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Am J Physiol Heart Circ Physiol 292: H2598-H2606, 2007. First published January 12, 2007; doi:10.1152/ajpheart.01393.2006
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Rho kinase activation plays a major role as a mediator of irreversible injury in reperfused myocardium

Shabaz A. Hamid, Hugo S. Bower, and Gary F. Baxter

Royal Veterinary College, University of London, London, United Kingdom

Submitted 20 December 2006 ; accepted in final form 12 January 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intracellular signal transduction events in reperfusion following ischemia influence myocardial infarct development. Here we investigate the role of Rho kinase (ROCK) activation as a specific injury signal during reperfusion via attenuation of the reperfusion injury salvage kinase (RISK) pathway phosphatidylinositol 3-kinase (PI3K)/Akt/endothelial nitric oxide (NO) synthase (eNOS). Rat isolated hearts underwent 35 min of left coronary artery occlusion and 120 min of reperfusion. Phosphorylation of the ROCK substrate protein complex ezrin-radixin-moesin, assessed by immunoblotting and immunofluorescence, was used as a marker of ROCK activation. Infarct size was determined by tetrazolium staining, and terminal dUTP nick-end labeling (TUNEL) positivity was used as an index of apoptosis. The ROCK inhibitors fasudil or Y-27632 given 10 min before ischemia until 10 min after reperfusion reduced infarct size (control, 34.1 ± 3.8%; 5 µM fasudil, 18.2 ± 3.1%; 0.3 µM Y-27632, 19.4 ± 4.4%; 5 µM Y-27632, 9.2 ± 2.9%). When 5 µM Y-27632 was targeted specifically during early reperfusion, robust infarct limitation was observed (14.2 ± 2.6% vs. control 33.4 ± 4.4%, P < 0.01). The protective action of Y-27632 given at reperfusion was attenuated by wortmannin (29.2 ± 6.1%) and N{omega}-nitro-L-arginine methyl ester (30.4 ± 5.7%), confirming a protective mechanism involving PI3K/Akt/NO. Ezrin-radixin-moesin phosphorylation in risk zone myocardium confirmed early and sustained ROCK activation during reperfusion and its inhibition by Y-27632. Inhibition of ROCK activation at reperfusion reduced the proportion of TUNEL-positive nuclei in the infarcted region. In conclusion, ROCK activation occurs specifically during early reperfusion. Inhibition of ROCK at reperfusion onset limits infarct size through an Akt/eNOS-dependent mechanism, suggesting that ROCK activation at reperfusion may be deleterious through suppression of the RISK pathway.

apoptosis; myocardial infarction; nitric oxide

DESPITE THE UNQUESTIONED NEED for reperfusion to prevent ischemic necrosis in acute myocardial infarction, there is accumulating experimental evidence that reperfusion is associated with the paradoxical activation of lethal signals that culminate in necrosis/apoptosis. Indeed, recent experimental evidence suggests that reperfusion may contribute to a far greater extent than was previously supposed (8, 9). Whereas this does not detract from the proven therapeutic value of reperfusion, it prompts a reassessment of reperfusion injury and its mechanisms. Rho kinase (ROCK) is a family of serine/threonine protein kinases, stimulated by G protein-coupled receptor activation of small plasma membrane-bound GTP-binding proteins, such as the Rho and Rac family. Relevant to the pathophysiology of myocardial infarction, potent stimuli for ROCK activation include hypoxia (25), pressor mediators such as endothelin-1, angiotensin II, and norepinephrine (29, 30), and mitochondrially derived reactive oxygen species (2). In the cardiovascular system, the diverse outcomes of ROCK signaling include effects on cardiac myocyte hypertrophy, vascular smooth muscle tone, and vascular smooth muscle cell proliferation and cell migration (20, 15, 24). In addition to modifying target protein function by phosphorylation, ROCK regulates at the transcriptional level inflammatory cytokines, thrombotic factors, NADPH oxidase, and endothelial nitric oxide (NO) synthase (eNOS) (18, 22, 23, 30).

