Apoptosis (2006) 11:1091–1100 DOI 10.1007/s10495-006-7110-y
Higenamine reduces apoptotic cell death by induction of heme oxygenase-1 in rat myocardial ischemia-reperfusion injury Young Soo Lee · Young Jin Kang · Hye Jung Kim · Min Kyu Park · Han Geuk Seo · Jae Heun Lee · Hye Sook Yun-Choi · Ki Churl Chang
Published online: 11 May 2006 C Springer Science + Business Media, LLC 2006
Abstract Pharmacological modulation of heme oxygenase (HO) gene expression may have significant therapeutic potential in oxidant-induced disorders, such as ischemia reperfusion (I/R) injury. Higenamine is known to reduce ischemic damages by unknown mechanism(s). The protective effect of higenamine on myocardial I/R-induced injury was investigated. Ligation of rat left anterior descending coronary artery for 30 min under anesthesia was done and followed by 24 h reperfusion before sacrifice. I/R-induced myocardial damages were associated with mitochondria-dependent apoptosis as evidenced by the increase of cytochrome c release and caspase-3 activity. Administration of higenamine (bolus, i.p) 1 h prior to I/R-injury significantly decreased the release of cytochrome c, caspase-3 activity, and Bax expression but up-regulated the expression of Bcl-2, HO-1, and HO enzyme activity in the left ventricles, which were inhibited by ZnPP IX, an enzyme inhibitor of HO-1. In addition, DNA-strand break-, immunohistochemical-analysis, and TUNEL staining also supported the anti-apoptotic effect of higenamine in I/R-injury. Most importantly, administration of ZnPP IX inhibited the beneficial effect of higenamine. Taken together, Y. S. Lee · Y. J. Kang · H. J. Kim · M. K. Park · H. G. Seo · J. H. Lee · K. C. Chang () Department of Pharmacology, College of Medicine and Institute of Health Sciences, Gyeongsang National University, Jinju 660-751, Korea e-mail:
[email protected] H. S. Yun-Choi Natural Products Research Institute, Seoul National University, Seoul 110-460, Korea Y. J. Kang Present Address: Department of Pharmacology, College of Medicine, Yeungnam University, Daegu, 705-717
it is concluded that HO-1 plays a core role for the protective action of higenamine in I/R-induced myocardial injury. Keywords Apoptosis . Heme oxygenase . Higenamine . Ischemia reperfusion injury Abbreviations I/R ischemia-reperfusion Hig higenamine HO-1 heme oxygenase-1 INF infarct area LAD left anterior descending coronary artery TTC triphenyltetrazolium chloride TUNEL terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling PN peroxynitrite ZnPP IX zinc protoporphyrin IX
Introduction Myocardial injury from ischemia-reperfusion (I/R) is a clinical problem associated with procedures such as thrombolysis, angioplasty and coronary bypass surgery, which are commonly used to establish the blood reflow to minimize the damage of the heart due to severe myocardial ischemia. Reperfusion of an occluded coronary artery is known to reduce infarct size, preserve left ventricular function, and reduce overall mortality in humans [1, 2], but it is now recognized that the readmission of oxygenated blood into previously ischemic myocardium can initiate a cascade of events that will paradoxically produce additional myocardial cell dysfunction and cell apoptosis [3, 4]. Although the long term consequences of this process are not completely
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understood, several lines of evidence have shown that myocardial I/R is associated with increase in apoptotic cells [5, 6]. The importance of heme oxygenase (HO)-1 expression and its association with anti-apoptotic pathway in myocardial I/R-induced injury has recently been stressed [7–10]. Thus, it is of great interest to investigate therapeutic potential of a chemical that induces HO-1 gene expression in oxidative stress-induced disorders. Previously, we reported that higenamine, an active ingredient of Aconite root, upregulated nitric oxide-mediated HO-1 expression in RAW 264.7 cells [11], and reduced oxidant-induced damages by inhibition of iNOS induction in lipopolysaccharide (LPS)treated animals [12]. We, therefore, investigated whether higenamine ameliorates myocardial I/R-induced injury in the rat by up-regulation of HO-1. We found that higenamine concentration dependently showed anti-apoptosis of the rat heart subjected to I/R thus reduced the I/R-induced injury. The beneficial effect of higenamine, however, was reversed by HO-1 inhibitor, ZnPP IX, suggesting that pharmacological modulation of HO-1 gene expression by higenamine may have significant therapeutic potential in myocardial injuries from I/R.
