Author + information
- Received May 24, 1999
- Revision received September 10, 1999
- Accepted October 21, 1999
- Published online February 1, 2000.
- Toshiaki Sato, MD, PhDa,1,
- Norihito Sasaki, MD, PhDa,
- Brian O’Rourke, PhDa and
- Eduardo Marbán, MD, PhD, FACCa,* ()
- ↵*Reprint requests and correspondence: Dr. Eduardo Marbán, Institute of Molecular Cardiobiology, Johns Hopkins University, Ross 844/720 Rutland Avenue, Baltimore, Maryland 21205
To determine the mechanism of cardioprotection afforded by nicorandil, an orally efficacious antianginal drug, we examined its effects on ATP-dependent potassium (KATP) channels.
Nicorandil can mimic ischemic preconditioning, while mitochondrial KATP(mitoKATP) channels rather than sarcolemmal KATP(surfaceKATP) channels have emerged as the likely effectors.
Flavoprotein fluorescence and membrane current in intact rabbit ventricular myocytes were measured simultaneously to assay mitoKATPchannel and surface KATPchannel activities, respectively. In a cell-pelleting model of ischemia, cells permeable to trypan blue were counted as killed by 60 and 120 min of ischemia.
Nicorandil (100 μmol/liter) increased flavoprotein oxidation but not membrane current; a 10-fold higher concentration recruits both mitoKATPand surfaceKATPchannels. Pooled dose-response data confirm that nicorandil concentrations as low as 10 μmol/liter turn on mitoKATPchannels, while surfaceKATPcurrent requires exposure to millimolar concentrations. Nicorandil blunted the rate of cell death in a pelleting model of ischemia; this cardioprotective effect was prevented by the mitoKATPchannel blocker 5-hydroxydecanoate but was unaffected by the surfaceKATPchannel blocker HMR1098.
Nicorandil exerts a direct cardioprotective effect on heart muscle cells, an effect mediated by selective activation of mitoKATPchannels.
Nicorandil, a hybrid ATP-dependent potassium (KATP) channel opener and nitrate compound (1), is used clinically for the treatment of angina pectoris (2). The cardioprotective effects of nicorandil in ischemic hearts have received much attention: nicorandil can improve the recovery of postischemic contractile dysfunction and can reduce infarct size in several animal models (3–5)and in humans (6–9). The initial hypothesis to explain these observations invoked sarcolemmal KATP(surfaceKATP) channels: opening of surfaceKATPchannels would abbreviate excitability such that calcium overload and energy consumption would be attenuated (10). However, recent studies provide evidence that mitochondrial KATP(mitoKATP) channels rather than surfaceKATPchannels are the dominant players (11,12).
The selective mitoKATPchannel inhibitor 5hydroxydecanoate (5HD) (13)abolishes the infarct size-limiting effect of nicorandil (5). Furthermore, it has been reported that nicorandil given orally to rats is preferentially distributed into heart mitochondria (14). Therefore, we hypothesized that nicorandil targets mitoKATPchannels and the nicorandil-induced cardioprotection is mediated by opening of mitoKATPchannels. To test this hypothesis, we simultaneously assayed the activity of surfaceKATPchannels and mitoKATPchannels by measuring membrane current and flavoprotein fluorescence in rabbit ventricular myocytes (12).
Preparation of rabbit myocytes
The investigation conforms with The Guide for the Care and Use of Laboratory Animalspublished by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). Isolated ventricular myocytes were obtained from New Zealand white rabbits (weighing 1 to 2 kg) by conventional enzymatic dissociation methods (15). Cells were then filtered through nylon mesh and washed several times with Ca2+-free solution. The calcium concentration was gradually brought back to 1 mmol/liter.