Appreciation of ROCK signaling in vascular smooth muscle contraction has fueled the development of selective pharmacological inhibitors as therapeutic vasodilators (29). One such compound, fasudil, was developed for the treatment of angina pectoris. Studies with fasudil (or its metabolite hydroxyfasudil) and the structurally unrelated inhibitor Y-27632 have indicated beneficial effects of selective ROCK inhibition in experimental models of left coronary artery occlusion that are independent of coronary vasodilatation. Bao et al. (3) reported that Y-27632 given to mice before coronary occlusion and reperfusion resulted in marked limitation of infarct size in a dose-dependent fashion. Wolfrum et al. (33) reported that fasudil given to rats before coronary occlusion and reperfusion similarly limited infarct size. They showed that the protective effect was abolished by inhibitors of phosphatidylinositol 3-kinase (PI3K)/Akt activation and NOS activation. The suggestion that ROCK acts as a negative regulator of PI3K activation is of particular interest since several lines of evidence support the proposal that prosurvival protein kinase pathways, or reperfusion injury salvage kinase (RISK) pathways, including PI3K/Akt, p42/p44 mitogen-activated protein kinases and protein kinase G (PKG) promote cell survival during reperfusion (9, 13, 36). Thus it is possible that ROCK is a negative regulator of at least one key RISK pathway.

The aim of the present study was to examine the pattern of ROCK activation during reperfusion and the potential for ROCK inhibition to modify infarct development when administered as an adjunct to reperfusion. We hypothesized that ROCK is activated specifically after reperfusion and that suppression of ROCK activity during early reperfusion limits infarct size through augmentation of Akt activation and NO synthesis in the myocardium.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Materials

Male Sprague-Dawley rats (250–350 g) were used in these studies. Their handling and use were in accordance with the UK Guidelines on the Operation of the Animals (Scientific Procedures) Act 1986 (The Stationery Office, UK). Procedures were approved by the institutional Ethical Review Board and regulated under UK Home Office Project license no. 70/5536. Fasudil and Y-27632 dihydrochloride were from Tocris. Antibodies against phosphorylated and total ezrin-radixin-moesin (ERM) were from Santa Cruz. All other reagents were of analytical standard.

Heart Perfusion

Rats were anesthetized with a mixture containing pentobarbital sodium (50 mg/kg ip) and heparin (1,000 IU/kg ip). Excised hearts were perfused retrogradely through the aorta at 11.3 kPa with Krebs-Henseleit buffer containing (in mmol/l) 118 NaCl, 25 NaHCO3, 11 glucose, 4.7 KCl, 1.2 MgSO4·7H2O, 1.2 KH2PO4, and 1.8 CaCl2·2H2O; aerated with carbogen, pH 7.3–7.5, at 37°C. Coronary flow rate (CFR) was determined by timed collection of the coronary effluent. A saline-filled latex balloon connected to a pressure transducer was inserted into the left ventricle (LV), and baseline end-diastolic pressure was set at 5–10 mmHg without any further adjustment of balloon volume. Heart rate, LV end-diastolic pressure, and LV developed pressure were recorded continuously (PowerLab System, AD Instruments, Abingdon, UK). After stabilization for 15–20 min, hearts were excluded from further study if they failed to sustain steady sinus rhythm, if coronary flow rate < 10 ml/min, heart rate < 200 beats/min, or LV systolic pressure < 60 mmHg.

Infarct Induction and Measurement

A 3-0 silk suture was positioned around the left main coronary artery and threaded through a plastic snare to permit reversible occlusion of the artery. Coronary occlusion was induced for 35 min by clamping the snare onto the heart. Reperfusion was achieved by releasing the snare. At 120 min after reperfusion, the coronary artery was reoccluded, and the risk zone was delineated by perfusing 0.5% Evans blue into the aortic cannula. Following freezing at –20°C, hearts were sectioned (2 mm) and incubated in 1% triphenyltetrazolium chloride in phosphate buffer (pH 7.4, 37°C) for 15 min to define white necrotic tissue when fixed in 10% formalin for 24 h. Risk zone areas and infarct-to-risk ratios were determined by computerized planimetry using J-Image version 1.6 software (National Institutes of Health, Bethesda, MD).

Treatment Protocols

Treatment protocols are illustrated in Fig. 1. All hearts were stabilized for 15–20 min and then randomized to a control or treatment group. Infarct study 1 was performed to determine cardioprotective concentrations of fasudil and Y-27632 (groups 1–5).


