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Diabetes Mellitus Reduces the Antiarrhythmic Effect of Ion Channel Blockers

Ito, Isao, MD; Hayashi, Yukio, MD; Kawai, Yusuke, MD; Iwasaki, Mitsuo, MD; Takada, Koji, MD; Kamibayashi, Takahiko, MD; Yamatodani, Atsushi, MD*; Mashimo, Takashi, MD

doi: 10.1213/01.ane.0000229709.29185.88
Cardiovascular Anesthesia: Research Report
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We designed the present study to examine whether diabetes mellitus affects the antiarrhythmic effect of flecainide, a sodium channel blocker, E-4031, a potassium channel blocker, and verapamil, a calcium channel blocker, in diabetic rats. The experiments were performed in intact and diabetic rats 2, 4, and 6 wk after administration of streptozotocin. Rats were anesthetized with halothane and monitored continuously for arterial blood pressure and premature ventricular contractions. The arrhythmogenic dose of epinephrine was defined as the smallest dose producing 3 or more premature ventricular contractions within a 15-s period. The arrhythmogenic doses of epinephrine in the presence of flecainide were 8.2 ± 2.2 (mean ± sd), 7.4 ± 6.1, 5.5 ± 2.8, and 2.0 ± 0.5 μg/kg in intact and diabetic rats 2, 4, and 6 wk after streptozotocin administration, respectively. Similarly, the arrhythmogenic doses of epinephrine in the presence of E-4031 were 7.7 ± 2.6, 2.3 ± 0.7, 2.0 ± 0.7, and 1.2 ± 0.5 μg/kg, and those in the presence of verapamil were 8.2 ± 2.1, 3.1 ± 1.2, 2.3 ± 0.9, and 1.5 ± 0.5 μg/kg. Insulin partially recovered the antiarrhythmic effect of the blockers. We concluded that diabetes mellitus reduces the antiarrhythmic effects of flecainide, E-4031, and verapamil.

IMPLICATIONS: We examined the effect of diabetes mellitus on the antiarrhythmic effect of sodium, potassium, and calcium channel blockers in halothane-epinephrine-induced arrhythmias in rats. Our results show that diabetes mellitus reduces the antiarrhythmic effect of ion channel blockers.

From the Department of Anesthesiology and *Department of Medical Physics, School of Allied Health Sciences, Osaka University Faculty of Medicine, Osaka, Japan.

Accepted for publication May 16, 2006.

Address correspondence and reprint requests to Yukio Hayashi, MD, Department of Anesthesiology, Osaka University Faculty of Medicine (D-7), 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan. Address e-mail to yhayashi@anes.med.osaka-u.ac.jp.

Diabetes mellitus (DM) is associated with myocardial electrical physiological changes and the prolongation of action potential duration is one of its most characteristic properties (1,2). Electrophysiological studies have reported that ion currents, such as potassium, sodium, and calcium current, are attenuated in DM (2–5). Furthermore, DM affects the pharmacological potency of various types of drugs including catecholamines, α2 agonists, and adenosine triphosphate (ATP)-sensitive potassium channel openers (6–8). Ion channel blockers have been widely used in clinical settings as antiarrhythmic drugs (9) and our previous report showed that these channel blockers are effective for preventing epinephrine-induced arrhythmias during halothane anesthesia (10). However, the antiarrhythmic efficacy of these agents in diabetic conditions is not well understood. We designed the present study to examine whether DM affects myocardial vulnerability to the arrhythmic effect of epinephrine during halothane anesthesia and modulates the antiarrhythmic effect of ion channels blockers in halothane-epinephrine-induced arrhythmias in rats.

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METHODS

The study protocol was approved by the Animal Care Committee of Osaka University Faculty of Medicine.

Male Sprague-Dawley rats, weighing 300–430 g, were used. The rats were housed in a temperature-controlled environment under 12-h light: 12-h dark cycles, with free access to food and water. Rats were randomly assigned to two groups, diabetic or control rats. Diabetic rats received a single intraperitoneal administration of streptozotocin, 50 mg/kg, which was dissolved in 50 mmol sodium citrate buffer at pH 4.5. Control rats did not receive streptozotocin. Serum glucose levels were checked by a commercial blood glucose analyzer (AccuCheck® ADVANTAGE blood glucose monitor) at 2-wk intervals after streptozotocin administration and on the day of the experiments. The experiments were performed 2, 4, and 6 wk after the administration of streptozotocin in diabetic rats.