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(6 mg/kg). The trachea was cannulated with a catheter, and artificial respiration was provided by a respirator with a frequency of 60 strokes/min and a tidal volume of 2 ml to maintain normal pO2 and pCO2 . Rectal temperature was monitored with a rectal probe and maintained within 36.5◦ C and 37◦ C. Coronary occlusion and reperfusion were performed as described by Zingarelli et al. [14]. The chest was opened by a cut along the left side of the sternum through the ribs. The animal was rotated to expose the left ventricle. Ligation proceeded with a 6-0 silk suture passed with a tapered needle underneath the left anterior descending branch (LAD) of the left coronary artery. After 30 min occlusion, reperfusion occurred by cutting the knot on top of the tubing with a surgical blade. One group of rat underwent this surgical procedure with the exception of LAD occlusion and reperfusion, and served as a sham (S) control. All animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised in 1996). The protocol was approved before the animal study by the Animal Research Committee of the Gyeongsang National University, Korea. Experimental protocol
Materials and methods Materials Antibodies to Bcl-2, Bax, cytochrome c and HO-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Ac-DEVD-pNA and peroxynitrite were obtained from Alexis (San Diego, CA). Enhanced chemiluminescence (ECL) detection reagents were from iNtRON Corporation (Seoul, Korea). Zinc protoporphyrin IX (ZnPP IX), an inhibitor of HO, was obtained from Calbiochem Corporation (La Jolla, CA). DNA laddering assay kits were obtained from Promega Life Science Corporation (Madison, WI). Ketamine was purchased from Yuhan Corporation (Seoul, Korea). Xylazine was obtained Bayer Korea Ltd (Seoul, Korea). Cytochrome c2+ was purchased from Sigma. Unless otherwise indicted, all other reagents used in this study were purchased from Sigma-Aldrich Company Ltd. (St. Louis, MO). Higenamine was prepared as previously described [13]. All chemicals for in vivo study were prepared freshly at the time of use in 0.9% (w/v) NaCl solution. In particular, ZnPP IX was dissolved in 0.2 M NaOH, subsequently adjusted to a pH 7.4, and diluted in 0.9% NaCl solution. Myocardial ischemia and reperfusion injury Adult male Sprague-Dawley rats (weighing 220–250 g) were anesthetized with ketamine (30 mg/kg) and xylazine Springer
Rats were randomly assigned to one of the four groups; Group A, sham (n = 5), Group B; ischemia (30 min) and subsequent reperfusion (24 h) and treatment with placebo (saline 0.3 ml, n = 5), Group C; pretreatment with higenamine (1 mg/kg; n = 7, 5 mg/kg; n = 12, 10 mg/kg, n = 9) before I/R injury, Group D; pretreatment with both higenamine (10 mg/kg) and zinc protoporphyrin IX (ZnPP IX, 2.5 mg/kg), an inhibitor of HO, before I/R injury (n = 7). Higenamine or vehicle (saline) was treated 1 h prior to I/R injury by intra peritoneally (i.p.) as a bolus dose. In group D animals, higenamine and Znpp IX were injected simultaneously 1 h prior to I/R injury. Tissue preparation After completion of the experiment according to the protocols, hearts from each group were rapidly harvested, and left ventricles were removed and used for both histological and biochemical studies. Quantification of myocardial injury The infarct size in the excised heart was evaluated by the staining using 1% triphenyltetrazolium chloride (TTC) as previously described by Yamashita et al. [15]. The noninfarcted (stained) and infarcted (not stained) areas were determined after incubation with 1% TTC solution. The
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sizes of corresponding area were calculated as a percentage normalized to the weight of the slice and expressed as ratio of infarcted area (INF) to left ventricle (LV).
were further centrifuged at 100,000 g for 1 h at 4◦ C in an ultracentrifuge (Beckman Coulter, Fullerton, CA) and the final supernatant is used as the soluble cytosolic fraction. Both mitochondrial and cytosolic fractions were then processed for immunoblotting.
