Takatani, Tomoka MS; Takahashi, Kyoko PhD; Jin, Chengshi MS; Matsuda, Takahisa MS; Cheng, Xinyao MS; Ito, Takashi MS; Azuma, Junichi MD
Myocardial ischemia causes depressed myocardial function and associated deleterious morphologic alterations that lead to heart failure. This injury is a pathologic process that results in extensive cell death, a significant portion of which can be attributed to apoptosis. 1 Myocyte apoptosis has been demonstrated in necropsy samples of humans suffering myocardial infarction as well as in rabbit, rat, and mouse models of continuous ischemia or transient ischemia followed by reperfusion. 2–5 Although the identities of the molecular signaling pathways that mediate ischemia-induced apoptosis are largely unknown, recent studies have shown that mitochondria play an important role in apoptosis. 6 Mitochondrial cytochrome c released into cytosol activates caspases, culminating in DNA fragmentation. 7 In contrast, another mitochondrial pathway promotes cell survival rather than cell death in ischemic preconditioning. 8 This endogenous process, well demonstrated to be operative in vivo in diverse species and tissues, refers to the paradoxical protection against lethal ischemia by brief episodes of prior “conditioning” ischemia. Mitochondrial K+ATP channel openers have continued to play a major role in the mechanisms of preconditioning. 9,10 Mitochondrial K+ATP channel opener-induced cardioprotection may be related to the inhibition of apoptosis. 11 Moreover, the infarct size-limiting effects by K+ATP channel openers have been shown to be as potent as those of preconditioning. 12,13
Minoxidil (2,4-diamino-6-piperidinylpyridine 3-oxide) is a potent vasodilator that also induces hypertrichosis of facial and body hair. 14,15 Characterization of minoxidil's effect in vivo and in vitro indicates that the sulfated metabolite opens the K+ATP channel pore coupled with sulfonylurea receptor subunits (SUR) in smooth muscle cells. 16 In cardiac myocytes, Hayashi et al 17 found that minoxidil activates K+ATP channels, causing shortening of the action potential duration by the patch-clamp technique. However, the precise mechanism and efficacy of minoxidil by K+ATP channel openers has not been completely elucidated.
To clarify the efficacy of minoxidil as a K+ATP channel opener, we examined the effect of minoxidil supplementation on ischemia-induced injury. Moreover, we focused on the relationship between mitochondrial K+ATP channels and apoptosis, and investigated possible mechanisms of minoxidil in car-dioprotection against ischemia damage. A newly developed stimulated ischemia model was used in which isolated cardio-myocytes were sealed within cultured flasks containing medium with varying concentrations of minoxidil. 18
Preparation of Cultured Cardiac Myocytes and New Simulated Ischemia Model
Primary cardiac myocyte cultures from 1-day-old Wistar rats were prepared according to the procedure described by Takahashi et al. 18 All experimental procedures were approved by the Animal Care Committee of Osaka University and conformed to international guidelines. This procedure yielded cultures with 90 to 95% myocytes, as assessed by microscopic observation of cell beating. The myocytes were maintained in serum containing culture medium, Dulbecco modified Eagle medium/F-12 (ICN Biomedicals; 1:1 vol/vol) supplemented with newborn calf serum (5%; ICN Biomedicals), 3 mM pyruvic acid, 100 μM ascorbic acid, 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium (Boehringer Mannheim; DMEM/F-12 medium) for 48 hours followed by serum-free medium. The ischemia experiments were performed 24 hours after transferring the cells to serum-free medium. To produce the ischemic condition, cells were sealed in a small flask-type culture vessel (12.5 cm2: FALCON). Prior to the sealing step, the flasks were filled with normal media (2.5 mL) and phosphate-buffered saline (PBS;+, 47.5 mL), which contained the following constituents (in mM): NaCl (137), Na2HPO4/12H2O (8.1), KCl (2.7), KH2PO4 (1.5), CaCl2/2H2O (0.9) and MgCl2/12H2O (0.3), and bubbled with 5% CO2−95% N2 for 2 minutes to fix the initial pH at 7.4 and eliminate oxygen from the remaining air space. Subsequently, the lid was tightly sealed, preventing gas from entering the flask. In the control group, the cells were incubated in 2.5 mL of medium, which was equilibrated with a 5% CO2 - 95% air atmosphere. Only the culture medium in the control group was changed daily. The simulated ischemia model combined the stress of hypoxia, acidosis, and stagnant incubation medium.
