Diabetes induces significant myocardial abnormalities, but the effects of halogenated anesthetics on this diseased myocardium remain a matter of debate.
Left ventricular papillary muscles and triton-skinned cardiac fibers were provided from control and streptozotocin-induced diabetic rats. The effects of halothane and sevoflurane were studied on inotropic and lusitropic responses, under low (isotony) and high (isometry) loads in papillary muscles and then on isometric tension-Ca2+ concentration (pCa) relations obtained in triton-skinned cardiac fibers. Data are presented as mean +/- SD.
Sevoflurane and halothane induced a negative inotropic effect that was more important in diabetic rats (active force: 1.5% halothane, 19+/-6 vs. 24+/-6% of baseline, P < 0.05; 3.6% sevoflurane, 47+/-14 vs. 69+/-17% of baseline, P < 0.05). However, when differences in minimum alveolar concentration were considered, no significant difference was observed between groups for halothane. The effects of halothane and sevoflurane on isotonic relaxation and postrest potentiation were not significantly different between groups. In contrast, the decrease in Ca myofilament sensitivity produced by each anesthetic agent was greater in diabetic rats than in control rats (0.65% halothane, -0.15+/-0.07 vs. -0.05+/-0.04 pCa unit, P < 0.05; 1.8% sevoflurane, -0.12+/-0.06 vs. -0.06+/-0.04 pCa unit, P < 0.05).
The negative inotropic effect of halothane and sevoflurane was greater in diabetic rats, mainly because of a significant decrease in myofilament Ca sensitivity.
SEVERAL complications may occur during the development of diabetes mellitus, related to hypertension, coronary artery disease, or a specific cardiomyopathy. 1Moreover, during anesthesia and surgical procedures, patients with diabetes have an increased incidence of intraoperative hypotension. 2To study the functional changes of diabetic cardiomyopathy, the streptozotocin-induced diabetic rat has been widely used as an animal model of diabetes. 3–7The most significant functional changes encountered in the diabetic myocardium are represented by a slowing of contraction and relaxation velocities without a change in peak developed tension. 5These alterations can be related to a variety of anomalies involving the sarcoplasmic reticulum (SR), mitochondria, sodium–calcium exchange, calcium channel currents, and intracellular calcium metabolism, together with a decrease in cross-bridge cycling. 4,6,8–11Previous studies have demonstrated that pathologic states such as myocardial ischemia, cardiac insufficiency, or cardiac hypertrophy may accentuate the negative inotropic response to halogenated anesthetics. 12On the other hand, Brian et al. 13have shown that the minimum alveolar concentration (MAC) values of halothane, isoflurane, and enflurane were diminished in diabetic rats compared with control rats. However, Hattori et al. 7have suggested that the myocardium of diabetic rats could be less sensitive to halogenated agents (halothane, enflurane, and isoflurane) than myocardium from healthy rats. Therefore, the effect of halogenated agents on myocardium from diabetic rats is still a matter of debate. In diabetic rats, the effect of halogenated anesthetics on myofilament calcium responsiveness is only known for halothane. 14This effect might be important because modification in myofilament calcium responsiveness has been described in diabetic myocardium, 15and halogenated anesthetics have been shown to act on myofilament Ca2+sensitivity. 16
Therefore, this experimental study was designed to compare the myocardial effects of halothane and sevoflurane in myocardium from diabetics and control rats. The experimental models used enabled us to investigate the effects on contraction (inotropy) and relaxation (lusitropy) at different loading conditions (isotony vs. isometry) and the direct effects on myofilament calcium sensitivity.
Materials and Methods
Care of the animals conformed to the recommendations of the Helsinki Declaration, and the study was performed in accordance with the regulations of the official edict of the French Ministry of Agriculture.
Six-week-old male Wistar rats (Iffa Credo, L’Arbresles, France) were each assigned to one of two groups, a control group and a diabetes mellitus group. In the diabetic group, streptozotocin was injected intravenously (65 mg/kg; Sigma Chemical, L’Isle d’Abeau Chesnes, France), and rats were studied 3–4 weeks later, as previously reported. 4,17All animals had continuous access to rat chow and were given water ad libitum . Transcutaneous determination of glucose blood concentration (Glucotrend®; Boehringer, Manheim, Germany) was performed to ensure that the animals experienced diabetes (i.e. , blood glucose concentration > 25 mm/l). At the moment of killing, blood samples were withdrawn from diabetic (n = 12) and control rats (n = 6) and were centrifuged at 5,000 g for 15 min; then, plasma fractions were collected and stored at −20°C for further determination of glucose and bicarbonate concentrations (Cobas integra 400; Roche Diagnostic, Manheim, Germany).
