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(Circulation. 2000;101:1319.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Inhibition of Myosin Phosphatase by Upregulated Rho-Kinase Plays a Key Role for Coronary Artery Spasm in a Porcine Model With Interleukin-1ß

Tadashi Kandabashi, MD; Hiroaki Shimokawa, MD, PhD; Kenji Miyata, MD; Ikuko Kunihiro, BS; Yoji Kawano, PhD; Yuko Fukata, MD, PhD; Taiki Higo, MD; Kensuke Egashira, MD, PhD; Shosuke Takahashi, MD, PhD; Kozo Kaibuchi, MD, PhD; Akira Takeshita, MD, PhD

From the Departments of Cardiovascular Medicine (T.K., H.S., K.M., I.K., T.H., K.E., A.T.) and Anesthesiology and Critical Care Medicine (T.K., S.T.), Graduate School of Medical Sciences, Kyushu University, Fukuoka, and the Division of Signal Transduction, Nara Institute of Science and Technology (Y.K., Y.F., K.K.), Ikoma, Japan.

Correspondence to Hiroaki Shimokawa, MD, PhD, Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail shimo{at}cardiol.med.kyushu-u.ac.jp

	Abstract

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Background—We recently demonstrated that the Rho-kinase–mediated pathway plays an important role for coronary artery spasm in our porcine model with interleukin-1ß (IL-1ß). In this study, we examined whether or not Rho-kinase is upregulated at the spastic site and if so, how it induces vascular smooth muscle hypercontraction.

Methods and Results—Segments of the left porcine coronary artery were chronically treated from the adventitia with IL-1ß–bound microbeads. Two weeks after the operation, as reported previously, intracoronary serotonin repeatedly induced coronary hypercontractions at the IL-1ß–treated site both in vivo and in vitro, which were markedly inhibited by Y-27632, one of the specific inhibitors of Rho-kinase. Reverse transcription–polymerase chain reaction analysis demonstrated that the expression of Rho-kinase mRNA was significantly increased in the spastic compared with the control segment. Western blot analysis showed that during the serotonin-induced contractions, the extent of phosphorylation of the myosin-binding subunit of myosin phosphatase (MBS), one of the major substrates of Rho-kinase, was significantly greater in the spastic than in the control segment and that the increase in MBS phosphorylations was also markedly inhibited by Y-27632. There was a highly significant correlation between the extent of MBS phosphorylations and that of contractions.

Conclusions—These results indicate that Rho-kinase is upregulated at the spastic site and plays a key role in inducing vascular smooth muscle hypercontraction by inhibiting myosin phosphatase through the phosphorylation of MBS in our porcine model.

Key Words: vasospasm • muscle, smooth • kinase • myosin • phosphorylation

	Introduction

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The clinical importance of coronary artery spasm in the pathogenesis of ischemic heart disease is now widely accepted.^{1 2 3 4} However, the intracellular mechanism for the spasm still remains to be elucidated. We previously developed a porcine model of coronary spasm in which the spasm was repeatedly induced by serotonin or histamine at the atherosclerotic lesions made by a combination of endothelial injury and high-cholesterol feeding.^{5 6 7} We subsequently developed a porcine model in which long-term adventitial treatment with interleukin-1ß (IL-1ß), one of the major inflammatory cytokines, induces arteriosclerotic changes and vasospastic responses of the coronary artery.^{8 9 10 11 12 13} Because the histological changes and vasospastic responses in our porcine models are similar to those observed in humans,^{14 15} our models may be useful to examine the molecular mechanism of the spasm in humans.^{6 8 9}

Phosphorylation of myosin light chain (MLC) is one of the most important steps for vascular smooth muscle contraction.^{16 17} The classic concept of the mechanism of vascular smooth muscle contraction includes an activation of MLC kinase (MLCK) that leads to the phosphorylation of MLC and subsequent smooth muscle contraction.¹⁸ However, because the intracellular Ca²⁺ concentrations were not always proportional to the levels of MLC phosphorylation and smooth muscle contraction, an additional mechanism to regulate Ca²⁺ sensitivity has been proposed.¹⁹ Recently, evidence for the involvement of the small GTPase Rho in Ca²⁺ sensitivity in smooth muscle contraction was reported from several laboratories.^{20 21 22}