Figure 1
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  		Fig. 1. Schematic representation of treatment protocols. For infarct size studies, all hearts were stabilized and subjected to 35-min coronary artery occlusion (CAO) and 120-min reperfusion, after which infarct size was assessed by tetrazolium staining. In groups 2–5, treatment with Rho kinase (ROCK) inhibitors fasudil or Y-27632 commenced before CAO and continued until 10 min after reperfusion. In groups 7, 8, and 10, perfusion with Y-27632 was commenced 5 min before reperfusion and stopped 10 min after reperfusion. Further hearts were prepared in groups 6 and 7 for immunoblotting (open arrows) or immunohistochemical analysis (solid arrows). L-NAME, N{omega}-nitro-L-arginine methyl ester.

 

Infarct study 1. Control hearts were subjected to 35 min of ischemia followed by 120 min reperfusion (group 1). To assess the effect of ROCK inhibition during ischemia-reperfusion, fasudil at 1 µmol/l (group 2) and 5 µmol/l (group 3) was present in the perfusion buffer from 10 min before coronary occlusion until 10 min after reperfusion. The ROCK inhibitor Y-27632 was also administered from 10 min before coronary occlusion until 10 min after reperfusion at 0.3 µmol/l (group 4) and 5 µM (group 5).

Subsequently, a further study (infarct study 2) examined the effects of Y-27632 given specifically during early reperfusion (groups 6–11).


Infarct study 2. After the stabilization period, control hearts were subjected to 35 min of ischemia followed by 120 min of reperfusion (group 6). To assess the effect of ROCK inhibition during the early stage of reperfusion, Y-27632 (5 µmol/l) was given 5 min before reperfusion until 10 min after reperfusion (group 7). The contribution of NO generation due to ROCK inhibition was tested using the NO synthase inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME; 100 µmol/l) given at reperfusion, commencing 10 min before treatment with 5 µM Y-27632 (group 8). To control for L-NAME treatment in group 8, 100 µmol/l L-NAME was perfused for 25 min commencing 15 min before reperfusion (group 9). The influence of ROCK inhibition on the PI3K/Akt pathway was assessed using wortmannin (10 µmol/l), given 10 min before treatment with 5 µM Y-27632 (group 10). Wortmannin (10 µmol/l) alone was administered from 25 min ischemia until 10 min of reperfusion (group 11) to control for group 10.

Western Blot Analysis

The phosphorylated and total levels of the ROCK substrate ERM were determined in naïve hearts and in hearts subjected to 35-min ischemia with or without 10-min reperfusion. Protein was extracted with lysis buffer from ischemic risk zone samples, and supernatants were used for Western blot analysis. Proteins were transferred to a nitrocellulose membrane and then blocked for 3 h with 4% milk-fat solution. Membranes were incubated with primary antibodies (1:1,000) in Tris-buffered saline/0.5% (vol/vol) Tween 20, and 1% (wt/vol) BSA solution for 1 h at room temperature. Bound antibodies were detected by a secondary antibody conjugated to horseradish peroxidase and visualized by electrochemiluminescence (Santa Cruz). Nitrocellulose-bound proteins were detected with anti-phospho-ERM, anti-eNOS (Ser1177) and anti-Akt (Thr308) antibodies followed by total ERM, eNOS, and Akt antibodies after dissociating membrane-bound probes with reblot solution (Pierce).

Immunofluorescence Analysis

Following perfusion fixation with 2% wet/vol paraformaldehyde, wax-embedded sections were examined by immunohistochemical and terminal dUTP nick-end labeling (TUNEL) analysis. To standardize the analysis, consecutive transverse 8-µm sections were cut from the same region, 4–6 mm above the cardiac apex, mounted on poly-L-lysine-coated slides, deparaffinized, and rehydrated before fluorescence labeling.