Western blot analysis of Bcl-2, Bax, cytochrome c, and HO-1 protein
Caspase-3 activity
Total proteins were extracted from left ventricles of the heart. One hundred mg of heart tissue was placed in 1 ml of lysis buffer (containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS] in 1 × PBS) and subsequently homogenized at 4◦ C for 20 s and incubated on ice for 30 min, and then centrifuged at 13,000 rpm for 20 min. Protein concentration was measured by the Bradford protein assay. Fifty to eighty µg of total protein was mixed with loading buffer (5% mercaptoethanol, 0.05% bromophenol blue, 75 mM Tris-HCl, pH 6.8, 2% SDS and 10% glycerol), boiled for 5 min and loaded onto 10% gradient SDS-polyacrylamide gel. Proteins were transferred to PVDF membrane in the presence of glycine/methanol transfer buffer (20 mM Tris base, 0.15 M glycine, 20% methanol) in semi-dry transfer r SD, BioRad). PVDF membranes were system (Trans-Blot blocked with 5% skim milk in 1 × TBS-T buffer (20 mM Tris-HCl pH 7.6, 137 mM NaCl, 0.05% Tween-20) for 1 h at room temperature. Membrane was subsequently exposed to mouse monoclonal anti-rat Bcl-2, mouse monoclonal anti-rat Bax (Santa Cruz), rabbit polyclonal anti-rat cytochrome c (Santa Cruz) and goat polyclonal anti-mouse HO-1 (Santa Cruz) at 1:500 concentration in 5% skim milk in TTBS for 3 h at room temperature, respectively. Bound antibody was detected by horseradish peroxidase conjugated with anti-mouse and anti-rabbit IgG. Finally, enhanced chemiluminescence (ECL) detection reagents were employed to visualize peroxidase reaction products (iNtRON). Protein detection was made at 15 kDa, 21 kDa, 26 kDa, and 32 kDa for cytochrome c, Bax, Bcl-2, and HO-1, respectively. Isolation of mitochondrial and cytosolic fraction Left ventricles were removed from the heart and washed in the PBS solution, washed again in the sucrose buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 250 mM sucrose) resuspended in the same buffer. After 1 h of incubation on ice, tissues were lysed by Dounce homogenizer with 30 strokes. Homogenates were centrifuged at 750 g at 4◦ C for 10 min and the supernatants were re-centrifuged at 1200 g at 4◦ C for 10 min. After decanting, they were recentrifuged at 10,000 g at 4◦ C for 15 min, and these pellets were used as the mitochondrial fraction. The supernatants
To measure caspase-3-like activity, the tissue was homogenized in buffer A (100 mM HEPES, pH 7.4, 140 mM NaCl, and protease inhibitors, including 0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml pepstatin, and 10 µg/ml leupeptin), and the crude homogenate was centrifuged at 13,000 rpm for 20 min at 4◦ C. The enzyme reaction mixture contained 0.04 µg of rh-caspase-3 (or 100 µg of cytosolic protein) and 200 µM Ac-DEVD-pNA in 50 µl of buffer B (100 mM HEPES pH 7.4, 20% glycerol, and protease inhibitors). The enzyme reaction was initiated by adding the substrate to a 96-well plate containing the enzyme solution and incubated at 37◦ C for 2 h. The caspase-3-like activity was calculated from the initial velocity by measuring the increased absorbance at 405 nm every 10 min. The reaction mixture without enzyme or substrate was served as a control. Assay for HO enzyme activity To determine HO enzyme activity, left ventricles were removed and homogenized on ice in a Tris-HCI lysis buffer (pH 7.4) containing 0.5% Triton X-100 and protease inhibitors. Samples were frozen in small aliquots until use. Homogenates (100 µl) were mixed with 0.8 mM NADPH, 0.8 mM