Measurement of Cell Viability
Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay according to the method of Mosmann. 19 Cardiomyocytes were plated onto a 96-well microplate (3 × 105 cells/well) and cultured for 72 hours. After incubation for 24 hours with 0 to 500 μM minoxidil in a serum-free medium, MTT solution (5 mg/ml) was added to each well (10 μL per 100 μL medium) for 4 hours. The MTT metabolites of dark blue crystals were then dissolved in DMSO (200 μL/well). The plates were read using a microplate reader (Model 450/550; Bio-Rad, New York, NY) with a test wavelength of 570 nm and a reference wavelength of 630 nm.
Evaluation of Cellular Morphology and Beating Status
The degenerated status (morphologic degeneration area-points) was quantified by measuring lysis, ballooning of cells, and morphologic changes of the same area before and after ischemia for 72 hours. Each experimental group was compared with the quantity of increasing in morphologic degeneration during 72 hours of ischemia insult.
The cell images were introduced into an intensified charged couple device camera and videotaped by a VHS recorder. The shape and location of each cell area were recorded before initiating the experiment by taking a photograph. Image processing software (NIH Image 1.59/Power Macintosh 7200) was used to determine alterations in the cell morphology. The ischemia-induced changes in beating status were estimated for each cell and expressed as a percentage of observed cells.
Determination of Creatine Phosphokinase Content and ATP Content
The creatine phosphokinase (CPK) and ATP content of the myocytes were measured according to the manufacture's instructions using a commercially available kit, the CPK-test Wako kit (Wako Chem.) and ATP-assay kit (Toyo inc.), respectively. The results were expressed as IU/μg protein or pmol/μg protein. The protein concentration was determined by the method of Lowry 20, using bovine serum albumin as a standard.
Determination of Apoptotic Cells
For visualizing fragmented nuclei, cells were fixed with 1% paraformaldehyde for 30 minutes at room temperature. After being rinsed in PBS, the cells were permeabilized in 70% ethanol. The cells were rinsed twice in PBS and stained with the fluorescent dye Hoechst 33258 (Sigma-Aldrich) for 15 minutes at room temperature. After a final rinse in PBS, the cells were mounted in the FlowFade antifade reagent (Molecular Probes), and visualized under ultraviolet light with the Olympus fluorescent microscopy system. More than 100 cardiac myocytes obtained from 3 different primary culture preparations were counted, and the number of fragmented nuclei was presented as a percentage of total cells. Further characterization of apoptosis was performed using a commercially available cell death detection kit to find DNA strand breaks using the terminal deoxynucleotidyl transferase-mediated dUDP nick end labeling (TUNEL) reagent according to the manufacturer's protocol (Promega).
Depending upon the design of the experiment, statistical significance was determined by either the Student t test, X2-test, or analysis of variance (ANOVA) with Bonferroni method used to compare individual data points for a significant F value. Each value was expressed as mean ± SEM. Differences were considered significant when the calculated P value was < 0.05.
Influence of Minoxidil on Viability of Cultured Cardiomyocytes
To assess whether minoxidil has cytotoxic effects on cardiomyocytes, cell viability using MTT assay was measured. MTT reductase activity was high in the cells exposed to minoxidil at a concentration of 1 to 500 μM for 24 to 48 hours (Table 1). Namely, minoxidil had no cytotoxicity at concentrations up to 500 μM.
