Cardiac mitochondrial cGMP stimulates ... - Semantic Scholar

Clinical Science (2007) 112, 113–121 (Printed in Great Britain) doi:10.1042/CS20060144

Cardiac mitochondrial cGMP stimulates cytochrome c release Kazuhiko SEYA, Shigeru MOTOMURA and Ken-Ichi FURUKAWA Department of Pharmacology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan

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Although the existence of cardiac mitochondrial cGMP has been reported previously [Kimura and Murad (1974) J. Biol. Chem. 249, 6910–6916], the physiological and pathophysiological properties of cGMP in cardiac mitochondria have remained unknown. The aim of the present study was to clarify whether cardiac mitochondrial cGMP regulates the apoptosis of cardiomyocytes. In the presence of GTP, the NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine; 1 mmol/l) and SNP (sodium nitroprusside; 1 mmol/l) each markedly increased the cGMP level in a highly purified mitochondrial protein fraction prepared from left ventricular myocytes of male Wistar rats, and these increases were inhibited by 1 µmol/l ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), an inhibitor of NO-sensitive guanylate cyclase. In purified mitochondria, both SNAP (1 mmol/l) and the membrane-permeant cGMP analogue 8-Br-cGMP (8-bromo-cGMP; 1 mmol/l), but not cGMP (1 mmol/l), increased cytochrome c release from succinate-energized mitochondria without inducing mitochondrial swelling and depolarization of the mitochondrial membrane as factors of activation of MPT (mitochondrial permeability transition). The cytochrome c release mediated by SNAP was inhibited in the presence of 1 µmol/l ODQ. On the other hand, 1 mmol/l SNAP induced apoptosis in primary cultured adult rat cardiomyocytes in a time-dependent manner, and this induction was significantly inhibited in the presence of ODQ. Furthermore, apoptosis induced in primary cultured cardiomyocytes by hypoxia/re-oxygenation was also inhibited by ODQ. These results suggest that the acceleration of cGMP production in cardiac mitochondria stimulates cytochrome c release from mitochondria in an MPT-independent manner, resulting in apoptosis.

INTRODUCTION Reperfusion therapy for acute myocardial infarction is known to reduce the infarct size, improve left ventricular function and reduce mortality. However, these beneficial effects are limited by acceleration of damage by reperfusion [1]. One of a variety of mechanisms of reperfusion injury is the activation of white blood cell migration, which induces severe myocardial inflammation [2]. Olivetti et al. [3] reported that, within 10 days of the onset of acute myocardial infarction in humans, 12 % of cardio-

myocytes were TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling)-positive, indicating the induction of apoptosis, in the border zone of the infarction compared with less than 1 % of cells in areas remote from the infarct zone. Apoptosis evoked by reperfusion after myocardial infarction may play an indispensable role in the development of cardiac remodelling. One of the important mechanisms for cardiac remodelling is the induction of apoptosis by NO (nitric oxide) produced by iNOS (inducible NO synthase), which is potentially activated by inflammation via

Key words: apoptosis, cytochrome c release, hypoxia/re-oxygenation, mitochondrial cGMP, mitochondrial permeability transition (MPT), nitric oxide, adult rat cardiomyocyte. Abbreviations: 8-Br-cGMP, 8-bromo-cGMP; DAF-2, 4,5-diaminofluorescein; DAF-2 DA, DAF-2 diacetate; diS-C3 (5), 3,3 dipropylthiadicarbocyanine iodide; NO, nitric oxide; iNOS, inducible NO synthase; MPT, mitochondrial permeability transition; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; RCR, respiratory control ratio; SNAP, S-nitroso-N-acetyl-dl-penicillamine; SNP, sodium nitroprusside. Correspondence: Dr Ken-Ichi Furukawa (email [email protected]).