glucose-6-phosphate, 1.0 U glucose-6-phosphate-1dehydrogenase, 1 mM MgCl2 , 10 ml of rat liver cytosol as a source of biliverdin reductase at 4◦ C. The reaction was initiated by the addition of hemin (final concentration 0.25 mM) and incubated at 37◦ C in the dark for 1 h and terminated by addition of chloroform (600 µl). The produced bilirubin was calculated by the difference in absorption between 460 and 530 nm using a quartz cuvette. Controls included naive samples in the absence of the NADPH generating system and all the ingredients of the reaction mixture in the absence of homogenates. Determination of myocardial apoptosis by DNA ladder and TUNEL assay Cell death by apoptosis was evaluated by DNA laddering and TUNEL assay. For DNA strand break assay, freshly frozen myocardium (20–30 mg) was minced in 600 µl of lysis buffer (Puregene DNA isolation kit) and was quickly homogenized with 30–50 strokes using a microfuge tube Springer
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pestle. The tissue was digested with 100 µg/ml of protease K at 65◦ C for 2 h and incubated with RNase A at 37◦ C for 1 h. After incubation, tissues were precipitated and centrifuged at 13,000 g for 5 min. Supernatant containing DNA were precipitated with isopropanol. After centrifugation at 13,000 g for 5 min, the resulting DNA pellets were washed with 75% ethanol and dissolved in DNA hydration solution (Promega, USA). After measuring the DNA concentration at 260 nm by spectrophotometry, 10 µg DNA was loaded onto 1.5% agarose gel containing 0.5 µg/ml ethidium bromide. DNA electrophoresis was carried out at 80 V for 1–2 h. DNA ladders, an indicator of tissue apoptotic nucleosomal DNA fragmentation, were visualized under ultraviolet light which was photographed for permanent records. For TUNEL assay, the hearts were perfused first with 0.9% NaCl for 5 min and then with 4% paraformaldehyde in PBS (pH 7.4) for 30 min. Fixed tissues were then embedded in a paraffin block and 4–5 slides at 1 µm thickness were cut from each tissue block. Immunohistochemical procedures for detecting apoptotic cardiomyocytes were performed by using a terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) kit (Roche Co., USA) according to the manufacturer’s instructions. Propidium iodide was used as a counter stain.
tion (50 µM) yields upon the addition of peroxynitrite (25 µM initial concentration after mixing) were determined by incubation of reaction mixtures in potassium phosphate (100 mM) plus DTPA (pH 7.2; 0.1 mM) at 22◦ C for 3 min in the absence or presence of higenamine (1 µM–1 mM). The oxidation of cytochrome c2+ was followed at 550 nm using a Beckman DU 640 spectrophotometer (Fullerton, CA, USA). Control experiments verified that the chemical used in these procedures did not reduce cytochrome c2+ .
Immunohistochemistry
Western blot analysis of the expression of pro-apoptotic or anti-apoptotic proteins showed that I/R stress significantly increased the expression of pro-apoptotic Bax protein but decreased Bcl-2 protein in the rat myocardium. However, by the treatment of higenamine prior to I/R injury, the expression of Bax or Bcl-2 protein was down-regulated or upregulated in a concentration-dependent manner, respectively (Fig. 1(A) and (B)). Immuno-histochemical analysis data further support that I/R injury increased pro-apoptotic gene expression, Bax, but decreased anti-apoptotic genes, Bcl-2, whereas, these effects were reversed by the administration of higenamine (Fig. 1(C)).