Mechanical Study of Papillary Muscles
After brief anesthesia with pentobarbital sodium, the hearts were quickly removed, and then the left ventricular papillary muscles were carefully excised and suspended vertically in a 200-ml jacketed reservoir with Krebs-Henseleit bicarbonate buffer solution (118 mm NaCl, 4.7 mm KCl, 1.2 mm MgSO4, 1.1 mm KH2PO4, 25 mm NaHCO3, 2.5 mm CaCl2, and 4.5 mm glucose) maintained at 29°C with a thermostatic water circulator. 18Preparations were field stimulated at 10 pulses/ min with rectangular wave pulses lasting 5 ms just above threshold. The bathing solution was bubbled with 95% oxygen and 5% carbon dioxide, resulting in a pH of 7.40. After a 60-min stabilization period at the initial muscle length at the apex of the length-active isometric tension curve (Lmax), papillary muscles recovered their optimal mechanical performance. The extracellular calcium concentration ([Ca2+]o) was decreased from 2.5 mm to 0.5 mm because rat myocardial contractility is nearly maximum at 2.5 mm. 19Halothane (Fluotec 3; Cyprane Ltd., Keighley, United Kingdom) and sevoflurane (Sevotec 3; Ohmeda, West Yorkshire, United Kingdom) were added to the carbon dioxide–oxygen mixture using calibrated vaporizers, as previously described. 18,20Anesthetic concentrations in the gas phase were measured continuously using an infrared calibrated analyzer (Artema MM206; Taema, Antony, France). Halothane concentrations ranged from 0.3 to 1.5 vol%, and those of sevoflurane ranged from 0.7 to 3.6 vol%, equivalent to 0.5–2.5 MAC in the healthy rat. 18–20A correction factor of −23% was applied to estimate real MAC values in diabetic rats. 13The correction factor for isoflurane (−17%) was also applied for sevoflurane. 13A 20-min equilibration period was allowed between each anesthetic concentration.
The electromagnetic lever system has been described previously. 19All analyses were made from digital records of force and length obtained with a computer. Conventional mechanical variables at Lmaxwere calculated from three twitches. The first twitch was isotonic and was loaded with the preload corresponding to Lmax. The second twitch was abruptly clamped to zero load just after the electrical stimulus with a critical damping. The third twitch was fully isometric at Lmax. We determined the maximum unloaded shortening velocity (Vmax) using the zero-load technique, and we determined maximum shortening (maxVc) and lengthening (maxVr) velocities and time to peak shortening of the twitch with preload only. In addition, the maximum isometric active force normalized per cross-sectional area (AF), the peaks of the positive (+dF · dt−1) and the negative (−dF · dt−1) force derivatives at Lmaxnormalized per cross-sectional area, and the time to peak force and time to half relaxation from the isometric twitch were recorded. Because changes in the contraction phase induce coordinated changes in the relaxation phase, indexes of contraction–relaxation coupling have therefore been developed to study lusitropy. 21Therefore, the R1 coefficient (=maxVc/maxVr) studies the coupling between contraction and relaxation under low load and thus lusitropy, in a manner that is independent of inotropic changes. R1 tests SR uptake function. 18,21The R2 coefficient (=+ dF · dt−1/−dF · dt−1) studies the coupling between contraction and relaxation under high load and thus lusitropy, in a manner that is less dependent on inotropic changes. 18
During rest in the rat myocardium, SR accumulates calcium, and the first beat after the rest interval is more forceful that the last beat before the rest interval. 18AF during postrest recovery was studied at a [Ca2+]Oof 0.5 mm and after a 1-min rest duration. At the end of the study, the muscle cross-sectional area was calculated from the length and weight of papillary muscle, assuming a density of 1.