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coagulation of white blood cells during reperfusion after myocardial infarction [4]. Two apoptotic pathways after NO production have been proposed, namely synthesis of peroxynitrite by reaction with reactive oxygen species, which activates the MPT (mitochondrial permeability transition), and activation of the NO/cGMP pathway [5,6]. However, the detailed apoptotic mechanism through the NO/cGMP pathway in the heart remains unknown. In cardiac muscle, the physiological role of the cytosolic NO/cGMP pathway is still controversial. Synthesized cGMP regulates various effector proteins, such as protein kinases, phosphodiesterases and ion channels. Some researchers have proposed that this pathway mainly shows cardioprotective effects, such as negative inotropic effects and electrophysiological stabilization of cardiomyocytes [7,8]. On the contrary, other reports have suggested that the cardiac NO/cGMP pathway induces apoptosis [5,9]. As mentioned above, the downstream mechanism of the cytosolic cGMP produced to induce apoptosis is currently unclear [10]. An mtNOS (mitochondrial NO synthase) has been discovered in cardiac muscle, but its nature remains unclear except for its Ca2+ -sensitivity and constitutive expression [11,12]. The best-characterized target for signal transduction by NO is a haem-containing enzyme, NO-sensitive guanylate cyclase, which catalyses the conversion of GTP into cGMP and pyrophosphate [13]. In 1974, Kimura and Murad [14] reported the existence of two forms of guanylate cyclase (soluble and membrane) in the rat heart. In that report, the authors also demonstrated that the 10 000-g precipitate, namely the cardiac mitochondrial fraction, had a cGMP synthetic activity in rats. Furthermore, Nakazawa et al. [15] reported that the major proportion of guanylate cyclase in rat cerebellum was present in the mitochondrial fraction. However, the physiological and pathophysiological properties of mitochondrial cGMP are currently unknown. In the present study, we hypothesized that another cGMP production system exists in cardiac mitochondria and plays a pivotal role in the induction of apoptosis. To determine whether the mitochondrial cGMP production system is constitutively present in rat cardiomyocytes and whether it plays a role in the response to apoptotic stimuli by NO, we investigated the effects of extrinsic NO and a membrane-permeant cGMP analogue on mitochondrial cGMP production, cytochrome c release from mitochondria, activation of MPT and induction of apoptosis in cardiac mitochondria and primary cultured adult cardiomyocytes.

MATERIALS AND METHODS Materials SNAP (S-nitroso-N-acetyl-dl-penicillamine), ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), DMSO, C


8-Br-cGMP (8-bromo-cGMP), annexin V–FITC and propidium iodide were obtained from Wako Pure Chemical Industries. SNP (sodium nitroprusside) was obtained from Sigma–Aldrich. diS-C3 (5) (3,3 -dipropylthiadicarbocyanine iodide) was obtained from LAMBDA Fluoreszenztechnologie. DAF-2 DA [DAF-2 (4,5diaminofluorescein) diacetate] was purchased from Daiichi Pure Chemical. All chemicals used were of the highest purity available commercially. All solutions were made fresh at sufficiently high concentrations that only very small aliquots had to be added to the assay tubes or culture medium. SNAP and ODQ were freshly dissolved in DMSO. The final concentration of DMSO never exceeded 0.4 % and had no effect on the cells or assays. The final concentration of DMSO was 0.1 % in the experiments using mitochondrial suspension or 0.11 % in those using primary cultured cardiomyocytes. The primary antibody against cytochrome c and Alexa Fluor® 680 goat anti-(mouse IgG) as the secondary antibody were purchased from Invitrogen. This study was carried out in accordance with the Guidelines for Animal Experimentation from Hirosaki University.

Isolation of mitochondria Left ventricular mitochondria were isolated in homogenization buffer [250 mmol/l sucrose and 10 mmol/l Tris/HCl (pH 7.4)] containing 1 mmol/l EGTA by differential centrifugation of heart homogenates from male Wistar rats (350–400 g; CLEA Japan), essentially as described previously [16]. The isolated mitochondria were resuspended in homogenization buffer and stored on ice. The viability of cardiac mitochondria was assessed by measuring the RCR (respiratory control ratio) using a Clark-type oxygen electrode (Gilson). We used mitochondria maintaining a RCR value of more than 6. The protein concentration in the mitochondrial suspension was determined by the Bradford method using BSA fraction V as a standard [17]. To obtain highly purified mitochondria, the isolated mitochondrial suspension was purified further by centrifugation (82 000 g for 200 min at 4 ◦ C) through a continuous sucrose gradient [1.1–1.6 mol/l sucrose in 5 mmol/l potassium phosphate (pH 7.4) and 1 mmol/l EGTA]. The resulting fractions were collected, resuspended in homogenization buffer, centrifuged (10 000 g for 15 min at 4 ◦ C) and resuspended again in homogenization buffer. The mitochondrial protein fraction was prepared from highly purified mitochondria (60–100 mg/ml) by sonication for 90 s at 150 W and 50 % duty cycles (Sonifier; Branson Sonic Power). After sonication and centrifugation (100 000 g for 15 min at 4 ◦ C), the mitochondrial protein fraction was obtained as a 100 000-g supernatant. The protein concentration was determined and adjusted to 10 mg/ml with homogenization buffer.

