Cardiovascular Research

Cardiovascular Research 2004 61(3):548-558; doi:10.1016/j.cardiores.2003.12.004
© 2004 by European Society of Cardiology

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Copyright © 2003, European Society of Cardiology

Inhibition of Rho-kinase protects the heart against ischemia/reperfusion injury

Weike Bao^*,a, Erding Hu^b, Ling Tao^c, Rogely Boyce^d, Rosanna Mirabile^d, Douglas T Thudium^d, Xin-ling Ma^c, Robert N Willette^a and Tian-li Yue^a

^aDepartments of Investigative and Cardiac Biology (UW-2510), GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406-0939, USA
^bVascular Inflammatory Diseases, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406-0939, USA
^cDepartment of Emergency Medicine, Thomas Jefferson University, Philadelphia, PA 19107-5004, USA
^dSafety Assessment, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406-0939, USA

^* Corresponding author. Tel.: +1-610-270-5366; fax: +1-610-270-5080. weike_2_bao{at}gsk.com

Received 28 May 2003; revised 2 December 2003; accepted 4 December 2003

	Abstract

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
Objective: To investigate the role of Rho A and Rho-kinase in acute myocardial ischemia/reperfusion injury and the protective effect of Rho-kinase inhibitor, Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide]. Methods and results: Male CD1 mice were subjected to 30 min of coronary occlusion and 24 h reperfusion. Ischemia/reperfusion upregulated expression of Rho A in ischemic myocardium, and subsequently activated Rho-kinase. Y-27632 significantly inhibited the activation of Rho-kinase following ischemia/reperfusion. Treatment with Y-27632 at 10 and 30 mg/kg oral administration, reduced infarct size by 30.2% and 41.1%, respectively (P<0.01 vs. vehicle). Y-27632 also enhanced post-ischemia cardiac function. Left ventricular systolic pressure, +dP/dt and –dP/dt were significantly improved by 23.5%, 52.3%, and 59.4%, respectively (P<0.01 vs. vehicle). Moreover, Y-27632 reduced ischemia/reperfusion-induced myocardial apoptosis. The apoptotic myocytes in ischemic myocardium after 4 h reperfusion were reduced from 13.1% in vehicle group to 6.4% in Y-27632-treated group (P<0.01). Meanwhile, ischemia/reperfusion-induced downregulation of Bcl-2 in myocardium was remarkably attenuated in the treated animals. Ischemia/reperfusion resulted in remarkable elevation in serum levels of proinflammatory cytokines, interleukin-6 (IL-6), keratinocyte chemoattractant (KC) and granulocyte colony-stimulating factor (G-CSF), which was significantly suppressed by Y-27632. In addition, Y-27632 decreased ischemia/reperfusion-induced accumulation of neutrophils in the heart by 45% (P<0.01). Conclusions: These results suggest that Rho-kinase plays a pivotal role in myocardial ischemia/reperfusion injury. The cardiac protection provided by treatment with a selective Rho-kinase inhibitor is likely via anti-apoptotic effect and attenuation of ischemia/reperfusion-induced inflammatory responses. The finding of this study suggest a novel therapeutic approach to the treatment of acute myocardial ischemia/reperfusion injury.

KEYWORDS Mouse; Ischemia; Reperfusion; Infarction; Apoptosis

	1. Introduction

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
Ischemic heart disease is the principal etiology for the development of congestive heart failure. Thus, effective treatment to reduce post-ischemic injury and minimize myocardial necrosis continues to be the major therapeutic goal of modern cardiology. Reperfusion of coronary flow is necessary to resuscitate the ischemic or hypoxic myocardium. Timely reperfusion facilitates cardiomyocytes salvage and decreases cardiac mortality and morbidity. With prolongation and severity of ischemia, reperfusion of an ischemic area may result, however, in paradoxical cardiomyocyte dysfunction, a phenomenon termed "reperfusion injury" [1]. Ischemia/reperfusion injury is commonly associated with procedures such as thrombolysis, angioplasty, coronary artery bypass and heart transplantation. Despite significant these therapeutic advances, myocardial ischemia/reperfusion injury still represents an important unmet medical need.

