Enhanced Myosin Light Chain Phosphorylations as a Central Mechanism for Coronary Artery Spasm in a Swine Model With Interleukin-1β
Background Although coronary artery spasm plays an important role in a wide variety of ischemic heart diseases, the intracellular mechanism for the spasm remains to be clarified. We examined the role of myosin light chain (MLC) phosphorylations, a key mechanism for contraction of vascular smooth muscle, in our swine model with interleukin-1β (IL-1β).
Methods and Results IL-1β was applied chronically to the porcine coronary arteries from the adventitia to induce an inflammatory/proliferative lesion. Two weeks after the operation, intracoronary serotonin repeatedly induced coronary hyperconstrictions at the IL-1β-treated site both in vivo and in vitro, which were markedly inhibited by fasudil, an inhibitor of protein kinases, including protein kinase C and MLC kinase. Western blot analysis showed that during serotonin-induced contractions, MLC monophosphorylation was significantly increased and sustained in the spastic segment compared with the control segment, whereas MLC diphosphorylation was noted only in the spastic segment. A significant correlation was noted between the serotonin-induced contractions and MLC phosphorylations. Both types of MLC phosphorylation were markedly inhibited by fasudil. In addition, MLC diphosphorylation was never induced by a simple endothelium removal in the normal coronary artery, whereas enhanced MLC phosphorylations in the spastic segment were noted regardless of the presence or absence of the endothelium.
Conclusions These results indicate that enhanced MLC phosphorylations in the vascular smooth muscle play a central role in the pathogenesis of coronary spasm in our swine model.
Coronary artery spasm plays an important role in a wide variety of ischemic heart diseases, not only in variant angina but also in unstable angina, myocardial infarction, and sudden death.1 However, the intracellular mechanism for the spasm remains to be clarified. We previously developed a swine model of coronary spasm, in which the spasm was repeatedly induced by serotonin or histamine at the atherosclerotic site made through a combination of endothelial injury and high-cholesterol feeding.2 We recently demonstrated that chronic treatment with IL-1β, one of the major inflammatory cytokines, causes arteriosclerosis-like changes and vasospastic responses of the coronary artery, which indicates the importance of inflammatory/proliferative changes of the coronary artery in the pathogenesis of the spasm.3,4 Because the spasm induced in our swine models has many similarities to that observed in humans, our models may be useful to elucidate the pathogenesis of the spasm in humans.2–5 We subsequently confirmed that the intracellular signaling pathway mediated by PKC in the vascular smooth muscle is substantially involved in the pathogenesis of the spasm in our models.5,6
Phosphorylation of MLC is one of the most important steps for vascular smooth muscle contraction.7–9 Vascular smooth muscle contraction is initiated by Ca2+/calmodulin-activated MLCK with subsequent phosphorylation of the 20-kD regulatory MLC.7–9 Phosphorylation of the regulatory MLC then activates myosin Mg2+-ATPase and permits cross-bridge cycling, which leads to force generation and contraction.7–9 It was reported that MLC phosphorylation was augmented in canine vasospastic cerebral artery after experimental subarachnoid hemorrhage10 or in hyperplastic rabbit carotid artery after balloon injury.11 However, it remains to be clarified whether MLC phosphorylations are quantitatively and/or qualitatively altered in the spastic coronary artery. It is important to clarify this point for understanding of the pathogenesis of coronary artery spasm.
In this study, we examined the possible alterations in the MLC phosphorylations at the spastic site of the coronary artery in our swine model with IL-1β.3,4,6 We also examined the inhibitory effects of fasudil, an inhibitor of MLCK and PKC, which inhibits both vascular contractions and MLC phosphorylations to a variety of agents.12,13 Fasudil is known to be a potent inhibitor of MLC phosphorylations and has been clinically used in Japan in the treatment of the cerebral vasospasm after subarachnoid hemorrhage.12,13
Twenty-five 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; oxygen was supplemented via 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 solution with recombinant human IL-1β (2.5 μg).3,4,6 We previously confirmed that the treatment with control beads alone caused no significant arteriosclerotic changes or vasospastic responses of the porcine coronary artery.3,4,6
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.
