1 School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK. 2 Department of Physiology, Faculty of Medicine & Health Sciences, United Arab Emirates University, PO Box 17666, Al Ain, United Arab Emirates
*Corresponding author. E-mail: S.M.Harrison@Leeds.ac.uk
Accepted for publication: July 30, 2003
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Abstract |
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Methods. Contractility and cytosolic calcium ion (Ca2+) 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 litre1 at 1 Hz stimulation. Sarcoplasmic reticulum (SR) Ca2+ content was assessed by rapid application of caffeine. All experiments were carried out at 3637°C.
Results. The amplitude of shortening, the electrically evoked Ca2+ transient, SR Ca2+ content and myofilament Ca2+ 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 Ca2+ 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 Ca2+ efflux from the cell, an effect reversed by halothane.
Conclusions. 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 Ca2+ transient, SR Ca2+ content and myofilament Ca2+ sensitivity were qualitatively similar in control and STZ myocytes and were all depressed to the same extent by halothane.
Br J Anaesth 2004; 92: 24653
Keywords: anaesthetics volatile, halothane; antibiotics, streptozotocin; complications, diabetes; heart, ventricular myocytes
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Introduction |
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One such condition is diabetic cardiomyopathy, where cardiac dysfunction has been well documented.16 17 For example, in experimental models of type I diabetes, a variety of alterations in the amplitude and time course of contraction and the intracellular Ca2+ ([Ca2+]i) transient have been reported in single ventricular myocytes.18 Changes in contractile proteins,19 intracellular Ca2+ homeo stasis20 secondary to altered gene and protein expression17 and cardiac electrophysiology21 have all been suggested to contribute to the alterations in contractile function in the diabetic heart. It is interesting to note that diabetic patients are reported to be more prone to intraoperative cardiovascular morbidity than non-diabetic patients.22
A routinely used experimental model of type I diabetes is that induced following the injection of streptozotocin (STZ), which effectively destroys pancreatic beta cell function,23 decreasing insulin secretion and elevating blood glucose. The cardiac defects observed with this model are consistent with those seen in the clinical condition of type I diabetes in that cardiomyopathy is induced independent of other complicating factors such as atherosclerosis or vascular disease.2426
Hattori and colleagues27 reported that the negative inotropic effect of halothane was blunted in papillary muscles isolated from the left ventricle of rats with STZ-induced diabetes. Amelioration of the halothane-induced negative inotropic effect in diabetic ventricle could only result from a reduced impact of halothane on the cytosolic Ca2+ transient and/or myofilament Ca2+ sensitivity. In this study we have investigated the effect of halothane on contraction amplitude, the cytosolic Ca2+ transient, SR Ca2+ content, fractional release of Ca2+ from the SR and myofilament Ca2+ sensitivity, in order to identify which mechanisms contribute to the previously reported27 reduced negative inotropic effect of halothane in diabetic rats.
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Methods |
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Ventricular myocyte isolation
Single ventricular myocytes were isolated according to previously described techniques28 with minor modifications. In brief, rats were killed humanely by stunning followed by cervical dislocation (schedule 1 procedure, UK Home Office), the heart removed quickly and mounted on a Langendorff perfusion apparatus. Hearts were perfused retrogradely at a constant flow of 8 ml g heart1 min1 with a HEPES-based salt solution (isolation solution see below) containing Ca2+ 0.75 mmol litre1. Perfusion flow rate was adjusted to allow for differences in heart weight between STZ-treated and control animals. When the coronary circulation had cleared of blood, perfusion was continued for 4 min with Ca2+-free isolation solution containing EGTA 0.1 mmol litre1, and then for 6 min with solution containing Ca2+ 0.05 mmol litre1, collagenase 0.75 mg ml1 (type 1; Worthington, NJ, USA) and protease (type XIV; Sigma) 0.075 mg ml1. After this time, the ventricles were excised from the heart, minced and gently shaken in collagenase-containing isolation solution supplemented with bovine serum albumin 1%. Cells were filtered from this solution at 4 min intervals and resuspended in isolation solution containing Ca2+ 0.75 mmol litre1.