Myofilament Calcium Sensitivity
Ventricular fiber bundles (approximately 200 μm in diameter) were dissected from papillary muscles of the left ventricle in a zero-Ca2+Krebs solution (pH 7.40) and were incubated for 1 h in a relaxing solution (see next paragraph) containing 1% Triton X-100 to dissolve the membranes without affecting the contractile proteins. After the skinning procedure, one bundle was immersed in a 0.8-ml chamber filled with the relaxing solution and mounted between a fixed end and a force transducer (Grass Model FT-03C; Quincy, MA), adjusted to slack length, stretched by 20%, and subjected to an activation–relaxation cycle. The muscle contracture was amplified on a differential amplifier (Biologic Amplifier 120; BioScience, Washington, DC) and was recorded on a Gould TA240 recorder (Gould Instruments, Valley View, OH). The length and diameter of the muscles were measured by use of a graticule in the dissecting microscope. The sarcomere length in our setup was verified by a calibrated micrometer for several bundles in each experimental group under a ×400 Zeiss lens (Carl Zeiss, Oberkochen, Germany). Values ranged between 2.2 and 2.4 μm for all bundles tested. For all experiments described below, the length of the fibers was kept constant to avoid sarcomere length-dependence changes in Ca2+sensitivity. All experiments were performed at constant temperature (22°C).
The relaxing solution contained 10 mm MOPS (3-[N-morpholino]propanesulfonic acid), 170 mm K-propionate, 2.5 mm Mg-acetate, and 5 mm K2-EGTA. Activating solutions had the same composition as the relaxing solution except that Ca2+-EGTA was substituted for K2-EGTA at various ratios. The concentrations of the different components in the solutions were calculated using program 3 of Fabiato and Fabiato to keep the ionic strength at 200 mm. The following stability constants were used in the calculations: KCaEGTA1.919 × 106· m−1, KCaATP5.0 × 103· m−1, KMgEgta40m−1, and KMgegta1.0 × 104· m−1.22Solution also contained adenosine triphosphate (ATP; 2.5 mm) and phosphocreatine (10 mm), and pH was 7.00 ± 0.01. Free Ca2+concentrations of activating solutions ranged from pCa 6.2 ([Ca2+]= 0.63 μm) to pCa 4.6 ([Ca2+]= 25.0 μm, maximally activating solution), where pCa =−log10[Ca2+]. All chemicals were obtained from Sigma Chemical Co.
To assess the effects of halothane and sevoflurane, the test solutions were equilibrated by continuous bubbling for 20 min with the chosen anesthetic agent. Halothane and sevoflurane were mixed with 100% nitrogen by means of calibrated vaporizers. The anesthetic concentrations in the gas phase were monitored with an infrared calibrated analyzer. The anesthetic concentrations used were 0.65 vol% and 1.80 vol% for halothane and sevoflurane, respectively. These concentrations were chosen to obtain anesthetic concentrations in the experimental solutions (22°C) roughly equivalent to those obtained using 0.90 vol% halothane and 2.20 vol% sevoflurane at 29°C (the temperature used in the papillary muscle experiments).
For each skinned cardiac bundle, a pCa–tension curve was obtained in baseline conditions by stepwise exposure of the fibers to solutions with increasing Ca2+concentrations and measurements of developed tension. Ca2+concentrations ranged from pCa 6.2 to pCa 4.6. Intermediate tensions were expressed as a percentage of the maximal tension obtained at pCa 4.6. Data were fitted using nonlinear fit of the Hill equation (EnzFitter 1.05; Biosoft, Cambridge, United Kingdom). The slope coefficient (nH) as well as the pCa for half maximal tension (pCa50) were calculated for each bundle.
Both anesthetics were then tested on the same fiber. Hence, pCa–tension curves were obtained for each anesthetic in a random order in each fiber. A final pCa–tension curve was obtained with solutions free of anesthetics. Because maximal activated tension decreased regularly in some fibers during the study and represented 75–95% of the initial developed force at the end of the overall experiment, tension values were normalized to their maximal value for each pCa–tension curve, and the mean value of the two control curves was used to assess the effects of each anesthetic studied.
In a second series of experiments, changes of tension at maximal Ca2+-activated force were examined using a pCa 4.6 solution. Each fiber was exposed in random order to test solutions equilibrated with halothane or sevoflurane. Each test was immediately preceded and followed by determination of maximal Ca2+-activated tension with the control test (i.e. , free of anesthetic) so that no significant differences between controls were observed. Isometric tension development from baseline to steady state was compared between test solutions and the mean of the two control measurements. Results were expressed as a percentage of these corresponding control values.