The molecular mechanism of MLC phosphorylation regulated by Rho was largely unknown, but recent analyses revealed that Rho regulates MLC phosphorylation through its target protein, Rho-kinase, and the myosin-binding subunit (MBS) of MLC phosphatase (MLCPh).^{23 24} Indeed, studies in vitro suggested that Rho activates Rho-kinase, which then phosphorylates MBS and results in the inhibition of MLCPh.²³ We have recently demonstrated in our porcine model with IL-1ß that MLC phosphorylations (on stimulation by serotonin) are enhanced at the spastic site^{12 13} and that hydroxyfasudil, a specific Rho-kinase inhibitor, exerts an inhibitory effect on the spasm both in vivo and in vitro.¹³ However, the molecular mechanism for the spasm in our model remains to be elucidated.

This study was thus designed to examine whether or not Rho-kinase is upregulated at the spastic site and if so, how it induces vascular smooth muscle hypercontraction.

	Results

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In Vivo Study
Two weeks after the operation, serotonin 10 µg/kg IC repeatedly caused hyperconstriction at the IL-1ß–treated site in vivo (Figures 1 and 2). Pretreatment with Y-27632 did not significantly change the baseline heart rate or blood pressure (data not shown). Y-27632 dose-dependently inhibited serotonin-induced coronary hyperconstriction at the IL-1ß–treated site in vivo, whereas at the control site, its inhibitory effect on serotonin-induced constriction was not evident (Figures 1 and 2).

View larger version (88K): [in this window] [in a new window]   Figure 1. Coronary arteriograms 2 weeks after application of IL-1ß under control conditions (Control) and after nitroglycerin 10 µg/kg IC (NG). Intracoronary serotonin 10 µg/kg (S10) induced hyperconstriction at IL-1ß–treated site (white arrow), which was dose-dependently inhibited by pretreatment with intracoronary Y-27632 (10, 30, and 100 µg/kg, Y10, Y30, and Y100, respectively) (bottom 3 panels).

View larger version (11K): [in this window] [in a new window]   Figure 2. Inhibitory effect of Y-27632 on serotonin-induced constrictions in vivo as evaluated by coronary arteriography. Y-27632 dose-dependently inhibited serotonin-induced hyperconstrictions at IL-1ß–treated segment, whereas its inhibitory effect was not evident in control segment. Results are expressed as mean±SEM.

Organ Chamber Experiment
Serotonin 1 µmol/L induced a contraction in the IL-1ß–treated and control coronary segments without endothelium, which rapidly developed and reached a maximum after 5 to 8 minutes. Serotonin caused hypercontractions in the IL-1ß–treated segments compared with the control segments, which were markedly inhibited by Y-27632 (Figure 3).

View larger version (12K): [in this window] [in a new window]   Figure 3. Inhibitory effect of Y-27632 on serotonin (1 µmol/L)–induced contractions in vitro. Y-27632 inhibited serotonin-induced hypercontractions of IL-1ß–treated segment, and it also significantly inhibited contractions in control segment. Results are expressed as mean±SEM.

RT-PCR Analysis
The expected sizes of the bands for Rho-kinase were detected in both the spastic and control coronary segments. However, the density of PCR products from Rho-kinase mRNA (normalized to that from ß-actin mRNA) was significantly higher in the spastic than in the control segment (Figure 4).

View larger version (8K): [in this window] [in a new window]   Figure 4. RT-PCR analysis for Rho-kinase mRNA expression in control and IL-ß–treated coronary segments. Results are expressed as mean±SEM.

MBS Phosphorylations
The extent of MBS phosphorylation was measured when the serotonin-induced contraction of each ring (without endothelium) reached a maximum. Western blot analysis showed that on stimulation by serotonin, MBS phosphorylation was significantly increased in the IL-1ß–treated coronary segment compared with the control segment (Figure 5). In the spastic coronary segments, the enhanced MBS phosphorylation was markedly inhibited by Y-27632 to levels under control conditions (Figure 5). Importantly, there was a highly significant positive correlation between the extent of MBS phosphorylations and that of serotonin-induced contractions (Figure 6).