TUNEL. The "DeadEnd" TUNEL labeling kit (Promega) was used according to the manufacturer's instructions. Briefly, tissue sections were permeabilized with 20 µg/ml proteinase at room temperature for 15 min. After sections were washed with PBS, they were further fixed with 4% paraformaldehyde. After tissue was washed with PBS, it was covered with equilibration buffer (200 mmol/l potassium cacodylate, pH 6.6, 25 mM Tris·HCl, 0.2 mmol/l DDT, 0.25 mg/ml BSA, and 2.5 mmol/l cobalt chloride) for 10 min. Sections were covered with dTDT, including a nucleotide mix containing fluorescein-12-dUTP and rTDT enzyme and incubated with dTDT for 60 min at 37°C in a humidified chamber. The dTDT reaction was stopped by addition of citrate buffer. After cell nuclei were washed with PBS, they were labeled with 1:2,000 TO-PRO-3 for 15 min at 4°C. Negative controls lacked dTDT treatment, and positive controls were produced by incubation with 10 U/ml DNAse.

Infarct and nonrisk zones were delineated by hematoxylin/eosin staining using sections adjacent to those used for TUNEL labeling. Infarcted cells did not take up the cytoplasmic dye and appeared white. Cells in the infarct region displayed irregular morphology and a loss of contact with adjacent cells.

Immunolabeling. Samples were permeabilized with 0.2% Triton X-100 in PBS for 15 min at room temperature, followed by a 30-min blocking step (2% goat serum, 1% bovine serum albumin, and 0.1% Triton X-100 in PBS). Sections were incubated with either 1:200 phospho- or total ERM for 12 h at 4°C in a humidified chamber. After being washed with PBS, 1:250 anti-rabbit FITC was incubated for 1 h in the dark at 4°C. Cells were counterstained for F-actin with 1:30 phalloidin-tetramethylrhodamine isothiocyanate (TRITC) and nuclear labeled with 1:2,000 TO-PRO-3 for 15 min. Images were observed and captured with the use of a confocal microscope (LSM-510, Zeiss).

Statistical Analysis

Data are expressed as means ± SE. Differences between means of continuous data were compared with one-way ANOVA followed by Tukey's post hoc analysis. Probability (P) values of <5% were accepted as being statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A total of 133 rats were used for these studies. Five hearts were excluded from further analysis because they failed to meet the baseline inclusion criteria (see METHODS), or because of failure to delineate the risk zone adequately, or failure of the tetrazolium stain. Thus we report data from animals: 87 successfully completed infarct experiments, and 40 hearts were used for immunoblotting and immunohistochemical analysis. Cardiac function data are shown in Table 1.


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  		Table 1. Cardiac function for treatment groups

 
ROCK Activation Occurs During Reperfusion

We tested the hypothesis that ROCK activation occurs following ischemia-reperfusion by assessing the expression of phosphorylated ERM (Fig. 2). Immunoblotting showed that 35-min ischemia alone did not influence the level of phosphorylated ERM. However, 10 min after reperfusion, a 1.9-fold increase in phospho-to-total ERM ratio was observed, while administration of Y-27632 (5 µmol/l) from 5 min before reperfusion until 10 min after reperfusion abolished the increase in phospho-ERM (Fig. 2A).


Figure 2
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  		Fig. 2. Activation of ROCK by ischemia-reperfusion was detected by assessing phosphorylation of the substrate proteins ezrin-radixin-moesin (ERM). A: immunoblot analysis of risk zone myocardium showed an intensified phospho-ERM (p-ERM) to total ERM signal 10 min after reperfusion. 80 kDa, ezrin/radixin; 75 kDa, moesin. Treatment with Y-27632 from 5 min before reperfusion until 10 min after reperfusion abrogated the increase in phospho-ERM. *P < 0.05 vs. preischemic baseline (n = 4 per group). B: representative fluorescence micrographs taken from risk zone myocardium showing increased phospho-ERM after 120 min of reperfusion compared with preischemic baseline. Treatment with Y-27632 (5 µM) from 5 min before reperfusion until 10 min after reperfusion was associated with significant attenuation of the phospho-ERM fluorescence. C: quantitative analysis of phospho-ERM fluorescence from nonrisk left ventricular myocardium and the infarct region, sampled 120 min after reperfusion. In control myocardium subjected to 35-min ischemia (35I) and 120-min reperfusion (120R), phospho-ERM fluorescence was significantly increased when compared with the preischemia values in infarct zone. Treatment with 5 µM Y-27632 significantly attenuated phospho-ERM intensity. AU, arbitrary units. *P < 0.05 vs. corresponding preischemia value; #P < 0.05 vs. corresponding control 120R value (1-way ANOVA, n = 4 consecutive sections from each region, with 4 hearts in each treatment group).