Measurement of shortening
Ventricular myocytes were allowed to settle on the glass bottom of a Perspex chamber mounted on the stage of an inverted microscope (Axiovert 35, Zeiss, Germany). Myocytes were superfused (35 ml min1) with a HEPES-based normal Tyrode (NT) solution containing Ca2+ 1 mmol litre1 at 3637°C. Myocyte contraction was induced by field stimulation (at 1 Hz) via platinum electrodes situated in the sides of the chamber. Unloaded shortening was used as an index of contractility.29 Shortening was followed using a video edge detection system (VED-114, Crystal Biotech, USA). The degree of shortening (expressed as a percentage of resting cell length (RCL)), the time to peak shortening (TPK) and time from peak to half relaxation (THALF) were recorded.
Measurement of the Ca2+ transient
Myocytes were loaded with the fluorescent indicator fura-2 AM (F-1221, Molecular Probes, USA) as described previously.30 In brief, 6.25 µl of a 1.0 mmol litre1 stock solution of fura-2 AM (dissolved in dimethylsulphoxide (DMSO)) was added to 2.5 ml of cells (final fura-2 concentration of 2.5 µmol litre1) and shaken gently for 10 min. Fura-2 loading was carried out at room temperature (24°C) to reduce compartmentation of the dye into intracellular organelles. After loading, myocytes were centrifuged, washed with NT to remove extracellular fura-2 and then left for 30 min to ensure complete hydrolysis of the intracellular ester. Following de-esterification, cells were superfused (as above) with NT at 3637°C. To measure [Ca2+]i, myocytes were alternately illuminated by 340 nm and 380 nm light using a monochromator (Cairn Research, UK) which changed the excitation light every 2 ms. The resultant fluorescent emission at 510 nm was recorded by a photomultiplier tube and the ratio of the emitted fluorescence at the two excitation wavelengths (340/380 ratio) was calculated to provide an index of [Ca2+]i.
SR Ca2+ release was assessed using previously described techniques.30 After establishing steady-state Ca2+ transients in electrically stimulated (1 Hz) myocytes loaded with fura-2, stimulation was stopped for a period of 5 s. Caffeine 20 mmol litre1 was then rapidly applied for 10 s using a solution switcher device.31 Electrical stimulation was restarted and the Ca2+ transients were allowed to recover to steady state. Fractional release of Ca2+ from the SR was assessed by comparing the amplitude of the steady-state electrically evoked Ca2+ transients with that of the caffeine-evoked Ca2+ transient.14
Solutions
The isolation solution was composed of NaCl 130 mmol litre1, KCl 5.4 mmol litre1, MgCl2 1.4 mmol litre1, NaH2PO4 0.4 mmol litre1, HEPES 5 mmol litre1, glucose 10 mmol litre1, taurine 20 mmol litre1, creatine 10 mmol litre1, pH 7.1 (NaOH) at 37°C. After dissociation, myocytes were perfused with NT solution of the following composition: NaCl 140 mmol litre1, KCl 5 mmol litre1, MgCl2 1 mmol litre1, glucose 10 mmol litre1, HEPES 5 mmol litre1, CaCl2 1 mmol litre1, pH 7.4. Halothane 0.6 mmol litre1 was added to the NT solution from a 0.5 mol litre1 stock solution made up in DMSO. The DMSO concentration in halothane-containing solutions was 0.12%, a concentration that had no significant effect on contractions (not shown).
Statistical analysis
Results are expressed as the mean (SEM) of n observations. Statistical comparisons were performed using independent t-test, paired t-test or two-way analysis of variance (ANOVA) as appropriate. P values less than 0.05 were considered significant.
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Results |
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Characteristics of myocyte shortening
Typical fast-time base recordings of shortening in control and STZ-treated myocytes, before and after application of halothane 0.6 mmol litre--1 are shown in Figure 1A. The amplitude of shortening, expressed as a percentage of RCL, was not significantly altered by STZ treatment (Fig. 1B). Halothane induced a significant sustained negative inotropic effect; however, contrary to an earlier report,27 the magnitude of this effect was not altered by STZ treatment (Fig. 1B). In the absence of halothane, the time course of contraction was significantly prolonged by STZ treatment (P<0.05); mean TPK shortening in control and STZ myocytes was 94 (3) ms (n=21) and 113 (4) ms (n=30), respectively (P<0.001 vs control). THALF relaxation in control and STZ myocytes was 41 (3) ms (n=21) and 51 (4) ms (n=30), respectively (P<0.05 vs control). However, exposure to halothane did not lead to any further significant changes in the time course of contraction (Fig. 1C and D).