On completion of experiment according to protocols, left ventricles were removed where a 2-mm section was sliced from the middle, fixed in 10% (v/v) neutral buffered formalin solution, and embedded in paraffin. Paraffin-embedded myocardial sections (5 µm) were mounted on super frost slides and dried overnight at 37◦ C. The tissue specimens were subjected to antigen retrieval by heating in 10 mM citrate (pH 6.0) in a microwave oven for 1 min. Immunostaining was carried out with a mouse Bcl-2 monoclonal antibody (1:100, Santa Cruz), or mouse Bax monoclonal antibody (1:100, Santa Cruz) at 4◦ C overnight. Antigen-antibody complexes were detected by the super-sensitive alkaline phosphatase kit (VECTASTAIN, Burlingame, CA). Hematoxyline was used as a counter stain. Antioxidant effects The peroxynitrite-dependent oxidation of cytochrome c2+ was measured as described previously [16]. Cytochrome c2+ was reduced with sodium dithionite immediately before use and purified by chromatography on Sephadex G-25 using potassium phosphate (100 mM) plus DTPA (pH 7.2, 0.1 mM) as the elution buffer. The concentration of cytochrome c2+ was determined spectrophotometrically at 550 nm in the same buffer (ε = 21 mM/cm). Cytochrome c2+ oxidaSpringer
Statistical evaluations Data were expressed as mean ± S.E.M with the number (n) of experiment. Differences between data sets were assessed by one-way ANOVA followed by Student-Newman-Keuls test. A level of P < 0.05 was accepted as statistically significant.
Resutls Effects of higenamine on the I/R-induced myocardial apoptosis
Higenamine up-regulates HO-1 expression and activity As shown in Fig. 2, higenamine up-regulated the HO-1 protein expression and activity in I/R rats in a dose-dependent manner. Western blot analysis revealed that HO-1 protein expression in the hearts was induced which peaked after 18 h injection (i.p.) of higenamine (10 mg/kg) in intact rats (data not shown). Curiously enough, the increased level of HO-1 expression due to higenamine (10 mg/kg) in I/R rats was significantly suppressed by administration of ZnPP IX, (2.5 mg/kg, i.p.), an inhibitor of HO-1 (Fig. 2(A)). As expected, the increased HO enzyme activity by higenamine administration was also inhibited by ZnPP IX in I/R rats (Fig. 2(B)).
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Fig. 1 Regulation of proteins associated with apoptosis by higenamine in rat I/R myocardium in vivo. A representative pattern showing expression of the Bax (A) and Bcl-2 (B) proteins by Western blot analysis in ischemic tissue after 30 min ischemia followed by 24 h reperfusion with or without higenamine pretreatment. (A) Ischemia and reperfusion increased the expression of Bax protein in the left ventricles about 3.5 fold compared to sham group, in which administration of higenamine (Hig) concentration dependently and statistically significantly reduced this pro-apoptotic gene expression. (B) Ischemia and reperfusion significantly decreased the expression of Bcl-2 protein in the left ventricles
compared to sham group, in which administration of higenamine (Hig) concentration-dependently and statistically significantly increased this anti-apoptotic gene expression. (C) A representative immunostaining picture showing of Bax and Bcl-2 antibodies in paraffin-embedded sections. Lane 1; sham, lane 2; I (30 min) + R (24 h), lane 3; I/R + higenamine (Hig, 10 mg/kg). (Magnification × 200). The expression pattern of Bax or Bcl-2 was consistently similar to the representative one in three to five independent experiments. Data represent the mean ± S.E.M. ∗ P < 0.05 for I/R vs. I/R + Hig, ‡ P < 0.05 for sham (S) vs. I/R
Higenamine blocks I/R-induced cytochrome c release and activation of caspase-3
shown in I/R group, in which treatment with higenamine suppressed activities that were almost compatible to those of sham animals (Fig. 3(B)). However, administration of ZnPP IX significantly inhibited the effect of higenamine on caspase-3 activity (Fig. 3(B))