Preparation of IL-1β Beads
IL-1β beads were prepared as follows3,4: 1 g of Sepharose microbeads (45 to 165 μm in diameter) was added to 50 mL of 1 mmol/L HCl solution and resuspended in 20 mL NaHCO3/NaCl solution with 1 mg of IL-1β. The beads were allowed to bind with IL-1β at room temperature for 1 hour and then at 4 OC overnight. After centrifugation at 1200 rpm for 5 minutes, the supernatant was separated, and the concentration of the remaining IL-1β in the supernatant was measured with an ELISA.14 The IL-1β–bound beads in the pellet were resuspended in 20 mL of NaHCO3/NaCl solution and centrifuged four times at 1200 rpm for 5 minutes. Then, the IL-1β–bound beads were resuspended with Tris-HCl buffer solution for 1 hour and finally washed and resuspended so the concentration of IL-1β was 50 μg/mL. All preparations were performed under sterile conditions.3,4,6
Because in our bead preparation most of the IL-1β molecules were bound inside the beads by a covalent bond at the amino residues of the proteins, ≤1.2% of the IL-1β molecules were actually bound to the surface of the beads and biologically active. Thus, when 2.5 μg of IL-1β bound to the beads was applied to the coronary artery, ≤30 ng of IL-1β was biologically active.3
In Vivo Experiment
Two weeks after the operation, we performed coronary arteriographic study in which the coronary artery vasomotion was examined in vivo.
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 under control conditions and after administration of 10 μg/kg nitroglycerin IC. ECGs (leads I, II, III, V1, and V6), along with mean arterial pressure and heart rate, were recorded continuously during the experiments. Coronary arteriography was repeated 2 minutes after the intracoronary administration of serotonin (10 μg/kg), when the serotonin-induced coronary vasoconstriction peaked.3,4,6 Then, intracoronary administration of fasudil at three different doses (1, 3, and 10 μg/kg) was performed, and the coronary vasomotion to serotonin was again evaluated after each dose of fasudil. In the previous studies, we have confirmed that the serotonin-induced vasospasm in the spastic segment and the serotonin-induced constriction in the control segment are reproducible at an interval of 20 minutes.3,4,6
The cineangiograms were projected on a screen using a cineprojector (ELX-35CB; Nishimoto Sangyou Inc), and an end-diastolic frame was selected and printed.3–6 The coronary luminal diameters were measured with computer-assisted quantitative coronary angiogram system (CAD 98, Elmo Co). The degree of constrictive response was expressed as the percent decrease in the luminal diameter from the control level. The coronary diameter was measured at the segments treated with IL-1β as well as at the untreated segments of a comparable baseline diameter.3–6
In Vitro Experiment
At 3 to 4 days after the in vivo experiments, when the effects of fasudil had totally disappeared, the animals were sedated with ketamine hydrochloride (12.5 mg/kg IM), killed with a lethal dose of sodium pentobarbital, and exsanguinated; then, the heart was excised. The coronary arteries at the IL-1β–treated and control sites were carefully dissected, cleaned of any perivascular tissue, and cut into rings measuring ≈4 mm in length. In some of the rings, the endothelium was removed by gently rubbing the luminal surface with a cotton swab.14 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. The hook anchoring the upper end of the strip was connected to the lever of a force transducer (Nihon-Kohden Kogyo). The resting tension was adjusted to 5 g of KCl solution (62 mmol/L) was applied every 15 to 20 minutes until the amplitude of the contraction reached a constant value. The tension was represented as a percentage of the tension attained in the last precontraction with 62 mmol/L KCl. The presence or absence of the endothelium was confirmed by the presence or absence of the relaxation to bradykinin (10−7 mol/L) during a contraction evoked by prostaglandin F2α. The contractions to serotonin were examined in the absence and presence of different doses of fasudil (10−6 and 10−5 mol/L), which was added 10 minutes before addition of serotonin.