Data are expressed as mean ± SD. Comparison of two means was performed using the Student t test. Comparison of several means was performed using analysis of variance and the Newman-Keuls test. All P values were two-tailed, and a P value less than 0.05 was required to reject the null hypothesis. Statistical analysis was performed with NCSS 6.0 software (Statistical Solutions Ltd., Cork, Ireland).
Papillary muscles and triton-skinned fibers were obtained from 17 control rats and 19 diabetic rats. Diabetic rats had significantly lower body and heart weight than control rats, but the ratio of the heart to body weight was not significantly different, indicating that no cardiac hypertrophy occurred. Blood glucose concentrations were five times higher in diabetic rats than in control rats (table 1).
The mean Lmaxand the mean ratio of resting force to total force were not significantly different between groups (table 2). We observed that shortening velocities in isotonic conditions were decreased in diabetic rats, whereas active force was not significantly modified compared with that of control rats (table 2). Prolongation of duration of contraction was observed in diabetic rats, as shown by the prolongation of time to peak force in isometric conditions and time to peak shortening in isotonic conditions. Time to half relaxation was also prolonged in diabetic rats (table 2). We observed significant differences in contraction–relaxation coupling in isometric (R2) but not in isotonic (R1) conditions.
A decrease in contractility was observed as [Ca2+]owas decreased from 2.5 mm to 0.5 mm. The decrease in Vmaxwas not significantly different between control and diabetic rats (64 ± 8 vs. 69 ± 5% of the value at a [Ca2+]oof 2.5 mm, not significant), but the decrease in AF was more pronounced in control rats (53 ± 10 vs. 65 ± 10% of the value at a [Ca2+]oof 2.5 mm, P < 0.05). At a [Ca2+]oof 0.5 mm, there was no significant difference between groups in baseline values of Vmax, AF, and R1, but R2 was significantly higher in diabetic rats in the halothane (2.56 ± 0.39 vs. 1.98 ± 0.36, P < 0.05) and sevoflurane groups (2.66 ± 0.62 vs. 2.04 ± 0.35, P < 0.05). At a [Ca2+]oof 0.5 mm, the postrest potentiation was significantly impaired in diabetic rats (117 ± 9 vs. 163 ± 25% of AF, P < 0.05).
In control rats, halothane induced a marked negative inotropic effect that was significantly greater than that of sevoflurane, under both isotonic and isometric conditions (fig. 1). At the highest concentration, AF was 24 ± 6% of baseline values for halothane and 69 ± 17% of baseline values for sevoflurane. In diabetic rats, the negative inotropic effects of halothane and sevoflurane were significantly more pronounced (fig. 1). At the highest concentration, AF was 19 ± 6% of baseline values for halothane and 47 ± 14% of baseline values for sevoflurane. Because the MAC of halogenated anesthetics is lower in diabetic rats, we also plotted the inotropic effect as a function of MAC values in each group, as previously reported. 12At equipotent anesthetic concentrations, the negative inotropic effect of halothane was no longer significantly different between the two groups, in contrast with sevoflurane (fig. 2).
Under isotonic conditions and in control rats, halothane induced a significant negative lusitropic effect (increase in R1), whereas sevoflurane did not (fig. 3). There were no significant differences between control and diabetic rats (fig. 3).
Under isometric conditions and in control rats, halothane induced a significant positive lusitropic effect (decrease in R2), whereas sevoflurane did not (fig. 3). At the highest concentration, R2 was 78 ± 11% of baseline values (P < 0.05) for halothane and 90 ± 12% of baseline values (not significant) for sevoflurane. These effects were significantly more pronounced in diabetic rats (fig. 3). At the highest concentration, R2 was 64 ± 11% of baseline values (P < 0.05) for halothane and 79 ± 13% of baseline values for sevoflurane (P < 0.05).
In control rats, halothane did not significantly modify the postrest potentiation, whereas sevoflurane enhanced it (fig. 4). These effects were not significantly different between control and diabetic rats (fig. 4).
