located within the cell (presumably at the level of the t-tubules).
European Journal of Biochemistry 08/2000; 267(16):5142-8. · 3.58 Impact Factor
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ABSTRACT: We have described the concentration-dependent inotropic effects of halothane, isoflurane and sevoflurane on rat ventricular cells and investigated the role of the sarcoplasmic reticulum (SR) in these inotropic actions. Single ventricular myocytes, isolated from rat hearts, were stimulated electrically at 1 Hz and contractions recorded optically. Cells were exposed to a range of concentrations of halothane, isoflurane or sevoflurane for a period of 1 min to determine the concentration-dependency of their inotropic actions. For each anaesthetic, the peak negative inotropic action was determined early during an exposure, and sustained negative inotropic action was measured at steady-state just before wash-off. In some experiments, cells were equilibrated with ryanodine 1 mumol litre-1 to investigate the role of the SR in these intropic effects. Halothane caused a concentration-dependent initial increase in contractions (to mean 130 (SEM 28)% at 10 mmol litre-1) followed by rapid onset of a negative inotropic effect (K0.5 0.34 mmol litre-1 for peak effect; K0.5 0.46 mmol litre-1 for sustained effect). Exposure to isoflurane induced a small potentiation of contractions in some cells, followed by a concentration-dependent decrease in contraction in all cells (K0.5 0.85 mmol litre-1 for peak effect; K0.5 1.92 mmol litre-1 for sustained effect); contractions recovered partially during a 1-min exposure. On wash-off, contractions were increased transiently above control. Sevoflurane caused a large initial decrease in contraction which then returned rapidly towards control (K0.5 0.2 mmol litre-1 for peak effect; K0.5 2.57 mmol litre-1 for sustained effect). In common with isoflurane, removal of sevoflurane caused a transient increase in contractions above control. After exposure to ryanodine, the positive inotropic effects of halothane and isoflurane did not occur, and recovery of contractions during exposure to isoflurane and sevoflurane was abolished as was the transient increase in contractions seen on wash-off, indicating that these effects were mediated via the SR. Halothane had the most potent sustained negative inotropic effect but there was little difference between the negative inotropic effects of isoflurane and sevoflurane at clinically relevant concentrations. At higher concentrations, sevoflurane caused a less potent negative inotropic effect than isoflurane. The SR plays a major role in the effects of all three anaesthetics. One possible mechanism underlying the initial potentiation of contraction by halothane (and isoflurane) may be sensitization of the Ca(2+)-induced Ca(2+)-release process of the SR.
BJA British Journal of Anaesthesia 06/1999; 82(5):723-30. · 4.24 Impact Factor
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ABSTRACT: The mechanisms contributing to the negative inotropic effect of halothane were studied in isolated rate ventricular myocytes. Contraction and intracellular Ca2+ transients were measured optically in these cells. The initial application of halothane (2% or 0.5 mmol litre-1) led to short-lived increases in the Ca2+ transient and contraction, which were abolished by ryanodine. Continued application of halothane led to a sustained decrease in contraction: this resulted from: (i) a decrease in myofilament Ca2+ sensitivity; (ii) a decrease in the Ca2+ transient; and (iii) a decrease in the Ca2+ content of the sarcoplasmic reticulum. Although halothane reduced action potential duration, the sustained negative inotropic effect was similar when action potentials or voltage clamp pulses of constant duration were used to trigger contractions. In cells exposed to nifedipine 0.5 mumol litre-1 (which decreases the L-type Ca2+ current, ICa), Ca2+ transients, sarcoplasmic reticulum Ca2+ content and fractional release (the fraction of sarcoplasmic reticulum Ca2+ content released during each stimulus) were reduced. Halothane 0.5 mmol litre-1 (which also decreases ICa) decreased Ca2+ transients to a lesser extent and reduced sarcoplasmic reticulum Ca2+ content to a greater extent than nifedipine, whereas fractional release was unchanged compared with control. These data suggest that halothane sensitizes Ca(2+)-induced Ca2+ release from the sarcoplasmic reticulum in addition to reducing ICa.
BJA British Journal of Anaesthesia 05/1999; 82(4):609-21. · 4.24 Impact Factor
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ABSTRACT: A method is described that enables the cell membrane of isolated rat ventricular myocytes to be permeabilized and resealed while maintaining cell viability. Streptolysin O, a cholesterol-binding cytolysin, was used to form pores in the surface membrane; subsequent incubation with 5% fetal bovine serum was used to reverse this permeabilization. The efficacy of membrane permeabilization and resealing was ascertained using a simultaneous double-staining technique using propidium iodide, a marker for cells with permeabilized membranes, and fluorescein diacetate, a marker for viable cells. This procedure allowed a distinction to be made between dead cells, unpermeabilized cells and viable cells that had been successfully permeabilized and resealed. The accessibility of the cell interior during permeabilization was investigated by including fluorescein isothiocyanate (FITC)-labelled dextrans (11, 38 and 148 kDa) and bovine serum albumin (67 kDa) in the permeabilization buffer, and localizing the FITC label using confocal microscopy following resealing. The confocal images showed that these molecules entered the cells and were retained after resealing. Following the permeabilization-resealing protocol, cells appeared to have both normal morphology and response to electrical stimulation. Thus this appears to be a cheap, simple and effective method to introduce relatively large molecules into cardiac myocytes.