Mitochondrial cGMP induces cytochrome c release

Measurement of mitochondrial cGMP Activation of the NO-sensitive guanylate cyclase in the mitochondrial protein fraction by NO donors was performed for 10 min at 30 ◦ C in a solution consisting of 10 mmol/l MgCl2 , 0.2 mmol/l isobutyl methylxanthine, a phosphodiesterase inhibitor, 2 mmol/l GTP and 100 mmol/l Tris/HCl (pH 7.5). The reaction was stopped by rapid addition of ice-cold 50 % trichloroacetic acid to the reaction tube. Following centrifugation, the supernatant was collected and the cGMP level produced was measured using an assay developed by our laboratory, as described previously [18]. The level of cGMP was expressed in terms of the amount of cGMP produced per mg of cellular protein per 10 min.

Determination of various mitochondrial functions Cytochrome c release from mitochondria was measured by Western blotting [19]. Specifically, initial mitochondrial suspensions (0.05–0.1 mg of protein/ml) were incubated in a high-potassium buffer [0.15 mol/l KCl, 5 mmol/l KH2 PO4 , 5 mmol/l succinic acid and 10 mmol/l Hepes (adjusted to pH 7.4 with 1 mol/l Tris)] at 30 ◦ C. At 10 min after drug administration, the suspension was centrifuged at 7000 g for 10 min at 4 ◦ C. The supernatant was treated with trichloroacetic acid and the cytochrome c level in the precipitate obtained following centrifugation at 20 000 g was measured by Western blotting. The protein concentration of supernatant was measured to assess the mitochondrial outer membrane integrity. Supernatant maintaining a protein concentration of up to 10 µg/ml was regarded as intact mitochondria, and was used further as described below. Precipitates were separated on SDS/14 % (w/v) polyacrylamide gels and transferred on to PVDF membranes (Immobilon-FL; Millipore). After blocking with PBST buffer [10 mmol/l Na2 HPO4 /HCl (pH 7.4), 150 mmol/l NaCl and 0.05 % Tween 20] containing 3 % (v/v) skimmed milk, membranes were incubated with the anti-(cytochrome c) antibody at room temperature for 1 h. Membranes were then washed three times with PBST (10 min each) and incubated with Alexa Fluor® 680 goat anti-(mouse IgG) secondary antibody for 1 h at room temperature. After three washes with PBST, proteins were detected using Odyssey® Imaging System (LI-COR Biosciences). Mitochondrial swelling was determined in highpotassium buffer containing the initial mitochondrial suspension (0.05 mg of protein/ml) by measuring the decrease in absorbance at 540 nm [20]. The mitochondrial membrane potential was measured in high-potassium buffer containing 0.5 µg/ml of diSC3 (5) and the initial mitochondrial suspension (0.1– 0.2 mg of protein/ml) by recording the fluorescence intensity at 670 nm in a fluorescence spectrophotometer (FP-750; Jasco) following excitation at 620 nm [16,21].

Cell culture and detection of apoptotic cells Ventricular myocytes were isolated from adult Wistar rats (350–400 g) by collagenase perfusion [22]. Each intact rectangular cardiomyocyte was very gently pipetted up and down with a micro-tip and transferred to another dish to give a total of approx. 40 intact cardiomyocytes per each dish. After 12 h of culture, the culture medium consisting of Medium 199 supplemented with 14.3 mmol/l NaHCO3 (pH 7.4), 5 mmol/l creatine, 5 mmol/l taurine, 100 international units/ml penicillin, 0.1 mg/ml streptomycin and 0.2 % BSA was exchanged for medium containing NO donors, 8-Br-cGMP and other reagents. To inhibit fibroblast growth, cytosine arabinofuranoside (10 µmol/l) was added to the culture medium. To mimic conditions of ischaemia/reperfusion, cells were exposed to hypoxia for 1– 4 h with nitrogen (2 % O2 and 5 % CO2 ) and subsequently re-oxygenated for 2 h under normoxia (20 % O2 and 5 % CO2 ). FITC-conjugated annexin V/propidium iodide staining was performed essentially as described previously [23]. Briefly, 80 µl of binding buffer (BioVision Research Products) containing 25 µg/ml annexin V–FITC and 1 µg/ml propidium iodide was added to cells and the mixture was incubated for 10 min at room temperature in the dark. After fixing in formaldehyde, cells were analysed using a fluorescence microscope (IX71; Olympus). Cells treated with 1 mmol/l H2 O2 served as a positive control. Apoptotic cells were stained with annexin V– FITC, which binds with high affinity to phosphatidylserine, but excluded propidium iodide, a DNA dye that is unable to pass through the plasma membrane. In contrast, necrotic cells were stained with both annexin V–FITC and propidium iodide, as they had lost the physical integrity of their plasma membrane. The number of apoptotic cells was calculated by comparing the rate of apoptotic cells relative to the total number of cells. In each experiment, the mean values of 40 cells were taken.