Rho-kinase has been identified as one of the effectors of the small GTP-binding protein Rho [2]. Accumulating evidence has demonstrated that the Rho/Rho-kinase-mediated signaling pathway plays an important role in the signal transduction initiated by many agonists such as angiotensin II, thrombin, endothelin-1, norepinephrine and urotensin, that have been implicated in many major cardiovascular diseases such as hypertension, heart failure, myocardial infarction and atherosclerosis [3]. It is also known that Rho/Rho-kinase functions as a molecular switch to exert various cellular activities such as smooth muscle contraction, cell–cell surface adhesion, motility and cytokinesis [4]. Recent animal studies suggest that inhibition of Rho-kinase protect the myocardium from pacing- or endothelin-1-induced ischemic injury [5,6]. In addition, chronic inhibition of Rho-kinase blunts the process of left ventricular hypertrophy leading to cardiac contractile dysfunction in hypertension-induced heart failure [7]. However, the role of Rho-kinase in no-flow ischemia/reperfusion-induced myocardial injury in vivo is thus-far unknown. The goal of the present study was to determine whether inhibition of Rho-kinase protects the heart against reperfusion injury. To test this hypothesis, we used a highly selective Rho-kinase inhibitor, Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide] [8], in an established mouse model of myocardial ischemia/reperfusion injury [9,10].

	2. Materials and methods

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Heath (NIH Publication No. 85-23, revised 1996).

2.1 Mouse myocardial ischemia/reperfusion injury
Male CD1 mice (Charles River, Raleigh, USA), 30–35 g, ranging in age from 8 to 10 weeks, were orally administrated with Y-27632 (Calbiochem, USA) at a dose of 30 mg/kg by gavage 1 h before ischemia, and subjected to 30 min coronary artery occlusion and 24 h reperfusion as described in detail previously [9,10]. Briefly, the animals were anesthetized with pentobarbital sodium (60 mg/kg, i.p.), supplemental doses of anesthesia were given as required. The animal was placed supine and trachea was intubated with PE-60 tubing. The cannula was connected to a rodent ventilator (Harvard apparatus), at a rate of 105/min and a tide volume of 1 ml room air supplemented with oxygen (1 l/min). The body temperature was maintained by T/pump heat pad. The electrocardiogram (ECG) and the body temperature of the mice were monitored throughout the experiment by the Gould 3P 6600 data acquisition system. The chest cavity was entered through right a midline sternotomy. With the help of a light source of two flexible fiber optic arms, an 8-0 nylon suture was passed under left anterior descending coronary artery, and balloon occluder was applied on the artery. Myocardial ischemia and reperfusion were induced by inflating and then deflating a balloon occluder. The successful performance of coronary occlusion and reperfusion was verified by apical palor of the myocardium and typical ECG changes. The animals were sacrificed 24 h after reperfusion unless otherwise indicated.

2.2 Infarct size
At the end of study, the mice were heparinized and anesthetized with pentobarbital sodium. The heart was excised and flushed blood with normal saline through a Langendorff apparatus, then perfused with 1% solution of 2,3,5-triphenyltetrazolium chloride (TTC) in phosphate buffer to differentiate necrotic and viable myocardium. The coronary artery was then retied at the site of the previous occlusion and heart was perfused with a 2% solution of Evans blue dye to delineate ischemic area (area at risk). The atrial and right ventricular tissues were then excised, and the heart was cut into 5 transverse slices, fixed in 10% neutral buffered formaldehyde, weighed and digitally photographed. The areas of the infarcted, ischemic and nonischemic myocardium were measured by computerized videoplanimetry (Adobe Photo-shop, version 5.5), and from these measurements, infarct size was calculated as a percentage of the area at risk.

2.3 Cardiac function
Cardiac function was evaluated after 30 min coronary artery occlusion and 24 h reperfusion. Mice were anesthetized with sodium pentobarbital (35 mg/kg). A 1.4 F Millar Mikro-tip catheter transducer (Millar) was passed through the right carotid artery into the left ventricle (LV). LV pressure was digitally processed via the Gould 3P 6600 data acquisition system. Diastolic and systolic blood pressure, LV systolic pressure (LVSP), LV end diastolic pressure (LVEDP), positive and negative maximal values of the first derivative of LVP (+dp/dt and –dp/dt), and heart rate were monitored or derived by computer algorithm [11].