Measurements of MLC Phosphorylations
The extent of MLC phosphorylation in the strips was measured through separation of nonphosphorylated, monophosphorylated, and diphosphorylated forms by glycerol-PAGE, followed by electrophoretic transfer of the proteins to a nitrocellulose membrane. The relative amounts of each form were quantified by immunoblot procedures, as described previously.13
Rings mounted for isometric studies were frozen by immersion in acetone containing 10% trichloroacetic acid and 10 mmol/L dithiothreitol cooled with dry ice. Frozen tissues were washed twice with acetone containing 10 mmol/L dithiothreitol to remove the trichloroacetic acid and then dried. The dried ring was cut into small pieces, exposed to 80 μL of glycerol-PAGE sample buffer for purposes of extraction, and then passed through a 0.45-μm membrane filter. The urea-solubilized samples were subjected to glycerol-PAGE/immunoblot analysis, using the specific MLC antibody.15 The region containing MLC was visualized using an ECL Western blotting system (Amersham). The extent of MLC phosphorylation is expressed as the percent MLC in the monophosphorylated and diphosphorylated forms, respectively. The validity of the MLC phosphorylation assay system was demonstrated by using MLC-specific phosphatase purified from chicken gizzard.15
We used the drugs 5-hydroxytryptamine (serotonin), prostaglandin F2α (Sigma Chemical), and fasudil [1–5-(isoquinoline-sulfonyl)-homopiperazine] (Asahi Chemical).12,13 Dilution was done with a physiological salt solution.
The results were expressed as mean±SEM. Throughout the text, n represents the number of animals tested. A repeated-measures ANOVA was performed to evaluate global statistical significance, and if a significant F value was found, Scheffé’s test was performed to identify the difference among the groups. A value of P<.05 was considered to be statistically significant.
In Vivo Study
Two weeks after the operation, serotonin (10 μg/kg IC) repeatedly caused hyperconstriction at the IL-1β–treated site in vivo (Fig 1⇓). The pretreatment with fasudil did not significantly change baseline heart rate or blood pressure (data not shown). Fasudil dose-dependently inhibited the serotonin-induced coronary spasm at the IL-1β–treated site in vivo, whereas at the control site its inhibitory effect on the serotonin-induced contraction was not evident (Figs 1⇓ and 2⇓).
Organ Chamber Experiments
In the organ chamber experiments, serotonin (1 μmol/L) induced a contraction of the IL-1β–treated and control coronary segments with endothelium, which rapidly developed and reached a maximum after the first 5 to 8 min, followed by a slight decrease and then by a sustained response. Serotonin caused hypercontractions in the IL-1β–treated coronary segments compared with the control segments in vitro (Fig 3⇓). Fasudil dose-dependently inhibited the serotonin-induced contractions in vitro (Fig 3⇓).
The extents of MLC monophosphorylation and diphosphorylation were measured when the serotonin-induced contraction of each ring (with endothelium) reached a maximum. Western blot analysis showed that MLC monophosphorylation was significantly increased in the IL-1β–treated coronary segment than in the control segment (Figs 4⇓ and 5⇓), whereas MLC diphosphorylation was noted only at the IL-1β–treated segment (Figs 4⇓ and 5⇓). In the spastic segment, the enhanced MLC monophosphorylations were markedly and dose-dependently inhibited by fasudil to the levels under control conditions, whereas the MLC diphosphorylations were abolished by fasudil (Fig 5⇓). In contrast, in the control segment fasudil inhibited the increased MLC monophosphorylations to the levels under control conditions (Fig 5⇓).
Correlation Between Serotonin-Induced Contractions and MLC Phosphorylations
A significant correlation was noted between the serotonin-induced contractions and the increase in MLC monophosphorylations (Fig 6⇓). Similarly, a significant correlation was noted between the serotonin-induced contractions and the increase in MLC diphosphorylations (Fig 6⇓).
