Coronary microvascular protection with Mg 2 : effects on intracellular calcium regulation and vascular function

Transcription

1 Coronary microvascular protection with Mg 2 : effects on intracellular calcium regulation and vascular function NARUTO MATSUDA, 1 MOTOHISA TOFUKUJI, 1 KATHLEEN G. MORGAN, 2 AND FRANK W. SELLKE 1 1 Division of Cardiothoracic Surgery, Department of Surgery of Beth Israel Deaconess Medical Center and Harvard Medical School, Boston 02215; and 2 Boston Biomedical Research Institute, Boston, Massachusetts Matsuda, Naruto, Motohisa Tofukuji, Kathleen G. Morgan, and Frank W. Sellke. Coronary microvascular protection with Mg 2 : effects on intracellular calcium regulation and vascular function. Am. J. Physiol. 276 (Heart Circ. Physiol. 45): H1124 H1130, The use of Mg 2 -supplemented hyperkalemic cardioplegia preserves microvascular function. However, the mechanism of this beneficial action remains to be elucidated. We investigated the effects of Mg 2 supplementation on the regulation of intracellular calcium concentration ([Ca 2 ] i ) and vascular function using an in vitro microvascular model. Ferret coronary arterioles ( µm in diameter) were studied in a pressurized (40 mmhg) no-flow, normothermic (37 C) state. Simultaneous monitoring of internal luminal diameter and [Ca 2 ] i using fura 2 were made with microscopic image analysis. The microvessels (n 6 each group) were divided into four groups according to the content of MgCl 2 (nominally 0, 1.2, 5.0, and 25.0 mm) in a hyperkalemic cardioplegic solution ([K ] 25.0 mm). After baseline measurements, vessels were subjected to 60 min of hypoxia with hyperkalemic cardioplegia (equilibrated with 95% N 2-5% CO 2 ) containing each concentration of Mg 2 ([Mg 2 ]) and were then reoxygenated. During hyperkalemic cardioplegia, [Ca 2 ] i increased in a time-dependent manner in all groups. In the lower [Mg 2 ] cardioplegia groups, [Ca 2 ] i was significantly increased at the end of the 60-min cardioplegic period ( nm and nm in [Mg 2 ] 0 and 1.2 mm groups, respectively; both P 0.05 vs. baseline) with % vascular contraction. Conversely, there was no significant [Ca 2 ] i increase in the higher [Mg 2 ] cardioplegia groups and less vascular contraction ( %, both P 0.05 vs. [Mg 2 ] 1.2 mm group). After reperfusion, agonist (U , thromboxane A 2 analog)-induced vascular contraction was significantly enhanced in the lower [Mg 2 ] cardioplegia groups (both P 0.05 vs. control) but was normalized in the higher [Mg 2 ] cardioplegia groups. Intrinsic myogenic contraction was significantly decreased in the lower [Mg 2 ] cardioplegia groups (both P 0.05 vs. control) but was preserved in the higher [Mg 2 ] cardioplegia groups. These results suggest that supplementation of the solution with 5.0 mm [Mg 2 ]may prevent hyperkalemic cardioplegia-related intracellular Ca 2 overloading and preserve vascular contractile function in coronary microvessels. cardioplegia; coronary microvessel; vasospasm THE CORONARY MICROCIRCULATION plays a central role in the regulation of myocardial perfusion, which in turn 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. may affect myocardial contractile function. Recently, the influence of various surgical cardioplegic solutions on coronary microvascular function has received attention. Hyperkalemic cardioplegic solutions have been used to achieve cardiac arrest and to protect the myocardium during cardiac operations. However, there is abundant evidence that ischemic cardiac arrest using a hyperkalemic cardioplegic solution significantly changes the response of coronary microvessels to various vasoactive agents (13, 15, 20, 25). The pathophysiology underlying this hyperkalemic cardioplegiarelated microvascular dysfunction is likely to be multifactorial and, at present, not fully understood. However, intracellular calcium ([Ca 2 ] i ) overloading in coronary vascular smooth muscle could play a critical role in the development of microvascular dysfunction. The cellular mechanism of Ca 2 accumulation has been related, in part, to the high K concentration and insufficient oxygen supply in conventional