2.4 Myocardial apoptosis
The myocardial tissue was collected after 30 min ischemia and 4 h reperfusion. The apoptotic myocytes were detected by terminal deoxy-nucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay using a Cell Death Detection Kit (Roche) according to the manufacturer's instructions as described previously [12]. Briefly, the tissue sections after permeability treatment were incubated with the TUNEL reaction mixture containing TdT and fluorescein-dUTP. For double-labeling, the tissue sections after incubation with TUNEL reaction mixture and blockade of endogenous peroxidase and non-specific binding sites, were incubated with the monoclonal antibody against -sarcomeric actin (Sigma). After washing, the slides were incubated with biotinylated secondary antibody, and the cardiomyocytes were finally visualized with Texas red Avidin. For detection of total nuclei, the slides were covered with the mounting medium containing DAPI. At least 6 slides per block were evaluated. For each slide 15 fields were randomly chosen, a total of 2.5–3x10³ myocytes per slide was counted. The index of apoptosis, ie, the number of TUNEL-positive myocytes divided by the total myocytes per field, was determined.

2.5 Western blot analysis for Bcl-2
Ischemic/reperfused myocardial tissue was homogenized on ice with M-PER protein extraction reagent (Pierce) followed by sonication. The lysates were centrifuged at 14,000xg for 5 min. Equal amount of proteins (determined by BCA protein assay) were loaded, separated by electrophoresis on SDS-PAGE, and transferred onto a polyvinylidene difluoride (PVDF)-plus membrane. Blots were incubated in Tris-buffered saline containing 0.05% (v/v) Tween 20 (TBST) and 5% (w/v) nonfat milk powder to block nonspecific binding. The immunoblots were probed with a mouse monoclonal anti-Bcl-2 antibody (Sigma) with a dilution of 1:1000 overnight at 4 °C. Blots were then washed (TBST, 3x5 min) and incubated with horse-radish peroxidase conjugated rabbit anti-mouse secondary antibody at room temperature for 1 h. Chemiluminescence was developed by Supersignal-West dura western substrate (Pierce) and bands were detected with a Kodak Image Station 400. The blot densities were analyzed with Kodak 1D software (version 3.6).

2.6 Determination of Rho-kinase activity
Ischemic/reperfusion myocardial tissue was homogenized in modified RIPA buffer (50 mM Tris ph7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% Na-deoxycholate, 1 mM PMSF, 1 µg/ml of pepstatin, leupeptin, aprotinin, 1 mM NaVO4, and 1 mM NaF). Lysates were centrifuged at 8000xg for 30 min and the supernatants were collected. Equal amount of proteins were denatured in SDS sample buffer and separated by electrophoresis on SDS-PAGE, and transferred onto a PVDF membrane. Blots were incubated with 5% nonfat milk, and then the anti-Thr 445-phosphorylated -adducin antibody (Santa Cruz, CA) (1:500) was added, and incubated at 4 °C overnight, followed by three TBST washes. The secondary antibody, horse-radish peroxidase conjugated goat anti-rabbit antibody, was added and incubation continued for 1 h at room temperature. ECL was developed according to manufacturer's protocol.

2.7 Multiplexed serum cytokines-immunoassays
Individual mouse cytokine capture antibodies (R&D Systems), including tumor necrosis factor (TNF-), keratinocyte chemoattractant (KC) [13], granulocyte colony-stimulating factor (G-CSF) and seven interleukins (ILs), were coupled covalently to uniquely fluorescent carboxylated polystyrene microspheres (Luminex). Recombinant mouse cytokines were reconstituted and serially diluted in assay buffer. Standards or assay buffer were added to wells of a 96-well filter bottom plate. Pooled mouse serum was added to standard wells to account for matrix effects. Serum samples, collected from left ventricle at 2 h following reperfusion, were added to the remaining wells. The microsphere mixture was added to all wells and the plate was incubated for 4 h. After washing, a mixture of matching biotinylated detection antibodies (R&D) was added to each well and incubated for 30 min. Following incubation and washing, a solution of reporter dye (streptavidin R-phycoerythrin, Molecular Probes) was added. After 30 min, the wells were washed and the microspheres were resuspended in assay buffer and analyzed with a Luminex 100^TM analyzer.

