INTRODUCTION

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COLD POTASSIUM CARDIOPLEGIA (usually a K⁺ concentration of 15-25 mM), whether crystalloid or blood containing, is the most common means of myocardial preservation and has proved to be an essential component of modern cardiac operations. Although protection of cardiac function and myocardial perfusion has been improved, suboptimal cardiac performance still occurs postoperatively in a small set of patients, especially when operations are performed on acutely ischemic patients. A reason for this myocardial dysfunction may be related to coronary vasoconstriction during or after cardiac operations in which the administration of K⁺ cardioplegia has been performed (18). Although the pathogenesis of vasospasm is not fully understood, altered vasomotor regulation in coronary smooth muscle associated with prolonged exposure to K⁺ cardioplegia seems to be one of the essential events in this setting. Recent works have shown that K⁺ cardioplegia causes a marked change in the coronary smooth muscle intracellular calcium concentration ([Ca²⁺]_i) (8, 23). In addition, it has been reported that enhanced phosphorylation of the myosin light chain (MLC) in vascular smooth muscle could play a central role in the pathogenesis of coronary vasoconstriction (6).

In an effort to improve on the shortcoming of high-K⁺ cardioplegic solutions, K⁺ channel openers have received more attention. To date, several studies have demonstrated that supplementation of K⁺ channel openers improved the cardioprotective effects afforded by K⁺ cardioplegia (4, 7). One important factor that may contribute to this beneficial phenomenon is possibly related to the K⁺ channel opener-evoked coronary vasorelaxation. However, there is little information available regarding the molecular and cellular mechanisms, particularly the role of Ca²⁺, and the sequence of signaling events such as MLC phosphorylation underlying the development of vasoprotection. For this reason, the present study was designed to investigate the effects of supplementation of pinacidil, a widely used K⁺ channel opener, on the coronary vasomotor regulation. Specifically, the relationship among coronary myoplasmic Ca²⁺, phosphorylation of MLC, and lumen diameter was examined with the use of an in vitro coronary microvessel model.

	MATERIALS AND METHODS

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Experimental Techniques

Isolated arteriole preparations. Male Sprague-Dawley rats (250-350 g) were anesthetized with chloroform. Heparin sodium (1,000 IU/kg) was then administered intravenously. The heart was removed into cold (4°C) Krebs physiological saline solution (Krebs-PSS), which consisted of the following ionic concentrations (in mM): 119.0 NaCl, 25.0 NaHCO₃, 5.0 KCl, 1.2 NaH₂PO₄, 1.2 MgSO₄, 1.8 CaCl₂, and 11.0 glucose. Immediately, coronary arterioles (100-260 µm in diameter) were dissected from the left anterior descending artery-dependent subepicardial region in the left ventricle with the use of a ×10-60 dissecting microscope (Olympus Optical, Tokyo, Japan). During dissection, care was taken to remove as much of the surrounding myocardium as possible, and the endothelium was intentionally preserved to maintain the vascular integrity. Vessels were transferred to an experimental chamber in which both ends of the microvessel were cannulated with dual glass micropipettes (tip interior diameter ~60 µm) and secured with 10-0 nylon monofilament suture (Ethicon, Somerville, NJ). The chamber was mounted on a transillumination system, and oxygenated (95% O₂-5% CO₂) Krebs-PSS (37°C) was continuously circulated through the vessel chamber. All of the animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Research Council.

Measurements of intraluminal diameter. The vessels were pressurized to 40 mmHg in a no-flow state with the use of a burette manometer filled with Krebs-PSS. The internal luminal diameter was measured with a microscopic image system (Zeiss IM35 and Hitachi CCD TV camera, model KP-115). The calibration of the measurement was performed by use of an 80-µm tungsten wire. The changes in internal luminal diameter were expressed as percent contraction of the baseline diameter.

