Lidocaine and Mexiletine Inhibit Mitochondrial Oxidation in Rat Ventricular Myocytes

This study was approved by the Animal Investigation Committee of Tokushima University (Tokushima, Japan) and followed the animal use guidelines of the American Physiological Society (Bethesda, MD). Forty-eight male Wistar rats (250–300 g) were anesthetized with ether, and 1.0 IU/g heparin was injected intraperitoneally 30 min before surgery. Myocytes were obtained enzymatically (0.2 mg/ml collagenase and 0.05 mg/ml protease) using a Langendorff apparatus. The enzymatic dissociation method was similar to that of our previous study. 18 

Rod-shaped, clear striated ventricular myocytes were cultured on laminin-coated coverslips in M199 culture medium with 5% fetal bovine serum at 37°C. Experiments were performed during the next day. The mitochondrial redox state was monitored by recording the fluorescence of flavin adenine nucleotide-linked enzymes in mitochondria and served as an index of mitochondrial K^ATPchannel activities. Myocytes were superfused with bath solution containing 140 mm NaCl , 5 mm KCl, 1 mm CaCl², 1 mm MgCl², and 10 mm HEPES (pH adjusted to 7.4 with NaOH) at room temperature (20 ± 2°C). Fluorescence was monitored microscopically (Eclipse TS100; Nikon, Tokyo, Japan) with a digital charge coupled device camera (ORCA; Hamamatsu Photonics, Hamamatsu, Japan) from one cell at a time by focusing on individual myocytes. Fluorescence of single cells was excited for 100 ms every 10 s. Excitation of flavoprotein was obtained from a Xenon arc lamp filtered at 450–490 nm and reflected to the microscope objective lens (×40) by a dichroic mirror centered at 505 nm. Emitted fluorescence was recorded to pass through the dichroic mirror to a 520-nm-long path filter and was stored on a computer. The redox signal images were analyzed for average pixel intensities of regions of interest on a myocyte using an image processing system (AQUACOSMOS; Hamamatsu Photonics). The change of fluorescence was normalized to the baseline flavoprotein fluorescence obtained after exposure to 5 μm 2,4-dinitrophenol (DNP), a protonophore that uncouples respiration from ATP synthesis and collapses the mitochondrial potential, at the end of the experiments. At least 30 normalized fluorescence images were averaged before (diazoxide alone) and at each concentration of drugs. In the first series of the experiments, the effects of diazoxide, a selective mitochondrial K^ATPchannel opener, 6and 5-hydroxydecanoic acid sodium (5-HD), a relatively selective mitochondrial K^ATPchannel blocker, on flavoprotein fluorescence were evaluated. Then we assessed the effects of diazoxide alone and in combination with lidocaine or mexiletine on flavoprotein fluorescence in the following series. In the same cell, flavoprotein fluorescence was recorded before (diazoxide alone) and at five concentrations of either lidocaine (10⁻³∼ 10 mm) or mexiletine (10⁻³∼ 10 mm). Six data points obtained in the same cell were plotted as drug concentration compared with the normalized flavoprotein fluorescence; then these data were converted to probits, and the concentration–response equation was calculated by least-square curve fitting. From this equation, the concentrations of lidocaine or mexiletine needed to induce 50% inhibition of diazoxide-induced flavoprotein oxidation (EC⁵⁰) were calculated in each cell.

Lidocaine and mexiletine were obtained from Sigma Chemical (St. Louis, MO). Diazoxide (Sigma) was dissolved in dimethyl sulfoxide (< 0.1%) and prepared as a stock solution. 5-HD was purchased from Biomol Research Laboratories (Plymouth Meeting, PA). All other solutions were made daily.

Data are expressed as mean ± SD. Differences among data sets were evaluated by analysis of variance followed by Student-Newman-Keuls post hoc  test. A P  value less than 0.05 was considered significant.

Figure 1shows the representative example of flavoprotein fluorescence in cells exposed to diazoxide and 5-HD. The flavoprotein fluorescence value was expressed as a percent of that exposed to 5 μm DNP at the end of the experiments (DNP value). Diazoxide (25 μm) caused reversible mitochondrial oxidation to 63 ± 19% of the DNP values (n = 7). 5-HD (100 μm) attenuated the oxidative effects of diazoxide to 2 ± 5% of the DNP value (P < 0.05 vs.  diazoxide, n = 7). After washout of these drugs, mitochondrial oxidation was restored to the baseline level.

