To elucidate the mechanisms related to contractile recovery after BPV-induced cardiac asystole, isolated guinea pig papillary muscles were treated with insulin either in the continued presence of BPV or during its washout, and their contributions to the improvement of contractile performance were examined.
Preparation of cardiac muscles
According to a procedure approved by the Yonsei University College of Medicine Animal Research Committee, hearts were removed from 400-450 g male guinea pigs (Dunkin Hartley) after inhalation of sevoflurane (3-4 vol%) in an enclosed cage. Guinea pigs were obtained from the Institutional Animal Care Facility (Yonsei University Health System, Seoul, Korea). The right ventricular papillary muscles were excised, mounted horizontally in a tissue bath, and superfused at 37°C with modified normal Tyrode solution (Tyrode solution) (118 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1.2 mM MgSO4, 25 mM NaHCO3-, 11 mM glucose, and 0.10 mM EDTA) . The solution was recirculated through the bath from a reservoir containing 100 ml of Tyrode solution through which 95% O2 and 5% CO2 were bubbled, maintaining a pH of 7.4 ± 0.05. The tendinous end of the papillary muscle was attached to a Grass FT03 force transducer (Grass Instruments, Quincy, MA, USA) while the other end of the muscle was pinned to the bottom of the tissue bath. For stabilization, muscles were field-stimulated using a Grass S44 stimulator (Grass Instruments, Quincy, MA, USA) at 0.5 Hz for 60 minutes. After stabilization, contractile forces were obtained at 1.2 Hz for 15 minutes as a baseline. The 1.2 Hz stimulation was continuously applied during the entire experimental period after stabilization. The excised papillary muscles were randomly allocated into 3 groups: control (n = 10) insulin-treated (n = 39), and insulin-treated in the presence of high concentration of glucose (33 mM) (n = 31) groups. Forces were continuously recorded and analyzed using a Powerlab® Data Analysis System (Chart v.7.0, ADInstruments, Sydney, Australia). Contractile function parameters [peak force (PF) and maximum rate of force development (dF/dt-max)] were also recorded. dF/dt-max values were used as a measure of contractility.
Effects of insulin on contractility
Following baseline measurements, muscles were exposed to each drug concentration (10-3, 10-2, 10-1, 2 x 10-1, and 5 x 10-1 units/ml) of insulin (Humulin insulin R, Eli Lilly and Company, Indianapolis, IN, USA) for 10 minutes. To further define the effect of insulin in the presence of a higher-than-baseline concentration of glucose, concentration-responses for insulin were also obtained following the 15-minute exposure to 33 mM glucose. The insulin concentrations were determined based on the concentrations used by Armin and Bolte  in their study using guinea pig papillary muscles. Considering that the circulating lines were a PVC tubing system, 30% more insulin was added to each concentration to compensate for absorption of insulin to the tubing  in all insulin-related experiments.
Recirculation experiments – continuing BPV
To determine the insulin concentrations to use in asystole experiments, changes in contractile force in response to increasing concentrations of insulin following application of 50 µM BPV were measured. After obtaining baseline measurements, 50 µM BPV was applied for 15 minutes followed by sequential exposure to each concentration of insulin (10-3, 10-2, 10-1, 2 x 10-1, and 5 x 10-1 units/ml). Each concentration of insulin was maintained for 10 minutes. To further define the additive effect of glucose, muscles were exposed to 33 mM glucose after obtaining contractile depression with 50 µM BPV. Cumulative concentration-responses for insulin were obtained following a 15-minute exposure to 33 mM glucose.
To assess recovery from BPV-induced cardiac asystole, 500 µM BPV was applied after obtaining baseline measurements. If asystole did not develop for 20 minutes, 100 µM BPV was increased. If asystole did not develop despite a 600-µM BPV application for 20 minutes, we stopped the experiment. Five minutes after obtaining asystole, muscles were exposed to 10-1, 2 x 10-1, and 5 x 10-1 units/ml of insulin. Each insulin concentration was maintained for 20 minutes. To further define the additive effect of glucose, muscles were exposed to 33 mM glucose in Tyrode solution after obtaining asystole, followed by application of 10-1, 2 x 10-1, and 5 x 10-1 units/ml of insulin.
Because we did not observe any recovery from asystole in muscles treated with insulin with or without 33 mM glucose, we performed further experiments to confirm whether intermittent contractile responses (conduction block) could be reversed by insulin. When conduction block did not develop after a 15-minute application of 200 µM BPV, the concentration was increased to 250 µM. If there was no conduction block, the BPV concentration was further increased to 300 µM; if conduction block did not develop after 15 minutes, the experiment was stopped. After obtaining regular intermittent contractile responses, 10-1, 2 x 10-1, and 5 x 10-1 units/ml of insulin were applied. To examine the additive effect of glucose, at 5 minute after obtaining regular intermittent contractile responses, muscles were exposed to 33 mM glucose in Tyrode solution for 5 minutes, and the insulin concentrations were then increased sequentially. Each insulin concentration was maintained for 20 minutes.
Measurements of BPV concentrations in solutions
BPV concentrations in solution were measured under the recirculating condition at 3 minutes after applying 500 µM BPV, 5 minutes after asystole, and after 15, 30, 45, and 60 minutes, in the control solution or the control solution containing 2 x 10-1 unit/ml of insulin. In the control solution- or insulin-treated group, one milliliter of solution was collected at each time point. The samples were stored at -80°C until analysis. BPV in Tyrode solution was extracted by methylene chloride. After shaking, the organic solution was separated by centrifugation at 13,000 rpm for 3 minutes. It was dried using N2 gas and reconstituted in 5 mM ammonium acetate:acetonitrile (40:60, v/v). A 0.5-µl aliquot of the solution was analyzed using an LS/MS/MS system. Lidocaine was used as the internal standard.
Non-recirculation experiments – BPV washout
Using the same experimental protocol as in recirculation experiments, 500-600 µM BPV was applied. Five minutes after obtaining asystole, muscles were washed out with Tyrode solution containing 2 x 10-1 unit/ml of insulin for 60 minutes. To define the additive effect of glucose, muscles were washed with Tyrode solution containing 33 mM glucose and 2 x 10-1 unit/ml of insulin for 60 minutes. Plain Tyrode solution was used as a control.
Insulin was purchased from Eli Lilly Company (Indianapolis, IN, USA). All other chemicals were purchased from Sigma-Aldrich Chemicals Company (St. Louis, MO, USA).
Because of variations in baseline values from one muscle to another, alterations in contractile function were expressed as a percentage of baseline. Differences in the time to the appearance of asystole (Tasystole), the time to the appearance of the first contractile response (T1stR), and the time to the first appearance of regular contraction (TRR) among treatment groups in the washout condition were analyzed using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests. Repeated measures ANOVA was used to test for differences between baseline and time-dependent changes in contractile forces in the recirculation condition. To compare the two groups at each concentration for 60 minutes, unpaired t-test was used. Linear mixed models (LMMs) were used to compare the BPV concentrations between control preparations and those treated with insulin for 60 minutes. LMMs were also used to compare the changes in contractile forces during recovery among control, insulin-, and insulin and 33 mM glucose-treated groups in the washout condition. In all analyses using LMMs, post hoc analysis was performed in which two-tailed P values were adjusted by the Bonferroni correction to control the significance level. All data are presented as the mean ± SD. A P value < 0.05 was considered significant. The Shapiro-Wilk test was used as a test of normality test. Non-parametric tests were used when data did not meet the assumption of normality. No statistical power calculation was conducted prior to the study. The sample size was based on our previous experiences with this design