Myofilament Calcium Sensitivity
Experiments were performed in 17 skinned fibers (length, 1.72 ± 0.67 mm; diameter, 212 ± 62 μm) from 8 diabetic rats and 14 fibers (length, 1.84 ± 0.31 mm; diameter, 204 ± 63 μm) from 7 control rats. In baseline conditions (i.e. , in the absence of anesthetics), maximal Ca2+-activated tension and pCa50were not significantly different between diabetic and control rats (table 3). In both groups, halothane and sevoflurane produced a decrease in maximal developed tension and in pCa50, with no significant difference between anesthetic agents (table 3). However, while their effects on maximal tension were moderate and identical in the two groups, myofilament Ca2+sensitivity decreased to a greater extent in fibers from diabetic rats than in those from control rats (figs. 5A and B). This was attested by a larger decrease in pCa50with each anesthetic in fibers from diabetic rats as compared with those from control rats, in the presence of halothane and sevoflurane (fig. 5C).
In the current study, we showed that (1) halothane and sevoflurane induced a greater negative inotropic effect in diabetic myocardium, but these differences were no longer significant for halothane when differences in MAC values were taken into account; (2) although the lusitropic effect under low load did not differ, the lusitropic effect under high load differed significantly between control and diabetic rats; (3) in both groups, halothane did not modify postrest potentiation, whereas sevoflurane enhanced it; and (4) halothane and sevoflurane decreased myofilament Ca2+sensitivity to a greater extent in diabetic than in control rats.
We observed important alterations in the myocardium of diabetic rats, as previously reported. 5We observed a decrease in Vmaxwithout change in AF, which was associated with a marked prolongation of the contraction phase (table 2). According to the Huxley theory, the absence of modification in AF suggests that there is no change in the total number of actomyosin cross-bridges, whereas the diminution in Vmaxsuggests a decrease in ATPase actomyosin activity. 23An isomyosin shift from V1 (αα dimer) to V3 (ββ dimer) has been consistently reported in diabetic myocardium, 24which results in a decreased ATPase activity and a decline in Vmax. 25Prolongation of contraction has also been consistently reported in diabetic myocardium and could be related to a slower cross-bridge cycling rate and slower Ca2+release from the SR. In our study, we also observed that postrest potentiation was markedly altered in diabetic myocardium (fig. 4), suggesting abnormalities of the cardiac SR calcium release channel (i.e. , ryanodine receptor), a decrease in the capacity of the SR to accumulate calcium during rest, or both. Abnormalities of SR, particularly those of the ryanodine receptor, have been already reported during diabetes. 3,4
We noted that relaxation was markedly prolonged only in isometry (increase in time to half relaxation), suggesting discrepancies between isotonic and isometric conditions (table 2). The results observed in isotonic conditions conflict with those seen in previous studies because they suggest that there was no modification or improvement in relaxation. It has been shown that abnormalities of the SR in diabetic myocardium are gradual and sequential. Zhong et al. 11showed that sarco(endo) plasmic reticulum ATPase protein concentration was unchanged in 4-week but decreased in 6-week diabetic rat hearts. Therefore, it is probable that in our 4-week streptozotocin-treated rats, modifications in SR were not sufficient to explain the discrepancies between isotony and isometry. A modification of Ca2+sensitivity, as assessed by R2 and pCa50, was probably not responsible for these discrepancies because we had shown that myofilaments calcium sensitivity was not different between diabetic and control rats (tables 2 and 3).
