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Transient and sustained changes in myofilament sensitivity to Ca2+ contribute to the inotropic effects of sevoflurane in rat ventricle
British Journal of Anaesthesia 94 (3): 279–86 (2005)
doi:10.1093/bja/aei053
Advance Access publication December 17, 2004
Transient and sustained changes in myofilament sensitivity to Ca2+ contribute to the inotropic effects of sevoflurane in rat ventricle M. D. Graham1, G. Bru-Mercier1, P. M. Hopkins2 and S. M. Harrison1* 1
School of Biomedical Sciences and 2Academic Unit of Anaesthesia, University of Leeds, Leeds LS2 9JT, UK *Corresponding author. E-mail: [email protected]
Methods. Contractility and cytosolic Ca2+ (fura-2) were recorded optically in ventricular myocytes stimulated electrically (1 Hz) at 30 C. Myofilament Ca2+ 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. Results. Isoflurane reduced mean (SD) myofilament Ca2+ sensitivity from 10.3 (1.9) to 5.9 (1.6) mm Fr 1 (P<0.001) throughout a 1 min exposure, which returned to control on removal. In contrast, on initial exposure to sevoflurane, Ca2+ sensitivity was reduced from 10.8 (1.3) to 4.3 (0.9) mm 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) mm Fr 1; P<0.001) before preanaesthetic levels were restored. Conclusions. These data show that both isoflurane and sevoflurane reduce apparent myofilament Ca2+ sensitivity at steady state. However, sevoflurane (but not isoflurane) induced transient changes in apparent myofilament Ca2+ sensitivity, which would contribute to its inotropic profile. Br J Anaesth 2005; 94: 279–86 Keywords: anaesthetics volatile, isoflurane; anaesthetics volatile, sevoflurane; heart, myocytes; physiology, cardiac Accepted for publication: October 22, 2004
The volatile anaesthetics isoflurane and sevoflurane induce both positive and negative inotropic effects in ventricular myocytes,1 2 however, the mechanisms underlying these effects are not fully understood. On application of isoflurane and sevoflurane, contraction is initially reduced but then recovers partially towards control. The extent to which contraction is reduced at steady-state by both anaesthetics is dose dependent.1 On removal of isoflurane and sevoflurane, contractions increase transiently above control before returning to preanaesthetic levels, an effect more pronounced with equimolar sevoflurane. Previous work in intact ventricular tissue2–5 has suggested that at steady-state, isoflurane reduces myofilament Ca2+ sensitivity, which would contribute to its sustained negative inotropic effect. In a previous study from our laboratory we reported that sevoflurane had
little effect on myofilament Ca2+ sensitivity in rat ventricular myocytes2 whereas Bartunek and colleagues6 showed sevoflurane reduced sensitivity at steady-state in ferret ventricular muscle and therefore further investigation is warranted. It is also apparent from previously published data, where contractility and cytosolic Ca2+ were recorded simultaneously, that proportionately greater changes in the magnitude of contraction compared to the cytosolic Ca2+ transient are induced on application and removal of sevoflurane but not with isoflurane.2 This may suggest that transient anaestheticinduced changes in the apparent sensitivity of the myofilaments to Ca2+ may occur on exposure and removal of sevoflurane. Therefore, the aim of this study was to examine the relationship between contraction and the cytosolic Ca2+ transient at various points before, during and after exposure
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Background. 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 Ca2+ sensitivity contribute to their sustained negative inotropic effects. In this study, the role of changes in myofilament Ca2+ sensitivity in both positive and negative inotropic effects of these agents was examined in intact ventricular myocytes.
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to isoflurane and sevoflurane to further elucidate the role of changes in myofilament Ca2+ sensitivity in the inotropic action of these two anaesthetics.
Concentrations of anaesthetics in the superfusate were verified with gas liquid chromatography7 and found to be stable over the course of an experiment.
Recording cell length
Methods Animal procedures conformed with regulations stated in the Animals (Scientific Procedures) Act 1986 of the UK government Home Office.
Cell isolation
Solutions The isolation solution was composed of sodium chloride 130 mM; potassium chloride 5.4 mM; magnesium chloride 1.4 mM; monosodium phosphate 0.4 mM; HEPES 5 mM; glucose 10 mM; taurine 20 mM; creatine 10 mM; pH 7.1 (sodium hydroxide) at 37 C. After dissociation, cells were stored in and subsequently perfused with a physiological salt solution of the following composition: sodium chloride 140 mM; potassium chloride 5.4 mM; magnesium chloride 1.2 mM; monosodium phosphate 0.4 mM; HEPES 5 mM; glucose 10 mM; calcium chloride 1 mM; pH 7.4 (sodium hydroxide) at 30 C. Isoflurane and sevoflurane were delivered from stock solutions made up in dimethyl sulfoxide (DMSO). After dilution of the stock solutions, the final concentration of DMSO in the superfusate never exceeded 0.2%, a concentration that had no significant effect on contractions (not shown). 0.6 mM approximates to twice the minimum alveolar concentration (MAC) of isoflurane and sevoflurane and therefore these concentrations are both clinically relevant and broadly equi-anaesthetic in rat ventricle.8
Recording Ca2+ transients Cells were loaded with fura-2 by gentle agitation of a 2 ml aliquot of cell suspension with 6.25 ml of 1 mM fura-2 AM in DMSO for 12 min. After centrifugation as before, the supernatant was removed by suction and the pellet of cells resuspended in 750 mM Ca2+ solution. The fura-2 loaded cells were left for at least 30 min before use to allow de-esterification of the dye to take place. These cells were then transferred to the tissue chamber and stimulated as above. To record Ca2+ transients, the fura-2 loaded cells were excited alternately with light at two wavelengths (340 and 380 nm) and fluorescence was detected at 510 (40) nm using a monochromator based system (Cairn, Kent, UK). The ratio of fluorescence at 510 nm in response to excitation at 340 and 380 nm was used as a measure of the intracellular Ca2+ concentration. Ca2+ transients were digitized at 1 kHz and analysed using Ionoptix software. Cells were exposed to either 0.6 mM isoflurane or sevoflurane for a period of 1 or 4 min before solution was returned to the control superfusate. If either contraction or Ca2+ transient magnitude did not return to within –10% of precontrol values, then the cell was excluded from analysis.
Statistical analysis Data are presented as mean (SEM) and statistical comparisons carried out with SigmaStat (Jandel Scientific, Erkrath, Germany) using one-way repeated measures ANOVA followed by Holm-Sidak tests for multiple comparisons or Friedman’s repeated measures ANOVA on ranks followed by Dunn’s method for multiple comparison if the data failed a normality test (Kolmogorov–Smirnov). All figures were prepared using SigmaPlot (Jandel Scientific).