 
Analysis of the sites of ROCK activation was undertaken by confocal fluorescence microscopy of left ventricular myocardial sections (Fig. 2, B and C). Under baseline preischemic conditions, phospho-ERM fluorescence was negligible in all areas of the LV. In control myocardium subjected to 35-min ischemia and 120-min reperfusion, a marked increase in phospho-ERM signal was observed in the infarct region, demonstrating sustained activation of ROCK 120 min after reperfusion. When Y-27632 was administered during the first 10 min of reperfusion, the increase in phospho-ERM was significantly attenuated in the central infarct region. Considered together, these data are consistent with an early and sustained activation of ROCK in the ischemic risk territory during the reperfusion period. They also confirm the efficacy of Y-27632 as a potent inhibitor of ROCK activation.

ROCK Inhibition During Ischemia and Reperfusion Limits Infarct Size

We examined the cardioprotective action of pharmacological ROCK inhibition in two dosing schedules. Risk zone volume, a major determinant of infarct size, was comparable in all groups in the range 0.32–0.46 cm3, with no statistically significant differences between the groups in either infarct study. In infarct study 1, the administration of fasudil or Y-27632 from 10 min before coronary occlusion until 10 min after reperfusion (i.e., bracketing ischemia and early reperfusion) resulted in a marked reduction in infarct size (Fig. 3A). Control infarct following 35-min coronary artery occlusion and 120-min reperfusion was 34.1 ± 3.8%, a value consistent with our previous experience of this model of ischemia-reperfusion injury. For both drugs, the protection afforded was concentration dependent: fasudil at 1 µM, 28.9 ± 3.9%; at 5 µM, 18.2 ± 3.1%; Y-27632 at 0.3 µM, 19.4 ± 4.4%; at 5 µM, 9.2 ± 2.9%.


Figure 3
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  		Fig. 3. Infarct size represented as percentage of infarction within the ischemic risk zone after 35-min coronary occlusion and 120-min reperfusion. A: in infarct study 1, hearts were treated with ROCK inhibitors fasudil or Y-27632 before coronary occlusion, throughout coronary occlusion, and for the first 10 min of reperfusion. Treatment with either inhibitor led to a concentration-dependent limitation in infarct size. B: in infarct study 2, hearts were treated with 5 µM Y-27632 only during the early reperfusion period (from 5 min before reperfusion until 10 min after reperfusion). Infarct limitation was similar to that observed in infarct study 1. The addition of L-NAME or wortmannin (wort) abrogated the protective effect of Y-27632 at reperfusion. *P < 0.01 vs. corresponding control (con) group (1-way ANOVA, n = 5–14 per group).

 
ROCK Inhibition Specifically During Reperfusion Limits Infarct Size Through an Akt/NO-Dependent Mechanism

On the basis of the observations above, 5 µM Y-27632 was selected for further study in which treatment was targeted during early reperfusion (infarct study 2; Fig. 3B). The treatment of hearts from 5 min before reperfusion until 10 min after reperfusion with 5 µM Y-27632 significantly limited infarct development (control, 33.4 ± 4.4%; Y-27632, 14.2 ± 2.6%, P < 0.01). To test the hypothesis that ROCK inhibition is associated with augmented NO synthesis, L-NAME was coadministered as an inhibitor of NO synthase. L-NAME blunted the protective effects of Y-27632 at reperfusion (30.4 ± 5.7%, P not significant versus group 6 control), suggesting that the protection afforded by ROCK inhibition at reperfusion is NO dependent. Wortmannin also prevented the myocardial salvaging properties of Y-27632 at reperfusion (29.2 ± 6.1%, P not significant versus group 6 control). Neither L-NAME (33.5 ± 6.4%) nor wortmannin (34.3 ± 4.8%) affected infarct size compared with group 6, consistent with our own and others' previous experience.