The animals were anesthetized with 2.0% halothane in oxygen. After tracheotomy, the lungs were mechanically ventilated with a tidal volume of 12 mL/kg at 40–50 breaths/min (Rodent Ventilator; Ugo Basile, Vasere, Italy). The ventilation rates were adjusted to maintain Paco2 at 35–45 mm Hg. The inspired concentration of halothane, 1.5%, was monitored continuously with an anesthetic gas analyzer (CAPNOMAC ULTIMA multiple gas monitor; Datex, Helsinki, Finland). Lead II of the electrocardiogram (ECG) and heart rate were monitored continuously by an ECG amplifier and pulse counter unit (AC-611G; Nihon Kohden, Tokyo, Japan). Polyethylene catheters (PE-50, PE-10) were inserted into a femoral artery for blood sampling and arterial blood pressure monitoring with a pressure transducer unit (AP-641G; Nihon Kohden) and into a femoral vein for administration of drugs. The ECG and arterial blood pressure were recorded continuously with a thermal array recorder (WS-641G; Nihon Kohden). A heating pad was used to maintain rectal temperature at 37.5°C–38.5°C. Arterial pH and oxygen tension were maintained at 7.35–7.45 and more than 100 mm Hg, respectively. After completion of the preparation, anesthesia was maintained for a further 30 min to achieve a steady state.

The arrhythmogenic dose of epinephrine was defined as the dose that produced 3 or more premature ventricular contractions within 15 s of injection. Epinephrine was injected at logarithmically spaced doses (0.5, 0.71, 1.0, 1.41, 2.20, 2.83, 4.0, 5.66, 8.0, 11.4 μg/kg) after an initial dose of 4.0 μg/kg (10). The 4.0 μg/kg dose of epinephrine served as an indicator for the direction of subsequent doses of epinephrine to establish the arrhythmogenic dose, i.e., lower or higher dose of epinephrine. This method reduces the number of epinephrine injections necessary to determine the arrhythmogenic dose. Ten to 30 min was allowed between injections until the arterial blood pressure and heart rate became stable.

When the criterion for arrhythmogenic dose was satisfied, a 2.0-mL arterial blood sample was collected for measurement of the plasma concentration of epinephrine. The blood samples were put into precooled plastic tubes containing 20 μL of 0.2 M EDTA-2Na and 0.2 M Na2S2O5, which were centrifuged at 4000 rpm for 10 min at 2°C to separate the plasma. For analysis of epinephrine, 0.5 mL plasma was acidified by the addition of 0.25 mL of 2.5% perchloric acid to precipitate protein. The samples were stored at −40°C for no longer than 7 days, until analysis. The plasma concentration of epinephrine was determined in a fully automated high-performance liquid chromatography-fluorometric system (HLC-8030 Catecholamine Analyzer; Tosoh, Tokyo, Japan), by a diphenyl ethylenediamine condensation method. This assay method has a limit of sensitivity of 10 pg/mL for epinephrine; the inter-assay and intra-assay variations were <3%.

Because a previous study (10) showed flecainide (3.0 mg/kg), a sodium channel blocker, significantly increased the arrhythmogenic dose of epinephrine, we used the same dose of flecainide in this study. Subsequently, we performed a pilot study to determine the dose of each blocker which increased the arrhythmogenic dose of epinephrine similar to flecainide (3.0 mg/kg). We examined the arrhythmogenic dose of epinephrine in the presence of 0.1 and 0.2 mg/kg of E-4031, a potassium channel blocker, and 0.05, 0.1, and 0.15 mg/kg of verapamil, a calcium channel blocker. Then, we compared with the arrhythmogenic dose in the presence of 3.0 mg of flecainide (n = 4 in each group). The arrhythmogenic doses of epinephrine in the presence of 3.0 mg/kg of flecainide, 0.1 and 0.2 mg/kg of E-4031, and 0.05, 0.1, and 0.15 mg/kg of verapamil were 7.7 ± 2.7, 5.0 ± 2.0, 7.7 ± 2.7, 6.2 ± 1.2, 8.2 ± 2.3, and 9.7 ± 1.9 μg/kg (mean ± sd), respectively.

Based on these results, we examined the arrhythmogenic dose and plasma concentration of epinephrine in the presence of saline (no channel blockers), 3.0 mg/kg of flecainide, 0.2 mg/kg of E-4031, and 0.1 mg/kg of verapamil in control (no streptozotocin) and diabetic rats. The experiments with diabetic rats were performed 2, 4, and 6 wk after streptozotocin administration. The first administration of epinephrine was started 30 min after saline or channel blocker administration. In addition, we examined the effect of insulin on the pharmacological potency of channel blockers in diabetic rats. Thirty-two diabetic rats started to received insulin treatment (14 U insulin zinc suspension per day subcutaneously) 2 wk after streptozotocin and the insulin treatment was continued for a subsequent 4 wk. The same experiments were performed 6 wk after streptozotocin administration (4 wk after insulin treatment) in the presence of saline, flecainide, E-4031, and verapamil.