As shown in Fig. 3(A), most of detectable cytochrome c was found in the mitochondrial fraction in the sham operated animals. In contrast, after I/R injury, the expression of cytochrome c was significantly increased in the cytosol fraction. Administration of higenamine resulted in a concentration-dependent decrease of the expression of cytochrome c in cytosol fraction. It should be noted that the decreased release of cytochrome c by higenamine was significantly counteracted by ZnPP IX. Higenamine also dose-dependently decreased the activity of caspase-3 in I/R rats (Fig. 3(B)). When compared to those in the sham group, more than two fold increases of caspase-3 activities were
Higenamine reduces apoptosis and infarct size by I/R injury As shown in Fig. 4(A), DNA strand break was evident in I/R hearts, where administration of higenamine resulted in disappearance of DNA ladders as shown in lane 3. It was, however, reappeared by the administration of ZnPP IX as shown in lane 4. I/R injury caused apoptosis in the rat heart was further supported by increased number of Springer
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Fig. 2 Effects of higenamine on the expression and enzyme activity of HO-1 in rat I/R myocardium in vivo. (A) A representative pattern of Western blot analysis indicates that higenamine (Hig) concentrationdependently up-regulated HO-1 expression. The increased expression of HO-1 protein by higenamine was significantly inhibited by administration with ZnPP IX. (B) HO enzyme activity indicates that administration of higenamine concentration-dependently increased enzyme activity like HO-1 protein expression, which was also significantly inhibited by ZnPP IX. Data are the mean ± S.E.M. of three to five independent experiments. ∗ P < 0.05 for I/R vs. I/R + Hig,# P < 0.05 for I/R + Hig (10 mg/kg) vs. I/R + Hig + ZnPP IX
TUNEL-positive cells (29.2 ± 6.1 vs. 6 ± 2.8 in sham I/R). As shown in Figs. 4(B) and (C) , the increased number of TUNEL-positive cells by I/R injury was significantly reduced by higenamine treatment, which was again reversed by ZnPP IX (2.5 mg/kg). These results suggest that higenamine has significant anti-apoptotic effects after I/R in vivo. Fig. 4(D) clearly shows higenamine significantly reduced the ratio of INF/LV. For example, at the dose of 10 mg/kg higenamine it reduced the ratio from 35 ± 5.0% to 13 ± 6.1%. Accordingly, higenamine (10 mg/kg) treatment resulted in about 37% reduction in infarct size, which was significantly (P < 0.05) reversed by treatment with ZnPP IX (2.5 mg/kg).
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Fig. 3 Effects of higenamine on cytochrome c release and caspase3 activity in rat I/R myocardium in vivo. As described in Method, mitochondrial and cytosolic fractions were isolated from the left ventricles. (A) Western blot analysis depicts that increased expression of cytochrome c was seen in the cytosolic fraction compared to mitochondria in I/R rat heart, the effect which was reversed by administration of higenamine. In the presence of ZnPP IX, the effect of higenamine was significantly inhibited. (B) Caspase-3 activity is shown in I/R myocardium with or without higenamine. Higenamine decreased the activity of caspase-3 in a dose-dependent manner, which was significantly inhibited by the presence of ZnPP IX. The activity of the caspase-3-like protein was represented as percentage to that of proteolytic activity of sham animals (S). Data are the mean ± S.E.M. of three independent experiments.‡ P < 0.05 for sham vs. I/R, ∗ P < 0.05 for I/R vs. I/R + Hig.# P < 0.05 for I/R + Hig (10 mg/kg) vs. I/R + Hig + ZnPP IX
concentration-dependent manner, in which the concentration to inhibit 50% (IC50 ) was 43.6 µM. Glutathione also concentration-dependently inhibited the oxidation of cytochrome c2+ by PN. The antioxidant effect of higenamine was as potent as glutathione (IC50 , 35 µM) in terms of IC50 .
