This study showed that insulin did not reverse BPV-induced conduction block or asystole in isolated cardiac tissues in the recirculation conditions. Although asystole was not reversed, insulin treatment is likely to replenish the energy source that was depleted by high concentrations of BPV.
In the present study, the recirculation condition was used to investigate the effect of insulin on the recovery of stimulated contractile responses, which are dependent on elimination of BPV from tissues. By contrast, the washout condition was used to determine if insulin could enhance or accelerate the time-dependent contractile recovery via its metabolic contributions.
In the recirculating condition, neither asystole nor conduction block was reversed by insulin. These findings suggest that insulin does not lower BPV content in cardiac tissues sufficiently to restore Na+ channel function to conduct action potentials. Simply improving metabolic energetics would be ineffective in restoring myocardial contractility when the continuing presence of BPV in the tissue prevents the conduction of action potentials. Considering that myocardial BPV content has a strong positive correlation with aqueous plasma concentration [11], our findings regarding BPV concentrations in aqueous phase indirectly suggest that insulin does not reduce BPV content sufficiently to dissociate BPV from Na+ channels in cardiac tissues. It has been reported that insulin had no effect on TTX-inhibited Na+ channel in rat myoballs [12].
Insulin increases glucose utilization by alterations in membrane kinetics so that glucose transport is accelerated by insulin [12]. With the addition of insulin, glucose uptake increases much more rapidly than increased perfusate concentrations of glucose in the absence of insulin [13]. However, regardless of increased glucose uptake, the positive inotropic effect of insulin has been reported to be independent on the external glucose concentration in the bathing medium [14, 15]. In the present study, our results demonstrated that insulin had a dose-related negative inotropic effect that also was not affected by external glucose concentration.
However, this may not be applicable for contractile depression induced by BPV application. While insulin concentration-dependently depressed contractility in our result, insulin application following contractile depression by 50 µM BPV did not depress contractility further, suggesting certain mechanisms that attenuate further depression by insulin. Of note, in the presence of 33 mM glucose, insulin application increased contractility modestly, but significantly, suggesting that, at least in part, enhanced glucose uptake by insulin is associated with this positive inotropic effect.
With elimination of BPV from the perfusate and its presumed diffusion from the tissue under the washout condition, the contractile responses to 1.2 Hz stimulation were completely and quickly restored in all muscles as shown in Table 1. This return of contractile responses is consistent with a much faster decrease in myocardial BPV content and restored Na+ channel function by washout. In this condition, we observed complete recovery of contractile forces in the insulin- or insulin combined with 33 mM glucose-treated groups while contractility in the control group recovered to approximately 60% of baseline levels, which indicates long-term injury in tissues by BPV. Of note, differences among the control, insulin, and insulin combined with 33 mM glucose groups for the restoration of contractility appeared to be dependent on whether insulin was used or not. Therefore, the contractile recovery to baseline after treatment with insulin or insulin combined with 33 mM glucose suggests improved myocardial energetics, possibly due to increased cytoplasmic glucose concentrations. These findings imply that although asystole persists under recirculation conditions, metabolic energy is replenished continuously due to enhanced glucose uptake by insulin.
Recently, Kim et al. [5] reported successful resuscitation of BPV-induced circulatory collapse in dogs by insulin. In this study, in association with external chest compression, all insulin/glucose-treated dogs were successfully resuscitated, however, none of the control dogs were resuscitated. Based on irreversible restoration of stimulated contractile responses by insulinunder recirculation conditions in our results, Kim et al.’s in vivo findings highly suggest the importance of ongoing circulation accomplished with chest compressions to restore cardiac rhythm. Ongoing circulation by chest compression results in gradual redistribution of BPV by increasing blood flow to other organs [5] and possible enhancement of hepatic extraction of BPV [16] would lower BPV concentrations in the myocardium, which contributes to dissociation of BPV from the Na+ channel to permit initiation of action potentials. However, a longer period of time was required to reverse impaired myocardial conduction [5]. Considering the redistribution of BPV by chest compression in whole animals or the clinical setting, our results in the recirculation and washout condition from an isolated rodent heart preparation have a limitation to extrapolate to whole animals or humans. However, our results may provide mechanistic insights into the delayed recovery of myocardial conduction and enhanced supply of myocardial energetics which contribute to hemodynamic recovery in whole animals.
Currently, as a mechanism of lipid rescue from BPV-induced cardiac toxicity, two important mechanisms such as lipid sink [17] and metabolic lipid flux effect [1] have been proposed. In the previous studies in in vitro using isolated guinea pig papillary muscles [8] and in an intact rat model of BPV-induced cardiovascular collapse [18], the contractile or hemodynamic recovery has been attributable to lipid sink effect, rather than the metabolic lipid flux effect. Additionally, the liver-targeting property [19] and accelerated clearance of BPV by lipid emulsions [20, 21] may add more benefit to decrease the BPV concentrations in cardiac tissues. Although insulin is likely to replenish the depleted ATP, it has no local anesthetic-binding effect, a major disadvantage to be rescued from BPV-induced cardiac toxicity. Based on these aspects, insulin/glucose does not appear to have more benefits than lipid emulsions for the recovery from BPV-induced cardiac arrest.