Measurement of intracellular NO NO generation in cardiomyocytes was monitored by labelling with DAF-2 DA, which is de-esterified intracellularly to DAF-2 [24]. NO provides the third nitrogen to form a triazo ring with the two amino groups of the non-fluorescent DAF-2 and converts it into DAF2T (diaminotriazolofluorescein), which can be monitored at 490 nm excitation and 530 nm emission. Following incubation of the cells with 10 µmol/l DAF-2 DA at 37 ◦ C for 1 h, fluorescence was measured using a fluorescence microscope (IX71; Olympus) and the images were analysed with QCapture PRO software (QImaging). For quantification of NO production, 12 cells distributed randomly were counted in each condition. C


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Cytochrome c release from cardiac mitochondria

Figure 1 Increases in cGMP in the mitochondrial protein fraction mediated by NO donors

The mitochondrial protein fraction was stimulated by the NO donors SNAP and SNP. cGMP levels in the mitochondrial protein fraction were measured in the absence (control) or presence of 1 mmol/l SNAP, 1 mmol/l SNP, 1 mmol/l SNAP + 1 µmol/l ODQ or 1 mmol/l SNP + 1 µmol/l ODQ for 10 min at 30 ◦ C. Values are the means + − S.E.M. of nine (control), four (SNAP and SNAP + ODQ) or five (SNP and SNP + ODQ) experiments. *P < 0.05 and **P < 0.01 compared with the control; #P < 0.05 and ##P < 0.01 compared with the cGMP level in the presence of each NO donor.

Statistical analysis Comparisons between two groups were carried out using a two-tailed Student t test. Multiple group comparisons were performed by one-way ANOVA, followed by Tukey’s procedure for comparison of means. Values are means + − S.E.M., and P < 0.05 was considered to indicate statistical significance.

RESULTS Level of cGMP in the mitochondrial protein fraction Figure 1 shows that cGMP production in the mitochondrial protein fraction in the presence of the NO donors SNAP and SNP after incubation for 10 min at 30 ◦ C was significantly induced compared with the control level (P < 0.05 and P < 0.01 respectively). ODQ (1 µmol/l), an NO-sensitive guanylate cyclase inhibitor, significantly inhibited the increase in cGMP induced by SNAP or SNP alone (P < 0.01 and P < 0.05 respectively). On the other hand, control molecules that lacked the NO-donating S-nitroso group (N-acetylpenicillamine) and NO group (sodium hexacyanoferrate) did not alter the cGMP level in the mitochondrial protein fraction (results not shown). These results suggest that a cGMP production system exists in cardiac mitochondria. Subsequently, a 10 min reaction time was employed for the remaining experiments using purified cardiac mitochondria. C


The levels of cytochrome c release from cardiac mitochondria induced by SNAP, 8-Br-cGMP and cGMP were determined by Western blotting. Bolus administration of 30 µmol/l Ca2+ as a positive control induced cytochrome c release compared with the control, and this release was completely inhibited in the presence of cyclosporin A, an inhibitor of mitochondrial cyclophilin D (Figures 2A and 2B). Activation of the cardiac mitochondrial NO/cGMP pathway by 1 mmol/l SNAP increased cytochrome c release from mitochondria, and this increase was significantly inhibited in the presence of 1 µmol/l ODQ (Figures 2A and 2B). We investigated further whether the membrane-permeant cGMP analogue 8-Br-cGMP also stimulated the release of cytochrome c from cardiac mitochondria. Cytochrome c release was increased by 8-Br-cGMP in a dose-dependent manner and reached 3.5 + − 0.6-fold of the control level at 1 mmol/l (Figure 2C). cGMP (1 mmol/l) did not stimulate cytochrome c release from mitochondria (Figures 2A and 2B). SNP (1 mmol/l), another NO donor, increased cytochrome c release in a similar manner to SNAP (results not shown). Control molecules that lacked the NO-donating S-nitroso group (N-acetylpenicillamine) and NO group (sodium hexacyanoferrate) did not induce cytochrome c release from mitochondria (results not shown).