Measurements of [Ca²⁺]_i. [Ca²⁺]_i of coronary smooth muscle was measured by the method described by Meininger et al. (9), an approach we have previously used (8, 23). Coronary arterioles in the vessel chamber were loaded with 5 µM fura 2-AM (acetoxymethyl ester of fura 2) in Krebs-PSS containing 0.05% DMSO and 0.01% pluronic F-127. In the preliminary experiments, we confirmed that these loading vehicles do not affect the coronary microvascular reactivity. The loading time was 45 min at room temperature (21-23°C) followed by a 30-min wash period at 37°C. The objective lens used was a Nikon Fluor ×20 (numerical aperture 0.75). Optimal focus was adjusted by the viewing of smooth muscle layer under bright-field microscopy. Excitation light at 350 ± 5 and 390 ± 6 nm was used. Emission at 510 ± 24 nm was monitored with a photomultiplier tube (Hamamatsu R928), digitized by a data acquisition analog-to-digital converter, and processed by use of DTVee version 3.0 (Data Translation). [Ca²⁺]_i was estimated from the ratio (R) of measured fluorescence signals (F) elicited at two wavelengths according to the following equation: R =[F_350(mv)  F_350(bg)]/[F_390(mv)  F_390(bg)], where F_350(mv) and F_390(mv) are the total measured fluorescence of the vessels at wavelengths of 350 and 390 nm, respectively, and F_350(bg) and F_390(bg) are the background fluorescence signals at each respective wavelength. Background signals were measured on vessels before the loading of fura 2-AM. In the preliminary experiments, the background autofluorescence intensities measured in the absence of fura 2 were not significantly altered by any of our experimental interventions, including administration of drugs and hypoxia. Despite the undoubted benefit of fura 2 in evaluating relative changes in [Ca²⁺]_i, it has become clear that there are a number of uncertainties in precise quantification of absolute [Ca²⁺]_i (2), and its dissociation constant (K_d) value is temperature sensitive (17). Therefore, we used the ratio of measured fluorescence signals elicited at wavelengths of 350 and 390 nm (R_350/390) as an index of [Ca²⁺]_i. As is well known, a ratiometric analysis of fura 2 is not linear to the absolute [Ca²⁺]_i; however, this makes it possible to evaluate relative changes in [Ca²⁺]_i independently of dye concentration, path length, excitation intensity, and photobleaching. Thus the ratios as presented would appear to be a reasonable index of the [Ca²⁺]_i.

Measurements of MLC phosphorylation. Phosphorylation of 20-kDa MLC was measured by the method using glycerol-urea minigels (22). At specific time points during the experimental protocol, the vessels were rapidly removed from the experimental apparatus and immediately frozen (within 3 s) by immersion for 60 min in an acetone dry-ice slurry containing 10% TCA and 10 mM dithiothreitol (DTT). Frozen vessels were gradually warmed up to room temperature, followed by three rinses with acetone containing 5 mM DTT to remove TCA, and then stored at 80°C before use. In general, pooled segments of three vessels (6-8 mm in total length) subjected to a given protocol were combined for one phosphorylation measurement. The samples were suspended in 20 µl of urea sample buffer [8.0 M urea, 20 mM Tris base, 23 mM glycine (pH 8.6), 10 mM DTT, 10% glycerol, and 0.04% bromphenol blue], applied to glycerol-urea minigels (10% acrylamide-0.8% bisacrylamide, 40% glycerol, 20 mM Tris base, and 23 mM glycine), and subjected to electrophoresis at a constant voltage of 400 V until the dye front ran off. Electrophoretic transfer of proteins from the gels onto polyvinylidene difluoride membranes was carried out. The membrane was blocked in 5% milk solution for 30 min and then incubated overnight with a 20-kDa MLC antibody (Sigma). We preliminarily confirmed that this monoclonal antibody could not show any reactivity to myocardial tissue in the absence of coronary vessels. The blot was then placed in an anti-mouse IgG (goat) conjugated with horseradish peroxidase (Calbiochem) and visualized with chemiluminescence (Super Signal; Pierce, Rockford, IL). The MLC bands were quantitated densitometrically with the use of NIH Image, and the MLC phosphorylation levels were expressed as the area of phosphorylated MLC divided by the total area of 20-kDa MLC times 100%. Care was taken to ensure that bands subjected to densitometry were not saturated.

