Sevoflurane is widely used as a pediatric anesthetic because of its rapid induction and emergence from anesthesia compared with halothane and isoflurane [1,2]. Sevoflurane is also suitable for inhaled anesthesia in children because it has a pleasant smell and causes minimal airway irritation [2,3].
In vivo studies in both animals and humans have demonstrated that neonates and infants have immature hearts that are quite sensitive to the cardiovascular depressant effect of the volatile anesthetics . However, the direct effects of anesthetics on the intrinsic myocardial contractility of these immature hearts are difficult to assess in vivo because multiple physiologic factors, including pharmacokinetic factors, central and autonomic nervous system-mediated responses, alterations in preload and afterload, and direct myocardial effects, are affected by the anesthetics. Although several in vitro studies using isolated cardiac muscles and perfused hearts have demonstrated the myocardial depressant effects of volatile anesthetics in immature animals [5-7], the direct effects of anesthetics in immature hearts have not been elucidated well at the cellular level .
We previously reported that halothane caused a direct myocardial depression in cultured neonatal rat ventricular myocytes . In addition, the use of these cells permits the direct effects of anesthetics on cardiac excitation-contraction coupling to be studied at the cellular level in the absence of hemodynamic, humoral, autonomic, and metabolic influences. The purpose of this study was to determine the effects of sevoflurane, isoflurane, and halothane on the intrinsic contraction of cultured neonatal rat ventricular myocytes. In addition, the effects of these anesthetics on cardiac excitation-contraction coupling were examined in the presence of an L-type Ca2+ channel agonist, Bay K 8644. Because the volatile anesthetics are known to inhibit Ca2+ influx in adult cardiac myocytes [10-12], it is reasonable to speculate that Bay K 8644 attenuates anesthetic-induced myocardial depression. However, the effects of Bay K 8644 on anesthetic-induced myocardial depression in adult myocardium are somewhat controversial and seem to be dependent on differences in species and/or experimental conditions [12-14]. In immature myocardium, little is known about the effects of Bay K 8644 on anesthetic-induced myocardial depression .
All experiments were performed under the supervision of the animal care committee of Sapporo Medical University. Primary cultured cardiac myocytes were prepared from ventricles of 1- to 3-day-old Wistar rat hearts by enzymatic digestion, as previously described [9,15,16]. Briefly, neonatal rat hearts were removed and incubated at 37[degree sign]C in a collagenase (200 U/mL; Dainihon Seiyaku, Tokyo, Japan)-Hanks' balanced salt solution (Ca2+ and Mg2+ free). The resulting cardiac myocytes were then seeded on 35 x 15-mm tissue culture dishes coated with collagen type I in 1.5 mL of Dulbecco's modified Eagle's minimum essential medium (DMEM; Dainihon Seiyaku) buffered with 5 mM HEPES (Sigma Chemical Company, St. Louis, MO) containing 10% fetal bovine serum (Flow Laboratory, London, UK) and antibiotics. Serum-containing medium was replaced with fresh medium every 2 days. All experiments were performed after 6-7 days of culture.
The contraction of cultured myocardial cells was measured by using a Fotonic sensor[trade mark sign] (MTI Co., New York, NY), a fiberoptic displacement measurement instrument, at 37[degree sign]C as previously described [9,15,16]. Briefly, the serum-containing medium was replaced with serum-free DMEM buffered with HEPES at least 2 h before each experiment. The cultured myocytes were then allowed to equilibrate for 10 min at 37[degree sign]C in a humidified CO2 incubator (Sanyo, Tokyo, Japan) containing a 5% CO2/95% air atmosphere. From our preliminary experiments, we found that these myocytes could maintain a constant beating rate and amplitude for up to 2 h under these experimental conditions.
To determine the direct myocardial depressant effect of sevoflurane, isoflurane, and halothane, changes in beating rate and amplitude during exposure to the anesthetics were observed. Baseline data were collected from cultured myocytes for 30 s in the absence of any intervention. Each volatile anesthetic was then passed over the serum-free medium in a plate of cells by using a carrier gas of 95% O2/5% CO2 for 7 min, as described previously . In preliminary experiments, a maximal myocardial depression was obtained within 5 min after exposure to anesthetics. The concentration of sevoflurane, isoflurane, and halothane in the carrier gas was monitored continuously by using a precalibrated anesthetic monitor (Model 303; Atom, Tokyo, Japan). Myocytes were randomly exposed to 1%, 2%, 3%, or 4% (vol/vol) of either sevoflurane, isoflurane, or halothane. Each dish of cultured myocytes was tested at only one concentration of anesthetic, and six dishes were analyzed for each concentration. Seven minutes after the anesthetic was introduced, the beating rate and amplitude were measured.