Time Course of MLC Phosphorylations
Analysis of the time course of the MLC phosphorylations demonstrated that the elevated levels of MLC monophosphorylation were sustained in the IL-1β–treated spastic segment compared with the control segment (Fig 7⇓), whereas the MLC diphosphorylation was again noted only in the spastic segment (Fig 7⇓).
Effect of Endothelium Removal
Because the above in vitro experiments were performed in coronary segments with endothelium, the effect of endothelium removal was examined in our present model. Endothelium removal augmented the serotonin-induced contractions significantly but equally in both the control and IL-1β–treated coronary segments (Fig 8⇓). Concerning the MLC phosphorylations, regardless of the presence or absence of the endothelium, MLC monophosphorylation was greater in the IL-1β–treated spastic segment than in the control segment, and MLC diphosphorylation was noted only in the spastic segment (Fig 9⇓).
The novel findings of the present study were that (1) coronary spasm was associated with an enhanced and sustained MLC monophosphorylation and an appearance of MLC diphosphorylation, (2) the occurrence of the spasm was inhibited by fasudil together with a reduction in MLC monophosphorylation and an abolishment of MLC diphosphorylation, and (3) those enhanced MLC phosphorylations at the spastic site were noted regardless of the presence or absence of the endothelium. Thus, the present study clearly demonstrated that the enhanced MLC phosphorylations in the vascular smooth muscle play a central role in the pathogenesis of coronary spasm. To our knowledge, this is the first report that demonstrated the intracellular mechanism for coronary spasm in relation to MLC phosphorylations.
Mechanism for the Enhanced MLC Phosphorylations in the Spastic Coronary Artery
The level of MLC phosphorylation is determined by a balance between MLC phosphorylation by MLCK and dephosphorylation by MLC phosphatase7,8 (Fig 10⇓). In the present study, diphosphorylation of MLC was noted only in the spastic coronary artery. We have previously suggested that the generation of diphosphorylated MLC may be caused in part by the inhibition of MLC phosphatase in smooth muscle cells.11 We showed that the treatment with 10 to 100 nmol/L calyculin A, a protein phosphatase inhibitor, potently induced MLC diphosphorylation in smooth muscle cells without an increase in intracellular calcium levels.16 We also found that the direct increase in intracellular calcium levels by the calcium ionophore did not result in an increase in diphosphorylated MLC (unpublished data). Noda et al17 reported that in permeabilized porcine aortic smooth muscle cells, the increase in intracellular calcium levels caused monophosphorylation of MLC alone, whereas additional treatment with GTP-γS, which is thought to inactivate MLC phosphatase, caused both monophosphorylation and diphosphorylation of MLC. These results suggest that inhibition of MLC phosphatase activity is essential for induction of MLC diphosphorylation in smooth muscle cells. We consider that regulatory mechanism of MLC phosphatase may be altered in the spastic coronary artery and a resultant inactivation of MLC phosphatase may cause both the enhanced and sustained MLC monophosphorylation and the appearance of MLC diphosphorylation, which result in the occurrence of coronary artery spasm (Fig 10⇓). In contrast, the contribution of Ca2+/calmodulin-MLCK pathway to the occurrence of coronary spasm may be minimal (Fig 10⇓).
We previously demonstrated that PKC-mediated pathway is substantially involved in the pathogenesis of the spasm, whereas the contribution of Ca2+ release from intracellular store site may be minimal5,6 (Fig 10⇑). Ikebe and Brozovich18 recently reported that direct injection of PKC into skinned smooth muscle cells induced force generation and MLC phosphorylation through inhibition of MLC phosphatase. PKC-mediated pathway (probably including inhibitory mechanism of MLC phosphatase) may be augmented in the spastic coronary artery compared with that in the control artery. However, inhibitory mechanism of MLC phosphatase remains to be clarified. Eto et al19 reported that a novel MLC phosphatase inhibitor that is potentiated by PKC was isolated from porcine aorta media. Kimura et al20 reported that rho kinase phosphorylated the 130-kD subunit of MLC phosphatase and reduced its activity. Further study is necessary to elucidate the inhibitory mechanism of MLC phosphatase in the spastic coronary artery. Our working hypothesis for the intracellular mechanism for coronary spasm is shown in Fig 10⇑.