Mitochondrial swelling and mitochondrial membrane potential As an index of mitochondrial swelling, the decrease in absorbance of an initial mitochondrial suspension (0.05 mg of protein/ml) was measured at 540 nm in high-potassium buffer. Bolus administration of 30 µmol/l Ca2+ as a positive control induced a marked swelling of mitochondria (Figure 3A, trace b), and this was completely inhibited in the presence of cyclosporin A (Figure 3A, trace c). However, activation of cardiac mitochondrial cGMP production by 1 mmol/l SNAP and 8-Br-cGMP did not induce mitochondrial swelling (Figure 3B, traces b and c respectively). The sudden increase in absorbance at 540 nm just after the administration of SNAP was caused by the self-absorbance of SNAP (Figure 3B, trace b). The mitochondrial membrane potential was measured using the cyanine dye diS-C3 (5) in high-potassium buffer. Bolus administration of 30 µmol/l Ca2+ as a positive control induced depolarization of the mitochondrial membrane potential (Figure 3C, trace b), and this was completely inhibited in the presence of cyclosporin A (Figure 3C, trace c). However, activation of cardiac mitochondrial cGMP production by 1 mmol/l SNAP and 8-Br-cGMP did not alter mitochondrial membrane potential (Figure 3D, traces b and c respectively).


Figure 3 Influences of SNAP and 8-Br-cGMP on MPT

Figure 2 Effects of SNAP, 8-Br-cGMP and cGMP on cytochrome c release from cardiac mitochondria

(A) Isolated cardiac mitochondria were incubated with SNAP, 8-Br-cGMP and cGMP in a high-potassium buffer for 10 min at 30 ◦ C. Cytochrome c release from mitochondria was determined by Western blotting. Lane 1, authentic cytochrome c (Cyt c; 1 µg); lane 2, control (Cont.); lane 3, 30 µmol/l Ca2+ (positive control); lane 4, 30 µmol/l Ca2+ +1 µmol/l cyclosporin A (CSA); lane 5, 1 mmol/l SNAP; lane 6, 1 mmol/l SNAP + 1 µmol/l ODQ; lane 7, 1 mmol/l cGMP; lane 8, 1 mmol/l 8-Br-cGMP. (B) Band intensity was normalized to the mean value obtained from the control. Values are the means + − S.E.M. of four (cGMP) or five experiments. *P < 0.05 and **P < 0.01 compared with control; #P < 0.05 compared with the presence of Ca2+ alone; ##P < 0.01 compared with the presence of SNAP alone. (C) Concentration-dependent effects of 8-Br-cGMP on cytochrome c release from cardiac mitochondria. Densitometric analysis of the band intensities of 8-Br-cGMP were normalized to the mean value obtained from the control. 8-Br-cGMP (0.1–1 mmol/l) accelerated the release of cytochrome c in a dose-dependent manner. Values are the means + − S.E.M. of five experiments. *P < 0.05 compared with the absence of 8-Br-cGMP.

SNAP- and 8-Br-cGMP-induced apoptosis in adult rat cardiomyocytes Figure 4(A) shows representative images of annexin V–FITC staining of apoptotic cardiac myocytes after treatment for 3 h with 1 mmol/l SNAP. The percentage of apoptotic cells at 6 h after addition of 1 mmol/l H2 O2 as a positive control was 73.2 + − 3.8 % (n = 3). SNAP significantly increased the number of apoptotic

(A and B) Absorbance of a mitochondrial suspension (0.05 mg of protein/ml) was measured at 540 nm in a high-potassium buffer. (A) Mitochondria were pre-incubated in the absence (control; trace a) or presence of 30 µmol/l Ca2+ (positive control; trace b) or 30 µmol/l Ca2+ + 1 µmol/l cyclosporin A (trace c). (B) Mitochondria were pre-incubated in the absence (control; trace a) or presence of 1 mmol/l SNAP (trace b) or 1 mmol/l 8-Br-cGMP (trace c). (C and D) Mitochondrial membrane potential was measured using the cyanine dye diS-C3 (5) in a high-potassium buffer. (C) Mitochondria (0.1 mg of protein/ml) were pre-incubated in the absence (control; trace a) or presence of 30 µmol/l Ca2+ (positive control; trace b) or 30 µmol/l Ca2+ + 1 µmol/l cyclosporin A (trace c). (D) Mitochondria were pre-incubated in the absence (control; trace a) or presence of 1 mmol/l SNAP (trace b) or 1 mmol/l 8-Br-cGMP (trace c).