Effect of Minoxidil Supplementation on Ischemia-Induced Necrosis of Cultured Myocytes
Figure 1 and Table 2A reveal the effect of minoxidil supplementation on myocyte morphology for the 72 hours in the sealed condition. The untreated ischemic cells showed pronounced injury, including ballooning and cellular lysis (Fig. 1). By contrast, myocytes treated with 5 μM of minoxidil exhibited less ischemia-induced morphologic degeneration (Fig. 1). The degenerated status was quantified by measuring lysis and morphologic by changed area with the NIH image software. The amount of morphologic degeneration in the untreated ischemic cardiomyocytes increased approximately 19-fold compared with the control group over the 0- to 72-hour ischemic period, while minoxidil treatment was reduced approximately 9-fold (Table 2A).
The effects of minoxidil on ischemia-induced CPK loss and impaired beating activity are shown in Table 3A. The CPK activity of the untreated ischemic cardiomyocytes decreased approximately 80% compared with that of the control group over the 48- to 72-hour ischemic period, while minoxidil treatment did not attenuate the decline in CPK activity. However, minoxidil treatment prevented the loss of beating function in many of the ischemic cells after a 48-hour ischemic incubation. After the same period of ischemia, approximately 70% of the untreated cells ceased their beating (Table 3B). The untreated ischemic cells had a decreased ATP content (44 pmol/μg protein) compared with that of the control group (176 pmol/μg protein), after a 48-hour ischemic incubation. In contrast, minoxidil treatment attenuated the decline in intracellular ATP content, with the content averaging 111 pmol/μg (Table 3C).
Effect of Minoxidil on Ischemia-Induced Apoptosis
Recent reports have indicated that apoptosis plays an important role in myocardial ischemic injury. 21 Apoptosis can also be induced in cultured neonatal cardiomyocytes during the course of a hypoxic insult. 22 In our simulated ischemic model, apoptosis was established by the Hoechst 33258 nuclear staining pattern and by DNA ladder analysis. 18
Figure 2A shows a typical fluorescent microphotograph of the controls and minoxidil-treated cells after a 72-hour ische-mic insult. Based on the specific DNA strain, Hoechst 33258, these cells also exhibited fragmented nuclei. The procedure revealed that simulated ischemia significantly increased the number of apoptotic cells, with the extent of apoptosis being 44% (Fig. 2A) (Table 2B). In contrast, myocytes treated with 5 μM of minoxidil were resistant to ischemia-induced apoptosis, as only 21% of the cells underwent apoptosis after a 72-hour ischemic insult. Similarly, this result obtained from the Hoechst 33258 fragmented nuclei staining pattern was also confirmed by TUNEL assay (Fig. 2B).
Inhibition of 5-Hydroxydecanoate on Attenuation of Ischemia-Induced Apoptosis by Minoxidil
Next, we investigated whether the cytoprotective effect of minoxidil is related to mitochondrial K+ATP channel activation. As an indicator of DNA fragmentation, Figure 3 demonstrates Hoechst 33258 nuclei staining, and shows a quantitative determination of apoptotic nuclei in each experimental group. After 48-hour ischemic insults, apoptotic nuclei significantly decreased from 34 to 28% in cells treated with 5 μM of minoxidil. However, this protective effect of minoxidil was blocked by the mitochondrial K+ATP channel antagonist 5-hydroxydecanoate (5-HD; 100 μM). 5-HD alone caused no effects on apoptosis in ischemic cells.
In the present study, we found that minoxidil reduced the extent of ischemia-induced injury in cultured neonatal rat cardiac myocytes. Moreover, minoxidil attenuated ischemia-induced apoptosis. This effect was blocked by the mitochondrial K+ATP channel antagonist 5-HD. These observations suggest that the activation of mitochondrial K+ATP channels inhibits apoptosis, contributing at least in part to the cardioprotective effect of minoxidil.