crystalloid cardioplegic solutions. It is well known that magnesium is an important ionic modulator of blood vessel tone. Indeed, Mg 2 has been characterized as an endogenous calcium channel blocker that relaxes vascular smooth muscle and attenuates the vasoconstriction induced by several vasoactive drugs (1). The importance of Mg 2 in cardioplegic solutions has been increasingly recognized in the maintenance of myocardial function after surgical ischemia (5, 8, 24). Recently, we demonstrated that hypermagnesium cardioplegia can preserve coronary microvascular function after surgical cardiac arrest compared with a magnesium-free hyperkalemic cardioplegic solution (23). However, the molecular basis for the action of magnesium in the vascular system is not well known. The relationship between extracellular Mg 2 concentration ([Mg 2 ]) and [Ca 2 ] i during or after hyperkalemic cardioplegia has not yet been well characterized. To simultaneously assess both intracellular Ca 2 changes in coronary smooth muscle cells and microvascular function, we established an in vitro microvascular model that could simulate the regulation of coronary microcirculation under reduced oxygen supply with crystalloid cardioplegia in the operating room. The aim of the present study was to investigate the effects of Mg 2 supplementation on [Ca 2 ] i regulation in coronary microvessels and vascular function during and after hyperkalemic cardioplegia. H /99 $5.00 Copyright 1999 the American Physiological Society

2 CORONARY MICROVASCULAR PROTECTION WITH MAGNESIUM H1125 METHODS AND MATERIALS Isolated Microvessel Preparations The methods for isolation of coronary microvessels were described previously (20). Briefly, male ferrets (8 12 wk old) were anesthetized with chloroform, and their hearts were removed into cold (4 C) Krebs-physiological saline solution (Krebs-PSS), which consisted of the following ionic concentrations (in mm): NaCl, 25.0 NaHCO 3, 4.6 KCl, 1.2 KH 2 PO 4, 1.2 MgSO 4, 1.8 CaCl 2, and 11.0 glucose. Coronary arterial microvessels ( µm ID) were dissected from the left anterior descending artery-dependent subepicardial region in the left ventricle using a dissecting microscope (Olympus Optical, Tokyo, Japan). During dissection, care was taken to remove as much of the surrounding myocardium as possible and to avoid stretching and rubbing the intimal surface against foreign material. Microvessels 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% O 2-5% CO 2 ). Krebs-PSS (37 C) was continuously circulated through the tissue chamber. In all experiments, the presence of intact endothelium was confirmed by determining the vasodilative response to acetylcholine (10 6 M) in microvessels precontracted with potassium ions (20 mm). All of the animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No , Revised 1985). Measurements Intraluminal diameter measurement. The vessels were pressurized to 40 mmhg in a no-flow state using a burette manometer filled with Krebs-PSS. The internal luminal diameter was measured with a video-monitored microscopic system (model KP-115, Zeiss IM35 and Hitachi CCD TV camera). The calibration of the measurement was performed using an 80-µm tungsten wire. The minimum resolution of the system was 1.5 µm. [Ca 2 ] i measurement. [Ca 2 ] i of coronary microvascular smooth muscle was measured using calcium-sensitive fluorescent dye fura 2. Coronary microvessels in the tissue chamber were loaded with 5 µm acetoxymethyl ester of fura 2 (fura 2-AM) in Krebs-PSS containing 0.05% dimethyl sulfoxide and 0.01% Pluronic F-127. The loading time was 45 min followed by a 30-min wash period at 37 C. The objective lens used was a Nikon Fluor 40 (NA 0.8). To avoid the influence of fluorescent signals from the endothelium, optimal focus was adjusted to the middle of the microvascular smooth muscle layer by viewing the microvascular wall under a bright microscopic field. Excitation light at and 390 6nm was used. Emission at nm was monitored with a photomultiplier tube (Hamamatsu R928) and digitized by a Data Acquisition-EZ A/D Converter. The digital signal of the two wavelengths