We assessed the influence of ROCK inhibition by Y-27632 on the Akt/eNOS salvage pathway by immunoblotting of risk zone myocardium following 35-min ischemia and 10-min reperfusion (Fig. 4). We observed that the application of 5 µmol/l Y-27632 during early reperfusion was associated with marked increases in the phospho-eNOS and phospho-Akt signals.


Figure 4
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  		Fig. 4. Western blot analyses of phosphorylated Akt and phosphorylated endothelial nitric oxide synthase (eNOS) in risk zone myocardium. Samples were harvested for analysis at baseline, after 35-min ischemia, or after 10-min reperfusion. Representative immunoblots present lanes in the same order (left to right) as in the histograms beneath. In the presence of Y-27632, phospho-eNOS and phospho-Akt signals were significantly augmented at 10-min reperfusion. *P < 0.05 vs. baseline (1-way ANOVA, n = 4 per group).

 
The application of ROCK inhibitors had little discernible effect on either heart rate or LV contractility (summarized as rate-pressure product). In infarct study 1, there was a tendency toward improved coronary flow rate during the early reperfusion period with fasudil and Y-27632 at all concentrations (Table 1). In infarct study 2, where treatment with Y-27632 was commenced only 5 min before reperfusion, there was a nonsignificant but noticeable effect of Y-27632 on coronary flow rate at 1-min reperfusion (16.4 ± 2.3 vs. 14.6 ± 1.5 ml/min in control group, P = 0.1). L-NAME treatment reduced coronary flow rate for 60 min after the onset of reperfusion. There was a 23% reduction in coronary flow rate measured at 1 min of reperfusion relative to that in the control; this was accompanied by an almost twofold reduction in the developed pressure at that time point (P < 0.05 vs. control). However, it is noteworthy that Y-27632, when coadministered with L-NAME, abrogated the depressant effects of L-NAME on coronary flow rate and rate-pressure product. Wortmannin alone depressed the hyperemic response at 1 min of reperfusion by 27% of its baseline value (P < 0.05).

ROCK Inhibition During Reperfusion Reduces Apoptotic Index

TUNEL positivity was assessed as an index of apoptosis following 35-min ischemia and 120-min reperfusion (Fig. 5). In preischemic LV myocardium sampled from areas corresponding to infarct regions, fewer than 0.5% of nuclei were TUNEL positive. Ischemia-reperfusion resulted in a significant increase in the number of TUNEL-positive cells in the infarct regions (3.4 ± 0.9% and 5.4 ± 1.3%, respectively; P < 0.05). Treatment with Y-27632 during early reperfusion significantly reduced the numbers of TUNEL-positive nuclei in the infarct regions.


Figure 5
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  		Fig. 5. Apoptotic nuclei were detected in myocardial sections by TUNEL reaction and confocal fluorescence microscopy. A: representative micrographs showing nuclear staining in risk zone myocardium corresponding to the central infarct zone before the onset of ischemia (A,a), after 35-min ischemia + 120-min reperfusion (A,b), and after 35-min ischemia + 120-min reperfusion with Y-27632 at early reperfusion (A,c). Nuclei were visualized with TO-PRO-3 (dark blue), and terminal dUTP nick-end labeling (TUNEL)-positive nuclei labeled with fluorescein appeared turquoise (marked with arrows). The addition of Y-27632 (5 µM) from 5 min before reperfusion until 10 min after reperfusion led to a significant reduction in the number of TUNEL-positive nuclei in the infarct region. B: quantitative analysis of nuclear fluorescence from nonrisk left ventricular myocardium and the central infarct region, sampled 120 min after reperfusion. In control myocardium subjected to 35-min ischemia and 120-min reperfusion, TUNEL fluorescence was significantly increased compared with the preischemia values in the infarcted zones. Treatment with 5 µM Y-27632 significantly attenuated TUNEL positivity. *P < 0.05 vs. corresponding preischemia value; #P < 0.05 vs. corresponding control 120R value (1-way ANOVA, n = 4 consecutive sections from each region, with 4 hearts in each treatment group).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These data provide definitive evidence that ROCK activation occurs during reperfusion. Although recent studies have shown that pharmacological inhibition of ROCK is cardioprotective against ischemia-reperfusion injury, the present study provides the first evidence that ROCK activation is a key determinant of irreversible tissue injury specifically during reperfusion. Moreover, inhibition of ROCK protects against ischemia-reperfusion injury exclusively during reperfusion through a mechanism dependent on constitutive PI3K/Akt and NO signaling. These findings lead us to suggest that ROCK activation could represent a reperfusion injury kinase by contributing to negative regulation of the PI3K/Akt/NO RISK pathway. This new appreciation of ROCK as a mediator of reperfusion injury has important implications for potential therapeutic manipulation of survival pathways in patients undergoing reperfusion for acute myocardial infarction.