Antioxidant effect of higenamine
Discussion
By measuring the capacity of inhibition of cytochrome c2+ oxidation by peroxynitrite (PN) in cell free system, antioxidant action of higenamine was evaluated and compared to glutathione. As shown in Fig. 5, higenamine inhibited PN-induced oxidation of cytochrome c2+ in a
The present study clearly demonstrated that higenamine significantly reduced infarct size in rat I/R hearts in vivo. The mechanism(s) underlying this beneficial effect of higenamine on I/R injury was closely related to anti-apoptotic action. This conclusion was based on the results of DNA
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fragmentation experiment, TUNEL assay and Western blot analysis. Many genes have been reported to be linked with the regulation of programmed cell death under physiological and pathological conditions, in which Bcl-2 and Bax genes are suggested to play a major role in determining cell’s survival or death after apoptotic stimuli [20–22]. Mitochondrial dysfunction is one of the most critical events associated with
myocardial I/R injury. The release of cytochrome c from mitochondria into cytosol is a critical initiation step in reactive oxygen species-triggered apoptosis. As expected, higenamine significantly reduced the expression of Bax, but increased that of Bcl-2 protein in those rat hearts that were subjected to I/R. In searching possible action sites of higenamine inhibition of I/R-induced myocardial apoptosis, we focused on the mitochondrial cytochrome c release and
Fig. 4 Anti-apoptotic effect of higenamine-induced HO-1 in I/R rat myocardium in vivo. (A) Increased DNA strand breaks in I/R rat hearts were significantly diminished by the administration of higenamine (10 mg/kg, i.p.) in lane 3, which reappeared by the presence of ZnPP IX (2.5 mg/kg) as shown in lane 4. (B) A representative photomicrographs of TUNEL staining of rat cardiac myocyte from rats rendered to I/R with or without pretreatment of higenamine. As TUNEL-positive nuclei were stained bright green/yellow, I/R significantly increased the positive nuclei, which were concentration-dependently decreased by the presence of higenamine. However, ZnPP IX antagonized the effect
of higenamine. (C) Percentage of TUNEL-positive nuclei in rat hearts subjected to I/R injury with different treatment. (D) Measurement of myocardial infarct size in rat hearts subjected to I/R with different treatment. Higenamine significantly and concentration-dependently reduced the infarct size in I/R rat myocardium, and the effect was antagonized by ZnPP IX. Data represent as the mean ± S.E.M. of three independent experiments.‡ P < 0.05 for sham vs. I/R, ∗ P < 0.05 for I/R vs. I/R + Hig,# P < 0.05 for I/R + Hig vs. I/R + Hig + ZnPP IX. INF; infarct area, LV; left ventricle Continue on next page Springer
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Fig. 4 Continued
Fig. 5 Inhibition of peroxynitrite-dependent oxidation of cytochrome c2+ by higenamine and glutathione. Oxidation of cytochrome c2+ by peroxynitrite (PN) in cell free system was determined with various concentrations of glutathione and higenamine. Both chemicals significantly and concentration-dependently inhibited the oxidation of cytochrome c2+ by PN. Data represent as the mean ± S.E.M of triple experiments. ∗ P < 0.05
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caspase-3 activation pathway. The detection of changes in cytochrome c between mitochondria and cytosol by Western blot showed that cytosolic cytochrome c increased in the I/R myocardium. This change was suppressed in the higenamine + I/R myocardium. Corresponding to this alteration, caspase-3 was also activated in the I/R myocardium; this activation was again suppressed in the higenamine + I/R myocardium. We believe this anti-apoptotic effect is due to modulation of genes related with apoptosis, such as Bcl2/Bax by higenamine. How is possible higenamine to modulate these genes? We speculate that HO-1 is responsible for this. Because it has been shown that upregulation of Bcl-2 is a possible pathway through which HO-1 exert their cytoprotective effects. More specifically, it was suggested that a down-regulation of HO-1 mRNA and a reduction in HO-1 activity resulted in the occurrence of I/R-induced ventricular apoptosis [24]. Up-regulation of HO-1 expression markedly reduced the infarct size due to I/R-induced injury in rat, and the effect was completely abolished by HO-1 inhibitor [25]. Indeed, a great number of studies have shown that the induction of HO-1 expression and activity represents an adaptive response to various injuries [23]. Thus, HO-1 has been implicated in several clinically relevant disease states, including transplant rejection, hypertension, acute renal injury, and myocardial I/R-induced injury [30, 31]. We found that the up-regulation of HO-1 protein occurred in I/R hearts or in intact hearts (data not shown) after treated with higenamine, which was related with reduction of infarct