cells, and this increase was almost inhibited in the presence of ODQ. Figure 4(B) shows the time course changes in the ratio of apoptotic cells after SNAP administration. The control ratio of apoptotic cells before incubation of isolated rat cardiomyocytes with SNAP was 33.5 + − 8.3 % (Figure 4B). The ratio did not change significantly after 6 h in the non-treatment group (Figure 4B). Treatment with SNAP induced apoptosis in adult rat cardiomyocytes in a time-dependent manner, and this induction was markedly inhibited in the presence of 1 µmol/l ODQ. The ratio of apoptotic cells at 3 h after the administration of 1 mmol/l SNAP was increased, and this was significantly inhibited in the presence of ODQ (P < 0.01). Furthermore, 0.3 mmol/l 8-Br-cGMP, but not 0.3 mmol/l cGMP, increased the number of apoptotic cells (Figure 5). The control ratio of apoptotic cells before incubation of rat isolated cardiomyocytes with these C


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Figure 5 8-Br-cGMP-induced apoptosis in primary cultured adult rat cardiomyocytes

Apoptotic cells induced by 8-Br-cGMP were monitored by annexin V–FITC/propidium iodide staining of cardiomyocytes. The time course changes in the rate of apoptotic cells in the absence (control; open bars) or presence of 8-Br-cGMP (0.3 mmol/l; closed bars) or cGMP (0.3 mmol/l; grey bars) are shown. The values for the apoptotic cells were calculated by comparison of the rates of the apoptotic cells relative to the total number of cells. Values are the means + − S.E.M. of five (control and 8-Br-cGMP) or four (cGMP) experiments. *P < 0.05 compared with the presence of cGMP.

Figure 4 SNAP-induced apoptosis in primary cultured adult rat cardiomyocytes

(A) Upper panel shows representative images of annexin V–FITC/propidium iodide staining of cardiomyocytes incubated for 3 h under the following conditions: (a) in the presence of H2 O2 ; (b) control (Cont.); (c) in the presence of SNAP; (d) in the presence of SNAP + ODQ. (e) A propidium-iodide-positive cardiomyocyte is shown, which was revealed as a necrotic cell. The lower panel shows the phase-contrast view of each cardiomyocyte. (B) The time course changes in the rate of apoptotic cells in the absence (control; open bars) or presence of SNAP (1 mmol/l; closed bars) or SNAP + ODQ (1 µmol/l; grey bars). The values for the apoptotic cells were calculated by comparing the rates of the apoptotic cells relative to the total number of cells. Values are the means + − S.E.M. of three (control and SNAP) or five (SNAP + ODQ) experiments. *P < 0.05 compared with the presence of SNAP alone. molecules was 21.7 + − 5.3 %, and the ratio of apoptotic cells in the non-treatment group did not change significantly after 6 h (n = 5). 8-Br-cGMP accelerated apoptosis in adult rat cardiomyocytes in a time-dependent manner, with a significant increase in the ratio of apoptotic cells observed at 3 h after the administration of 8Br-cGMP (Figure 5). Treatment with 0.3 mmol/l cGMP did not increase the ratio of apoptotic cells (Figure 5).

Protective effect of ODQ on hypoxia/re-oxygenation-induced apoptosis To define a pathophysiological role for the mitochondrial cGMP production in hypoxia/re-oxygenation-induced C


Figure 6 Hypoxia/re-oxygenation-induced apoptosis in primary cultured adult rat cardiomyocytes

Apoptotic cells induced by hypoxia/re-oxygenation were monitored by annexin V–FITC/propidium iodide staining of cardiomyocytes. The time courses of changes in the rate of apoptotic cells under hypoxia are shown. Horizontal-lined bar, normoxia (6 h); hatched bar, normoxia in the presence of 1 µmol/l ODQ; open bars, hypoxia; grey bars, hypoxia followed by re-oxygenation (2 h); closed bars, hypoxia followed by re-oxygenation (2 h) in the presence of ODQ. The values for apoptotic cells were calculated relative to the total cells. Values are the means + − S.E.M. of five experiments. *P < 0.05 compared with hypoxia followed by re-oxygenation in the absence of ODQ. cardiomyocyte apoptosis, primary cultured cardiomyocytes were subjected to hypoxia for 1–4 h, followed by 2 h of re-oxygenation. Hypoxia induced apoptosis in cardiomyocytes after re-oxygenation (2 h) in a timedependent manner, and this effect was markedly inhibited in the presence of 1 µmol/l ODQ (Figure 6). When