View larger version (19K): [in this window] [in a new window]   Figure 5. Western blot analysis for MBS phosphorylation of porcine coronary artery with or without serotonin (1 µmol/L). MBS phosphorylation was significantly increased in response to serotonin in IL-1ß–treated segment compared with control segment. Y-27632 significantly suppressed MBS phosphorylation in response to serotonin in IL-1ß–treated segment. Results are expressed as mean±SEM.

View larger version (14K): [in this window] [in a new window]   Figure 6. Correlation between extent of MBS phosphorylations (arbitrary units) and that of serotonin-induced contractions (percent of contraction to 62 mmol/L KCl). There was a highly significant positive correlation between the 2 values.

	Discussion

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The novel findings of the present study were that (1) the expression of Rho-kinase was upregulated at the spastic coronary segment, (2) coronary spasm was associated with an enhanced MBS phosphorylation that should have resulted in the inhibition of MLCPh, and (3) Rho-kinase mediated this MBS phosphorylation, resulting in the occurrence of smooth muscle hypercontraction. Thus, the present study clearly demonstrates that enhanced inhibition of MLCPh by upregulated Rho-kinase plays a key role in the molecular mechanisms of coronary spasm in our porcine model (Figure 7).

View larger version (25K): [in this window] [in a new window]   Figure 7. Working hypothesis on intracellular mechanisms for coronary artery spasm. PLC indicates phospholipase C; DG, diacylglycerol; IP3, inositol 1,4,5-triphosphate; CaM, calmodulin; Cat., catalytic subunit; M20, 20-kDa regulatory subunit; +, stimulation; and -, inhibition. For occurrence of spasm, Rho-kinase–mediated pathway may play an important role, whereas contribution of intracellular Ca2+ release may be minimal. Rho-kinase appears to be upregulated at spastic site and suppresses MLCPh through its MBS phosphorylations, resulting in enhanced MLC phosphorylations and subsequent hypercontractions or spasms of coronary artery.

Inhibition of MLCPh Through MBS Phosphorylation in the Spastic Coronary Artery
We recently reported that coronary artery spasm is associated with an enhanced and sustained MLC monophosphorylation and the appearance of MLC diphosphorylation.^{12 13} The level of MLC phosphorylation is determined by a balance between MLC phosphorylation by MLCK and dephosphorylation by MLCPh.^{17 25} Seto et al²⁶ previously suggested that the generation of diphosphorylated MLC may be caused in part by inhibition of MLCPh in smooth muscle cells. They also showed that treatment with calyculin A, an inhibitor of phosphatases, including MLCPh,^{27 28} potently induced MLC diphosphorylation in smooth muscle cells without an increase in intracellular Ca²⁺ levels.²⁶ Furthermore, we recently found that direct increase in the intracellular Ca²⁺ levels by calcium ionophore does not result in an increase in diphosphorylated MLC (unpublished data). These lines of evidence suggest that inhibition of MLCPh activity is essential for the occurrence of coronary artery spasm.

In the present study, MBS phosphorylation at the IL-1ß–treated site in response to serotonin was significantly increased, suggesting that MLCPh activity is significantly suppressed in the spastic compared with the control segment, resulting in an increase in MLC phosphorylations on stimulation by serotonin (Figure 7). In contrast, the extent of the MBS phosphorylations under control conditions was comparable between the spastic and the control segments, which was consistent with our previous findings that the extents of MLC monophosphorylation under control conditions were comparable between the 2 sites.^{12 13}