2.8 Immunohistochemical analysis for Rho A and Bcl-2
Immunohistochemical staining for Rho A, Bcl-2 and neutrophils was performed on multiple transverse sections from ischemic myocardial tissues fixed with 10% neutral buffered formalin. The primary antibodies were: monoclonal anti-human Rho A (5 µg/ml) (Cytoskeleton), monoclonal anti-mouse Bcl-2 (1:100) (Sigma) and absorbed rabbit anti-mouse neutrophil polyclonal antibody (0.75 µg/ml) (Inter-Cell Tech). As a negative control, an isotype matched mouse IgG or rabbit IgG was used to replace the primary antibody, respectively. The primary antibody was labeled using a biotinylated secondary antibody, and visualized with Vectastain ABC reagent and DAB (Vector Lab).

2.9 Statistical analysis
Data are presented as mean±S.E.M. Differences between treatment and control groups were compared by paired and unpaired Student's t-test or by ANOVA followed by Bonferroni test. P values of <0.05 were considered statistically significant.

	3. Results

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
3.1 Rho A expression
Rho A expression in the ischemic myocardium was detected by immunohistochemical analysis. In the sham-operated heart, minimal Rho-A immunoreactivity was noted in interstitial cells (resident macrophages/fibroblasts) (Fig. 1A,B). In contrast, Rho A immunoreactivity was clearly increased in the heart subjected to 30 min ischemia and 4 h reperfusion (data not shown) or 24 h reperfusion (Fig. 1C). Rho A expression was mainly located in clusters of infiltrating inflammatory cells, primarily neutrophils (Fig. 1D) compared with the sham operated heart (Fig. 1A,B). Fig. 1E and F are negative controls of Fig. 1C and D.

View larger version (145K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Ischemia/reperfusion upregulated of Rho A expression. (A) Sham; (C) Ischemia/reperfusion; (E) Ischemia/reperfusion (negative control). B, D and F are a part of corresponding A, C and E with higher magnification. The arrow in B indicates that Rho A immunoreactivity is restricted to interstitial cells (resident macrophages or fibroblasts). The arrow in D indicates that Rho A immunoactivity is markedly increased in inflammatory cells consisting primarily of neutrophils. Scale bars: 85 um (E); 40 um (F).

 
3.2 Rho-kinase activity
To determine whether ischemia/reperfusion induced Rho-A upregulation subsequently activates Rho-kinase, phosphorylation of a Rho-kinase substrate, -adducin, was measured by Western blot analysis using the anti-Thr 445-phosphorylated -adducin antibody. As shown in Fig. 2, myocardial ischemia/reperfusion resulted in 8.7-fold increase in the amount of phosphorylated -adducin (Thr 445) compared with the sham-operated heart, indicating the activation of Rho-kinase in ischemic myocardium following ischemia/reperfusion. In contrast, treatment with Y-27632 significantly attenuated the amount of p-adducin (Thr 445) by 63.4% (P<0.05 vs. the vehicle-treated group) (Fig. 2).

View larger version (30K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Y-27632 inhibits activation of Rho-kinase following myocardial ischemia/reperfusion. Top panel. Representative Western blots from two separate experiments. Bottom panel. Quantitative results. The mean density value of p-adducin (Thr 445) in sham-operated mice was defined as 100%. Data are mean±S.E.M. (n=6–8), *P<0.001 versus sham; #P<0.05 versus I/R.