Figure 7 Intracellular NO production in primary cultured adult rat cardiomyocytes

NO generation in cardiomyocytes was monitored by labelling with DAF-2 DA. Changes in the rate of intracellular NO production under conditions of normoxia, hypoxia or hypoxia (4 h) followed by re-oxygenation (2 h) in the absence or presence of ODQ (1 µmol/l) are shown. The values of the fluorescence intensity were calculated as percentages of normoxia. Values are the means + − S.E.M. of six experiments. *P < 0.05 compared with normoxia; #P < 0.05 compared with hypoxia. NS, not significant. cardiomyocytes were cultured for 6 h under normoxia in the absence or presence of ODQ, the ratios of apoptotic cells were similar. The ratio of apoptotic cells increased after 4 h of hypoxia followed by 2 h re-oxygenation, and this was significantly inhibited in the presence of ODQ (P < 0.05 compared with hypoxia/re-oxygenation in the absence of ODQ for 6 h). Hypoxia alone for 4 h did not increase the ratio of apoptotic cells. Propidium-iodidepositive necrotic cells were also detected, but the ratio was small (6.0 + − 2.4 % after 4 h of hypoxia followed by 2 h re-oxygenation; n = 4). We investigated NO generation during hypoxia/reoxygenation further by using DAF-2, which fluoresces in combination with NO. As shown in Figure 7, hypoxia significantly increased DAF-2 fluorescence after re-oxygenation compared with normoxia; however, the increase in DAF-2 fluorescence was not inhibited by 1 µmol/l ODQ.

DISCUSSION Preventing reperfusion injury after reperfusion therapy for acute myocardial infarction is very important in inhibiting further progression of cardiac remodelling. Although it has been proposed that the cytosolic cardiac NO/cGMP pathway contributes to the induction of cardiac remodelling, the downstream mechanism of cGMP-dependent protein kinase activation by this pathway is unknown [10]. In the present study, we have demonstrated the existence of another cGMP production

system in cardiac mitochondria using a fluorometric assay developed in our laboratory [18]. Furthermore, we have also confirmed that cardiac mitochondrial cGMP stimulates cytochrome c release from mitochondria independent of MPT. In rat cardiac mitochondria, the two distinct NO donors, SNAP and SNP, both increased mitochondrial cGMP, and these increases were inhibited in the presence of ODQ, an NO-sensitive guanylate cyclase inhibitor. The influence of contaminating cytosolic NO-sensitive guanylate cyclase can be ignored, because cytochrome c release from cardiac mitochondria was stimulated by the membrane-permeant cGMP analogue 8-Br-cGMP, but not by membrane-impermeant cGMP. Furthermore, mitochondrial NO-sensitive guanylate cyclase in the mitochondrial protein fraction, obtained as a 100 000-g supernatant by sonication and subsequent centrifugation of highly purified mitochondria, did not react with an antibody against cytosolic soluble guanylate cyclase, suggesting distinct molecular species (K. Seya, S. Motomura and K.-I. Furukawa, unpublished work). The genetic origin of cardiac mitochondrial NO-sensitive guanylate cyclase remains to be characterized and identified. In the present study, the mechanism of cardiac mitochondrial cGMP-induced cytochrome c release from mitochondria was not clearly identified. Bolus administration of 30 µmol/l Ca2+ induced cytochrome c release from mitochondria and cardiac apoptosis through Ca2+ -induced MPT, which was completely inhibited in the presence of cyclosporin A. On the other hand, cytochrome c release from mitochondria induced by mitochondrial cGMP was independent of MPT, because cyclosporin A failed to inhibit this release. This phenomenon suggests that a downstream process of mitochondrial cGMP production involves a signalling pathway in the cardiac mitochondrial intermembrane space which is separate from MPT. Generally, bolus administration of Ca2+ (20–30 µmol/ l) induces cytochrome c release (approx. 30–40 % of total cytochrome c) and activates MPT, resulting in apoptosis [25]. In the present study, cGMP production did not affect the activation of MPT, and it was also confirmed that the net amount of cytochrome c in 100 µg of mitochondria/ml was approx. 0.5 µg and the control value was up to 0.05 µg (less than 10 % of the total cytochrome c pool). Furthermore, when cytochrome c (more than 0.2 µg) is released from mitochondria, cardiomyocyte apoptosis is induced. From these phenomena, we hypothesize that cardiac apoptosis induced by mitochondrial cGMP production is accompanied by cytochrome c release of more than 30–40 % of the total cytochrome c pool. Another apoptotic mechanism mediated by NO that does not involve the NO/cGMP pathway has been proposed. Some reports have asserted that NO-induced apoptosis is mediated by peroxynitrite production C