Enhanced MBS Phosphorylation Caused by Upregulated Rho-Kinase
In the present study, we demonstrated that the expression of Rho-kinase mRNA was significantly upregulated in the spastic compared with the control segment. Smooth muscle MLCPh consists of a 38-kDa catalytic subunit, the 130-kDa MBS, and a 21-kDa subunit.^{29 30} MBS serves as a targeting subunit of MLCPh to myosin and enhances the activity of the enzyme toward myosin.²⁹ Recently, we reported that Rho-kinase phosphorylates MBS and reduces the MLCPh activity in vitro.²³ When an activated mutant of Rho was expressed in NIH3T3 fibroblasts, the extent of MLC phosphorylation was increased together with an increase in MBS phosphorylation.²³ Taken together, these findings suggest that MBS phosphorylation is mediated by Rho-kinase, resulting in inhibition of MLCPh and a subsequent increase in MLC phosphorylations (Figure 7).

Indeed, in the present study, the hypercontractions to serotonin were dose-dependently inhibited by Y-27632, one of the specific inhibitors of Rho-kinase both in vivo and in vitro.³¹ Furthermore, there was a highly significant positive correlation between the extent of MBS phosphorylations and that of the serotonin-induced contractions. We also recently demonstrated that hydroxyfasudil, another specific inhibitor of Rho-kinase, also dose-dependently inhibited the hypercontractions to serotonin both in vivo and in vitro.¹³ Thus, it is highly possible that in our porcine model, upregulated Rho-kinase inhibits MLCPh through MBS phosphorylation, resulting in the occurrence of coronary artery spasm (Figure 7).

We have previously shown in the present model that the coronary constriction in response to prostaglandin F₂ is resistant to the blockade of protein kinase C (PKC) and is not augmented at the IL-1ß–treated site, whereas that to serotonin or histamine is sensitive to the blockade of PKC and is augmented at the IL-1ß–treated site.¹⁰ Thus, the PKC-mediated pathway for vascular smooth muscle contraction is apparently involved in the molecular mechanism for coronary artery spasm in our model, although the relationship between PKC and Rho-kinase remains to be elucidated (Figure 7).

Although Rho-kinase is one of the major regulators of vascular smooth muscle contraction,^{13 22 23 32} other regulators, including the C-kinase–potentiated inhibitor of myosin phosphatase (CPI 17)^{33 34} and arachidonic acid,³⁵ might also be involved in smooth muscle hypercontraction (Figure 7). The possible involvement of those mechanisms in the pathogenesis of coronary spasm remains to be examined.

In summary, we were able to demonstrate that Rho-kinase is upregulated at the spastic site and mediates coronary spasm by inhibiting MLCPh through its MBS phosphorylation. The detailed molecular mechanism(s) for the upregulation of Rho-kinase in the inflammatory/arteriosclerotic coronary segment remains to be examined in a future study.

	Methods

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This experiment was reviewed by the Committee on Ethics in Animal Experiments of the Kyushu University School of Medicine and was carried out according to the Guidelines for Animal Experiments of the Kyushu University School of Medicine and the Law (No. 105) and Notification (No. 6) of the Japanese Government.

Animal Preparation
Male Yorkshire pigs weighing 25 to 30 kg were sedated with ketamine hydrochloride (12.5 mg/kg IM) and anesthetized with sodium pentobarbital (25 mg/kg IV). The animals were then intubated and ventilated with room air, and oxygen was supplemented by a positive-pressure respirator (Shinano Inc). Under aseptic conditions, a left thoracotomy was performed, and the proximal segments of the left anterior descending and circumflex coronary arteries were carefully dissected. The dissected segments of the coronary arteries were gently wrapped with cotton mesh that had absorbed 0.05 mL of sepharose bead suspension with recombinant IL-1ß (2.5 µg).^{8 9 10} We previously confirmed that treatment with control beads alone causes no significant arteriosclerotic changes or vasospastic responses of the porcine coronary artery.^{8 9 10}