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The technique used to prepare rat ventricular myocytes has been described previously in full.7 Briefly, rats (200–250 g) were killed by a blow to the head followed by cervical dislocation (schedule 1 techniques sanctioned by the UK government Home Office) and the heart rapidly excised into an ‘isolation solution’ (see below for composition), supplemented with calcium chloride 750 mM and equilibrated with oxygen 100%. The coronary arteries were flushed of blood by retrograde perfusion of the heart via the aorta with the above solution and then perfused for 4 min with the isolation solution, to which sodium EGTA 100 mM was added. The heart was then perfused for 9 min with the isolation solution supplemented with collagenase 1 mg ml 1 (type 1, Worthington Biochemical Corp., NJ, USA) and protease 0.1 mg ml 1 (Sigma, type XIV), after which the ventricles were cut from the heart, finely chopped and shaken in the collected enzyme solution (supplemented with 1% BSA) for 5 min periods. Dissociated cells were harvested by filtration at the end of each 5 min digestion and the remaining tissue returned for further enzyme treatment. The dissociated cells were centrifuged at 30 g for 40 s and resuspended in the calcium chloride 750 mM solution and stored at room temperature until required.
Freshly dissociated cells were transferred to a tissue chamber (volume 0.1 ml) attached to the stage of an inverted microscope (Nikon Diaphot). The cells were allowed to settle for several minutes onto the glass bottom of the chamber before being superfused continuously at a rate of 3 ml min 1 with the physiological salt solution. Solutions were delivered to the experimental chamber by magnetic drive gear metering pumps (Micropump, Concord, CA, USA) and solution level and temperature maintained by feedback circuits.9 All experiments were carried out at 30 C to maximize the retention of cytosolic fura-2, which is crucial in experiments of this nature as good signal to noise ratio is essential. Cells were stimulated electrically at a frequency of 1 Hz (stimulus duration 2 ms) via two platinum electrodes situated in the sides of the chamber. Cell length was continuously digitized at 200 Hz and analysed using Ionoptix software (Ionoptix).
Sevoflurane and myofilament Ca2+ sensitivity
Results Figure 1 illustrates slow time base records of cell length and the cytosolic Ca2+ transient before during and after a 1 min exposure to 0.6 mM isoflurane (A) or sevoflurane (B), respectively. In a group of 13 cells, upon addition of
isoflurane, the Ca2+ transient and contraction were reduced to 81 (2)% and 42 (4)%, respectively. During exposure, Ca2+ transient and contraction magnitude recovered towards control by 6 and 19%, respectively, such that at the end of a 1 min exposure, the Ca2+ transient and contraction
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However, it is also plausible that the relationship between cytosolic Ca2+ and contraction is modified at the points of MN and immediately after removal of anaesthetic. To address this, myofilament Ca2+ sensitivity was assessed at a variety of time points during an exposure to anaesthetic: (i) under control conditions, (ii) at the point of MN, (iii) after 30 s of exposure, (iv) at 1 min (and additionally 2, 3 and 4 min for sevoflurane, see below), (v) at the peak of the positive inotropic effect (overshoot), and (vi) post-control. Figure 2A illustrates plots of cell length vs fura-2 fluorescence ratio from a representative cell under control conditions, at the point of MN and at the overshoot after removal of sevoflurane. During the final phase of relaxation, the myofilaments come into quasi-equilibrium with cytosolic Ca2+ and therefore the final trajectory of this relationship provides an index of the sensitivity of the myofilaments to Ca2+.10 Figure 2B illustrates the final phase of relaxation on expanded scales and the solid lines represent linear regression of these data. The gradient of the relationship provides an index of myofilament Ca2+ sensitivity.10 These data
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Fig 2 (A) Plots of cell length against fura-2 fluorescence ratio under control conditions, following addition of 0.6 mM sevoflurane at the point of MN and at the peak of the positive inotropic effect (overshoot) on removal of sevoflurane. (B) As above on expanded scales to show the final phase of relaxation. Data are fitted by linear regression, the gradient of which provides an index of the responsiveness of the myofilaments to Ca2+.
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magnitude were 87 (3)% and 61 (7)% of control. In a separate group of 10 cells, sevoflurane induced a more pronounced initial decrease in the Ca2+ transient and contraction (to 68 (3) and 31 (2)%, respectively), which then recovered towards control (by 18 and 43%, respectively) such that after a 1 min exposure, the Ca2+ transient and contraction were 87 (3) and 74 (9)% of control, respectively. On removal of anaesthetic, contractions increased (to 143 (6) and 172 (6)% for isoflurane and sevoflurane, respectively) associated with a modest increase in the Ca2+ transient (to 105 (1) and 110 (2)% of control for isoflurane and sevoflurane, respectively). These data illustrate that contraction is reduced to a proportionately greater extent than the Ca2+ transient at the maximum negative inotropic effect (MN) and on removal, contraction increases to a greater extent than the Ca2+ transient before returning to control levels (–10%), especially with sevoflurane. It is possible that these proportionate differences can be explained by the non-linearity between either cytosolic Ca2+ and fura-2 fluorescence or between cytosolic Ca2+ and contraction.
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relationship was initially reduced as with isoflurane but a new steady state was not achieved and so the duration of exposure was extended to 4 min. Figure 3B illustrates that application of sevoflurane significantly reduced myofilament Ca2+ sensitivity (from 10.8 (1.3) to 4.3 (0.9) mm Fr 1; P<0.001) but the extent of this decrease was not maintained. After 2 min of exposure, the gradient of the relationship had increased to 6.2 (1.3) (P=0.051 vs MN). After 3 min, a new steady state was achieved with data at 3 and 4 min being significantly different (P<0.05) from that at the point of MN, but still significantly different from control. This suggests that sevoflurane induces both a transient (over 2 min of exposure) but also a sustained reduction in the myofilament responsiveness to Ca2+ as at steady state, the gradient is still significantly reduced compared with control. On removal of sevoflurane, myofilament Ca2+ sensitivity is increased significantly above control (to 17.7 (2.2) mm Fr 1; P<0.001) before returning to preanaesthetic levels. Previous work11 12 has suggested that anaesthetics may have a variety of effects at the myofilamental level resulting in apparent changes in myofilament Ca2+ sensitivity and analysis of the time-course of contraction may help elucidate the mechanisms involved. Figures 4 (isoflurane) and 5 (sevoflurane) illustrate mean data for the time to peak (A and B) and time from peak to 50% relaxation (C and D) of the Ca2+ transient and contraction, respectively, at the various time points studied (data for sevoflurane is only shown up to 1 min as no further significant changes in time course occurred subsequent to this time). The time-course of the cytosolic Ca2+ transient was not affected by isoflurane either during or after exposure (Fig. 4A and C), however, the time to peak contraction was significantly reduced (P<0.001 compared with control) throughout the exposure (Fig. 4B) before returning to preanaesthetic level on removal. Sevoflurane led to a significant (P<0.001) sustained decrease in the time to peak contraction during exposure with no change in the time-course of decay of the Ca2+ transient (Fig. 5A and B). On removal of sevoflurane, time to peak of both the Ca2+ transient and contraction were prolonged. There were no significant changes in the time to 50% relaxation of the Ca2+ transient during or after an exposure. However, relaxation was slowed significantly (P<0.01) at MN, and this effect was ameliorated over time; at 1 min, the time to 50% relaxation was not significantly different from control but was significantly different from data at MN (P<0.05).