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via reaction with reactive oxygen species in cardiac mitochondria [26,27]. In the present study, we have demonstrated that NO-induced apoptosis in cultured cardiomyocytes was strongly inhibited in the presence of ODQ. Furthermore, 8-Br-cGMP, but not cGMP, induced cardiac apoptosis. These results suggest that NO-induced apoptosis mainly progresses via the mitochondrial cGMP production system and/or cytosolic NO/cGMP pathway in rat cardiomyocytes and support a previous report that NO induces apoptosis in a cGMP-dependent manner in isolated adult cardiomyocytes [5]. To determine whether the mitochondrial cGMP production system or cytosolic NO/cGMP pathway contributes to the cardiac apoptosis, it is necessary to identify the unknown mitochondrial guanylate cyclase and to clearly define the details of the downstream signalling process in cardiac mitochondrial cGMP production that induces cytochrome c release. There are many reports regarding the relationship between the cytosolic NO/cGMP pathway and apoptosis in many cell types. However, there has been no direct proof of a relationship between the NO/cGMP pathway and cardiomyocyte apoptosis in association with reperfusion injury after reperfusion therapy for myocardial infarction. The cardiac cGMP concentration during ischaemia is possibly increased despite the decrease in guanylate cyclase activity, which is clearly decreased upon reperfusion [28]. One of the important mechanisms is the induction of apoptosis by NO produced by iNOS, which is activated by the inflammation caused by reperfusion after myocardial infarction [4,29,30]. To mimic the condition of reperfusion after myocardial infarction, we investigated the use of a high concentration of the NO donors SNAP and SNP [4]. The increases in mitochondrial cGMP, cytochrome c release from adult cardiac mitochondria and apoptosis in isolated and cultured rat adult cardiomyocytes evoked by the NO donors were significantly inhibited in the presence of ODQ. Furthermore, in primary cultured rat adult cardiomyocytes, 2 h of reperfusion after hypoxia (2 % O2 for 1–4 h) stimulated NO generation and induced cardiac apoptosis, and these effects were strongly inhibited in the presence of ODQ. These results also suggest that NO largely generated by re-oxygenation plays an important role in the development of cardiac remodelling after reperfusion therapy for myocardial infarction by inducing apoptosis partly via mitochondrial cGMP production. Finally, our results raise the possibility that chronic inhibition of mitochondrial cGMP production will be of value in the treatment and prevention of cardiac remodelling induced by NO after myocardial infarction. In summary, extrinsic NO markedly increased the level of mitochondrial cGMP and cytochrome c release from mitochondria, and these effects were significantly inhibited in the presence of ODQ. However, these phenomena did not involve mitochondrial swelling or depolarization C


of the mitochondrial membrane. A membrane-permeant cGMP analogue, but not membrane-impermeant cGMP, also mimicked the effects of a high NO concentration. Furthermore, hypoxia/re-oxygenation-induced cardiac apoptosis in primary cultured cardiomyocytes isolated from adult rats was also inhibited by ODQ. These results suggest that another cGMP production system exists in cardiac mitochondria and stimulates cytochrome c release from mitochondria in an MPT-independent manner resulting in apoptosis.

ACKNOWLEDGMENTS This work was supported in part by grants from the Aomori Bank for young researchers, a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan, The Karohji-memorial Aid of Medical Study, and Hirosaki University Educational Improvement and Promotional Aid. We thank Dr Teruko Takeo, Hirosaki University, for technical advice, and to Ms Megumi Suzuki and Ms Emi Okubo, Hirosaki University, for technical support.

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Received 8 June 2006/9 August 2006; accepted 8 September 2006 Published as Immediate Publication 8 September 2006, doi:10.1042/CS20060144

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