Preparation of IL-1ß Beads
IL-1ß beads were prepared as previously reported.^{8 9} Briefly, 1 g of sepharose microbeads (45 to 165 µm in diameter, Pharmacia) was added to 50 mL of 1 mmol/L HCl solution and resuspended in 20 mL NaHCO₃/NaCl solution with 1 mg IL-1ß. The beads were allowed to bind with IL-1ß at room temperature for 1 hour and then at 4°C overnight. After centrifugation at 1200 rpm for 5 minutes, the supernatant was separated, and the concentration of the remaining IL-1ß in the supernatant was measured by ELISA.^{8 9} The IL-1ß–bound beads in the pellet were resuspended in 20 mL NaHCO₃/NaCl solution and centrifuged 4 times at 1200 rpm for 5 minutes. Then IL-1ß–bound beads were resuspended with Tris-HCl buffer solution for 1 hour and finally washed and resuspended so that the final concentration of IL-1ß was 50 µg/mL. All preparations were performed under sterile conditions.^{8 9 10}

In Vivo Experiment
Two weeks after the operation, the animals were again anesthetized and ventilated as described above, and selective coronary arteriography was performed. A preshaped Judkins catheter was inserted into the right or left femoral artery, and then coronary arteriography in a left anterior oblique view was performed. During the experiments, heparin (3000 U IV) was administered every 60 minutes, and ECGs (leads I, II, III, V₁, and V₆), along with mean arterial pressure and heart rate, were continuously monitored and recorded on a pen recorder (NEC San-Ei).

Coronary arteriography was performed with the Toshiba cineangiography system (DG-15GB/CAS-CA, Toshiba Medical Inc). The angiograms were recorded on 35-mm cine film (Varicath I) at 48 frames per second. The angle of the projection, the posture of the animal, and the distance from the x-ray focus to the animal and that from the animal to the image intensifier were carefully kept constant during the experiment.

The cineangiograms were projected onto a screen with a cine projector (ELX-35CB, Nishimoto Sangyo), and an end-diastolic frame was selected and printed. The coronary luminal diameters were measured with a caliper. Excellent correlations between repeated measurements and between different observers with this technique were previously confirmed.⁸ The degree of vasoconstricting response was expressed as the percent decrease in the luminal diameter from the control level.

The following protocols were examined in the coronary angiographic study in vivo (n=6). First, coronary arteriography was performed under control conditions. Second, coronary vasoconstricting responses to intracoronary serotonin (10 µg/kg) were examined. Coronary arteriography was performed 2 minutes after intracoronary administration of serotonin. Third, intracoronary administration of Y-27632, one of the specific inhibitors of Rho-kinase,³¹ was performed at 3 different doses (10, 30, and 100 µg/kg). Two minutes after the intracoronary administration of Y-27632, the coronary vasomotion to serotonin was again evaluated at each dose of the Rho-kinase inhibitor. Coronary diameter was measured at the segments treated with IL-1ß as well as at untreated segments of comparable diameter.^{7 8 9 10}

Organ Chamber Experiment
Three to 4 days after the in vivo experiments, when the effects of the angiographic study (including that of Y-27632) had completely disappeared, the animals were sedated with ketamine hydrochloride (12.5 mg/kg IM), euthanized with a lethal dose of sodium pentobarbital, and exsanguinated, and then the heart was excised. The coronary arteries at the IL-1ß–treated and control sites were carefully dissected and cleaned of any perivascular tissue, and the endothelium was removed by gentle rubbing of the luminal surface with a cotton swab and cut into rings measuring 4 mm in length.³⁶ The strips were fixed vertically between hooks in an organ bath of 20-mL capacity containing Krebs-Henseleit solution, which was maintained at 37°C and aerated with a mixture of 95% O₂/5% CO₂.^{12 13} The hook anchoring the upper end of the strip was connected to the lever of a force transducer (Nihon-Kohden). The resting tension was adjusted to 5 g.^{12 13} KCl solution (62 mmol/L) was applied every 15 to 20 minutes until the amplitude of the contraction reached a constant value. The contractions to serotonin were then examined in the absence and presence of Y-27632 (10^-5 mol/L), which was added 5 minutes before addition of serotonin. The developed tension was represented as a percentage of that attained in the last contraction with 62 mmol/L KCl.^{12 13} The coronary specimens were removed from the hook at the maximal contractions and were immediately frozen by immersion in acetone containing 10% trichloroacetic acid (TCA) cooled with dry ice for later Western blot analysis of MBS phosphorylations.