The number of rats in the present study was based on another preliminary experiment. The primary variable was the arrhythmogenic dose of epinephrine and we hypothesized that this dose in the presence of flecainide would be reduced after induction of DM. We examined the arrhythmogenic dose of epinephrine in the presence of flecainide (3.0 mg/kg) in control (no streptozotocin) and diabetic rats (2, 4, and 6 wk after administration of streptozotocin). The preliminary data showed that the arrhythmogenic doses of epinephrine in the presence of flecainide were 7.7 ± 2.7, 5.8 ± 1.6, 5.0 ± 1.4, 2.9 ± 0.8 μg/kg in intact and diabetic rats 2, 4, and 6 wk after receiving streptozotocin, respectively (n = 4 and data were expressed as mean± sd). Based on these primary data, we performed a power of analysis with a significance level of 0.05 and a power level of 0.9. The required sample size was 8 in. each group for a total of 32.

All data were expressed as mean ± sd. Data were analyzed by one-way analysis of variance and comparisons between groups were assessed by Student-Newman-Keuls test. P < 0.05 was considered statistically significant.

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RESULTS

Treatment with streptozotocin results in chronic hyperglycemia and decreased body weight (Table 1). Insulin treatment normalized blood glucose and increased body weight (Table 1). Figure 1 shows the arrhythmogenic threshold of epinephrine in control, diabetic, and insulin-treated diabetic rats. The arrhythmogenic dose of epinephrine was 2.21 μg/kg (95% confidence interval, 1.89–2.53 μg/kg), 1.98 μg/kg (1.10–2.85 μg/kg), 1.71 μg/kg (1.02–2.39 μg/kg), 1.94 μg/kg (1.23–2.65 μg/kg), and 3.02 μg/kg (1.96–4.07 μg/kg) in control rats, diabetic rats 2, 4, and 6 wk after receiving streptozotocin, and insulin-treated diabetic rats, respectively. Although induction of DM tended to decrease the arrhythmogenic dose, it did not significantly affect the arrhythmogenic dose (P = 0.07 by analysis of variance). Similarly, the plasma concentration of epinephrine did not significantly change (P = 0.11 by analysis of variance). Figures 2–4 show the change of antiarrhythmic thresholds (dose and plasma concentration) of epinephrine in the presence of flecainide, E-4031, and verapamil in control, diabetic (2, 4, and 6 wk after receiving streptozotocin), and insulin-treated diabetic rats. Flecainide, E-4031, and verapamil in control rats had similar arrhythmogenic thresholds for epinephrine and significantly increased arrhythmogenic thresholds compared with control rats without any blocker (Fig. 1–4). Although the arrhythmogenic thresholds for epinephrine in the presence of flecainide did not change 2 wk after induction of DM, the thresholds were significantly reduced 6 wk after induction of DM (Fig. 2). Insulin treatment recovered the arrhythmogenic thresholds in the presence of flecainide (Fig. 2). The arrhythmogenic thresholds of epinephrine in the presence of E-4031 were significantly reduced 2 wk after induction of DM (Fig. 3). Insulin treatment partially recovered the arrhythmogenic thresholds, but the arrhythmogenic thresholds of insulin-treated rats were significantly smaller than those in control rats (Fig. 3). Similarly, the arrhythmogenic thresholds of epinephrine in the presence of verapamil significantly reduced 2 wk after induction of DM (Fig. 4). Insulin treatment partially recovered the arrhythmogenic thresholds, but the arrhythmogenic thresholds in insulin-treated rats were significantly smaller than those in control rats (Fig. 4). The hemodynamic data obtained at the onset of arrhythmias were not significantly different among control and diabetic rats (Table 2). The mean arterial blood pressure during the arrhythmias 6 wk after induction of DM was significantly lower than that before DM in the flecainide study (Table 2). The mean arterial blood pressure during the arrhythmias 2, 4, and 6 wk after induction of DM and insulin treatment after DM was significantly lower than that before DM in E-4031 and verapamil studies (Table 2).

Table 1

Table 1

Figure 1.

Figure 1.

Figure 2.

Figure 2.

Figure 3.

Figure 3.

Figure 4.

Figure 4.

Table 2

Table 2

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DISCUSSION

The present study showed that streptozotocin-induced diabetes did not affect myocardial sensitization to epinephrine during halothane anesthesia, but reduced the antiarrhythmic effect of flecainide, E-4031, and verapamil.