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Discussion
Stage of exposure Fig 3 Mean (SEM) data describing the gradient of the relationship between cell length and fura-2 fluorescence from the type of analysis shown in Figure 2B at various points during (A) 1 min exposure to isoflurane, and (B) 4 min exposure to sevoflurane. ***Signifies a significant difference (P<0.001) from control (Con); +indicates that these data sets are significantly different from that recorded at the MN (P<0.05). Fr, fluorescence ratio.
Effects of isoflurane and sevoflurane on myofilament Ca2+ sensitivity These data represent a comprehensive description of the effects of isoflurane and sevoflurane on apparent myofilament Ca2+ sensitivity in intact ventricular myocytes.
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illustrate that the gradient is reduced at the point of MN and increased after removal of sevoflurane (overshoot) compared with control. Figure 3A illustrates mean data describing the gradients of relationships such as those shown in Figure 2B for a series of time points during a 1 min exposure to isoflurane. The gradient of the relationship under control conditions was 10.3 (1.9) mm Fr 1, which was significantly reduced (P<0.001) on application of isoflurane such that, at the point of MN, the gradient was reduced to 5.9 (1.6) mm Fr 1. The extent of this reduction was maintained during the 1 min exposure. On removal of isoflurane, at the peak of the positive inotropic effect (or overshoot) myofilament sensitivity returned to a level not significantly different from control (11.0 (1.8) mm Fr 1) and no further changes were observed post control. Initial experiments carried out with sevoflurane suggested that over a 1 min exposure (not shown), the gradient of the
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Fig 4 Mean (SEM) data describing (A and B) the time to peak, and (C and D) the time from peak to 50% relaxation of the Ca2+ transient and contraction, respectively, at various times during exposure to 0.6 mM isoflurane. *P<0.05, ***P<0.001 vs pre-control.
The method employed here10 effectively allows the determination of myofilament Ca2+ sensitivity on a beatto-beat basis such that changes in this parameter can be detected during and after anaesthetic exposure rather than just at steady state as in previous studies in intact tissue.2–5 Both isoflurane and sevoflurane (see below) induce a sustained decrease in myofilament Ca2+ sensitivity but also suggest that upon application of sevoflurane (but not isoflurane) apparent myofilament Ca2+ sensitivity is initially reduced to a greater extent than is observed at steady state, i.e. there appear to be both transient and sustained components to this effect. On removal of sevoflurane (but not isoflurane), myofilament Ca2+ sensitivity is increased transiently above control. These data are in agreement with previous reports on the effects of isoflurane on myofilament Ca2+ sensitivity at steady state but the present data for sevoflurane differ from our previous study. Davies and colleagues2 reported that, unlike halothane and isoflurane, sevoflurane had little or no effect on myofilament Ca2+ sensitivity at steady state. That study also utilized the method described by Spurgeon and colleagues10 to assess the responsiveness of the myofilaments to Ca2+ and so the reason for the differences between this and the previous study
are not clear (species, temperature, solution composition and sevoflurane concentration were the same in both studies). Further analysis of our previous data (Fig. 5 from Davies and colleagues2) identified that in eight of the nine cells studied, the gradient of the regression line was reduced by sevoflurane. Statistical comparisons (Wilcoxon signed rank) of all nine cells yielded a P value of 0.22, however, excluding the cell where the gradient did not decrease gives P=0.031. Therefore in the majority of cells reported by Davies and colleagues,2 sevoflurane led to a decrease in apparent myofilament sensitivity at steady state, which agrees with the present study. It is possible that in some cells, myofilament Ca2+ sensitivity is affected to a lesser extent at steady state than the majority. As cells used in studies of this nature are derived from both ventricles, this may reflect regional differences between right and left ventricular tissue though this would have to be investigated by further experimentation. The effects on apparent myofilament Ca2+ sensitivity may result from interaction of the anaesthetics with a variety of targets at the myofilament level such as the affinity of Ca2+ binding to the regulatory site of troponin C, the kinetics of myosin cross-bridge attachment and detachment, the force per cross-bridge as well as the kinetics of myosin ATPase
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hydrolysis. However, it is likely, as stated by others,6 11 that anaesthetics have multiple sites of action at the level of the myofilaments that contribute to their observed effects.
Effects of isoflurane and sevoflurane on the time-course of contraction Figures 4B and 5B illustrate that both isoflurane and sevoflurane induced a sustained acceleration of the time to peak contraction (with no associated changes in the time to peak of the Ca2+ transient). This result is similar to that observed with isoflurane in guinea-pig papillary muscle13 and with sevoflurane in ferret ventricular muscle under isometric conditions.6 A decrease in the time to peak contraction (in the absence of changes in the time-course of the Ca2+ transient) is consistent with an increase in the apparent on-rate constant for actin–myosin interaction. However, this is at odds with data from skinned ventricular muscle,12 which showed that sevoflurane (at 2 MAC) decreased the apparent on-rate constant ( fapp) for cross-bridge attachment with no effect on detachment rate (gapp) such that sevoflurane would reduce the number of cross-bridges in a force-generating state.11 12 Whereas this would reduce contractility at any cytosolic
Ca2+ (i.e. reduce apparent myofilament Ca2+ sensitivity) it may also be expected to increase the time to peak contraction, an effect opposite to that observed here with both isoflurane and sevoflurane. It is possible that such discrepancies could arise from the different preparations used in these studies, i.e. Triton X-100 skinned ventricular strips under isometric conditions, stretched to Lmax12 compared with intact ventricular myocytes in which resting sarcomere length is closer to that in the intact heart. At high load (e.g. a sarcomere length of 2.1–2.2 mm) contractile time-course is controlled at the level of the myofilaments whereas in isolated myocytes, sarcomere length is 1.8 mm, intrinsic myofilament Ca2+ sensitivity is lower14 and contractile timecourse affected by the dynamics of cytosolic Ca2+.10 Therefore, extrapolation of mechanical data from skinned muscle under high load to the intact isolated myocyte is not always appropriate. Furthermore, in skinned preparations, essential co-factors are lost, which could also help explain different effects between skinned and intact muscle. Figure 5B also shows that the time to peak contraction is prolonged during washout (overshoot) of sevoflurane (but not isoflurane, Fig. 4B); this could occur purely as a consequence of the prolongation of the time to peak of the Ca2+ transient
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Acknowledgements This work was supported by the British Heart Foundation, London, UK and by the Medical Research Council, UK.