Roles of MLC Monophosphorylations and Diphosphorylations in the Vascular Smooth Muscle Hypercontraction
In the present study, because a significant correlation was noted between serotonin-induced contractions and MLC monophosphorylations and diphosphorylations, both types of MLC phosphorylation may contribute to the occurrence of coronary spasm. Ikebe and Hartshorne21 found that phosphorylation of the second site of MLC further increased the actin-activated Mg2+-ATPase activity of myosin in vitro. We also reported that the second site of phosphorylation of MLC augmented the tension generation of the rabbit aorta.22 Indeed, in the present study, diphosphorylated MLC was noted only in the spastic segment during coronary spasm. However, the relative contribution of MLC monophosphorylations and diphosphorylations to the occurrence of coronary spasm remains to be further clarified.
Phenotype modulation of vascular smooth muscle cells (from growth-arrested type to actively growing type) was noted in the neointimal regions of the atherosclerotic artery.23 We found that in cultured smooth muscle cells, MLC diphosphorylation induced by prostaglandin F2α was augmented in actively growing smooth muscle cells rather than in growth-arrested smooth muscle cells.16 We recently found in our swine model with IL-1β that phenotype of vascular smooth muscle cells (myosin heavy chain isoforms) is altered toward dedifferentiation.24 These results suggest that MLC diphosphorylation occurs mainly in the actively growing cells in the spastic coronary artery. Phenotype change of arterial smooth muscle cells may thus be one of the important mechanisms for coronary artery spasm.
Inhibitory Effects of Fasudil
In the present study, fasudil was used as a pharmacological tool to inhibit MLC phosphorylations in the vascular smooth muscle.12,13 Fasudil is 10 times more potent against PKC (Ki=3.3 μmol/L) than against MLCK (Ki=36.0 μmol/L)13 (Fig 10⇑). Because Ca2+/calmodulin-MLCK pathway may not play a major role in the pathogenesis of coronary spasm in our model,6 the major inhibitory site of fasudil may be at the PKC level (or other fasudil-sensitive protein kinases pathway) (Fig 10⇑).
Interestingly, fasudil preferentially inhibited the enhanced components of coronary artery contraction and MLC phosphorylations at the spastic site, whereas at the control site. its inhibitory effect on the contraction and MLC phosphorylations was less prominent (Figs 2 through 5⇑⇑⇑⇑). These results also support our hypothesis that PKC-mediated pathway, including inhibitory mechanism of MLC phosphatase, may be augmented only in the spastic coronary artery. Fasudil may preferentially inhibit the augmented PKC pathway and may inhibit the enhanced components of contraction and MLC phosphorylations in the spastic coronary artery.
Endothelial Function and Coronary Artery Spasm
In the present study, the enhanced MLC phosphorylations in the spastic coronary segments were noted regardless of the presence or absence of the endothelium. In addition, simple endothelium removal never induced MLC diphosphorylation in the normal coronary artery. These results further support our notion that coronary spasm is caused primarily by hypercontraction of vascular smooth muscle but not by reduced vasodilating function of the endothelium.5,6 We recently confirmed that endothelium-dependent relaxations are fairly preserved in our previous model with endothelial denudation (7 days after the procedure)25 as well as in our present model with IL-1β (unpublished observations).
In summary, we were able to demonstrate for the first time that the enhanced MLC phosphorylations play a central role in the pathogenesis of coronary spasm. The molecular mechanisms for the enhanced MLC phosphorylations at the spastic coronary artery remain to be elucidated.
Selected Abbreviations and Acronyms
|MLC||=||myosin light chain|
|MLCK||=||myosin light chain kinase|
|PAGE||=||polyacrylamide gel electrophoresis|
|PKC||=||protein kinase C|
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 S. Tomita and E. Gunshima for their excellent technical assistance.