 
3.3 Infarct size
The ischemic area, determined by negative staining after perfusion with Evans blue dye and expressed as percent of LV, showed no difference between vehicle- and Y27632-treated groups (Fig. 3A, left side). However, infarct size, expressed as percent of ischemic area, was remarkably reduced in Y-27632-treated groups in a dose-dependent manner (Fig. 3A, right side). At 10 and 30 mg/kg of Y-27632, the infarct size was reduced by 30.2% and 41.1%, respectively (P<0.01 vs. vehicle, n=8). To further determine whether the anti-infarct effect of Rho-kinase inhibition sustains after 24 h reperfusion, infarct size was measured after a 30-min ischemia and 72 h reperfusion. Infarct size averaged 58.6±2.1% of ischemic area in control group. Treatment with Y-27632 significantly reduced the infarct size to 35.1±2.0% of ischemic area (P<0.01 vs. vehicle) (Fig. 3B). Y-27632 at 30 mg/kg had no effect on the systemic blood pressure, as shown in Table 1. Also, body temperature and heart rate did not differ significantly between treated mice and untreated mice before ischemia, during the 30 min coronary occlusion and following reperfusion (Table 2). The mortality after a 30-min ischemia and 24 h reperfusion was 18.1% and 11.1% in vehicle and Y-27632-treated mice, respectively (P>0.05) and most of mice died during early reperfusion.

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 (A) Y-27632 reduces myocardial infarct size after 30-min ischemia and 24 h reperfusion. Mice were orally administered with Y-27632 at dose indicated 1 h prior to ischemia, and subjected to 30 min of coronary occlusion and 24 h reperfusion. Left side of the figure shows the myocardial ischemic area, expressed as % of left ventricle. Right side is the myocardial infarct size, expressed as % of myocardial ischemic area. Data are mean±S.E.M., n=8 per group, *P<0.01 versus vehicle. (B) Y-27632 reduces myocardial infarct size after 30-min ischemia and 72 h reperfusion. Mice were orally administered with Y-27632 at dose of 30 mg/kg 1 h prior to ischemia, and subjected to 30 min of coronary occlusion and 72 h reperfusion. Infarct size was measured with TTC staining after 72 h of reperfusion. , Vehicle (n=6); , Y-27632 (n=7). Data are mean±S.E.M., *P<0.01 versus vehicle.

 

View this table: [in this window] [in a new window]   Table 1 Blood pressure after oral administration of 30 mg/kg of Y-27632

 

View this table: [in this window] [in a new window]   Table 2 Body temperature and heart rate during myocardial ischemia/reperfusion

 
3.4 Cardiac function
Myocardial ischemia/reperfusion caused a marked decrease in LVSP, +dP/dt and –dP/dt. Treatment with Y-27632 (30 mg/kg) significantly improved cardiac function recovery (Fig. 4). The values of LVSP, +dp/dt and –dp/dt at 24 h after reperfusion were enhanced from (% of sham) 61.3, 49.1, and 46.4 in vehicle to 75.7, 74.7, and 74 in Y-27632-treated group, respectively (P<0.05, n=8) (Fig. 4). There was no difference in the heart rate between two groups (data not shown).

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Y-27632 improves cardiac function following ischemia/reperfusion. Mice were administered with Y-27632 at dose 30 mg/kg, and subjected to 30-min ischemia and 24 h reperfusion. LVSP, +dP/dt, and –dP/dt were determined at 24 h after reperfusion. , Sham (n=8); , Vehicle (n=8); , Y-27632 (n=8). Data are mean±S.E.M., *P<0.01 versus sham, #P<0.01 versus vehicle.

 
3.5 Myocardial apoptosis
Heart tissue from sham-operated mice exhibited very low levels of staining for TUNEL (Fig. 5C). In contrast, a significant number of TUNEL-positive myocytes were detected in ischemic myocardial tissue from the hearts subjected to 30 min ischemia and 4 h reperfusion in the vehicle group (Fig. 5F). Most notably, the number of TUNEL-positive myocytes in the ischemic myocardium in Y-27632-treated animals significantly decreased (Fig. 5I). Quantitative measurement depicts a 51% reduction in TUNEL-positive myocytes in Y-27632-treated group compared with the vehicle group (P<0.01, n=6) (Fig. 5, lower panel).