References
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1 Davies LA, Hamilton DL, Hopkins PM, Boyett MR, Harrison SM. Concentration-dependent inotropic effects of halothane, isoflurane and sevoflurane on rat ventricular myocytes. Br J Anaesth 1999; 82: 723–30 2 Davies LA, Gibson CN, Boyett MR, Hopkins PM, Harrison SM. Effects of isoflurane, sevoflurane, and halothane on myofilament Ca2+ sensitivity and sarcoplasmic reticulum Ca2+ release in rat ventricular myocytes. Anesthesiology 2000; 93: 1034–44 3 Housmans PR, Wanek LA, Carton EG, Bartunek AE. Effects of halothane and isoflurane on the intracellular Ca2+ transient in ferret cardiac muscle. Anesthesiology 2000; 93: 189–201 4 Hanley PJ, Loiselle DS. Mechanisms of force inhibition by halothane and isoflurane in intact rat cardiac muscle. J Physiol 1998; 506: 231–44 5 Jiang YD, Julian FJ. Effects of isoflurane on [Ca2+]i, SR Ca2+ content, and twitch force in intact trabeculae. Am J Physiol 1998; 275: H1360–9 6 Bartunek AE, Housmans PR. Effects of sevoflurane on the intracellular Ca2+ transient in ferret cardiac muscle. Anesthesiology 2000; 93: 1500–8 7 Harrison SM, Robinson M, Davies LA, Hopkins PM, Boyett MR. Mechanisms underlying the inotropic action of halothane on intact rat ventricular myocytes. Br J Anaesth 1999; 82: 609–21 8 Franks NP, Lieb WR. Temperature dependence of the potency of volatile general anesthetics. Anesthesiology 1996; 84: 716–20 9 Cannell MB, Lederer WJ. A novel experimental chamber for single-cell voltage-clamp and patch-clamp applications with low electrical noise and excellent temperature and flow control. Pflugers Archiv 1986; 406: 536–9 10 Spurgeon HA, duBell WH, Stern MD, et al. Cytosolic calcium and myofilaments in single-rat cardiac myocytes achieve a dynamic equilibrium during twitch relaxation. J Physiol 1992; 447: 83–102 11 Murat I, Lechene P, Ventura-Clapier R. Effects of volatile anesthetics on mechanical properties of rat cardiac skinned fibers. Anesthesiology 1990; 73: 73–81 12 Prakash YS, Cody MJ, Hannon JD, Housmans PR, Sieck GC. Comparison of volatile anesthetic effects on actin-myosin cross-bridge cycling in neonatal versus adult cardiac muscle. Anesthesiology 2000; 92: 1114–25 13 Bosnjak ZJ, Aggarwal A, Turner LA, Kampine JM, Kampine JP. Differential effects of halothane, enflurane, and isoflurane on Ca2+ transients and papillary muscle tension in guinea pigs. Anesthesiology 1992; 76: 123–31 14 Hofmann PA, Fuchs F. Bound calcium and force development in skinned cardiac muscle bundles: effect of sarcomere length. J Mol Cell Cardiol 1988; 20: 667–77 15 Hattori Y, Azuma M, Gotoh Y, Kanno M. Negative inotropic effects of halothane, enflurane, and isoflurane in papillary muscles from diabetic rats. Anesth Analg 1987; 66: 23–8 16 Housmans PR, Murat I. Comparative effects of halothane, enflurane, and isoflurane at equipotent anesthetic concentrations on isolated ventricular myocardium of the ferret. II. Relaxation. Anesthesiology 1988; 69: 464–71
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(Fig. 5A) and may not reflect sevoflurane-induced effects at the level of the myofilaments. There were no significant changes in the time to 50% decay of the Ca2+ transient or 50% relaxation of the contraction during exposure to isoflurane (Fig. 4D) whereas, with sevoflurane, time to 50% relaxation was significantly prolonged at both MN and after 30 s. Figure 5D illustrates that the initial prolongation of relaxation at MN was ameliorated over time; at 30 s exposure, relaxation is still prolonged but this effect is reduced compared with MN. After 1 min of exposure, time to 50% relaxation returned to control values and data at this time was significantly different from that at MN. These changes occurred in the absence of changes in the time-course of the cytosolic Ca2+ transient suggesting sevoflurane-induced effects at the level of the myofilaments. These results contrast with other reports that demonstrate accelerated relaxation under isometric conditions,3 6 15 but agree with data from papillary muscles under isotonic conditions.16 A reduction in relaxation rate would be consistent with sevoflurane decreasing the cross-bridge off rate constant or myosin ATPase hydrolysis rate, or may result from interaction with other sites on the myofilament proteins, which cannot be distinguished by experiments of this nature. These data suggest that sevoflurane (but not isoflurane) has both transient and sustained effects on apparent myofilament Ca2+ sensitivity, and with sevoflurane this is accompanied temporally by transient changes in relaxation rate and it is tempting to speculate that these two are linked. The increase in apparent myofilament Ca2+ sensitivity on removal of sevoflurane is not associated with any significant change in the time to 50% decay of the Ca2+ transient or contraction, although time to peak of both parameters is prolonged (but see above). Therefore, mechanisms underlying this effect will require further experimentation. However, it should be noted that sevoflurane (but not isoflurane) affected the time-course of the Ca2+ transient during and after exposure suggesting that sevoflurane also affects cytosolic Ca2+ regulation in a complex manner, which would contribute to its inotropic effects in addition to those induced at the level of the myofilaments. In summary, these data show that both isoflurane and sevoflurane (in the majority of cells) reduce apparent myofilament Ca2+ sensitivity at steady state. In addition, sevoflurane (but not isoflurane) induces a transient further decrease in apparent myofilament Ca2+ sensitivity upon application and a transient sensitization upon removal. This may explain the greater proportionate effects of sevoflurane on contraction than the Ca2+ transient on application and removal and will also contribute to the recovery of contractions during an exposure to sevoflurane.