was processed using a program written using the DTVee version 3.0 programming environment (Data Translation). [Ca 2 ] i was estimated from the ratio (R) of measured fluorescence signals (F) elicited at two wavelengths according to the equation: R (F 350mv F 350bg )/(F 390mv F 390bg ), where F 350mv and F 390mv are the total measured fluorescence of the microvessels at wavelengths of 350 and 390 nm, respectively, and F 350bg and F 390bg are the background fluorescence signals at the respective wavelengths. The background signals were measured on microvessels before fura 2-AM was loaded. Particular care was taken to minimize possible photobleaching of fura 2 molecules. Quantification of [Ca 2 ] i. With the ratio method applied, intermicrovascular differences in fura 2 loading, microvascular thickness, light path length, and camera gain were canceled; therefore, the ratio values reflected true [Ca 2 ] i differences. For quantification of [Ca 2 ] i, we used an in situ dual-wavelength calibrating equation of Grynkiewicz et al. (7): [Ca 2 ] i K d [(R R min )/(R max R)], where K d (224 nm) is the effective dissociation constant, is the ratio of the fluorescence at 390 nm with 0 Ca 2 to the 390 nm fluorescence with 1.2 mm Ca 2. The maximum fluorescence ratio (R max )of fura 2 is observed when the dye is completely bound by Ca 2 in a solution containing 1.2 mm CaCl 2 plus 50 µm 4-bromo- A After that, EGTA (5 mm) was added to achieve the minimum fluorescence ratio (R min ). In these in situ calibration experiments, R min was highly consistent and reproducible with little variation; R max, however, was more variable. Values from the in situ microvessel calibration were R min 0.35, R max 1.22, and Experimental Protocols After a 60-min stabilizing period, measurements of [Ca 2 ] i and internal luminal diameter were taken (baseline control). Microvessels were divided into four groups according to the content of MgCl 2 (nominally 0, 1.2, 5.0, and 25.0 mm) in hypoxic, hyperkalemic cardioplegic solution. Hypoxia was induced by switching bubbling gas from 95% O 2-5% CO 2 to 95% N 2-5% CO 2. The composition of hyperkalemic cardioplegic solution was (in mm) 121 NaCl, 25 KCl, 12 NaHCO 3, 1.2 CaCl 2, and 11.1 glucose; ph 7.45, oxygen tension 5 30 mmhg. True anoxic condition was not achieved because a small amount of oxygen continuously diffused into the hyperkalemic cardioplegia from the atmosphere. The temperature was maintained at 37 C. All microvessels were subjected to 60 min of hypoxic, hyperkalemic cardioplegic solutions with each concentration of MgCl 2 and then reperfused with oxygenated Krebs-PSS for 60 min. During 60 min of hypoxic, hyperkalemic cardioplegia and after 60 min of normoxic reperfusion, [Ca 2 ] i and internal luminal diameter were measured every 15 min. After 60 min of reperfusion, a cumulative concentration-response curve to a stable thromboxane A 2 analog (U-46619, M) was constructed to evaluate the agonist-induced vascular contractility. The vessels were washed with a drug-free Krebs-PSS for 30 min. After the vessels were equilibrated for at least 30 min, the active pressure-diameter relation was studied. Initially, the pressure was reduced to 10 mmhg to stabilize for 10 min. Then the pressure was increased in increments of 10 mmhg up to 100 mmhg. At each pressure increment, the change in internal luminal diameter was measured after microvessel diameter had stabilized (generally after 3 min). Once the determination of the active pressure-diameter relation was completed, the pressure was returned to 50 mmhg, and finally, papaverine (100 µm) was applied in the tissue chamber to normalize the vascular diameter. The normalized diameter was defined as the ratio of the diameter observed at a given transmural pressure to the diameter of the same vessel at 50 mmhg pressure in the presence of papaverine. To test the osmotic influence of supplemented MgCl 2 in the hyperkalemic cardioplegic solutions, a subset (n 6) of experiments was performed in which adequate sucrose was added to the 1.2 mm [Mg] group to raise the osmolarity equal to that in the 25 mm [Mg] group ( mosmol/l).