View larger version (77K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Y-27632 reduces myocardial apoptosis following ischemia/reperfusion. Top panel, representative photomicrographs of in situ detection of nuclear DNA fragments (TUNEL assay) in heart tissue from the mice subjected to 30 min ischemia and 4 h reperfusion and treated with vehicle (D–F) or Y-27632, 30 mg/kg (G–I), or sham-operated (A–C). The C, F and I depict the apoptotic nuclei stained with FITC (TUNEL-positive nuclei); A, D and G are the same sections of C, F and I, respectively, were double-labeled with anti--sarcomeric actin, and visualized with Texas Red Avidin; B, E and H depict the total nuclei stained with DAPI. Bottom panel, quantitative measurement of percentage of apoptotic cardiomyocytes (TUNEL positive cells) in ischemic myocardium from , Vehicle- or , Y-27632-treated mice. Data are means±S.E.M., n=5 per group, *P<0.01 versus vehicle.

 
3.6 Expression of Bcl-2
As shown in Fig. 6, panel I-a, the basal expression of Bcl-2 immunoreactivity was associated with cardiac myofibrils in sham-operated mice. Bcl-2 expression was significantly downregulated in vehicle-treated ischemic/reperfused hearts (Fig. 6, panel I-b). However, this downregulation of Bcl-2 expression was attenuated in Y-27632-treated hearts (Fig. 6, panel I-c). Expression of Bcl-2 protein in myocardium was further determined by Western blot analysis (Fig. 6, panel II, III), showing that ischemia/reperfusion reduced BCL-2 level by 46.3% (P<0.01 vs. sham), and this downregulation in BCL-2 expression was attenuated by 61% in Y-27632-treated animals.

View larger version (47K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Panel I: Immunohistochemical detection of Bcl-2 expression in ischemic myocardium from mice subjected to 30 min ischemia and 4 h reperfusion and treated with vehicle or Y-27632. (A) Sham; (B) vehicle+I/R; C, Y-27632+I/R. Panel II: Representative Western blot analysis showing that Y-27632 attenuates ischemia/reperfusion-induced downregulation of Bcl-2 in ischemic myocardium. Panel III: Quantitative measurement. Bcl-2 level in sham (n=4), I/R (n=6), and Y+I/R (n=5). Data are means±S.E.M., *P<0.01 versus sham; #P<0.01 versus I/R.

 
3.7 Multiplexed serum cytokines
Among the tested cytokines, IL-6, KC and G-CSF were found to be substantially increased in the blood collected from the left ventricle following 30 min ischemia and 2 h reperfusion. However, the increase in these cytokines in blood was significantly attenuated by treatment with Y-27632 (P<0.05 vs. vehicle, n=8–12) (Fig. 7).

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Y-27632 suppresses ischemia/reperfusion-induced proinflammatory cytokines production. Serum samples were collected from the left ventricle at 2 h after reperfusion. , Sham (n=8); , Vehicle (n=12); , Y-27632 (n=12). Data are means±S.E.M., *P<0.01 versus vehicle.

 
3.8 Neutrophil accumulation
The number of neutrophils in sham ischemic myocardium was very low (data not shown). However, in the hearts subjected to ischemia/reperfusion, infiltrated neutrophils were markedly increased, frequently concentrated in the subepicardial regions, and extended into the area of injured myocardium of the left ventricle (Fig. 8, upper panel). Treatment with Y-27632 reduced neutrophils accumulation by 45% in the ischemic reperfusion myocardium (P<0.01, n=6) (Fig. 8, lower panel).

View larger version (68K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 Y-27632 reduces neutrophil infiltration into ischemic myocardium following 30 min ischemia and 24 h reperfusion. Top, representative micrographs of infiltration neutrophils staining in the heart tissue from mice subjected to 30 min ischemia and 24 h reperfusion. (A) Vehicle; (B) Y-27632-treated heart. Original magnification: x10. Arrow indicates the enlarged region shown on right up corner (x20), MI; myocardial infarction. Bottom, quantitative measurement of infiltration neutrophils in the tissue sections from , Vehicle (n=6) and , Y-27632-treated mice (n=6). Data are means±S.E.M., *P<0.01 versus vehicle.