Flavoprotein fluorescence and electrophysiologic recording
After isolation, cells were cultured on laminin-coated coverslips in M199 with 5% fetal bovine serum at 37°C and experiments were performed over the next day. Cells were mounted in a recording chamber and were superfused with external solution containing (in mmol/liter): NaCl, 140; KCl, 5; MgCl2, 1; CaCl2, 1; and HEPES, 10 (pH 7.4 with NaOH) at room temperature (approximately 22°C). Whole-cell current and flavoprotein fluorescence were recorded simultaneously. The internal pipette solution contained (in mmol/liter) the following: potassium glutamate, 120; KCl, 25; MgCl2, 0.5; potassium EGTA, 10; HEPES, 10; and MgATP, 1 (pH 7.2 with KOH). Whole-cell currents were elicited every 6 s from a holding potential of −80 mV by two consecutive steps to −40 (for 100 ms) and 0 mV (for 380 ms). Currents at 0 mV were measured 200 ms into the pulse. Endogenous flavoprotein fluorescence was excited with a xenon arc lamp with a bandpass filter centered at 480 nm, but only during the 100-ms step to −40 mV to minimize photobleaching. Emitted fluorescence was recorded at 530 nm by a photomultiplier tube and digitized. By focusing on individual myocytes with a ×40 objective, whole-cell current and fluorescence were monitored simultaneously from one cell at a time. The redox signal was averaged during the excitation window and calibrated with the values after exposure to 2,4-dinitrophenol (DNP), which uncouples respiration from ATP synthesis, collapses the mitochondrial potential and induces maximal oxidation. Therefore, the values of flavoprotein fluorescence were expressed as a percentage of the DNP-induced fluorescence.
Cell pelleting model of ischemia
The cell pelleting model of ischemia modified from Vander Heide et al. (16)was used to quantify cell injury. In brief, cells were washed with incubation buffer (in mmol/liter): NaCl2, 119; NaHCO3, 25; KH2PO4, 1.2; KCl, 4.8; MgSO4, 1.2; CaCl2, 1; HEPES, 10; glucose, 11; creatine, 24.9; taurine, 58.5; and supplemented with 1% BME amino acids and 1% MEM nonessential amino acids (pH 7.4 with NaOH). An aliquot of each cell suspension (0.5 ml) was placed into a microcentrifuge tube and centrifuged for 15 s into a pellet. Approximately 0.25 ml of excess supernatant was removed to leave a thin fluid layer above the pellet, and 0.2 ml of mineral oil was layered on the top of the pellet to prevent gaseous diffusion. After 60 and 120 min of pelleting, 5 μl of cell pellet was sampled through the oil layer and mixed with 75 μl of 85 mosm/liter hypotonic staining solution (in mmol/liter): NaHCO3, 11.9; KH2PO4, 0.4; KCl, 2.7; MgSO4, 0.8; CaCl2, 1 with 0.5% glutaraldehyde and 0.5% trypan blue. Microscopic examination was performed 2 to 5 min after mixing to determine the permeability of the cells to trypan blue. Cells permeable to trypan blue were counted as killed and expressed as a percentage of the total cells counted (>200 for each sample). Four groups of experiments were performed. In the control group (CONT), cells were pelleted and sampled at 60 and 120 min. For the nicorandil-treated group (NICO), nicorandil at a concentration of 100 μmol/liter was added to the solution 15 min before the pelleting. Cells treated with nicorandil in the presence of 500 μmol/liter of 5HD (NICO+5HD) or in the presence of 30 μmol/liter HMR1098 (NICO+HMR1098) were likewise pelleted and sampled. Once applied, drugs were not washed out and thus were present throughout the period of simulated ischemia. Experiments were performed at 37°C. Individual experiments in each group were performed on cells isolated from different hearts.
DNP was obtained from the manufacturer (Sigma Chemical; St. Louis, Missouri), as was sodium 5HD (Research Biochemicals International; Natick, Massachusetts). Nicorandil was a gift (Chugai Pharmaceutical Co., Ltd; Tokyo, Japan), as was HMR1098 (Hoechst Marion Roussel Chemical Research; Frankfurt, Germany).
Data are presented as mean ± SEM, and the number of cells or experiments is shown as n. Analysis of variance combined with Fisher post-hoc test was used to test for significance among groups. A value of p < 0.05 was considered significant.
Figure 1shows representative results from simultaneous measurements of flavoprotein fluorescence and surfaceKATPcurrent (IK,ATP) in a single ventricular myocyte. Nicorandil at a concentration of 100 μmol/liter reversibly oxidized the flavoproteins but did not activate IK,ATP. A second exposure to nicorandil at a concentration of 1 mmol/liter increased both flavoprotein fluorescence and IK,ATP. As summarized in Figure 2, nicorandil increased flavoprotein fluorescence in a concentration-dependent manner. Nicorandil at concentrations of 10 and 100 μmol/liter reversibly increased flavoprotein oxidation to 14 ± 2% (n = 4) and 31 ± 4% (n = 5) of the DNP value, respectively, without affecting IK,ATP. However, a very high concentration (1 mmol/liter) of nicorandil was required to increase not only flavoprotein oxidation but also IK,ATP. The selective mitoKATPchannel blocker 5HD (500 μmol/liter) (13)virtually abolished the nicorandil (100 μmol/liter)-induced flavoprotein oxidation. However, nicorandil (1 mmol/liter)-induced IK,ATPwas completely inhibited by 30 μmol/liter HMR1098, a selective surfaceKATPchannel blocker (17). These results suggest that nicorandil primarily activates mitoKATPrather than surfaceKATPchannels in rabbit ventricular cells.