doi:10.1093/bja/aei053
Advance Access publication December 17, 2004
Transient and sustained changes in myofilament sensitivity to Ca2+ contribute to the inotropic effects of sevoflurane in rat ventricle M. D. Graham1, G. Bru-Mercier1, P. M. Hopkins2 and S. M. Harrison1* 1
School of Biomedical Sciences and 2Academic Unit of Anaesthesia, University of Leeds, Leeds LS2 9JT, UK *Corresponding author. E-mail: [email protected]
Methods. Contractility and cytosolic Ca2+ (fura-2) were recorded optically in ventricular myocytes stimulated electrically (1 Hz) at 30 C. Myofilament Ca2+ 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. Results. Isoflurane reduced mean (SD) myofilament Ca2+ sensitivity from 10.3 (1.9) to 5.9 (1.6) mm Fr 1 (P<0.001) throughout a 1 min exposure, which returned to control on removal. In contrast, on initial exposure to sevoflurane, Ca2+ sensitivity was reduced from 10.8 (1.3) to 4.3 (0.9) mm 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) mm Fr 1; P<0.001) before preanaesthetic levels were restored. Conclusions. These data show that both isoflurane and sevoflurane reduce apparent myofilament Ca2+ sensitivity at steady state. However, sevoflurane (but not isoflurane) induced transient changes in apparent myofilament Ca2+ sensitivity, which would contribute to its inotropic profile. Br J Anaesth 2005; 94: 279–86 Keywords: anaesthetics volatile, isoflurane; anaesthetics volatile, sevoflurane; heart, myocytes; physiology, cardiac Accepted for publication: October 22, 2004
The volatile anaesthetics isoflurane and sevoflurane induce both positive and negative inotropic effects in ventricular myocytes,1 2 however, the mechanisms underlying these effects are not fully understood. On application of isoflurane and sevoflurane, contraction is initially reduced but then recovers partially towards control. The extent to which contraction is reduced at steady-state by both anaesthetics is dose dependent.1 On removal of isoflurane and sevoflurane, contractions increase transiently above control before returning to preanaesthetic levels, an effect more pronounced with equimolar sevoflurane. Previous work in intact ventricular tissue2–5 has suggested that at steady-state, isoflurane reduces myofilament Ca2+ sensitivity, which would contribute to its sustained negative inotropic effect. In a previous study from our laboratory we reported that sevoflurane had
little effect on myofilament Ca2+ sensitivity in rat ventricular myocytes2 whereas Bartunek and colleagues6 showed sevoflurane reduced sensitivity at steady-state in ferret ventricular muscle and therefore further investigation is warranted. It is also apparent from previously published data, where contractility and cytosolic Ca2+ were recorded simultaneously, that proportionately greater changes in the magnitude of contraction compared to the cytosolic Ca2+ transient are induced on application and removal of sevoflurane but not with isoflurane.2 This may suggest that transient anaestheticinduced changes in the apparent sensitivity of the myofilaments to Ca2+ may occur on exposure and removal of sevoflurane. Therefore, the aim of this study was to examine the relationship between contraction and the cytosolic Ca2+ transient at various points before, during and after exposure
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Background. 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 Ca2+ sensitivity contribute to their sustained negative inotropic effects. In this study, the role of changes in myofilament Ca2+ sensitivity in both positive and negative inotropic effects of these agents was examined in intact ventricular myocytes.
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to isoflurane and sevoflurane to further elucidate the role of changes in myofilament Ca2+ sensitivity in the inotropic action of these two anaesthetics.
Concentrations of anaesthetics in the superfusate were verified with gas liquid chromatography7 and found to be stable over the course of an experiment.
Recording cell length
Methods Animal procedures conformed with regulations stated in the Animals (Scientific Procedures) Act 1986 of the UK government Home Office.
Cell isolation
Solutions The isolation solution was composed of sodium chloride 130 mM; potassium chloride 5.4 mM; magnesium chloride 1.4 mM; monosodium phosphate 0.4 mM; HEPES 5 mM; glucose 10 mM; taurine 20 mM; creatine 10 mM; pH 7.1 (sodium hydroxide) at 37 C. After dissociation, cells were stored in and subsequently perfused with a physiological salt solution of the following composition: sodium chloride 140 mM; potassium chloride 5.4 mM; magnesium chloride 1.2 mM; monosodium phosphate 0.4 mM; HEPES 5 mM; glucose 10 mM; calcium chloride 1 mM; pH 7.4 (sodium hydroxide) at 30 C. Isoflurane and sevoflurane were delivered from stock solutions made up in dimethyl sulfoxide (DMSO). After dilution of the stock solutions, the final concentration of DMSO in the superfusate never exceeded 0.2%, a concentration that had no significant effect on contractions (not shown). 0.6 mM approximates to twice the minimum alveolar concentration (MAC) of isoflurane and sevoflurane and therefore these concentrations are both clinically relevant and broadly equi-anaesthetic in rat ventricle.8
Recording Ca2+ transients Cells were loaded with fura-2 by gentle agitation of a 2 ml aliquot of cell suspension with 6.25 ml of 1 mM fura-2 AM in DMSO for 12 min. After centrifugation as before, the supernatant was removed by suction and the pellet of cells resuspended in 750 mM Ca2+ solution. The fura-2 loaded cells were left for at least 30 min before use to allow de-esterification of the dye to take place. These cells were then transferred to the tissue chamber and stimulated as above. To record Ca2+ transients, the fura-2 loaded cells were excited alternately with light at two wavelengths (340 and 380 nm) and fluorescence was detected at 510 (40) nm using a monochromator based system (Cairn, Kent, UK). The ratio of fluorescence at 510 nm in response to excitation at 340 and 380 nm was used as a measure of the intracellular Ca2+ concentration. Ca2+ transients were digitized at 1 kHz and analysed using Ionoptix software. Cells were exposed to either 0.6 mM isoflurane or sevoflurane for a period of 1 or 4 min before solution was returned to the control superfusate. If either contraction or Ca2+ transient magnitude did not return to within –10% of precontrol values, then the cell was excluded from analysis.
Statistical analysis Data are presented as mean (SEM) and statistical comparisons carried out with SigmaStat (Jandel Scientific, Erkrath, Germany) using one-way repeated measures ANOVA followed by Holm-Sidak tests for multiple comparisons or Friedman’s repeated measures ANOVA on ranks followed by Dunn’s method for multiple comparison if the data failed a normality test (Kolmogorov–Smirnov). All figures were prepared using SigmaPlot (Jandel Scientific).