Measurement of Rho-Kinase mRNA
The total RNA was isolated from smooth muscle cells of the spastic and control coronary segments after removal of the endothelium and the adventitia. Possible contaminating genomic DNA was digested by RNase-free DNase. Total RNA (1 µg) was incubated for 60 minutes at 37°C for reverse transcription (RT) reaction in a total volume of 33 µL. An aliquot (5 µL) of RT product was used for polymerase chain reaction (PCR) amplification in a total volume of 100 µL. The thermal cycle profile used in this study was (1) denaturing for 30 seconds at 94°C, (2) annealing primers for 90 seconds at 55°C, and (3) extending the primers for 30 seconds at 72°C. The sequence of the primer for RT-PCR analysis of porcine Rho-kinase used in this study has been reported previously by Nishimura et al.³⁷ The PCR amplification was performed for Rho-kinase for 30 cycles and for ß-actin (as an internal control) for 25 cycles. These amplifications were performed in the linear relationship range between signal cycle number and intensity of RT-PCR products (data not shown). A portion (10 µL) of the PCR mixture was electrophoresed in 2% agarose gel in TAE buffer (40 mmol/L Tris-acetate, pH 8.5, 2 mmol/L EDTA).The gel was stained with ethidium bromide and then photographed. For the quantitative analysis, the density of bands was measured with an NIH image analyzer, then the levels of PCR products were normalized to PCR products for ß-actin.

Western Blot Analysis of MBS Phosphorylations
One of the major sites for phosphorylation of MBS by Rho-kinase both in vitro and in vivo has been identified as Ser-854, and we have developed an antibody that specifically recognizes MBS phosphorylated at Ser-854 (Kawano et al, unpublished observations). The extent of MBS phosphorylation in the strip was measured by SDS-PAGE, followed by electrophoretic transfer of the proteins to a nitrocellulose membrane.²³ The amounts of phosphorylated MBS (MBS-P) in each sample were quantified by immunoblot procedures.²³

The frozen coronary specimens obtained in the organ chamber experiments were washed 3 times with acetone containing 10 mmol/L dithiothreitol (DTT) to remove the TCA and dried. The dried ring was cut into small pieces and exposed to 200 µL of SDS-PAGE sample buffer for extraction. The extracted samples (20 µg protein in each sample) were subjected to SDS-PAGE/immunoblot analysis with the specific MBS-P antibody.²³ The region containing MBS-P was visualized with an ECL Western blotting luminol reagent (Santa Cruz Biotechnology).

Drugs
The following drugs were used: recombinant human IL-1ß (Otsuka Pharmaceutical Co), 5-hydroxytryptamine (serotonin), nitroglycerin (Nihon-Kayaku Pharmaceutical), and Y-27632 (Yoshitomi Pharmaceutical).

Statistical Analysis
The results are expressed as mean±SEM. Throughout the text, n represents the number of animals tested. A repeated-measures ANOVA was performed to evaluate the global statistical significance, and if a significant F value was found, Scheffé’s test was performed to identify the differences among the groups. The relationship between the extent of MBS phosphorylation and that of the serotonin-induced contraction was analyzed by linear regression analysis. A value of P<0.05 was considered to be statistically significant.

	Acknowledgments

 
This work was supported in part by grants from the Japanese Ministry of Education, Science, Sports, and Culture, Tokyo, Japan, and the Japanese Ministry of Health and Welfare, Tokyo, Japan. The authors wish to thank Dr J. Nishimura, Department of Molecular Cardiology, Kyushu University School of Medicine, for a generous gift of RT-PCR primers for Rho-kinase, and Drs T. Yamawaki, E. Tanaka, Y. Etoh, and K. Morishige for cooperation in this study. We also thank S. Tomita and E. Gunshima for their excellent technical assistance.

	Footnotes

 
The Methods section of this article can be found at http://www.circulationaha.org

Received August 5, 1999; revision received September 23, 1999; accepted October 6, 1999.

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