In the next series of experiments, we tested the idea that mitoKATPrather than surfaceKATPchannels act as the effectors for cardioprotection afforded by nicorandil, using a cell-pelleting model of ischemia. Figure 3plots the fraction of cells killed by 60 and 120 min of simulated ischemia as a percentage of the total number of viable cells before ischemia. Pelleting for 60 and 120 min killed 35 ± 4% (n = 4) and 48 ± 4% (n = 4) of cells, respectively (CONT). Inclusion of nicorandil (100 μmol/liter) significantly decreased the percentage of cells killed during ischemia to 22 ± 3% (n = 4) after 60 min and 31 ± 3% (n = 4) after 120 min ischemia (NICO, p < 0.01 vs. CONT). The cardioprotective effects of nicorandil were abolished by 500 μmol/liter 5HD (38 ± 4% after 60 min and 49 ± 4% after 120 min ischemia, respectively). In contrast, the selective surfaceKATPchannel inhibitor HMR1098 (30 μmol/liter) did not abolish the cardioprotection by nicorandil (NICO+HMR1098, p < 0.01 vs. CONT). These results indicate that nicorandil-induced cardioprotection against ischemic damage is mediated by opening of mitoKATPchannels but not surfaceKATPchannels.
Nicorandil targets mitoKATPchannels
Previous studies in our laboratory have demonstrated that the mitoKATPchannel opener diazoxide oxidizes flavoproteins without affecting IK,ATPin rabbit ventricular myocytes (12). Using the same experimental design, our present results demonstrate that nicorandil reversibly oxidized the mitochondrial matrix in a concentration-dependent manner, while millimolar concentrations are required to elicit IK,ATP. The oxidative effects of nicorandil actually reflect the opening of mitoKATPchannels, because the mitoKATPchannel blocker 5HD completely abolished the nicorandil-induced flavoprotein oxidation. These results indicate that nicorandil primarily activates mitoKATPchannels in intact rabbit ventricular cells.
MitoKATPchannels serve as effectors of cardioprotection
KATPchannel openers may shorten the action potential duration, thereby reducing cellular calcium overload and preserving viability in ischemic myocardium: this was initially proposed as the mechanism for protection of ischemic myocardium. Nevertheless, this hypothesis cannot account for the mechanism of cardioprotection, because abbreviation of action potentials is not necessary for protection (18–20). Alternatively, recent pharmacologic evidence hints that mitoKATPchannels are the dominant players. The mitoKATPchannel opener diazoxide protects rabbit ventricular myocytes in a cell pelleting model of ischemia (12)and improves functional recovery after ischemia in isolated rat and rabbit hearts (11); this diazoxide-induced protection is prevented by 5HD (11,12). In the present study, the cardioprotective effect of nicorandil was examined in a cellular ischemia model. Previous studies have shown that simulated ischemia preconditions myocytes in this model and that the underlying mechanisms for the protection are similar to those in intact hearts (21,22). Critz et al. (23)reported that nicorandil caused neither surfaceKATPchannel opening nor cardioprotection in rabbit myocytes. However, we found that nicorandil protects against cell death to the same degree as does genuine ischemic preconditioning. Although the reason for this discrepancy is unknown, the level of nucleotide diphosphates or intracellular pH during ischemia may affect the nicorandil-induced cardioprotection. To probe the final effector for cardioprotection, we used selective blockers of either mitoKATPor surfaceKATPchannels. HMR1098, a potent surfaceKATPchannel blocker (17), completely inhibited the nicorandil-induced IK,ATP(Fig. 2B). However, the cardioprotective effects of nicorandil were not blocked by HMR1098. In contrast, the mitoKATPchannel blocker 5HD completely abolished the nicorandil-induced cardioprotection. These results indicate that mitoKATPrather than surfaceKATPchannels are involved in the cardioprotection afforded by nicorandil. In separate experiments not shown herein, we determined that nitric oxide donors only weakly favor the opening of mitoKATPchannels (24). Thus, it seems unlikely that the protective effect of nicorandil is solely conferred by its nitrate moiety.