Figure 2shows the relation between lidocaine concentration and diazoxide (25 μm)-induced flavoprotein oxidation (n = 7). Flavoprotein oxidation was 63 ± 5% in the presence of diazoxide alone. With lidocaine, flavoprotein oxidation was 63 ± 7% at 0.001 mm, 42 ± 14% at 0.01 mm (P < 0.05 vs.  diazoxide alone), 25 ± 7% at 0.1 mm (P < 0.05), 9 ± 9% at 1 mm (P < 0.05), and 1 ± 1% at 10 mm (P < 0.05). Lidocaine induced 50% inhibition of diazoxide-induced flavoprotein oxidation (EC⁵⁰) at 98 ± 63 μm concentration.

Figure 3shows that mexiletine, like lidocaine, reduced the diazoxide-induced oxidation of flavoproteins in a concentration-dependent manner. Flavoprotein oxidation was 64 ± 6% in the presence of diazoxide alone. With mexiletine, flavoprotein oxidation was 61 ± 8% at 0.001 mm, 51 ± 5% at 0.01 mm (P < 0.05 vs.  diazoxide alone), 34 ± 10% at 0.1 mm (P < 0.05), 23 ± 5% at 1 mm (P < 0.05), and 1 ± 4% at 10 mm (P < 0.05;fig. 3B; n = 10). The EC⁵⁰for mexiletine to induce 50% inhibition of diazoxide-induced flavoprotein oxidation was 107 ± 89 μm.

The major findings in the current study are that both drugs inhibit diazoxide-induced flavoprotein fluorescence, which correlates with mitochondrial oxidation and depolarization. Activation of K^ATPchannels produces cardioprotective effects in cardiac myocytes, 19but the underlying mechanisms for such cardioprotection are poorly understood. One early hypothesis proposed that opening sarcolemmal K^ATPchannels shortens the action potential duration and then depresses contractility, 19,20which is a major source of ^ATPconsumption. Recent evidence, however, contradicts this hypothesis. Yao and Gross 21found that a low dose of the K^ATPchannel opener bimakalim had a minimal effect on action potential duration but still reduced infarction size. Such dissociation has also been shown in other studies. 22–25These data suggest that abbreviation of action potential duration may not be necessary for cardiac protection. The opening of mitochondrial rather than sarcolemmal K^ATPchannels may be a major contributor to cardiac protection against ischemia. 5Although the physiologic and pathophysiologic roles of the mitochondrial K^ATPchannel are not yet clear, opening mitochondrial K^ATPchannels results in K⁺entry and intramitochondrial depolarization. 1Therefore, a possible mechanism for the cardioprotective action of mitochondrial K^ATPchannels is that dissipation of inner mitochondrial membrane potential decreases the driving force for Ca²⁺influx through the Ca²⁺uniporter. 6This would reduce mitochondrial Ca²⁺overload and cause matrix swelling, which has been shown to enhance ATP synthesis and stimulate mitochondrial respiration. 6Another possibility is that opening mitochondrial K^ATPchannels, by decreasing the membrane potential, could promote binding of the endogenous mitochondrial ATPase inhibitor 26and thus conserve ATP during ischemia.

The mitochondrial redox state can be monitored by recording the fluorescence of flavin adenine nucleotide–linked enzymes in the mitochondria. 27,28To test the hypothesis that mitochondrial K^ATPchannels have an important role in cardioprotection, Liu et al.  6examined the effects of diazoxide on both mitochondrial and sarcolemmal K^ATPchannel activities using flavoprotein fluorescence, an index of mitochondrial redox state, and sarcolemmal K^ATPcurrents as indicators in intact rabbit ventricular myocytes and showed that diazoxide induced reversible oxidation of flavoproteins but did not activate sarcolemmal K^ATPchannels. They also found that diazoxide decreased the rate of cell death in a cellular model of simulated ischemia to approximately half that of controls. They concluded that diazoxide targets mitochondrial but not sarcolemmal K^ATPchannels and that the opening of mitochondrial rather than sarcolemmal K^ATPchannels might contribute to cardiac protection against ischemia. 6These results are similar to those obtained in the current study. We also studied the effects of diazoxide alone or in combination with 5-HD on flavoprotein oxidation in isolated rat ventricular myocytes and found that diazoxide caused reversible mitochondrial oxidation and that 5-HD attenuated the oxidative effects of diazoxide (fig. 1). Therefore, the results reported by Liu et al.  6and the results of the current study led us to conclude that the flavoprotein fluorescence we measured reflects the redox state of mitochondria.