3 H1126 CORONARY MICROVASCULAR PROTECTION WITH MAGNESIUM Drugs Fura 2-AM and Pluronic F-127 were obtained from Molecular Probes (Eugene, OR). U was purchased from Sigma Chemical (St. Louis, MO). Papaverine was obtained from Eli Lilly (Indianapolis, IN). All solutions were prepared on the day of the study. Statistical Analysis The response of microvessels to each intervention was examined only once in each animal. Therefore, each animal served as one sample, and n refers to the number of animals from which microvessels were taken in all experiments. In the microvessel contraction experiments, vessels not having cardioplegic intervention were taken as control, and changes in internal luminal diameter were expressed as the percent contraction of the baseline diameter. Results are expressed as means SE. The paired Student s t-test was applied for within-group comparisons with baseline. ANOVA followed by a multiple-comparison Fisher s test was used to test the differences among groups with different interventions (Stat- View 4.0; Abacus Concepts, Berkeley, CA). The probability was considered to be significant if the P value was RESULTS Characterization of Isolated Coronary Microvessels Before exposure of microvessels to hyperkalemic cardioplegia, there were no significant differences in either the baseline vascular diameter or [Ca 2 ] i in smooth muscle among the four groups (vascular diameter: to µm; [Ca 2 ] i :74 17 to nm). After reperfusion, there were no significant differences in the papaverine-applied vascular diameter among the groups ( to µm). With respect to the osmotic influence of hyperkalemic cardioplegia ([Mg] 1.2 mm), the sucrose-added cardioplegia caused changes in microvascular diameter and [Ca 2 ] i similar to the no-sucrose-added cardioplegia (peak vascular contraction during cardioplegia: % vs %; peak [Ca 2 ] i during cardioplegia: vs nm; sucrose added vs. no sucrose added, respectively; both P 0.05). These observations effectively ruled out a nonspecific osmotic effect of increased concentration of MgCl 2 in the present study protocols. Intracellular Ca 2 Dynamics Figure 1 shows time-course dynamics in [Ca 2 ] i during and after hyperkalemic cardioplegia. On exposure to the hyperkalemic cardioplegic solution, [Ca 2 ] i in the lower [Mg 2 ] groups increased gradually in a timedependent manner, and these increases were significantly different from the baseline level at the end of the 60-min cardioplegic period ( and nm, in [Mg 2 ] 0 and 1.2 mm groups, respectively; both P 0.01 vs. baseline; between-group differences were not significant). However, a slight increase in the [Ca 2 ] i occurred in the higher [Mg 2 ] groups during hyperkalemic cardioplegia, but these increases were not statistically significant compared with the baseline value ( and nm, in [Mg 2 ] 5.0 and 25.0 mm groups, respectively; both P 0.05 vs. baseline). After Fig. 1. Changes in intracellular Ca 2 concentration ([Ca 2 ] i ) during 60-min exposure to a hypoxic, hyperkalemic cardioplegic solution containing each [Mg 2 ] and followed by 60-min reperfusion with oxygenated Krebs-physiological saline solution (PSS). [Ca 2 ] i (nm) was measured using fura 2. All values are shown as means SE; n 6 ferrets from which microvessels were taken for each group. **P 0.01 vs. baseline control; P 0.05 vs. [Mg 2 ] 1.2 mm group. reperfusion with oxygenated Krebs-PSS, the [Ca 2 ] i returned to its baseline level within 15 min in all groups. Changes in Diameter As shown in Fig. 2, in the lower [Mg 2 ] groups percent contraction increased gradually during the cardioplegic period, and at the end of the 60-min cardioplegic period the percent contraction reached % and % in 0 and 1.2 mm [Mg 2 ] groups, respectively. In the higher [Mg 2 ] groups, per- Fig. 2. Changes in vascular diameter during 60-min exposure to a hypoxic, hyperkalemic cardioplegic solution containing each [Mg 2 ] and followed by 60-min reperfusion with oxygenated Krebs-PSS. Contractile responses are expressed as percent contraction of the baseline diameter. All values are shown as means SE; n 6 ferrets from which microvessels were taken for each group. P 0.05 vs. [Mg 2 ] 1.2 mm group.