Cardiac K+ATP channels present a complex picture in which the regulatory sulfonylurea-binding subunits, SUR2A, are large, multitransmembrane-helix, ATP-binding cassette proteins, usually illustrated surrounding a tetrameric pore composed of Kir6.2 subunits. 23,24 This architecture is expressed at very high levels in cardiac myocytes, coupling the metabolic state of the myocyte to its electrical activity. 25,26 Recent studies have indicated that the mitochondrial K+ATP channel, not the sarcolemmal K+ATP channel, is crucial effector in the mechanism of ischemic preconditioning. 27,28 The mito-chondrial K+ATP channel activation resulting from a decline in intracellular ATP by oxidative stress is one of the factors that activates cardioprotective signaling.
The effective dose of minoxidil differs according to various tissues. 29 In the present experiment, no cytotoxicity was identified by MTT assay at concentrations up to 500 μM of minoxidil within 48 hours.
First, we examined the effect of minoxidil on ischemia-induced injury. Five μM of minoxidil reduced the extent of ischemia-induced injury, as revealed by the involvement in morphologic degeneration, CPK release, the loss of beating function, and the decline in intracellular ATP content. Although the morphologic damage and CPK release after 72 hours of ischemia were not significantly suppressed by minoxidil treatment, 5 μM of minoxidil prevented the loss of beating function in many of the ischemic cells, and attenuated the decline in intracellular ATP content after a 48-hour ischemic incubation.
Next, we investigated the effect of minoxidil on ischemia-induced apoptosis and a mechanistic link between mitochondrial K+ATP channels and apoptosis. It is likely that apoptosis contributes to the deterioration of myocardial function in patients with end-stage heart failure. 22,30 It is also speculated that it plays a role in the process of cellular remodeling following a myocardial infarction. 31 In a simulated ischemia model, apoptosis becomes increasingly important during prolonged periods of ischemia. In contrast to our study, Veinot et al 32 found a different pattern, with apoptosis assuming primary importance during the early stages of an ischemic insult. Yet, our study is in agreement with Akiyama et al, 33 who found that apoptosis was a later event in the ischemic heart. Minoxidil appears to be effective at inhibiting ischemia-induced apoptosis, implying an antiapoptotic effect of minoxidil.
It is possible that the cardiac protection of minoxidil was closely associated to K+ATP channel opening. Remme et al 34 showed that activation of the K+ATP channel by cromakalim during ischemia postponed the onset of ventricular arrhythmias in isolated rabbit hearts, contributing to the cardioprotective potential of K+ATP channel openers during myocardial ischemia. It has also been demonstrated in adult rat hearts in both tissue culture and in vivo that the opening of mitochondrial K+ATP channels is involved in the cardioprotective mechanism. 35,36 Furthermore, cardioprotection can be recruited by drugs such as diazoxide that open mitochondrial K+ATP channels; conversely, mitochondrial K+ATP channel blockers (5-HD or glibenclamide) prevent both preconditioning and pharmacological cardioprotection. 9,37 Thus, the cytoprotective effect of mitochondrial K+ATP channel activation is related to inhibition of myocardial damage.
Selective inhibitors of mitochondrial K+ATP channels (5-HD) were used to examine the effect of minoxidil on cell death by K+ATP channel opening. 5-HD abolishes the anti-apoptotic effect of minoxidil. This result supports the link between the antiapoptotic effect of minoxidil and mitochondrial K+ATP channel opening. Our findings are in agreement with previous studies using K+ATP openers such as diazoxide. 11
Akao et al 11 found that the opening of mitochondrial K+ATP channels may act quite early in the apoptotic cascade by inhibiting cytochrome c release and mitochondrial membrane potential depolarization. However, the precise mechanisms by which mitochondrial K+ATP channel opening protects against apoptosis are still unknown. It was found that minoxidil induces some cell growth factors such as VEGF, HGF, and IGF-1. 29,38–40 It is possible that minoxidil protects ischemia-induced apoptosis through this action.
Our findings reveal that the activation of mitochondrial K+ATP channels inhibits apoptosis, contributing at least in part to the cardioprotective effect of minoxidil. Exploring the mechanisms by which mitochondrial K+ATP channel opening protects against ischemia-induced injury would likely result in novel strategies for the management of ischemic heart disease and could positively influence clinical outcome.
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