To determine whether the myocardial depressant effect of sevoflurane, isoflurane, and halothane was affected by sarcolemmal L-type Ca2+ channel function, we examined the concentration-response curves of the anesthetics in the presence of the Ca2+ channel agonist Bay K 8644 (1 [micro sign]M). Concentration studies were conducted in the same random fashion used previously. Five minutes before the administration of the anesthetic, the myocytes were exposed to Bay K 8644. The myocytes served as their own controls: baseline data were collected 5 min after Bay K 8644 administration, before anesthetic administration. Six culture dishes were analyzed for each anesthetic concentration.
Data are expressed as mean +/- SEM. The data for contractile variables are expressed as a percentage of the control values (0% anesthetics), and each dish served as its own control. The dose-response curves were compared by performing a one-way analysis of variance (ANOVA) followed by a Bonferroni/Dunn test. Comparisons between datasets were made by performing two-way ANOVAs. When appropriate, statistical significance was estimated by using an unpaired Student's t-test. Values were considered significantly different at P < 0.05.
There were no differences in beating rate and amplitude among the groups before and 5 min after drug administration (Table 1).
The addition of sevoflurane, isoflurane, or halothane to cultured neonatal rat ventricular myocytes reduced the beating rate and contractile amplitude of these cells in a concentration-dependent manner; spontaneous beating returned to control levels after the washout period (Figure 1). The diastolic amplitude did not change during the exposure to anesthetics. Summarized data for anesthetic-induced myocardial depression are shown in Figure 2. All three anesthetics caused concentration-dependent decreases in beating rate and amplitude in cultured neonatal rat ventricular myocytes (P < 0.001). Notably, halothane treatment yielded the largest decrease in myocyte function relative to isoflurane and sevoflurane. This difference was statistically significant (P < 0.01). Compared with sevoflurane, isoflurane treatment yielded significantly larger decreases in beating rate at concentrations of 3% and 4% (P < 0.05). However, despite these reduced beating rates, the amplitude of the observed contractions was maintained at concentrations up to 4%. At concentrations of 4%, sevoflurane, isoflurane, and halothane produced a 20% +/- 4%, 64% +/- 4%, and 92% +/- 3% reduction in beating rate, along with an 18% +/- 4%, 33% +/- 4%, and 97% +/- 3% reduction in amplitude, respectively.
The L-type Ca2+ channel agonist Bay K 8644 was used to examine what effect this drug had on myocytes exposed to volatile anesthetics. The beating rate and amplitude were 105% +/- 5% and 98% +/- 5% of preadministration values at 5 min (0% anesthetic) after 1 [micro sign]M Bay K 8644 administration, respectively (Table 1). Bay K 8644 significantly improved the anesthetic-depressed amplitude with sevoflurane (Figure 3), isoflurane (Figure 4), and halothane (Figure 5) (P < 0.05 versus control levels for each drug). Although the depressive effects of sevoflurane and isoflurane were not as pronounced as those of halothane, Bay K 8644 prevented the decreases in contractile amplitude associated with each anesthetic. Conversely, the decreases in beating rates observed in the presence of each anesthetic were only partially prevented by Bay K 8644 treatment, with statistically significant differences only at 4% halothane and isoflurane (Figure 3, Figure 4 and Figure 5) (P < 0.05).
To compare the anesthetic-induced myocardial depression at equianesthetic potencies, we replotted the decreases in beating rate and amplitude as a function of the minimum alveolar anesthetic concentration (MAC) (Figure 6). Because the MAC values for sevoflurane, isoflurane, and halothane in neonatal rat have not been reported, the adult rat MAC values  (sevoflurane 2.4%, isoflurane 1.64%, and halothane 1.0%) were used. When plotted this way, the MAC-dose response values for inhibition of beating rate and amplitude in cultured myocytes were similar for sevoflurane, isoflurane, and halothane.
This study demonstrated the direct myocardial depression of sevoflurane, isoflurane, and halothane in cultured neonatal rat ventricular myocytes. At equal concentrations, the negative chronotropic and inotropic potencies were halothane >> isoflurane > sevoflurane. The negative chronotropic effects of these anesthetics were only affected by the L-type Ca2+ channel agonist, Bay K 8644, in the presence of the highest concentrations in halothane and isoflurane. Conversely, Bay K 8644 prevented any anesthetic-induced negative inotropic effect.