4 CORONARY MICROVASCULAR PROTECTION WITH MAGNESIUM H1127 Fig. 3. Concentration-response curve to receptor-mediated vasoconstrictor U after 60-min exposure to a hypoxic, hyperkalemic cardioplegic solution containing each [Mg 2 ] and followed by 60-min reperfusion with oxygenated Krebs-PSS. Vessels not exposed to cardioplegia were taken as control, and contractile responses are expressed as percent contraction of the baseline diameter. *P 0.05 vs. Control. cent contraction reached a maximum level within the initial 15 min of cardioplegia, which sustained during the cardioplegic period. The peak values were % and % in 5.0 and 25.0 mm [Mg 2 ] groups, respectively (both P 0.05 vs. [Mg 2 ] 0 and 1.2 mm groups). After reperfusion, the vascular diameter recovered to its initial value within 15 min of reperfusion in all groups. Vascular Contractility After Reperfusion The agonist-induced contractile responses to U were significantly increased in the 0 and 1.2 mm [Mg 2 ] groups (both P 0.05 vs. control), whereas those in the 5.0 and 25.0 mm [Mg 2 ] cardioplegic groups were not altered compared with control. These differences were most pronounced at the higher concentrations of U tested (Fig. 3). Intrinsic myogenic contraction was observed to a stepwise increase in the transmural pressure 50 mmhg in control vessels. In vessels from higher [Mg 2 ] groups, similar myogenic contractions were observed, but cardioplegia caused an upward shift in the active pressure-diameter relation (both P 0.05 vs. control). However, the myogenic contractions were abolished in the lower [Mg 2 ] groups (both P 0.05 vs. [Mg 2 ] 5.0 and 25.0 mm; Fig. 4). DISCUSSION Changes in [Ca 2 ] i and Vascular Contraction During Hyperkalemic Cardioplegia Despite the fact that magnesium has been shown to protect the myocardium and coronary vasculature from hyperkalemic cardioplegia-related cardiac injury, its effect on regulating intracellular Ca 2 in coronary microvessels has not been well elucidated. The present study provides experimental evidence that a hyperkalemic cardioplegia containing a physiological concentration of Mg 2 (1.2 mm) causes a marked intracellular Ca 2 accumulation and significant vascular contraction, whereas a higher concentration of Mg 2 ( 5.0 mm) can prevent this Ca 2 overloading and contraction during hyperkalemic cardioplegia. The mechanism responsible for the hyperkalemic cardioplegia-related [Ca 2 ] i accumulation in vascular smooth muscle is most likely related to membrane depolarization on the basis of Nernst s equation (17). Membrane depolarization promotes Ca 2 influx through voltage-dependent Ca 2 channels. Ca 2 influx also induces release of Ca 2 from intracellular Ca 2 stores (4). In addition, during surgical cardioplegia, especially using nonoxygenated crystalloid cardioplegic solutions, coronary microvessels are exposed to conditions of hypoxia. This is associated with a lower production of ATP compared with a normoxic state. It is widely recognized that [Ca 2 ] i is elevated during and after periods of hypoxia. In previous studies, it was suggested that transsarcolemmal Ca 2 influx via Na /Ca 2 exchange may play an important role in hypoxia/ reoxygenation-mediated Ca 2 accumulation (12, 22). Recently, other investigators have reported that Ca 2 release from the sarcoplasmic reticulum may contribute to hypoxic pulmonary vasoconstriction (6, 10). High Mg 2 concentration has been suggested to inhibit Ca 2 entry into the cell by displacing Ca 2 from binding sites in the calcium channels and by hyperpolarization of sarcolemmal membrane (9). It has also been postulated that extracellular Mg 2 acts by raising intracellular Mg 2 concentration, thereby reducing the release of Ca 2 from the sarcoplasmic reticulum (3, 24). In addition, supplementation of Mg 2 in cardioplegic solutions may diminish the depletion of ATP stores, Fig. 4. Active pressure-diameter relations after 60-min exposure to a hypoxic, hyperkalemic cardioplegic solution containing each [Mg 2 ] and followed by 60-min reperfusion with oxygenated Krebs-PSS. Vessels not exposed to cardioplegia were taken as control, and vessel diameters were normalized to diameters at 50 mmhg after application of papaverine. All values are shown as means SE; n 6 ferrets from which microvessels were taken for each group. *P 0.05 vs. Control ( mmhg); P 0.05 vs. [Mg 2 ] 5.0 mm group ( mmhg).