Previous studies examining the protective effects of pharmacological ROCK inhibition during experimental myocardial infarction have shown benefits in a variety of treatment protocols including preischemic treatment (3, 7, 33) and intracoronary infusion during ischemia (26). In the present study, we confirmed that fasudil and Y-27632 conferred marked protection when administered throughout both ischemia and early reperfusion. However, we show for the first time that 5 µM Y-27632 is almost equally efficacious when administered only at the onset of reperfusion. There are several important implications of this observation. First, it is possible to conclude that the major deleterious actions of ROCK relevant to the development of early irreversible injury are mediated almost exclusively during reperfusion, rather than during ischemia. Second, in the absence of blood and extrinsic autonomic innervation, the buffer-perfused ex vivo heart model used here demonstrates that, during reperfusion, ROCK activation exhibits deleterious effects exclusively in the myocardial cell types rather than in blood-borne elements. Third, the brief administration of the ROCK inhibitor as an adjunct to reperfusion highlights an effective and realistically achievable time window for a potential therapeutic regimen.

Currently, much research is focused on the signal transduction pathways that are activated at reperfusion. An emerging paradigm of tissue salvage from ischemia-reperfusion injury invokes a role for the activation of salvage kinases (RISKs) that augment cellular resistance to the detrimental metabolic effects of abrupt reperfusion (9, 8, 13, 36). There is accumulating evidence that the PI3K/Akt/eNOS cassette is a prosurvival signal when recruited within the first few minutes of reperfusion (12, 32). Although the precise sequence of the distal mechanisms of protection is unknown, there is increasing evidence that this RISK pathway may be augmented by diverse protective maneuvers at reperfusion, including insulin (17), peptide growth factors (6), erythropoietin (14), 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) (34), adrenomedullin (10), natriuretic peptides (16, 35), and ischemic postconditioning (32). The present data are consistent with the proposal that ROCK inhibition encourages the downstream activation of constitutive NOS during the earliest moments of reperfusion and that this is a pivotal event in the cardioprotective cascade. A primary consequence of ROCK activation in blood vessels is attenuation of NO-mediated actions through inhibition of eNOS expression and translation (33), as well as arginase upregulation (22). In vascular smooth muscle cells, the use of fasudil ameliorated hypoxia-induced downregulation of eNOS expression (31). Both hydroxyfasudil and Y-27632 were reported to stimulate Akt serine 473 phosphorylation in human saphenous endothelial cells (33), hence suggesting ROCK also negatively regulates PI3K, upstream of Akt/eNOS. The suggestion that ROCK acts as a negative regulator of PI3K activation is of particular interest in relation to the present work. The mechanism through which ROCK inhibits PI3K is not known; however, the ROCK phosphorylation motif is found in the p110 catalytic subunit of PI3K and is also present in the plekstrin-homology (PH) binding domain of the p85 regulatory subunit (4, 33). Whereas there is some evidence that ROCK inhibitors increase NO production in cultured endothelial cells, their direct effects on vascular smooth muscle cell relaxation do not require NO participation since ROCK inhibits myosin light chain phosphatase, maintaining myosin light chain phosphorylation. Thus there may be dissociation between the vasodilator effects of Y-27632 and the cardioprotective action. Although NOS inhibition and PI3K/Akt inhibition per se reduced coronary flow in the earliest moments of reperfusion, these blockers did not affect the coronary flow response to Y-27632, although they effectively blocked the cardioprotective effects of Y-27632.