In the current study, both lidocaine and mexiletine reduced diazoxide-induced oxidation of flavoprotein in a concentration-dependent manner, suggesting that both drugs attenuate mitochondrial K^ATPchannel activities. If the opening of mitochondrial rather than sarcolemmal K^ATPchannels contributes to cardiac protection against ischemia, blockade of mitochondrial K^ATPchannels by lidocaine and mexiletine may produce impairment of mitochondrial oxidation mediated by mitochondrial K^ATPchannels. That is, our results suggest that both drugs may attenuate cardioprotective effects of mitochondrial K^ATPchannels. In contrast, blockade of sarcolemmal K^ATPchannels by lidocaine and mexiletine may be advantageous in the prevention of arrhythmia. During myocardial ischemia, extracellular myocardial K⁺concentration in the ischemic zone increases, and the resultant slowing of impulse propagation has a pivotal role in the pathogenesis of ventricular arrhythmia. 29In heart cells, K^ATPchannels are activated by depletion of intracellular ATP, hypoxia, or exposure to metabolic inhibitors 18and cause an increase in K⁺efflux. The activation of K^ATPchannels is at least partially responsible for the increase in outward K⁺currents, shortening of action potential duration, and increase in extracellular K⁺concentration during anoxic or globally ischemic conditions. 30–32Bekheit et al.  30reported that glibenclamide, a K^ATPchannel blocker, decreases the K⁺loss from ischemic myocardium and reduces the incidence of arrhythmia. These findings suggest a significant contribution of sarcolemmal K^ATPchannels to the formation of cardiac arrhythmia during ischemia. 30–33 

Both lidocaine and mexiletine are known to exert their therapeutic effects by selectively blocking voltage-dependent Na⁺channels in a rate- and concentration-dependent manner. 11,12In addition, many reports have evaluated the effects of these drugs on sarcolemmal K^ATPchannel activities. Using voltage clamp techniques, Yoneda et al.  13reported that lidocaine inhibits K^ATPchannel activities in a concentration-dependent manner in Xenopus  oocytes. Using patch clamp techniques, Olschewski et al.  14also reported that lidocaine blocked K^ATPchannels in rat cardiomyocytes (EC⁵⁰= 43 μm). In contrast, the effects of mexiletine on K^ATPchannel activities are controversial. Tricarico et al.  15reported that mexiletine was a state-dependent K^ATPchannel inhibitor in skeletal muscle. In ventricular muscles, Wu et al.  16found that 30 μm mexiletine did not significantly affect K^ATPcurrent, whereas Sato et al.  17reported that mexiletine shortened action potential duration via  partial activation of K^ATPchannels.

In the current study, the EC⁵⁰s for both lidocaine and mexiletine were higher than those in clinical use. The EC⁵⁰s obtained in the current study were 98 μm for lidocaine and 107 μm for mexiletine, whereas the therapeutic ranges of plasma concentration of lidocaine and mexiletine used as antiarrhythmic drugs have been reported as approximately 5–30 and 2–9 μm, respectively. 34–36In addition, we studied the effects of drugs in isolated rat ventricular myocytes. The effects of these drugs on rat myocardium may be different from the effects on human myocardium. Therefore, we should be careful in extending the current results to the human heart.

In conclusion, both lidocaine and mexiletine reduced flavoprotein fluorescence induced by diazoxide in rat ventricular myocytes, indicating that these antiarrhythmic drugs may produce impairment of mitochondrial oxidation mediated by mitochondrial K^ATPchannels.