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Halothane significantly decreased the caffeine-induced Ca2+ transient in both control and STZ myocytes (to 0.39 (0.05) and 0.34 (0.04) ratio units, respectively; Fig. 2C); however, fractional release of Ca2+ from the SR was significantly increased by halothane in both control and STZ myocytes (to 0.69 (0.06) and 0.67 (0.07), respectively; Fig. 2D).
Figure 3 illustrates the effect of STZ treatment and halothane on the rate of decay of the caffeine-evoked Ca2+ transient, which is primarily determined by the activity of the Na+/Ca2+ exchanger (NCX). This was significantly slower in STZ myocytes (Fig. 3A), the rate constant being 0.69 (0.04) fura-2 fluorescence units (FU) s1 compared with 0.89 (0.07) FU s1 in control myocytes. Halothane significantly increased the rate of decay of the caffeine transient in both control and STZ myocytes (to 1.08 (0.12) and 0.87 (0.09) FU s1, respectively; Fig. 3A).
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Characteristics of myofilament Ca2+ sensitivity
Figure 4A and B illustrates plots of cell length vs fura-2 ratio for representative control and STZ myocytes in the absence and presence of halothane. The gradient of the late phase of relaxation (right panels), when the myofilaments are in dynamic equilibrium with cytosolic Ca2+, gives an index of myofilament Ca2+ sensitivity.32 Figure 4C shows that this gradient was unaffected by STZ treatment (6.4 (1.5) and 7.8 (0.9) µm FU1 in 9 control and 12 STZ myocytes, respectively); however, halothane significantly decreased this gradient (P<0.05) in both control and STZ myocytes (to 4.3 (1.1) and 5.9 (0.6) µm FU1, respectively).
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Discussion |
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The main conclusion from this study is that we could find no evidence of an amelioration of the negative inotropic effect of halothane on contractile activity in myocytes from diabetic animals, in contrast to an earlier report.27 In the present study, halothane had qualitatively the same effect in control and STZ cells on a variety of parameters such as the magnitude of contraction and the Ca2+ transient, SR Ca2+ content, fractional release of Ca2+ from the SR and myofilament Ca2+ sensitivity.
Many similarities exist between the earlier27 and the current study, such as the dose of STZ administered to induce diabetes, resultant plasma glucose levels, the age of the animals at induction of diabetes and the period of time following induction before the animals were used. Furthermore, qualitatively similar effects of diabetes on the magnitude and time course of contraction were observed in this compared with other reports. Therefore, the reason for this disparity is not evident, but could reflect different experimental conditions employed in the two studies (e.g. papillary muscles vs isolated myocytes or the temperature at which the two studies were carried out: 3637°C in the present study vs 30°C27).
Figure 3 illustrates that the rate of decay of the caffeine-evoked Ca2+ transient, which primarily reflects Ca2+ extrusion from the cell via NCX, was reduced in STZ myocytes. This result is consistent with decreased NCX function in STZ myocytes.17 This could contribute to the significantly faster recovery of the Ca2+ transient after washout of caffeine (Fig. 3B) as Ca2+ efflux from the cell would be reduced in STZ myocytes, which would accelerate recovery of the SR Ca2+ content. Halothane significantly enhanced the rate of decay of the caffeine-evoked Ca2+ transient in both control and STZ myocytes and slowed the refilling of the SR with Ca2+. These results are consistent with halothane-induced enhancement of Ca2+ extrusion from the cell in both cell types. This result is intriguing as the major pathway for extrusion of Ca2+ from the cell in the presence of caffeine is via NCX, which others have reported to be inhibited by halothane.35 36 These conflicting data suggest that further studies are required to assess the direct effect of halothane (and other volatile anaesthetics) on NCX function. For example, the effects of volatile anaesthetics on whole-cell NCX currents under voltage-clamp conditions or the measurement of NCX current induced during the decline of a caffeine-evoked Ca2+ transient would help elucidate these discrepancies. It should also be noted that in contrast to the results observed here, halothane was found to have no effect on the decline of caffeine-induced Ca2+ transients in ferret ventricular myocytes at room temperature.37 However, NCX is temperature dependent and its activity is reduced at 23°C compared with 37°C.38
In conclusion, we could find no evidence that halothane had a less potent negative inotropic effect in ventricular myocytes from STZ-treated rats. Furthermore, a number of parameters describing Ca2+ regulation were studied but halothane had a similar effect in control and STZ myocytes. Some of the experimental results found in the present study differ from those in the literature, but these may well result from different experimental conditions between studies.
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Acknowledgements |
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