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The technique used to prepare rat ventricular myocytes has been described previously in full.7 Briefly, rats (200–250 g) were killed by a blow to the head followed by cervical dislocation (schedule 1 techniques sanctioned by the UK government Home Office) and the heart rapidly excised into an ‘isolation solution’ (see below for composition), supplemented with calcium chloride 750 mM and equilibrated with oxygen 100%. The coronary arteries were flushed of blood by retrograde perfusion of the heart via the aorta with the above solution and then perfused for 4 min with the isolation solution, to which sodium EGTA 100 mM was added. The heart was then perfused for 9 min with the isolation solution supplemented with collagenase 1 mg ml 1 (type 1, Worthington Biochemical Corp., NJ, USA) and protease 0.1 mg ml 1 (Sigma, type XIV), after which the ventricles were cut from the heart, finely chopped and shaken in the collected enzyme solution (supplemented with 1% BSA) for 5 min periods. Dissociated cells were harvested by filtration at the end of each 5 min digestion and the remaining tissue returned for further enzyme treatment. The dissociated cells were centrifuged at 30 g for 40 s and resuspended in the calcium chloride 750 mM solution and stored at room temperature until required.
Freshly dissociated cells were transferred to a tissue chamber (volume 0.1 ml) attached to the stage of an inverted microscope (Nikon Diaphot). The cells were allowed to settle for several minutes onto the glass bottom of the chamber before being superfused continuously at a rate of 3 ml min 1 with the physiological salt solution. Solutions were delivered to the experimental chamber by magnetic drive gear metering pumps (Micropump, Concord, CA, USA) and solution level and temperature maintained by feedback circuits.9 All experiments were carried out at 30 C to maximize the retention of cytosolic fura-2, which is crucial in experiments of this nature as good signal to noise ratio is essential. Cells were stimulated electrically at a frequency of 1 Hz (stimulus duration 2 ms) via two platinum electrodes situated in the sides of the chamber. Cell length was continuously digitized at 200 Hz and analysed using Ionoptix software (Ionoptix).
Sevoflurane and myofilament Ca2+ sensitivity
Results Figure 1 illustrates slow time base records of cell length and the cytosolic Ca2+ transient before during and after a 1 min exposure to 0.6 mM isoflurane (A) or sevoflurane (B), respectively. In a group of 13 cells, upon addition of
isoflurane, the Ca2+ transient and contraction were reduced to 81 (2)% and 42 (4)%, respectively. During exposure, Ca2+ transient and contraction magnitude recovered towards control by 6 and 19%, respectively, such that at the end of a 1 min exposure, the Ca2+ transient and contraction
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However, it is also plausible that the relationship between cytosolic Ca2+ and contraction is modified at the points of MN and immediately after removal of anaesthetic. To address this, myofilament Ca2+ sensitivity was assessed at a variety of time points during an exposure to anaesthetic: (i) under control conditions, (ii) at the point of MN, (iii) after 30 s of exposure, (iv) at 1 min (and additionally 2, 3 and 4 min for sevoflurane, see below), (v) at the peak of the positive inotropic effect (overshoot), and (vi) post-control. Figure 2A illustrates plots of cell length vs fura-2 fluorescence ratio from a representative cell under control conditions, at the point of MN and at the overshoot after removal of sevoflurane. During the final phase of relaxation, the myofilaments come into quasi-equilibrium with cytosolic Ca2+ and therefore the final trajectory of this relationship provides an index of the sensitivity of the myofilaments to Ca2+.10 Figure 2B illustrates the final phase of relaxation on expanded scales and the solid lines represent linear regression of these data. The gradient of the relationship provides an index of myofilament Ca2+ sensitivity.10 These data
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Fig 2 (A) Plots of cell length against fura-2 fluorescence ratio under control conditions, following addition of 0.6 mM sevoflurane at the point of MN and at the peak of the positive inotropic effect (overshoot) on removal of sevoflurane. (B) As above on expanded scales to show the final phase of relaxation. Data are fitted by linear regression, the gradient of which provides an index of the responsiveness of the myofilaments to Ca2+.
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magnitude were 87 (3)% and 61 (7)% of control. In a separate group of 10 cells, sevoflurane induced a more pronounced initial decrease in the Ca2+ transient and contraction (to 68 (3) and 31 (2)%, respectively), which then recovered towards control (by 18 and 43%, respectively) such that after a 1 min exposure, the Ca2+ transient and contraction were 87 (3) and 74 (9)% of control, respectively. On removal of anaesthetic, contractions increased (to 143 (6) and 172 (6)% for isoflurane and sevoflurane, respectively) associated with a modest increase in the Ca2+ transient (to 105 (1) and 110 (2)% of control for isoflurane and sevoflurane, respectively). These data illustrate that contraction is reduced to a proportionately greater extent than the Ca2+ transient at the maximum negative inotropic effect (MN) and on removal, contraction increases to a greater extent than the Ca2+ transient before returning to control levels (–10%), especially with sevoflurane. It is possible that these proportionate differences can be explained by the non-linearity between either cytosolic Ca2+ and fura-2 fluorescence or between cytosolic Ca2+ and contraction.
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relationship was initially reduced as with isoflurane but a new steady state was not achieved and so the duration of exposure was extended to 4 min. Figure 3B illustrates that application of sevoflurane significantly reduced myofilament Ca2+ sensitivity (from 10.8 (1.3) to 4.3 (0.9) mm Fr 1; P<0.001) but the extent of this decrease was not maintained. After 2 min of exposure, the gradient of the relationship had increased to 6.2 (1.3) (P=0.051 vs MN). After 3 min, a new steady state was achieved with data at 3 and 4 min being significantly different (P<0.05) from that at the point of MN, but still significantly different from control. This suggests that sevoflurane induces both a transient (over 2 min of exposure) but also a sustained reduction in the myofilament responsiveness to Ca2+ as at steady state, the gradient is still significantly reduced compared with control. On removal of sevoflurane, myofilament Ca2+ sensitivity is increased significantly above control (to 17.7 (2.2) mm Fr 1; P<0.001) before returning to preanaesthetic levels. Previous work11 12 has suggested that anaesthetics may have a variety of effects at the myofilamental level resulting in apparent changes in myofilament Ca2+ sensitivity and analysis of the time-course of contraction may help elucidate the mechanisms involved. Figures 4 (isoflurane) and 5 (sevoflurane) illustrate mean data for the time to peak (A and B) and time from peak to 50% relaxation (C and D) of the Ca2+ transient and contraction, respectively, at the various time points studied (data for sevoflurane is only shown up to 1 min as no further significant changes in time course occurred subsequent to this time). The time-course of the cytosolic Ca2+ transient was not affected by isoflurane either during or after exposure (Fig. 4A and C), however, the time to peak contraction was significantly reduced (P<0.001 compared with control) throughout the exposure (Fig. 4B) before returning to preanaesthetic level on removal. Sevoflurane led to a significant (P<0.001) sustained decrease in the time to peak contraction during exposure with no change in the time-course of decay of the Ca2+ transient (Fig. 5A and B). On removal of sevoflurane, time to peak of both the Ca2+ transient and contraction were prolonged. There were no significant changes in the time to 50% relaxation of the Ca2+ transient during or after an exposure. However, relaxation was slowed significantly (P<0.01) at MN, and this effect was ameliorated over time; at 1 min, the time to 50% relaxation was not significantly different from control but was significantly different from data at MN (P<0.05).