Halogenated anesthetic agents induce myocardial depression through different mechanisms, including Ca2+homeostasis (inhibition of L-type Ca2+channels and Na+–Ca2+exchanger, 26,27sarcolemmal Ca2+-ATPase and SR functions) and changes in Ca2+sensitivity or cross-bridge cycling. 16,28We found that halothane was the more potent negative inotropic agent, as previously described, 20,29and that the effects of halogenated agents were more pronounced in diabetic rats (fig. 1). This suggests a greater sensitivity of diabetic myocardium for the same concentration of halogenated agent, although the mechanism for this difference remains unclear. It is interesting to note that diabetes mellitus and halogenated anesthetics share a common target (Ca2+homeostasis, mitochondria, Ca2+sensitivity and cross-bridge cycling) and that diabetes could have potentiated the action of halogenated anesthetics. For example, halothane might have accentuated depletion of SR calcium stores seen in diabetes by increasing the opening duration of the ryanodine receptor or may have depleted the amount of ATP available in the cell. In our study, we observed that, when [Ca2+]owas decreased to 0.5 mm, AF decreased less in diabetic than in control rats, suggesting a calcium overload of the cytoplasm. It has been suggested that calcium overload occurs in diabetes. 4,30Because diabetes might alter SR uptake of Ca2+in rat myocardium, 31it may have contributed to the negative inotropic effect of halogenated anesthetics in diabetes. However, it is difficult to define the contribution of each component implied in the different mechanisms responsible of the action of halogenated because our experimental model does not allow us to study directly L-type Ca2+channels or the Na+–Ca2+exchanger. Nevertheless, the study gave some insight into the SR and the sensitivity of myofilaments to calcium (table 3and fig. 5). The fact that the responses of isotonic relaxation and postrest potentiation to halogenated anesthetic agents were not significantly modified in diabetic rats suggests that the SR is not the target and explains the observed difference in the inotropic effect of halogenated anesthetics.
The absence of difference in Ca2+sensitivity and maximal Ca2+-activated tension between diabetic and control skinned fibers in basal conditions is in accord with the findings of recent studies. 30Different results have also been reported, 4but the reason for these discrepancies is not clear. Nevertheless, the main finding of our experiments in skinned fibers was that the decrease in Ca2+sensitivity produced by each anesthetic agent was more important in diabetic rats. This effect was also suggested by the more important decrease in R2 observed in diabetic rats (fig. 3), 21although changes in R2 are complex and influenced by the changes in force and not only by myofilament calcium sensitivity. 18This result may reflect a faster rate of release of Ca2+by troponin on withdrawal of Ca2+in diabetic myocardium or could be the result of a modification of the ratio of the two troponin T isoforms. 15Taken together, these results strongly suggest that volatile anesthetics alter the function of regulatory proteins (i.e. , the troponin–tropomyosin complex) to a greater extent in diabetic than in normal hearts. Murat et al. 14found no difference in the decrease in myocardial Ca2+sensitivity produced by halothane in control and diabetic animals. The apparent discrepancy with our results may be accounted for by differences in halothane concentrations used. The effects of volatile anesthetics on Ca2+sensitivity of cardiac skinned fibers have been shown to be dose dependent, with a major shift occurring at the lowest concentrations, suggesting some sort of saturating phenomenon. 32It is therefore conceivable that the difference between diabetic and control fibers observed using 0.65% halothane (in the current study) might have been missed with the higher concentrations (1 and 2%) used by Murat et al. 14Finally, a significant increase in the Hill coefficient was observed during halothane exposure in diabetic rats. This reflected the fact that halothane effects were more important at low Ca2+concentrations, as found in other studies. 33The reason for this observation remains unclear because Hill coefficients have no simple relation to the number of Ca2+binding sites and cannot be attributed simply to the binding properties of the troponin complex.
The expression of AF as functions of MAC showed that the negative inotropic effects of halothane were not significantly different between control and diabetic rats, whereas the difference remained significant for sevoflurane. Because little information is available about anesthetic potency in diabetes, we believe that it is important to report our results both from a pharmacologic (fig. 1) and a clinical perspective (fig. 2). 12
The following points must be considered when assessing the clinical relevance of our results. First, this in vitro study only dealt with intrinsic myocardial contractility. Observed changes in cardiac function also depend on modifications in venous return, afterload, and compensatory mechanisms. Second, this study was conducted at low temperature and at a low-stimulation frequency. However, papillary muscles and skinned fibers must be studied at low temperature because stability of mechanical parameters is not sufficient at 37°C, and papillary muscles must be studied at a low frequency because high-stimulation frequency induces core hypoxia. 34Third, this study was performed in rat myocardium, which differs from human myocardium, and the effects of halogenated anesthetics can differ between species. 35Nevertheless, the myocardial effects of volatile anesthetics on the myocardium seem to be similar between rats and humans. 20,29
In conclusion, we found that the myocardial effects of halogenated anesthetics were more significant in diabetic rats, and these differences are probably related to a greater decrease in Ca2+sensitivity produced by each anesthetic agent.
The authors thank David Baker, D.M., F.R.C.A. (Department of Anesthesiology and Critical Care, CHU Necker-Enfants Malades, Paris, France), for reviewing the manuscript.