Important questions yet to be resolved relate to the temporal dynamics, mechanisms, and significance of ROCK activation following an ischemic insult. At present, the routine method for estimating ROCK activity in intact tissues is the assessment of phosphorylation of specific target proteins. The most commonly studied substrates are myosin phosphatase targeting subunit-1 (MYPT-1), myosin light chain, {alpha}-adducin, and ERM, the latter protein complex being used in this study. ERM proteins regulate actin filament interactions and are phosphorylated on carboxy-terminus threonine residues by ROCK (21). Previously, Sanada et al. (26) showed that, following a prolonged 60-min ischemic episode in the canine heart, there was marked elevation of phospho-MYPT-1. In the present study, our observation that phospho-ERM was upregulated during early reperfusion, assessed by immunoblotting, supports the general consensus that acute ischemia-reperfusion is a potent signal for ROCK activation. Furthermore, our data clearly show that ROCK activation occurred specifically after reperfusion and was sustained for 120 min after reperfusion in the infarct regions of the LV. Previous studies have shown prolonged phosphorylation of ROCK substrates following infarction, phospho-{alpha}-adducin, or phospho-ERM being upregulated 24 h (3) and 4 wk (31) after restoration of coronary blood flow. Thus the present study and earlier studies together suggest that ROCK activation occurs as an early feature of reperfusion, likely to be associated with the acute evolution of irreversible tissue injury, and is sustained during ventricular remodeling phase (19, 29).

At the end of the 2-h reperfusion period, immunohistochemical analysis of the region at risk also highlighted the antiapoptotic effects and ablated ROCK activity by reperfusion treatment with Y-27632. Whether the reduced TUNEL positivity at the end of 120-min reperfusion in Y-27632-treated hearts represents an unequivocal and biologically relevant reduction in apoptosis as a result of ROCK inhibition is arguable since TUNEL may not be an exclusive marker of apoptotic nuclei. Nevertheless, the rate of TUNEL positivity in control myocardium is broadly comparable to values reported in similar animal models of infarction (3, 27), and we believe that the reduction in TUNEL-positive nuclei after ROCK inhibition parallels the reduction in infarct size, representing a valid independent marker of irreversible cell injury. Recent studies in murine cardiomyocytes with overexpressed active ROCK-1 led to an increased caspase-3 dependent apoptosis (5). Investigations in noncardiac cells have elucidated roles for ROCK in mediating apoptotic events through actions on the cytoskeleton. For example, caspase-3 was shown to cleave and activate ROCK in a human T-cell line. ROCK activation then mediated the blebbing process through increased intracellular tension provided by inhibition of myosin light chain kinase and the resultant increase in myosin-actin interactions (28). However, any mechanistic links between ROCK and apoptosis in ischemia-reperfusion remain poorly understood. Speculatively, inhibition by ROCK of PI3K/Akt activity and NO generation might alter the balance of proapoptotic and antiapoptotic members of the Bcl protein family that are regulated by Akt (1), and this is currently the subject of more detailed study.

In conclusion, we have shown that ROCK activation in early reperfusion is a major contributory mechanism in irreversible reperfusion injury and that pharmacological inhibition of ROCK in early reperfusion markedly limits irreversible injury. Although this protective action of ROCK inhibition is apparently dependent on NO generation, the precise mechanism by which ROCK inhibition leads to NO generation, and the subcellular localization of this NO, is the focus of further studies. However, it is now clear that manipulation of the molecular events occurring at reperfusion could represent a therapeutic approach to maximize the benefits of reperfusion in acute myocardial infarction. We propose that ROCK activation is a deleterious reperfusion injury kinase and speculate that the balance of activities of injurious factors, such as ROCK, and RISK signals, such as PI3K/Akt, is a crucial determinant of tissue survival in reperfusion. The activation of ROCK at reperfusion predisposes to cell death by influencing the net balance of prosurvival and proinjury signals.


   ACKNOWLEDGMENTS
 
We express our gratitude to Helen Smith for valuable advice regarding the use of the confocal microscope and subsequent image analysis. We gratefully acknowledge the support provided by the British Heart Foundation (PG03/030).


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. F. Baxter, Division of Pharmacology, School of Pharmacy, Univ. of Cardiff, King Edward VII Ave., Cardiff CF10 3XF, UK (e-mail: baxtergf{at}cardiff.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 RESULTS
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