Experimental Design

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4

Experimental Protocols

Initially, the time course of changes in myoplasmic [Ca²⁺]_i, MLC phosphorylation, and vascular diameter during cardioplegia/reperfusion was examined. On the basis of the results, eight groups were studied. Vessels in group I (control group) were subjected to the K⁺ cardioplegia supplemented with vehicle (DMSO) alone. For assessment of the dose responsiveness to pinacidil (Pin), vessels in groups II, III, and IV were supplemented with 10, 50, and 200 µM Pin into the K⁺ cardioplegia, respectively. To determine whether the endothelium-derived nitric oxide (NO) was involved in the Pin-induced vasomotor changes, in group V an NO synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME; 10 µM), was coadministered with 200 µM Pin. To investigate the cellular mechanisms of Pin supplementation, in group VI an ATP-sensitive K⁺ (K_ATP) channel blocker glibenclamide (Glib; 10 µM) was coadministered with 200 µM Pin. Group IV served as the internal control for groups V and VI. To further investigate the coronary vasomotor regulation during K⁺ cardioplegia, we examined the effects of Glib alone (10 µM) in group VII and the administration of a voltage-dependent L-type Ca²⁺-channel blocker nicardipine (Nic; 10 µM) in group VIII.

Drugs

Data Analysis

	RESULTS

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Time-Course Studies

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Time-course profiles of coronary smooth muscle intracellular calcium concentration ([Ca2+]i) (A), myosin light chain (MLC) phosphorylation (B), and lumen diameter (C) during and after 60 min of K+ cardioplegic solutions in vehicle alone (control) and in groups supplemented with 200 µM pinacidil (Pin200). Internal vessel diameter and [Ca2+]i (loaded with fura 2) were simultaneously monitored with microscopic image analysis (n = 6 vessels for each group). Changes in lumen diameter were expressed as percent contraction of baseline diameter. [Ca2+]i was expressed as the ratio of measured fluorescence signals elicited at 350 and 390 nm (R350/390). Phosphorylation of 20-kDa MLC was measured in pooled vessels by glycerol-urea gels and expressed as percentage of area of phosphorylated MLC over total area of MLC (n = 4-7 measurements at each time point). B, top: representative immunoblots for MLC (U, unphosphorylated; P, phosphorylated). Values are means ± SE. * P < 0.05 vs. control.

Dose Dependency of Pin

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effects of pinacidil (Pin) supplementation on coronary smooth muscle [Ca2+]i (A), MLC phosphorylation (B), and lumen diameter (C) at end of 60 min of K+ cardioplegic period. Control, vehicle alone; Pin10, Pin50, and Pin200, groups supplemented with 10 µM Pin, 50 µM Pin, and 200 µM Pin, respectively; Pin200+L-NAME, 200 µM Pin + 10 µM N-nitro-L-arginine methyl ester (L-NAME); Pin200+Glib, 200 µM Pin + 10 µM glibenclamide (Glib); Glib, 10 µM Glib alone; Nic, 10 µM nicardipine (Nic). As described in Fig. 1, changes in [Ca2+]i (A) and vessel diameter (C) were simultaneously measured (n = 6 vessels for each group), and MLC phosphorylation level (B) was determined in pooled vessels (n = 4-6 measurements for each group). Each bar represents mean ± SE. * P < 0.05 vs. control. # P < 0.05 vs. Pin200.

Antagonism by Glib

Minimal Effect of L-NAME

	DISCUSSION

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The major findings of the present study were that 1) K⁺ cardioplegia-induced coronary constriction is associated with an elevation of [Ca²⁺]_i and an increase in the level of MLC phosphorylation; 2) this potentiation of the Ca²⁺-MLC phosphorylation pathway was inhibited by supplementation of Pin in a dose-dependent manner; 3) a selective blocker of K_ATP channel, Glib, antagonized the effects of Pin; 4) the ability of Pin to attenuate the Ca²⁺-MLC phosphorylation cascade was noted, regardless of the activity of NO synthase; and 5) Nic produced findings similar to those of Pin.