 

	4. Discussion

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
Recent studies have implicated the Rho/Rho-kinase-mediated pathway in variety of cardiovascular diseases, and Rho-kinase has been suggested as a potential therapeutic target for inhibition in treatment of cardiovascular diseases [3]. However, the role of Rho-kinase in myocardial ischemia/repefusion injury is unknown. Using well-established murine model of myocardial ischemia/reperfusion injury [9,10], we have demonstrated that ischemia/reperfusion upregulated Rho A expression in ischemic myocardium, and increased Rho-kinase activity. In present study, the activity of Rho-kinase was determined by phosphorylation of -adducin (Thr 445). -Adducin is one of down-stream substrates of Rho-kinase [14], it is expressed in many tissues including the heart [7,15]. It is also known that -adducin is specifically phosphorylated by Rho-kinase at Thr 445 and Thr 480 [15]. Myocardial ischemia/reperfusion resulted in 8.7-fold increase in the amount of p-adducin (Thr445), indicating the activation of Rho-kinase in the myocardium following ischemia/reperfusion. These results are consistent with a recent study showing that hypoxia upregulated Rho-kinase expression in endothelial cells [16]. We also demonstrated that the selective Rho-kinase inhibitor, Y-27632 produced a dose-dependent attenuation of myocardial infarction associated with ischemia/reperfusion injury and a concomitant improvement in cardiac function. Moreover, the anti-infarct effect of Rho-kinase inhibition sustained over 24 h reperfusion. The dose of Y-27632 that reduced infarct size also significantly inhibited Rho-kinase activity induced by myocardial ischemia/reperfusion injury, suggesting that the cardioprotection is likely through inhibition of Rho-kinase activation. Furthermore, ischemia/reperfusion-induced myocardial apoptosis was significantly reduced in Y-27632-treated animals. To the best of our knowledge, this is the first in vivo study demonstrating that Rho/Rho-kinase mediated pathway is potentially involved in acute myocardial ischemia/reperfusion injury. Our results support the novel concept that activation of the Rho/Rho-kinase pathway can be detrimental to the heart [17].

Both necrosis and programmed cell death (apoptosis) are known to occur following ischemia/reperfusion. Recent evidence suggests that myocardial apoptosis is initiated shortly after ischemia and amplified by the reperfusion, and contributes in part to overall cardiomyocyte death [18]. In this study, we examined apoptotic myocardial cell death with TUNEL assay. The percentage of TUNEL-positive cardiomyocytes after 30 min ischemia and 4 h reperfusion was 13.1±0.15%, similar to the data reported previously in rabbit hearts subjected to ischemia/reperfusion [19]. Treatment with Y-27632 resulted in a 51% reduction in apoptotic cardiomyocytes, consistent with the infarct-limiting effect of Y-27632, suggesting that the anti-apoptotic effect of Y-27632 in early phase of reperfusion could contribute to the reduction of later myocardial necrosis (myocardial infarction). The anti-infarct effect of Rho-kinase inhibition lasting to 72 h following reperfusion indicated that cardioprotection of Rho-kinase inhibition is mediated by anti-apoptosis not delaying the apoptosis, although we determined one early time point of myocardial apoptosis following ischemia/reperfusion in this study. To further investigate the anti-apoptotic effect of Y-27632, Bcl-2 expression in the heart was determined by immunohistochemical and further quantitated by Western blot analysis. Bcl-2 is an oncogene; its expression inversely regulates apoptosis. Expression of Bcl-2 in cardiomyocytes has been documented previously by immunohistochemistry analysis [20,21]. Overexpression of Bcl-2 protects cardiomyocytes from apoptotic cell death induced by p53 [22] and attenuates ischemia/reperfusion-induced myocardial injury in transgenic mice [23]. It was revealed in the present study that ischemia/reperfusion markedly downregulated expression of Bcl-2 in ischemic myocardium, consistent with the previous results [24–26]. Meanwhile, treatment with Y-27632 clearly attenuated ischemia/reperfusion-induced downregulation of Bcl-2 in myocardium, suggesting that the anti-apoptosis effect of Y-27632 was likely through regulation of Bcl-2 in myocardium.