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Discussion
Stage of exposure Fig 3 Mean (SEM) data describing the gradient of the relationship between cell length and fura-2 fluorescence from the type of analysis shown in Figure 2B at various points during (A) 1 min exposure to isoflurane, and (B) 4 min exposure to sevoflurane. ***Signifies a significant difference (P<0.001) from control (Con); +indicates that these data sets are significantly different from that recorded at the MN (P<0.05). Fr, fluorescence ratio.
Effects of isoflurane and sevoflurane on myofilament Ca2+ sensitivity These data represent a comprehensive description of the effects of isoflurane and sevoflurane on apparent myofilament Ca2+ sensitivity in intact ventricular myocytes.
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illustrate that the gradient is reduced at the point of MN and increased after removal of sevoflurane (overshoot) compared with control. Figure 3A illustrates mean data describing the gradients of relationships such as those shown in Figure 2B for a series of time points during a 1 min exposure to isoflurane. The gradient of the relationship under control conditions was 10.3 (1.9) mm Fr 1, which was significantly reduced (P<0.001) on application of isoflurane such that, at the point of MN, the gradient was reduced to 5.9 (1.6) mm Fr 1. The extent of this reduction was maintained during the 1 min exposure. On removal of isoflurane, at the peak of the positive inotropic effect (or overshoot) myofilament sensitivity returned to a level not significantly different from control (11.0 (1.8) mm Fr 1) and no further changes were observed post control. Initial experiments carried out with sevoflurane suggested that over a 1 min exposure (not shown), the gradient of the
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Fig 4 Mean (SEM) data describing (A and B) the time to peak, and (C and D) the time from peak to 50% relaxation of the Ca2+ transient and contraction, respectively, at various times during exposure to 0.6 mM isoflurane. *P<0.05, ***P<0.001 vs pre-control.
The method employed here10 effectively allows the determination of myofilament Ca2+ sensitivity on a beatto-beat basis such that changes in this parameter can be detected during and after anaesthetic exposure rather than just at steady state as in previous studies in intact tissue.2–5 Both isoflurane and sevoflurane (see below) induce a sustained decrease in myofilament Ca2+ sensitivity but also suggest that upon application of sevoflurane (but not isoflurane) apparent myofilament Ca2+ sensitivity is initially reduced to a greater extent than is observed at steady state, i.e. there appear to be both transient and sustained components to this effect. On removal of sevoflurane (but not isoflurane), myofilament Ca2+ sensitivity is increased transiently above control. These data are in agreement with previous reports on the effects of isoflurane on myofilament Ca2+ sensitivity at steady state but the present data for sevoflurane differ from our previous study. Davies and colleagues2 reported that, unlike halothane and isoflurane, sevoflurane had little or no effect on myofilament Ca2+ sensitivity at steady state. That study also utilized the method described by Spurgeon and colleagues10 to assess the responsiveness of the myofilaments to Ca2+ and so the reason for the differences between this and the previous study
are not clear (species, temperature, solution composition and sevoflurane concentration were the same in both studies). Further analysis of our previous data (Fig. 5 from Davies and colleagues2) identified that in eight of the nine cells studied, the gradient of the regression line was reduced by sevoflurane. Statistical comparisons (Wilcoxon signed rank) of all nine cells yielded a P value of 0.22, however, excluding the cell where the gradient did not decrease gives P=0.031. Therefore in the majority of cells reported by Davies and colleagues,2 sevoflurane led to a decrease in apparent myofilament sensitivity at steady state, which agrees with the present study. It is possible that in some cells, myofilament Ca2+ sensitivity is affected to a lesser extent at steady state than the majority. As cells used in studies of this nature are derived from both ventricles, this may reflect regional differences between right and left ventricular tissue though this would have to be investigated by further experimentation. The effects on apparent myofilament Ca2+ sensitivity may result from interaction of the anaesthetics with a variety of targets at the myofilament level such as the affinity of Ca2+ binding to the regulatory site of troponin C, the kinetics of myosin cross-bridge attachment and detachment, the force per cross-bridge as well as the kinetics of myosin ATPase
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hydrolysis. However, it is likely, as stated by others,6 11 that anaesthetics have multiple sites of action at the level of the myofilaments that contribute to their observed effects.
Effects of isoflurane and sevoflurane on the time-course of contraction Figures 4B and 5B illustrate that both isoflurane and sevoflurane induced a sustained acceleration of the time to peak contraction (with no associated changes in the time to peak of the Ca2+ transient). This result is similar to that observed with isoflurane in guinea-pig papillary muscle13 and with sevoflurane in ferret ventricular muscle under isometric conditions.6 A decrease in the time to peak contraction (in the absence of changes in the time-course of the Ca2+ transient) is consistent with an increase in the apparent on-rate constant for actin–myosin interaction. However, this is at odds with data from skinned ventricular muscle,12 which showed that sevoflurane (at 2 MAC) decreased the apparent on-rate constant ( fapp) for cross-bridge attachment with no effect on detachment rate (gapp) such that sevoflurane would reduce the number of cross-bridges in a force-generating state.11 12 Whereas this would reduce contractility at any cytosolic
Ca2+ (i.e. reduce apparent myofilament Ca2+ sensitivity) it may also be expected to increase the time to peak contraction, an effect opposite to that observed here with both isoflurane and sevoflurane. It is possible that such discrepancies could arise from the different preparations used in these studies, i.e. Triton X-100 skinned ventricular strips under isometric conditions, stretched to Lmax12 compared with intact ventricular myocytes in which resting sarcomere length is closer to that in the intact heart. At high load (e.g. a sarcomere length of 2.1–2.2 mm) contractile time-course is controlled at the level of the myofilaments whereas in isolated myocytes, sarcomere length is 1.8 mm, intrinsic myofilament Ca2+ sensitivity is lower14 and contractile timecourse affected by the dynamics of cytosolic Ca2+.10 Therefore, extrapolation of mechanical data from skinned muscle under high load to the intact isolated myocyte is not always appropriate. Furthermore, in skinned preparations, essential co-factors are lost, which could also help explain different effects between skinned and intact muscle. Figure 5B also shows that the time to peak contraction is prolonged during washout (overshoot) of sevoflurane (but not isoflurane, Fig. 4B); this could occur purely as a consequence of the prolongation of the time to peak of the Ca2+ transient
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Acknowledgements This work was supported by the British Heart Foundation, London, UK and by the Medical Research Council, UK.