5 H1128 CORONARY MICROVASCULAR PROTECTION WITH MAGNESIUM thereby protecting the intracellular metabolic function of microvascular smooth muscle. Accordingly, it seems that multiple cellular mechanisms may be involved in these beneficial actions of Mg 2 supplementation. Indeed, it should be noted that the present study examined the effects of hyperkalemic cardioplegic solutions containing a physiological concentration of calcium (CaCl mm). Therefore, it is possible that a reduced Ca 2 concentration in a hyperkalemic cardioplegia might require a lower level of Mg 2 supplementation to achieve microvascular protection from intracellular Ca 2 overload. In clinical practice, however, it is more difficult to precisely regulate the cardioplegic Ca 2 concentration because of transient variables such as ph and temperature. As a result, patients are at risk of exposure to higher than originally intended Ca 2 levels, which may increase the likelihood of the Ca 2 - mediated vascular injury. In addition, further decreasing the Ca 2 level may cause a calcium paradox in coronary microvessels. Therefore, we emphasize that the addition of Mg 2 may solve this dilemma by allowing for the safe use of higher cardioplegic Ca 2 concentrations, and we recommend sufficient supplementation of Mg 2 ( 5.0 mm) in hyperkalemic cardioplegic solutions. Changes in Vascular Contractility After Hyperkalemic Cardioplegia One purpose of this study was to develop a better understanding of the pathophysiology in microvascular contractile dysfunction after hyperkalemic cardioplegia. In the present study, we demonstrated that in the lower [Mg 2 ] groups the agonist (U-46619)-induced vascular contraction was markedly enhanced, whereas the intrinsic myogenic contractile response was significantly diminished. Conversely, in higher [Mg 2 ] groups the agonist-induced and myogenic responses were preserved. (Figs. 3 and 4). Although the explanations for these phenomenon remain unclear, they may be attributed to endothelial dysfunction or altered contractile properties of vascular smooth muscle, or both. We and others (15, 20) have previously showed a progressive deterioration of the endothelial-dependent relaxation in the coronary microcirculation after surgical cardioplegia. Furthermore, our observations in this study are in agreement with the findings of Pearson and associates (16), who showed that hypomagnesemia could impair the release of nitric oxide from the coronary endothelium and promote vasoconstriction. In addition, we have previously reported that a Mg 2 - based cardioplegic solution ([Mg 2 ] 25.0 mm) prevents much of the impairment in endothelium-dependent relaxation observed after exposure of microvessels to a purely hyperkalemic cardioplegic solution (23). Therefore, it is likely that hyperkalemic cardioplegia without sufficient Mg 2 supplementation may impair the endothelial function, including the release of nitric oxide, and predispose the patient to vascular hypercontraction in response to a vasoconstrictive agonist. Accordingly, it would be tempting to speculate that sufficient Mg 2 supplementation would afford endothelial protection, although the present study was not designed to provide direct evidence about functional implication of the endothelium. It may also be possible that enhanced Ca 2 accumulation in the vascular smooth muscle may activate a Ca 2 -dependent intracellular signaling pathway and alter the Ca 2 sensitivity of the contractile apparatus. Previous studies have demonstrated that an agonistinduced vascular tone is regulated by myosin light chain kinase, the activity of which is governed by a Ca 2 -calmodulin-mediated phosphorylation (19). There is some evidence that [Ca 2 ] i may also directly activate the Ca 2 -dependent isoforms of protein kinase C (conventional protein kinase C) and lead directly or indirectly to the phosphorylation of an entirely different subset of cellular proteins, including caldesmon, a number of intermediate filament proteins (desmin, synemin), and a few cytosolic proteins (18). Therefore, it is reasonable to propose that the [Ca 2 ] i overloading during cardioplegia is a strong trigger for enhancement of agonist-induced vascular contraction after reperfusion. Myogenic tone is a property of vascular smooth muscle manifested by contraction in response to the increase in transmural pressure and is involved in the autoregulation of coronary perfusion. Although not completely understood, the contribution of adenosine triphosphate-sensitive potassium (K ATP ) channels to myogenic contraction was recently implicated (11). The channels open when the