Lethal injury to the heart can be dramatically blunted by brief periods of prior ischemia (25). Such ischemic preconditioning (IPC) exists in most species, including human (26–28). Diazoxide mimics IPC and reduces infarct size in rabbit hearts (29). However, 5HD abolishes genuine IPC (30,31). These results implicate mitoKATPchannels as effectors of IPC. Nicorandil can mimic IPC by reducing infarct size in rabbit hearts, and this cardioprotection is abolished by 5HD (5). Interestingly, Sakai et al. (14)reported subcellular localization of nicorandil in myocardial mitochondria. Therefore, taken together, it is reasonable to consider that nicorandil targets mitoKATPchannels and that cardioprotective effects of nicorandil are mediated by opening of mitoKATPchannels.
Despite their favorable cardioprotective property, enthusiasm for KATPchannel openers has been tempered by the fear that they may promote the development of ventricular arrhythmias (32). This potential drawback limits the clinical utility of surfaceKATPchannel openers. In contrast, the selective mitoKATPchannel opener diazoxide protects the myocytes from ischemia (11, 12, 29), suggesting that mitoKATPchannels might be useful targets for the ischemic cardioprotection. We found that nicorandil, a clinically available anti-ischemic agent, appears to be a fairly selective mitoKATPchannel opener. It has been approved for human use (2)and has cardioprotective effects in humans (6–9). The clinical utility of nicorandil indicates drugs that target mitoKATPchannels, without activating surfaceKATPchannels, may be safe and effective for the protection of ischemic myocardium.
Our results indicated that nicorandil exerts a direct cardioprotective effect on heart muscle cells, an effect mediated by the selective activation of mitoKATPchannels. It links, for the first time, the basic phenomenon of ischemic preconditioning with the existing pharmacopeia for ischemic syndromes. Our findings support the principle that mitoKATPchannels are valuable new targets for anti-ischemic drug development.
↵1 Dr. Toshiaki Sato’s present address: Department of Physiology, Oita Medical University, 1-1 Idaigaoka, Hasama, Oita 879-5503, Japan.
☆ This study was supported by NIH R37HL36957 (to Dr. Marbán), Banyu Fellowship in Lipid Metabolism and Atherosclerosis (to Dr. Sato), and an unrestricted gift from Chugai Pharmaceutical Co.
- control group
- ischemic preconditioning
- ATP-dependent potassium
- mitochondrial KATP
- nicorandil-treated group
- sarcolemmal KATP
- Received May 24, 1999.
- Revision received September 10, 1999.
- Accepted October 21, 1999.
- American College of Cardiology
- ↵Krumenacker M, Roland E. Clinical profile of nicorandil: an overview of its haemodynamic properties and therapeutic efficacy. J Cardiovasc Pharmacol 1992;20:S93–102.
- Gross G.J.
- Mizumura T.,
- Nithipatikom K.,
- Gross G.J.
- Ito H.,
- Taniyama Y.,
- Iwakura K.,
- et al.
- Patel D.J.,
- Purcell H.J.,
- Fox K.M.
- Nichols C.G.,
- Lederer W.J.
- Garlid K.D.,
- Paucek P.,
- Yarov-Yarovoy V.,
- et al.
- Liu Y.,
- Sato T.,
- O’Rourke B.,
- Marban E.
- Sato T.,
- O’Rourke B.,
- Marban E.
- Liu Y.,
- Gao W.D.,
- O’Rourke B.,
- Marban E.
- ↵Sato T, Sasaki N, Seharaseyon J, O’Rourke B, Marbán E. Selective pharmalogical agents implicate mitochondrial, but not sarcolemmal, Katpchannels in ischemic cardioprotection. Circulation. In Press.
- Yao Z.,
- Gross G.J.
- Grover G.J.,
- Dalonzo A.J.,
- Dzwonczyk S.,
- Parham C.S.,
- Darbenzio R.B.
- Armstrong S.C.,
- Downey J.M.,
- Ganote C.E.
- ↵Sasaki N, Sato T, Ohler A, et al. Activation of mitochondrial ATP-dependent potassium channels by nitric oxide. Circulation. In Press.
- Murry C.E.,
- Jenings R.B.,
- Reimer K.A.
- Kloner R.A.,
- Yellon D.M.
- Baines C.P.,
- Liu G.S.,
- Birincioglu M.,
- Critz S.D.,
- Cohen M.V.,
- Downey J.M.
- Auchampach J.A.,
- Grover G.J.,
- Gross G.J.
- Hide E.J.,
- Thiemermann C.