This work was presented in part at the annual scientific meeting of the American Heart Association, New Orleans, La, 1996.
- Received June 19, 1997.
- Revision received August 18, 1997.
- Accepted September 12, 1997.
- Copyright © 1997 by American Heart Association
Maseri A, Severi S, DeNes DM, L’Abbate A, Chierchia S, Marzilli M, Ballestra AM, Parodi O, Biagini A, Distante A. ‘Variant’ angina: one aspect of a continuous spectrum of vasospastic myocardial ischemia: pathogenic mechanisms, estimated incidence and clinical and coronary angiographic findings in 138 patients. Am J Cardiol. 1978;42:1019–1035.
Shimokawa H, Tomoike H, Nabeyama S, Yamamoto H, Araki H, Nakamura M, Ishii Y, Tanaka K. Coronary artery spasm induced in atherosclerotic miniature swine. Science. 1983;221:560–562.
Shimokawa H, Ito A, Fukumoto Y, Kadokami T, Nakaike R, Sakata M, Takayanagi T, Egashira K, Takeshita A. Chronic treatment with interleukin-1β induces coronary intimal lesions and vasospastic responses in pigs in vivo: the role of platelet-derived growth factor. J Clin Invest. 1996;97:769–776.
Ito A, Shimokawa H, Kadokami T, Fukumoto Y, Owada MK, Shiraishi T, Nakaike R, Takayanagi T, Egashira K, Takeshita A. Tyrosine kinase inhibitor suppresses coronary arteriosclerotic changes and vasospastic responses induced by chronic treatment with interleukin-1β in pigs in vivo. J Clin Invest. 1995;96:1288–1294.
Ito A, Shimokawa H, Nakaike R, Fukai T, Sakata M, Takayanagi T, Egashira K, Takeshita A. Role of protein kinase C–mediated pathway in the pathogenesis of coronary artery spasm in a swine model. Circulation. 1994;90:2425–2431.
Kadokami T, Shimokawa H, Fukumoto Y, Ito A, Takayanagi T, Egashira K, Takeshita A. Coronary artery spasm does not depend on the intracellular calcium store but is substantially mediated by the protein kinase C–mediated pathway in a swine model with interleukin-1β in vivo. Circulation. 1996;94:190–196.
Murphy RA. What is special about smooth muscle? The significance of covalent cross bridge regulation. FASEB J. 1994;8:311–318.
Asano T, Ikegaki I, Satoh S, Suzuki Y, Shibuya M, Takayasu M, Hidaka H. Mechanism of action of a novel antivasospasm drug, HA1077. J Pharmacol Exp Ther. 1987;241:1033–1040.
Shimokawa H, Aarhus LL, Vanhoutte PM. Porcine coronary arteries with regenerated endothelium have a reduced endothelium-dependent responsiveness to aggregating platelets and serotonin. Circ Res. 1987;61:256–270.
Seto M, Sasaki Y, Sasaki Y. Alteration in the myosin phosphorylation pattern of smooth muscle by phorbol ester. Am J Physiol. 1990;259:C769–C774.
Eto M, Ohmori T, Suzuki M, Furuya K, Morita F. A novel protein phosphatase-1 inhibitory protein potentiated by protein kinase C: isolation from porcine aorta media and characterization. J Biochem. 1995;118:1104–1107.
Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A, Kaibuchi K. Regulation of myosin phosphatase by rho and rho-associated kinase (rho-kinase). Science. 1996;273:245–248.
Ikebe M, Hartshorne DJ. Phosphorylation of smooth muscle myosin at two distinct sites by myosin light chain kinase. J Biol Chem. 1985;260:10027–10031.
Fukumoto Y, Shimokawa H, Ito A, Kadokami T, Yonemitsu Y, Aikawa M, Owada MK, Egashira K, Sueishi K, Nagai R, Yazaki Y, Takeshita A. Inflammatory cytokine cause coronary arteriosclerosis-like changes and alterations in the smooth-muscle phenotypes in pigs. J Cardiovasc Pharamcol.. 1997;29:222–231.