intracellular ATP concen- K ATP tration falls to 1 mm (21). Opening of the K ATP channels causes membrane hyperpolarization and relaxes the vascular smooth muscle by preventing Ca 2 entry via voltage-sensitive Ca 2 channels. In a previous study, the K ATP channel blocker glibenclamide preserved myogenic reactivity after hyperkalemic cardioplegia (26). Taken together these observations and the results of the present study strongly suggest that supplementation with a higher concentration of Mg 2 ( 5.0 mm) may inhibit activation of K ATP channels by preventing intracellular ATP depletion in vascular smooth muscle and preserving the intrinsic myogenic contraction. Methodological Considerations The present study was designed to monitor intracellular Ca 2 accumulation and vascular function during and after hyperkalemic cardioplegia. This in vitro microvascular model uses a hypoxic, hyperkalemic cardioplegic solution that simulates the insufficient oxygen supply with crystalloid cardioplegia in the operating room. In this study, we aimed to address the efficacy of Mg 2 supplementation with the view of coronary microcirculatory protection. Coronary microvascular injury in stressed hearts is extremely important, because this microvascular function may be more vulnerable and sensitive to surgical hyperkalemic cardioplegia compared with myocardial function. Hyperkalemic cardioplegia-related coronary microcirculatory dysfunction in the setting of minimal changes in myo-

6 CORONARY MICROVASCULAR PROTECTION WITH MAGNESIUM H1129 cardial contractile function using a cardiopulmonary bypass model has recently been reported (23). There are a number of Ca 2 -sensitive indicators, such as aequorin, indo-1, and fura 2. In the present study, we used the fura 2 microscopic technique to monitor the real-time changes of [Ca 2 ] i in microvascular smooth muscle. Although fura 2 is useful for the evaluation of [Ca 2 ] i, it must be noted that determination of the absolute value of [Ca 2 ] i from the fura 2 fluorescence ratio is still problematic; it cannot be totally excluded that changes in background autofluorescence of microvessels, compartmentation of fura 2 into intracellular organelles such as sarcoplasmic reticulum, or the deesterized form of fura 2-AM cause fluorescence signals unrelated to [Ca 2 ] i. For quantification of [Ca 2 ] i, we used an in situ dual-wavelength calibration by obtaining R max and R min and thus estimated cytosolic [Ca 2 ] i. In these calibration experiments, R min was highly consistent and reproducible with little variation; R max, however, was more variable. Similar variability in R max values has been previously reported (14). The variation in R max values may be related to limited effectiveness of the calcium ionophore 4-bromo-A23187 in this microvascular model. Although the [Ca 2 ] i values in the present study were similar to those reported by other investigators (2, 14), they may have to be revised when better methods to calibrate fura 2 fluorescence become available. In conclusion, we have demonstrated that supplementation of 5 mm[mg 2 ] possibly prevents hyperkalemic cardioplegia-related intracellular Ca 2 overloading and preserves vascular function in coronary microvessels. To protect coronary microcirculation from Ca 2 - related vasospasm under cardiac surgery, these findings appear to be significant and clinically relevant and require further investigation for the development and manipulation of pharmacological strategies. The authors express gratitude to Prof. Shigetsugu Ohgi (Tottori, Japan) for the continuous encouragement. This study was supported by National Heart, Lung, and Blood Institute Grants HL (to F. W. Sellke) and HL (to K. G. Morgan). Address for reprint requests and other correspondence: F. W. Sellke, Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, East Campus, Dana 905, 330 Brookline Ave., Boston, MA ( fsellke@bidmc.harvard.edu). Received 13 July 1998; accepted in final form 9 December REFERENCES 1. Altura, B. M., and B. T. Altura. New perspectives on the role of magnesium in the pathophysiology of the cardiovascular system. I. Experimental aspects. Magnesium 4: , Batlle, D. C., R. Peces, M. S. LaPointe, M. Ye, and J. T. Daugirgas. Cytosolic free calcium regulation in response to acute changes in intracellular ph in vascular smooth muscle. Am. J. Physiol. 264 (Cell Physiol. 33): C932 C943, Boland, R., A. Martonosi, and T. W. Tillack. Developmental changes in the comparison and function of sarcoplasmic reticulum. J. Biol. Chem. 249: , Fabiato, A. Simulated calcium current can both cause calcium loading in and trigger the calcium release from the sarcoplasmic reticulum of skinned cardiac cell. J. Gen. Physiol. 