References
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1 Davies LA, Hamilton DL, Hopkins PM, Boyett MR, Harrison SM. Concentration-dependent inotropic effects of halothane, isoflurane and sevoflurane on rat ventricular myocytes. Br J Anaesth 1999; 82: 723–30 2 Davies LA, Gibson CN, Boyett MR, Hopkins PM, Harrison SM. Effects of isoflurane, sevoflurane, and halothane on myofilament Ca2+ sensitivity and sarcoplasmic reticulum Ca2+ release in rat ventricular myocytes. Anesthesiology 2000; 93: 1034–44 3 Housmans PR, Wanek LA, Carton EG, Bartunek AE. Effects of halothane and isoflurane on the intracellular Ca2+ transient in ferret cardiac muscle. Anesthesiology 2000; 93: 189–201 4 Hanley PJ, Loiselle DS. Mechanisms of force inhibition by halothane and isoflurane in intact rat cardiac muscle. J Physiol 1998; 506: 231–44 5 Jiang YD, Julian FJ. Effects of isoflurane on [Ca2+]i, SR Ca2+ content, and twitch force in intact trabeculae. Am J Physiol 1998; 275: H1360–9 6 Bartunek AE, Housmans PR. Effects of sevoflurane on the intracellular Ca2+ transient in ferret cardiac muscle. Anesthesiology 2000; 93: 1500–8 7 Harrison SM, Robinson M, Davies LA, Hopkins PM, Boyett MR. Mechanisms underlying the inotropic action of halothane on intact rat ventricular myocytes. Br J Anaesth 1999; 82: 609–21 8 Franks NP, Lieb WR. Temperature dependence of the potency of volatile general anesthetics. Anesthesiology 1996; 84: 716–20 9 Cannell MB, Lederer WJ. A novel experimental chamber for single-cell voltage-clamp and patch-clamp applications with low electrical noise and excellent temperature and flow control. Pflugers Archiv 1986; 406: 536–9 10 Spurgeon HA, duBell WH, Stern MD, et al. Cytosolic calcium and myofilaments in single-rat cardiac myocytes achieve a dynamic equilibrium during twitch relaxation. J Physiol 1992; 447: 83–102 11 Murat I, Lechene P, Ventura-Clapier R. Effects of volatile anesthetics on mechanical properties of rat cardiac skinned fibers. Anesthesiology 1990; 73: 73–81 12 Prakash YS, Cody MJ, Hannon JD, Housmans PR, Sieck GC. Comparison of volatile anesthetic effects on actin-myosin cross-bridge cycling in neonatal versus adult cardiac muscle. Anesthesiology 2000; 92: 1114–25 13 Bosnjak ZJ, Aggarwal A, Turner LA, Kampine JM, Kampine JP. Differential effects of halothane, enflurane, and isoflurane on Ca2+ transients and papillary muscle tension in guinea pigs. Anesthesiology 1992; 76: 123–31 14 Hofmann PA, Fuchs F. Bound calcium and force development in skinned cardiac muscle bundles: effect of sarcomere length. J Mol Cell Cardiol 1988; 20: 667–77 15 Hattori Y, Azuma M, Gotoh Y, Kanno M. Negative inotropic effects of halothane, enflurane, and isoflurane in papillary muscles from diabetic rats. Anesth Analg 1987; 66: 23–8 16 Housmans PR, Murat I. Comparative effects of halothane, enflurane, and isoflurane at equipotent anesthetic concentrations on isolated ventricular myocardium of the ferret. II. Relaxation. Anesthesiology 1988; 69: 464–71
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(Fig. 5A) and may not reflect sevoflurane-induced effects at the level of the myofilaments. There were no significant changes in the time to 50% decay of the Ca2+ transient or 50% relaxation of the contraction during exposure to isoflurane (Fig. 4D) whereas, with sevoflurane, time to 50% relaxation was significantly prolonged at both MN and after 30 s. Figure 5D illustrates that the initial prolongation of relaxation at MN was ameliorated over time; at 30 s exposure, relaxation is still prolonged but this effect is reduced compared with MN. After 1 min of exposure, time to 50% relaxation returned to control values and data at this time was significantly different from that at MN. These changes occurred in the absence of changes in the time-course of the cytosolic Ca2+ transient suggesting sevoflurane-induced effects at the level of the myofilaments. These results contrast with other reports that demonstrate accelerated relaxation under isometric conditions,3 6 15 but agree with data from papillary muscles under isotonic conditions.16 A reduction in relaxation rate would be consistent with sevoflurane decreasing the cross-bridge off rate constant or myosin ATPase hydrolysis rate, or may result from interaction with other sites on the myofilament proteins, which cannot be distinguished by experiments of this nature. These data suggest that sevoflurane (but not isoflurane) has both transient and sustained effects on apparent myofilament Ca2+ sensitivity, and with sevoflurane this is accompanied temporally by transient changes in relaxation rate and it is tempting to speculate that these two are linked. The increase in apparent myofilament Ca2+ sensitivity on removal of sevoflurane is not associated with any significant change in the time to 50% decay of the Ca2+ transient or contraction, although time to peak of both parameters is prolonged (but see above). Therefore, mechanisms underlying this effect will require further experimentation. However, it should be noted that sevoflurane (but not isoflurane) affected the time-course of the Ca2+ transient during and after exposure suggesting that sevoflurane also affects cytosolic Ca2+ regulation in a complex manner, which would contribute to its inotropic effects in addition to those induced at the level of the myofilaments. In summary, these data show that both isoflurane and sevoflurane (in the majority of cells) reduce apparent myofilament Ca2+ sensitivity at steady state. In addition, sevoflurane (but not isoflurane) induces a transient further decrease in apparent myofilament Ca2+ sensitivity upon application and a transient sensitization upon removal. This may explain the greater proportionate effects of sevoflurane on contraction than the Ca2+ transient on application and removal and will also contribute to the recovery of contractions during an exposure to sevoflurane.