Alteration of the Ca²⁺-MLC Phosphorylation Pathway in Coronary Arterioles

Several factors probably modulate the coronary myoplasmic Ca²⁺ regulation during hypothermic K⁺ cardioplegia. It has been suggested that extracellular high K⁺-induced membrane depolarization promotes Ca²⁺ influx through voltage-operated Ca²⁺ channels, which can also lead to the release of Ca²⁺ from intracellular Ca²⁺ stores (1). In fact, in the present study, we demonstrated the potential role of the voltage-operated Ca²⁺ channels in the response to K⁺ cardioplegia using Nic, a selective L-type Ca²⁺ channel blocker. In addition, during surgical cardioplegia, especially nonoxygenated crystalloid cardioplegia, the coronary smooth muscle is exposed to conditions of insufficient oxygen supply, with a lower production of ATP compared with a normal state. It is widely recognized that hypoxia causes [Ca²⁺]_i elevation in various cell types. A detailed discussion of the mechanism of hypoxia-related Ca²⁺ accumulation is beyond this brief review, but several intracellular organelles that have ATP-regulated Ca²⁺-handling mechanisms, i.e., the sarcoplasmic reticulum and mitochondria, may be considered as candidates for the Ca²⁺ release (3). Moreover, a number of thermosensitive Ca²⁺ transport systems may be modified in a hypothermic condition (11, 20, 24). We previously evaluated the individual effects of extracellular K⁺ concentration and temperature on the intracellular Ca²⁺ homeostasis during cardioplegia by use of this model (23). We observed that high-K⁺ concentration (25 mM) itself caused a marked increase in [Ca²⁺]_i, and this high K⁺-induced intracellular Ca²⁺ accumulation was attenuated in Ca²⁺-free hypothermia (4°C) but was minimally affected in hypothermia in the presence of extracellular Ca²⁺ (1 mM). Thus the net effects of cold K⁺ cardioplegia on the intracellular Ca²⁺ regulation seem to be complex and interactive and need to be clarified in further studies.

Exposure to the K⁺ cardioplegia resulted in a transient elevation of MLC phosphorylation levels, which remained at significantly suprabasal levels during the 60-min cardioplegic period. This pattern of MLC phosphorylation is similar to that previously reported in swine carotid arteriolar smooth muscle with high-K⁺ stimulation (14), which might be consistent with the muscle entering the "latch" state, a condition whereby force generation is maintained by attached, slowly cycling cross bridges (15). The lower, but sustained, phosphorylation level during cardioplegia might be explained by the fact that the increase in [Ca²⁺]_i is maintained and will continue to increase the MLC kinase activity relative to phosphatase activity (22).

Effects of Pin Supplementation on the Vasomotor Regulation

In line with the membrane potential-regulated influences on the Ca²⁺ influx, it has been reported that membrane hyperpolarization induced by a K⁺ channel opener, cromakalim, inhibits not only the sarcolemmal Ca²⁺ influx but also the inositol 1,4,5-trisphosphate-induced Ca²⁺ release from sarcoplasmic reticulum in porcine coronary artery (26). In addition, there is also a report that demonstrated that a K⁺ channel opener, nicorandil, directly stimulated the synthesis of cGMP in vascular smooth muscle, which consequently reduces [Ca²⁺]_i because of acceleration of Ca²⁺ extrusion via the sarcolemmal Ca²⁺ pump, and may prevent the MLC phosphorylation (21). Thus several mechanisms may underlie the effect of K⁺ channel openers on preventing the cardioplegia-induced potentiation of the Ca²⁺-MLC phosphorylation pathway.

Inhibitory Effects of Glib

Role of Endothelium-Derived NO

Clinical Implications and Limitations

We acknowledge the potential limitation inherent in our in vitro microvascular model, which is isolated from blood, neural activity, and hormonal influences. It is possible that the effect of Pin on the coronary Ca²⁺-MLC phosphorylation pathway may be different from warm or tepid cardioplegic solutions, because temperature substantially modulates ion-channel activity, membrane ATPases, and the activity of a number of enzymes regulating contractile protein interactions (24).

In conclusion, this study clarified that K⁺ cardioplegia augments the Ca²⁺-MLC pathway in coronary smooth muscle and results in vasoconstriction. Supplementation of >50 µM Pin effectively reduces the intracellular Ca²⁺ accumulation and maintains adequate vasorelaxation during the cardioplegic period through a K_ATP channel mechanism.