Experimental Physiology 06/1998; 83(3):293-303. · 3.21 Impact Factor
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ABSTRACT: Exposure of cardiac muscle to metabolic poisons reduces the availability of cellular ATP and cardiac dysfunction ensues. In this study rat ventricular myocytes were exposed to 2-deoxyglucose, iodoacetate and cyanide to induce complete metabolic blockade. Changes in contraction, cytosolic Ca2+ and pH were determined during metabolic blockade and following restoration of mitochondrial ATP production. Metabolic blockade resulted in a rapid failure of contractions and Ca2+ transients, a rise of diastolic Ca2+, a cytosolic acidosis and ultimately a rigor contracture. Washing out cyanide during the development of the rigor contracture led to a rapid relaxation of the contracture, a fall in cytosolic Ca2+ and a rapid, partial reversal of the cytosolic acidosis. The partial reversal of the cytosolic acidosis and fall of cytosolic Ca2+ were abolished in the presence of oligomycin. This suggests that the rapid partial recovery of cytosolic acidosis could result from the rephosphorylation of ADP to ATP by the mitochondrial F1,F0-ATPase (a reaction that consumes protons).
Experimental Physiology 06/1998; 83(3):349-60. · 3.21 Impact Factor
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ABSTRACT: We have investigated the effect of temperature upon the rate-dependent decrease in the L-type Ca2+ current (iCa) in isolated rat ventricular myocytes. Increasing the rate of stimulation from 0.5 to 3.0 Hz for 30s induced a reversible decrease in iCa which was temperature dependent. Compared to control (0.5 Hz), the first beat at 3 Hz was decreased by 38 +/- 7% at 22 degrees C and by 9 +/- 1% at 37 degrees C (mean +/- S.E.M., n = 5, P < 0.05) and, after 30 s of 3 Hz stimulation, iCa was reduced by a further 26 +/- 4 and 21 +/- 2% at 22 and 37 degrees C, respectively. The magnitude of this secondary decline was not significantly different at the two temperatures (P = 0.29). Corroboratory results were obtained from cell-attached patches which also illustrated that the rate-dependent decrease in iCa resulted from a reduction of open channel probability. Paired pulse experiments showed that the greater initial rate-dependent decrease in iCa at 22 degrees C occurred as a result of slower recovery from fast inactivation processes at 22 than at 37 degrees C. Recovery of the channel from fast inactivation was very temperature sensitive with a Q10 of 5.6. In contrast, the secondary, progressive decrease in iCa, which results from incomplete recovery from ultra-slow voltage-dependent inactivation, was similar at the two temperatures and appears to be much less temperature dependent.
Experimental Physiology 02/1998; 83(1):49-63. · 3.21 Impact Factor
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ABSTRACT: 1. The mechanisms underlying electrical restitution (recovery of action potential duration after a preceding beat) were investigated in ferret ventricular cells. The time to 80% recovery (t80) of action potential duration was approximately 204 ms. 2. At a holding potential of -80 mV, the Ca2+ current (ICa) reactivated and the delayed rectifier K+ current (IK) deactivated very rapidly (t80: approximately 32 and approximately 93 ms, respectively). The kinetics of both currents are too fast to account for electrical restitution alone. 3. The putative inward Na(+)-Ca2+ exchange current (INa-Ca) produced by the Na(+)-Ca2+ exchanger in response to the intracellular Ca2+ transient reprimed (t80: 189 ms) with the same time course as mechanical restitution (recovery of contraction) and with a similar time course to electrical restitution. 4. Substantial reduction of inward INa-Ca, by buffering intracellular Ca2+ with the acetyl methyl ester form of BAPTA, shortened the action potential and greatly altered the electrical restitution curve. Subsequent addition of nifedipine (to block ICa) or 4-aminopyridine (4-AP) (to block the transient outward current, ITO) further altered the electrical restitution curve. 5. Any time-dependent current that contributes to the action potential is likely to affect the time course of electrical restitution. Although ICa, IK and ITO were previously thought to be the only currents involved in electrical restitution, we conclude that inward INa-Ca also plays an important role.
The Journal of Physiology 11/1997; 504 ( Pt 2):301-14. · 4.72 Impact Factor
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ABSTRACT: 1. Inward Na(+)-Ca2+ exchange current (iNaCa) was either blocked in ferret ventricular cells by replacing extracellular Na+ with Li+ or substantially reduced by the almost complete elimination of the Ca2+ transient by buffering intracellular Ca2+ with the acetoxymethyl ester form of BAPTA (BAPTA AM). 2. During square wave voltage clamp pulses to 0 mV, replacing extracellular Na+ with Li+ or buffering intracellular Ca2+ with BAPTA AM resulted in the loss of a transient inward current. This current was increased by the application of isoprenaline (expected to increase the underlying Ca2+ transient) and displayed the voltage-dependent characteristics of inward iNaCa. 3. Replacing extracellular Na+ with Li+ or buffering intracellular Ca2+ caused a significant shortening of the action potential (at -65 mV, 44 +/- 2% with Li+ and 20 +/- 2% with BAPTA AM). The shortening can be explained by changes in iNaCa. 4. The action potential clamp technique was used to measure the BAPTA-sensitive current (putative iNaCa) and the Ca2+ current (ica; measured using nifedipine) during the action potential. Under control conditions, the inward BAPTA-sensitive current makes approximately the same contribution as iCa during much of the action potential plateau. These results suggest an important role for inward iNaCa in the ferret ventricular action potential.
The Journal of Physiology 03/1997; 498 ( Pt 3):611-25. · 4.72 Impact Factor