The neonatal (immature) myocardium has different characteristics compared with adult (mature) myocardium . For instance, immature myocardium generates less tension than the adult myocardium because neonatal contractile proteins are isoforms (lower ATPase activity) of the adult proteins, and maximal myofibrillar ATPase activity is reduced [4,19]. Furthermore, unlike mature myocardium, neonatal myocardium depends predominantly on transsarcolemmal Ca2+ influx to produce contraction, rather than on Ca2+ from the sarcoplasmic reticulum (SR), because the SR is sparse and functionally undeveloped [4,19]. Because proper functioning of the immature heart greatly depends on heart rates , it is important to evaluate anesthetics' effects on both inotropic and chronotropic cardiac function. The spontaneous beating in cultured neonatal rat ventricular myocytes allows us to examine the direct effects of anesthetics on cardiac excitation-contraction coupling in the absence of any hemodynamic, autonomic, humoral, or metabolic influences.
Although the volatile anesthetics seem to have a direct myocardial depressant effect in mature hearts [10,20-23], the direct effects of the anesthetics on intrinsic myocardial contractility in immature hearts are unclear. In newborn swine, sevoflurane lowered the systolic arterial blood pressure and cardiac index less than halothane or isoflurane . In humans, noninvasive echocardiography revealed that sevoflurane depressed cardiac contractility less than halothane or isoflurane, a feature consistent in neonates, infants, and young children [25,26]. However, it is difficult to assess the direct effects of volatile anesthetics on hearts in vivo because of concomitant changes in preload, afterload, and coronary flow after the induction of anesthesia.
To our knowledge, no attempt has been made to examine the direct effect of sevoflurane on cardiac contractility in immature myocardium at the cellular level and to compare it with that of isoflurane or halothane. Palmisano et al.  compared the direct myocardial effects of halothane and isoflurane at equal doses based on adult rabbit MAC (1.4% for halothane and 2% for isoflurane) in isolated infant and adult rabbit hearts. They found that halothane was a more potent depressant of cardiac function than isoflurane: 1 MAC halothane and isoflurane decreased the peak systolic left ventricular pressure by 25% and 15%, respectively. In contrast, Baum and Klitzner  reported that halothane (0.9 MAC) and isoflurane (1.25 MAC) decreased the peak developed force by 76% and 80%, respectively, in neonatal rabbit papillary muscle. In our study, sevoflurane and isoflurane decreased myocardial contractility less than halothane at equal concentrations. However, the myocardial depressant effects of the anesthetics were not different when the study concentrations (gas phase) were expressed as adult rat MAC values. The differences in the myocardial depressant potency among the anesthetics and among studies are likely attributable to interspecies variation or experimental preparation. In addition, the use of adult MAC values may be associated with potential error for comparisons at equianesthetic potency because MAC varies with age. Potential differences among anesthetic concentrations in the gas phase and tissue phase also confound comparisons among anesthetics. Further study is required to compare the myocardial depression in immature myocardium using neonatal MAC values and tissue-phase concentrations.
Heart rate is believed to be a key factor for maintaining cardiac output in neonatal hearts . In humans, halothane anesthetic induction decreased the heart rate in infants from 18% to 30% of baseline levels . In contrast, heart rate was not reduced by sevoflurane anesthesia in infants and children [25,26]. Sevoflurane anesthesia also results in a lower incidence of bradycardia and arrhythmias in infants and children compared with halothane [2,3]. In addition, rapid increases in isoflurane concentration are associated with periods of tachycardia . In this study, all three anesthetics decreased the beating rate in a concentration-dependent manner. Thus, the effects of isoflurane and sevoflurane on heart rate in vivo are most likely due to their indirect effects on the autonomic nervous system.
More specifically, these drugs decrease the pacemaker rate by decreasing the rate of diastolic depolarization and increasing the action potential duration on the sinoatrial node in adult guinea pig hearts . In addition, both halothane and isoflurane decrease the heart rate and increase the atrioventricular conduction time in isolated infant rabbit hearts . Because the decreases of spontaneous beating rate by anesthetics were less affected than amplitude by Bay K 8644, an L-type Ca2+ channel agonist, other ionic channels may contribute to the decreases in beating rate. In adult myocardium, there is controversy regarding the ionic events underlying the pacemaker process, but several ion channels seem to be involved, including the delayed rectifier K+ current, the hyperpolarization-activated current, and the T- and L-type Ca2+ currents. Because blockade of the K+ channel prolongs the action potential duration, resulting in a decrease in beating rate , it is possible that anesthetics directly decrease the beating rate by blocking K+ channels. However, it is not clear what mechanism is involved in regulating the beating rate of cultured cardiac myocytes. For example, we observed that an increase in extracellular Ca2+ or the addition of a beta-agonist increased the beating rate, whereas alpha-agonist and hypoxia decreased the beating rate in cultured myocytes [15,31,32]. We also reported that development of gap junctional intercellular communication plays a critical role in establishing spontaneous beating . Therefore, it is difficult to explain which mechanism is involved in the beating rate changes in the present results.