DM facilitates several functional changes in the myocardium (11). The diabetic heart decreases contractile response to β-adrenergic stimulation and impairs the positive inotropic response of epinephrine (6). Because stimulation of myocardial β adrenoceptors is exclusively involved in the genesis of halothane-epinephrine-induced arrhythmias (12), this decreased pharmacological responsiveness in diabetic myocardium led us to expect that the arrhythmogenic potency of epinephrine may be attenuated in diabetic rats. However, unexpectedly, we found that the arrhythmogenic threshold of epinephrine was not significantly different, regardless of induction of DM (Fig. 1). Presumably, some anatomical, physiological, and metabolic changes associated with DM may facilitate the myocardium to be sensitized to the arrhythmogenic action of epinephrine. For example, potassium abnormalities and acidosis associated with DM may reduce the arrhythmogenic threshold of epinephrine. Furthermore, one previous study (13) suggested that increased collagen concentration and electrophysiological sensitivity to epinephrine in the diabetic myocardium may enhance susceptibility to the arrhythmogenic effect of epinephrine.

Myocardial ionic currents are significantly affected by the pathophysiological process of DM (11). A marked increase in the action potential is consistently observed, and a decease in potassium current may be involved in this alternation (2,4). Thus, it may be expected that the antiarrhythmic potency of potassium channel blockers is attenuated and the present results confirmed this. Our data showed a significant attenuation of action of E-4031 even 2 weeks after induction of DM (Fig. 3). This is consistent with a previous electrophysiological study by Pacher et al. (14), who showed that action potential duration was significantly lengthened from 2 weeks of diabetes.

The influences of DM on myocardial calcium ion homeostasis were well examined and impairment of calcium ion homeostasis may be involved in the development of diabetic cardiomyopathy (11). The Ca2+-ATPase activity of the sarcoplasmic reticulum and Na+-Ca2+ exchanger are compromised, resulting in Ca2+ overload in cytoplasm (4,15,16). In addition, diabetes facilitates a decrease in L-type calcium current (1,4), although there is controversy in this field (2). The present study showed that the antiarrhythmic effect of verapamil was significantly attenuated even 2 weeks after induction of DM (Fig. 4). Calcium entry after β-adrenoceptors stimulation by epinephrine may play an important role in the genesis of halothane-epinephrine-induced arrhythmias in intact animals (17,18). On the other hand, the calcium entry was attenuated by the DM-induced intracellular calcium overload, so the calcium entry did not contribute to the genesis of arrhythmias, resulting in hyporesponsiveness of the antiarrhythmic effect of verapamil.

The effect of DM on myocardial sodium channel is limited compared with potassium and calcium channels. Myocardial sodium current consists of early, large, and transient inward current followed by a much smaller, slow, and persistent inward current (19). Chattou et al. (5) demonstrated that the early, large, and transient inward sodium current was not affected, but the small and persistent current was decreased 3–4 weeks after injection of streptozotocin. Although physiological significance of the small and persistent current has not been well elucidated, the current may play a role in the generation of cardiac arrhythmias (19). We showed that arrhythmogenic thresholds of epinephrine in the presence of flecainide were significantly reduced 6 weeks after DM, although it was not reduced 2 weeks after DM (Fig. 2). Our results suggest that the small and persistent sodium current may be involved in the antiarrhythmic action of flecainide after DM.

Diabetes may affect biotransformation and pharmacokinetics of many drugs (20). We did not determine whether the pharmacokinetics of the ion channel blockers used in this study are affected in the streptozotocin-induced diabetic state. Thus, we acknowledge the possibility that the pharmacokinetic difference among the ion channel blockers in a diabetic state is partially involved in the DM-induced pharmacological difference of ion channel blockers in the present study.

Our study also showed that insulin treatment partially recovered the arrhythmogenic thresholds of epinephrine in the presence of the ion channel blockers (Fig. 2–4). Insulin treatment of diabetes improves myocardial dysfunction (21,22). Presumably, insulin treatment may facilitate recovery of the myocardial ionic channel, resulting in the pharmacological recovery of ionic channel blockers.

Surgical stress increased sympathetic activity and endogenous catecholamine release. These physiological responses activate myocardial adrenoceptors (mainly β-adrenoceptors). Stimulation of myocardial adrenoceptors augments cardiac function and increases myocardial arrhythmogenicity. Because epinephrine is a potent β-agonist and halothane induces potent myocardial sensitization to epinephrine (23), exogenous administration of epinephrine under halothane anesthesia is a good model for studying arrhythmias during anesthesia. The present data, if applicable to the clinical setting, may have clinical relevance. The antiarrhythmic potency of ion channel blockers attenuates in the diabetic state, but insulin treatment makes antiarrhythmic agents work better.

In conclusion, DM reduced the antiarrhythmic action of ion channel blockers for halothane-epinephrine-induced arrhythmias in rats and control of blood glucose by insulin was effective for recovering the antiarrhythmic effect of the ion channel blockers.

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