size, Bax gene expression, cytochrome c release and caspase-3 activity. Moreover, the beneficial effects exerted by higenamine in I/R model such as decreased infarct size, reduction of cytochrome c release and caspase-3 activity were suppressed by the presence of ZnPP IX, which clearly suggest that HO-1 enzymatic activity is required for the protective mechanism of higenamine. The fact that ZnPP IX increases the incidence of reperfusion-induced ventricular fibrillation in I/R myocardium [25] and hemin-induced HO-1 overexpression has reduced infarct size after cardiac I/R injury [32] supports our present data and underscores the importance of HO-1 in prevention of oxidative damage. However, it should be noted that the concentration of ZnPP IX is critical for selective inhibition of HO-1 enzyme activity, because ZnPP IX inhibited NOS at concentrations of 10 µM and higher [34, 35], in brain tissues. With this in mind, we used deliberately choose the dose of ZnPP IX (2.5 mg/kg) for the present study. In fact, Katori et al. [36] reported that infusion of 1.5 mg/kg ZnPP IX (i.v.) was sufficient to prevent the expression of Bcl-2 and delay the expression of Bag-1 antiapoptotic protein in rats, of which cardiac transplantation were performed using hearts that were harvested from LEW rats and stored for 24 h at 4◦ C before being transplanted into LEW recipients. Then, how HO-1 can modulate Bcl-2 or Bax during I/R injury? At the present time, the exact mechanism(s) by
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which HO-1 modulates genes associated with apoptosis is not clear. However, we speculate that by products of HO1, such as carbon monoxide (CO) or bilirubin may regulate Bcl-2/Bax ratio by activating mitogen activated protein kinase (MAPK) signal pathways when myocardium is under I/R. Because CO is reported to have an antiapoptotic effect by inhibiting Fas/Fas ligand, caspases, proapoptotic Bcl-2 proteins, and cytochrome c release via the MKK3/p38α MAPK pathway during I/R lung injury (Zhang et al., 2004). These possibilities are under investigation. In addition, strong antioxidant action may have influenced to modulate apoptosis during I/R-induced injury in vivo, although we did not measure antioxidant effect of higenamine in vivo at the present experiment. Since higenamine reduced peroxynitrite (PN)-induced cytochrome c2+ oxidation in cell free system, in which the potency was almost equivalent to glutathione, and it was shown to increase MnSOD expression, antioxidant enzyme, in vivo [38], the antioxidant action of higenamine in vivo may have played some roles for the protective effect against oxidant-induced injury. Although the relationship among the events of I/R, ROS generation, and apoptosis are not well defined, oxidative stress can induce cardiac myocyte apoptosis in vitro [40, 41]. The ability of higenamine to scavenge free radicals [43], to reduce NO production [12], and to quench peroxynitrite may additionally contribute to the protective role to reduce infarct size from I/R injury. Furthermore, these antioxidant actions may lessen secondary oxidative damage, such as neutrophil infiltration, following I/R-induced myocardial injury [10]. At the moment, it remains to be elucidated how much portions of each component, between HO-1 expression and antioxidant action, contribute to anti-apoptotic action of higenamine. Finally, hemodynamic effects may also contribute the protective role of higenamine from I/R damage. Because higenamine increased cardiac contractility as well as vasodilating action in vivo [13], this hemodynamic property of higenamine may play a role in the protection seen in this model.
Conclusions In summary, we investigated the protective effect of higenamine on myocardial I/R injury. We found that higenamine reduced the infarct size of heart in rats rendered ischemia followed by reperfusion. Up-regulation of HO-1 by higenamine was related with the reduced infarct size in I/R-induced injury, modulation of genes associated with apoptosis, decrease in DNA strand breaks, reduction of TUNEL positive cells, and reduction of cytochrome c release and caspase-3 activity. These beneficial effects were reversed by the presence of ZnPP IX, which strongly suggest that HO-1 plays a core role for protective action of higenamine in I/R-induced
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injury. In addition, radical scavenging activity of higenamine may have contributed some degrees to reduce the apoptosis and infarct size. Thus, chemicals to induce HO-1 seem to be a rational therapeutic strategy to reduce the risk injury during I/R. We, thus, concluded that administration of higenamine protects I/R-induced rat heart injury by reducing mitochondria-dependent apoptosis in vivo. Therefore, higenamine may be beneficial in preventing I/R injury. Acknowledgments This work was supported by grant from the Ministry of Health and Welfare (01-PJ2-PG4-J201PT01-0002).
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