85: , Geffin, G. A., T. R. Love, W. G. Hendren, D. F. Torchiana, J. S. Titus, B. E. Redonnett, D. D. O Keefe, and W. M. Daggett. The effect of calcium and magnesium in hyperkalemic cardioplegic solutions on myocardial preservation. J. Thorac. Cardiovasc. Surg. 98: , Gelband, C. H., and H. Gelband. Ca 2 release from intracellular stores is an initial step in hypoxic pulmonary vasoconstriction of rat pulmonary artery resistance vessels. Circulation 96: , Grynkiewicz, G., M. Poenie, and R. Y. Tsien. A new generation of Ca 2 indicators with greatly improved fluorescent properties. J. Biol. Chem. 260: , Kronon, M., K. S. Bolling, B. S. Allen, S. Rahman, T. Wang, A. Halldorsson, and H. Feinberg. The relationship between calcium and magnesium in pediatric myocardial protection. J. Thorac. Cardiovasc. Surg. 114: , Lansman, J. B., P. Hess, and R. Tsien. Blockade of current through single calcium channels by Cd 2, Mg 2, and Ca 2 : voltage and concentration dependence of calcium entry into the pore. J. Gen. Physiol. 88: , Liu, X., R. M. Engelman, Z. Wei, N. Maulik, J. A. Rousou, J. E. Flack, III, D. W. Deaton, and D. K. Das. Postischemic deterioration of sarcoplasmic reticulum: warm vs. cold blood cardioplegia. Ann Thorac Surg 56: , Loutzenhiser, R., and M. Parker. Hypoxia inhibits myogenic reactivity of renal afferent arterioles by activating ATP-sensitive K channels. Circ. Res. 74: , Matsuda, N., T. Mori, H. Nakamura, and M. Shigekawa. Mechanisms of reoxygenation-induced calcium overload in cardiac myocytes: dependence on ph i. J. Surg. Res. 59: , McDonagh, P. F., and H. Laks. Use of cold blood cardioplegia to protect against coronary microcirculatory injury due to ischemia and reperfusion. J. Thorac. Cardiovasc. Surg. 84: , Meininger, G. A., D. C. Zawieja, and J. C. Falcone. Calcium measurement in isolated arterioles during myogenic and agonist stimulation. Am. J. Physiol. 261 (Heart Circ. Physiol. 30): H950 H959, Nakanishi, K., Z. Q. Zhao, J. Vinten-Johansen, J. C. Lewis, D. S. McGee, and J. W. Hammon, Jr. Coronary artery endothelial dysfunction after global ischemia, blood cardioplegia, and reperfusion. Ann. Thorac. Surg. 58: , Pearson, P. J., P. R. Evora, J. F. Seccombe, and H. V. Schaff. Hypomagnesemia inhibits nitric oxide release from coronary endothelium; protective role of magnesium infusion after cardiac operations. Ann. Thorac. Surg. 65: , Powell, T., P. E. R. Tatham, and V. W. Twist. Cytosolic free calcium measured by quin-2 fluorescence in isolated ventricular myocytes at rest and during potassium-depolarization. Biochem. Biophys. Res. Commun. 122: , Rasmussen, H., Y. Takuwa, and S. Park. Protein kinase C in the regulation of smooth muscle contraction. FASEB J. 1: , Sasaguri, T., T. Itoh, M. Hirata, K. Kitamura, and H. Kuriyama. Regulation of coronary artery tone in relation to the activation of signal transducers that regulate calcium homeostasis. J. Am. Coll. Cardiol. 9: , Sellke, F. W., T. Shafique, F. J. Schoen, and R. M. Weintraub. Impaired endothelium-dependent coronary microvascular relaxation following cold potassium cardioplegia and reperfusion. J. Thorac. Cardiovasc. Surg. 105: 52 58, Standen, N. B., J. M. Quayle, N. W. Davies, J. E. Brayden, Y. Huang, and M. T. Nelson. Hyperpolarizing vasodilators activate ATP-sensitive K channel in arterial smooth muscle. Science 245: , Tani, M., and J. R. Neely. Role of intracellular Na in Ca 2 overload and depressed recovery of ventricular function of reperfused ischemic rat hearts. Possible involvement of H -Na and Na -Ca 2 exchange. Circ. Res. 65: , Tofukuji, M., A. Stamler, J. Y. Li, A. Franklin, S. Y. Wang, M. D. Hariawala, and F. W. Sellke. Effects of magnesium cardioplegia on regulation of the porcine coronary circulation. J. Surg. Res. 68: , 1997.

7 H1130 CORONARY MICROVASCULAR PROTECTION WITH MAGNESIUM 24. Tsukube, T., J. D. McCully, M. Federman, I. B. Krukenkamp, and S. Levitsky. Developmental differences in cytosolic calcium accumulation associated with surgically induced global ischemia: optimization of cardioplegic protection and mechanism of action. J. Thorac. Cardiovasc. Surg. 112: , Wang, S. Y., M. Friedman, R. G. Johnson, R. M. Weintraub, and F. W. Sellke. Adrenergic regulation of coronary microcirculation after extracorporeal circulation and crystalloid cardioplegia. Am. J. Physiol. 267 (Heart Circ. Physiol. 36): H2462 H2470, Wang, S. Y., M. Friedman, A. Franklin, and F. W. Sellke. Myogenic reactivity of coronary resistance arteries after cardiopulmonary bypass and hyperkalemic cardioplegia. Circulation 92: , 1995.