Neonatal myocardium, unlike mature myocardium, relies predominantly on transsarcolemmal Ca2+ influx rather than Ca2+ released from the SR to support contraction. Bay K 8644, a dihydropyridine derivative, acts on L-type Ca2+ channels by increasing the opening time and the probability of channel opening . If a reduction of Ca2+ influx through the sarcolemma is involved in volatile anesthetic-induced myocardial depression in immature hearts, pretreatment with Bay K 8644 should attenuate the depression. In our study, Bay K 8644 improved the anesthetic-induced reduction in contractile amplitude, but not in beating rate, in all but at the highest concentrations of halothane and isoflurane. Baum and Klitzner  evaluated the effect of Bay K 8644 on volatile anesthetic-mediated depression in neonatal rabbit papillary muscle. They found that Bay K 8644 restored the peak developed tension from 24% to 54% of the baseline values obtained with halothane and from 20% to 45% of baseline with isoflurane treatment. The different degree of restored myocardial depression by Bay K 8644 in our study relative to theirs may be due to interspecies or preparation differences. In fact, neonatal rabbit myocardial contraction greatly depends on Ca (2+) influx through Na+/Ca2+ exchange, rather than through L-type Ca (2+) channels . Volatile anesthetics may induce myocardial depression by inhibiting Na+/Ca2+ exchange in neonatal rabbit myocytes . Therefore, Bay K 8644 could only partially restore the halothane and isoflurane-induced myocardial depression in neonatal rabbit papillary muscle .
Extrapolating our data to a clinical setting must be done with caution because of possible species differences and differences between in vivo and in vitro conditions. Because our study did not make any comparisons of anesthetic-induced myocardial depression between adult and neonatal myocytes, transference of our results to a clinical situation may be limited. However, the direct effects of the anesthetics are unclear in immature myocardium, and few attempts have been made to make comparisons at the cellular level . In addition, it is very difficult to compare myocardial contraction between adult and cultured myocardium because of the differences in cell shape (rod versus round type) and contractile style (required field stimulation versus spontaneous beating). Moreover, our measurement device did not allow us to quantitate myocardial contraction in adult myocardium. Further study is required to compare the myocardial depression at the cellular level using freshly isolated neonatal and adult myocytes.
There is interspecies variation in immature myocardium, as well as in adult myocardium. Although the extracellular Ca2+-dependent contraction seems to be common among species, the entry of extracellular Ca2+ is handled differently. In rat ventricular cardiomyocytes, the number of Ca2+ channels increases significantly during the first week after birth and throughout their development in primary culture . In neonatal rabbit hearts in which the L-type Ca2+ channel is not fully developed, it has been suggested that the activation of contraction is mediated primarily by the influx of Ca2+ via the Na+/Ca2+ exchanger . To our knowledge, most of these findings are derived from animals, and little is known about the Ca (2+) regulatory mechanisms in humans. It has been reported that the Ca2+ channel-gated Ca2+ release from the SR is the primary mechanism regulating the signaling of contraction in early postnatal human myocytes . Therefore, these interspecies differences in cardiac excitation-contraction coupling mechanism(s) may limit translation of our results to a clinical situation. Despite these limitations, this model allows us to examine the direct effects of anesthetics on myocardial contraction independent of hemodynamic, neural, or locally derived factors.
In conclusion, this study documents that sevoflurane, isoflurane, and halothane have direct myocardial depressant effects on cultured neonatal rat ventricular myocytes. The depressant effects in order of potency are halothane, isoflurane, and sevoflurane when given at equal concentrations. Bay K 8644, an L-type Ca2+ channel agonist, prevented the volatile anesthetic-induced myocardial depression, which implies that the reduction of Ca2+ currents via sarcolemmal L-type Ca2+ channels plays an important role in volatile anesthetic-induced myocardial depression in immature hearts.
The authors thank Dr. Derek S. Damron for his helpful comments.
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