The inflammatory reaction has been recognized as a hallmark of myocardial reperfusion injury. Inflammatory cells and proinflammatory cytokines are two important components known to be implicated in reperfusion injury. Neutrophils cause reperfusion injury by obstruction of capillary vessels, production of vasoactive substances, and release of cytotoxic agents. Proinflammatory cytokines promote further inflammatory cell adhesion and infiltration into myocardium, and influence acute tissue injury. Treatment with Y-27632 resulted in a significant reduction in the accumulation of neutrophils in ischemic myocardium, consistent with a recent in vivo study showing that Rho-kinase inhibitor reduced infiltration of inflammatory cells into the lung tissue in an animal model in which activation of eosinophils and neutrophils was induced by lysophosphatidic acid [27]. Our data were also supported by a study demonstrating that the Rho-kinase inhibitor suppressed chemotactic peptide-induced increases in phosphorylation of myosin light chain, shape changes and locomotion in human neutrophils, accompanied with reduction of cell migration [28]. In the Y-27632-treated group, the serum levels of IL-6, KC and G-CSF were significantly decreased compared with the vehicle group. IL-6 is a major cytokine responsible for induction of adhesion molecules [29]. KC, a murine homologue of human IL-8, is a CXC chemokine, produced by leukocytes and vascular cells. A recent in vivo study has revealed that KC provoked a dose- and time-dependent increase in leukocyte rolling, adhesion and tissue recruitment [15]. G-CSF is known to stimulate neutrophils production and mobilization. In addition, recent study showed that high level of circulating cytokines caused cardiac function [30]. The improvement of postischemic cardiac function by Y-27632 may also mediated by inhibition cytoines release. Also, we did not find protective effect of Y-27632 in our preliminary study using isolated perfused rat heart. Those results further demonstrated that reduction of proinflammatory cytokine release and attenuation of neutrophils infiltration would be important mechanisms by which Y-27632 protects the heart against ischemia/reperfusion injury.

Y-27632 is known as an antihypertensive agent and potentially useful for treatment of cardiovascular diseases associated with vascular smooth muscle cell hypercontraction [3]. Y-27632 at a dose of 30 mg/kg oral administration did not affect systemic blood pressure in the mice (Table 1). Also, Y-27632 did not significantly change heart rate before ischemia, during the 30 min coronary occlusion and following reperfusion (Table 2), suggesting that the cardioprotective effect observed in the present study was independent of a systemic hemodynamic action. Our data was consistent with a previous study showing that Y-27632 at 30 mg/kg orally caused only a slight and transient fall in blood pressure in normotensive rats [8]. This could be due to the fact that the expression and activity of Rho-kinase are remarkably augmented in the hypertensive not the normotensive animals [31]. However, recent studies have shown that Rho-kinase inhibitor improved brain blood flow after ischemia [32] and attenuated post-ischemic coronary microvascular spasm [33,34]. Therefore, the local vascular effects of the Rho-kinase inhibitor on cardioprotection warrants further exploration. Also, recent studies have shown that Na/H exchanger in fibroblasts is phosphorylated and activated by Rho-kinase [35], and inhibition of Na/H exchanger has been demonstrated to protect the heart from ischemia/reperfusion injury [36,37]. Therefore, whether the protective effect of Rho-kinase inhibitor is mediated by inhibition of Na/H exchanger needs further investigation.

In conclusion, myocardial ischemia/reperfusion upregulated Rho A expression, and Rho-kinase activity in ischemic myocardium. Treatment with Rho-kinase inhibitor, Y-27632, significantly inhibited Rho-kinase activity, reduced myocardial infarction, and improved post-ischemic cardiac dysfunction. The cardioprotective effect of Y-27632 could be attributed to the reduction of myocardial apoptosis, suppression of proinflammatory cytokine production and attenuation of neutrophil accumulation in the ischemic myocardium. The data suggest that the Rho/Rho-kinase mediated pathway plays an important role in myocardial reperfusion injury and inhibition of Rho-kinase could be a potential therapeutic intervention for prevention and treatment of acute myocardial ischemia/reperfusion injury.

	Notes

 
Time for primary review 00 days

This work was presented at AHA 2003 meeting, Orlando, FL, USA.

	References

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 

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