INTRODUCTION

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THE CORONARY MICROCIRCULATION plays a central role in the regulation of myocardial perfusion, which in turnmay 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 cardioplegia-related microvascular dysfunction is likely to be multifactorial and, at present, not fully understood. However, intracellular calcium ([Ca²⁺]_i) overloading in coronary vascular smooth muscle could play a critical role in the development of microvascular dysfunction. The cellular mechanism of Ca²⁺ 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, Mg²⁺ 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 Mg²⁺ 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 Mg²⁺ concentration ([Mg²⁺]) and [Ca²⁺]_i during or after hyperkalemic cardioplegia has not yet been well characterized.

To simultaneously assess both intracellular Ca²⁺ 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 Mg²⁺ supplementation on [Ca²⁺]_i regulation in coronary microvessels and vascular function during and after hyperkalemic cardioplegia.

	METHODS AND MATERIALS

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Isolated Microvessel Preparations

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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. 85-23, Revised 1985).

Measurements

[Ca²⁺]_i measurement. [Ca²⁺]_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 350 ± 5 and 390 ± 6 nm was used. Emission at 510 ± 24 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). [Ca²⁺]_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 [Ca²⁺]_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 [Ca²⁺]_i differences. For quantification of [Ca²⁺]_i, we used an in situ dual-wavelength calibrating equation of Grynkiewicz et al. (7): [Ca²⁺]_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 Ca²⁺ to the 390 nm fluorescence with 1.2 mM Ca²⁺. The maximum fluorescence ratio (R_max) of fura 2 is observed when the dye is completely bound by Ca²⁺ in a solution containing 1.2 mM CaCl₂ plus 50 µm 4-bromo-A23187. 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  = 1.10.

Experimental Protocols

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To test the osmotic influence of supplemented MgCl₂ 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 (390-400 mosmol/l).

Drugs

Statistical Analysis

	RESULTS

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Characterization of Isolated Coronary Microvessels

With respect to the osmotic influence of hyperkalemic cardioplegia ([Mg] 1.2 mM), the sucrose-added cardioplegia caused changes in microvascular diameter and [Ca²⁺]_i similar to the no-sucrose-added cardioplegia (peak vascular contraction during cardioplegia: 15.6 ± 2.8% vs. 17.2 ± 3.6%; peak [Ca²⁺]_i during cardioplegia: 218 ± 50 vs. 236 ± 49 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 MgCl₂ in the present study protocols.

Intracellular Ca²⁺ Dynamics

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Changes in intracellular Ca2+ concentration ([Ca2+]i) during 60-min exposure to a hypoxic, hyperkalemic cardioplegic solution containing each [Mg2+] and followed by 60-min reperfusion with oxygenated Krebs-physiological saline solution (PSS). [Ca2+]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. [Mg2+] 1.2 mM group.

Changes in Diameter

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Changes in vascular diameter during 60-min exposure to a hypoxic, hyperkalemic cardioplegic solution containing each [Mg2+] 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. [Mg2+] 1.2 mM group.

Vascular Contractility After Reperfusion

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Concentration-response curve to receptor-mediated vasoconstrictor U-46619 after 60-min exposure to a hypoxic, hyperkalemic cardioplegic solution containing each [Mg2+] 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.

Intrinsic myogenic contraction was observed to a stepwise increase in the transmural pressure >50 mmHg in control vessels. In vessels from higher [Mg²⁺] 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 [Mg²⁺] groups (both P < 0.05 vs. [Mg²⁺] 5.0 and 25.0 mM; Fig. 4).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Active pressure-diameter relations after 60-min exposure to a hypoxic, hyperkalemic cardioplegic solution containing each [Mg2+] 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 (10-100 mmHg); ¶ P < 0.05 vs. [Mg2+] 5.0 mM group (60-100 mmHg).

	DISCUSSION

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Changes in [Ca²⁺]_i and Vascular Contraction During Hyperkalemic Cardioplegia

The mechanism responsible for the hyperkalemic cardioplegia-related [Ca²⁺]_i accumulation in vascular smooth muscle is most likely related to membrane depolarization on the basis of Nernst's equation (17). Membrane depolarization promotes Ca²⁺ influx through voltage-dependent Ca²⁺ channels. Ca²⁺ influx also induces release of Ca²⁺ from intracellular Ca²⁺ 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 [Ca²⁺]_i is elevated during and after periods of hypoxia. In previous studies, it was suggested that transsarcolemmal Ca²⁺ influx via Na⁺/Ca²⁺ exchange may play an important role in hypoxia/reoxygenation-mediated Ca²⁺ accumulation (12, 22). Recently, other investigators have reported that Ca²⁺ release from the sarcoplasmic reticulum may contribute to hypoxic pulmonary vasoconstriction (6, 10).

High Mg²⁺ concentration has been suggested to inhibit Ca²⁺ entry into the cell by displacing Ca²⁺ from binding sites in the calcium channels and by hyperpolarization of sarcolemmal membrane (9). It has also been postulated that extracellular Mg²⁺ acts by raising intracellular Mg²⁺ concentration, thereby reducing the release of Ca²⁺ from the sarcoplasmic reticulum (3, 24). In addition, supplementation of Mg²⁺ in cardioplegic solutions may diminish the depletion of ATP stores, 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 Mg²⁺ supplementation.

Indeed, it should be noted that the present study examined the effects of hyperkalemic cardioplegic solutions containing a physiological concentration of calcium (CaCl₂ 1.2 mM). Therefore, it is possible that a reduced Ca²⁺ concentration in a hyperkalemic cardioplegia might require a lower level of Mg²⁺ supplementation to achieve microvascular protection from intracellular Ca²⁺ overload. In clinical practice, however, it is more difficult to precisely regulate the cardioplegic Ca²⁺ concentration because of transient variables such as pH and temperature. As a result, patients are at risk of exposure to higher than originally intended Ca²⁺ levels, which may increase the likelihood of the Ca²⁺-mediated vascular injury. In addition, further decreasing the Ca²⁺ level may cause a calcium paradox in coronary microvessels. Therefore, we emphasize that the addition of Mg²⁺ may solve this dilemma by allowing for the safe use of higher cardioplegic Ca²⁺ concentrations, and we recommend sufficient supplementation of Mg²⁺ (>5.0 mM) in hyperkalemic cardioplegic solutions.

Changes in Vascular Contractility After Hyperkalemic Cardioplegia

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 Mg²⁺-based cardioplegic solution ([Mg²⁺] 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 Mg²⁺ 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 Mg²⁺ 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 Ca²⁺ accumulation in the vascular smooth muscle may activate a Ca²⁺-dependent intracellular signaling pathway and alter the Ca²⁺ sensitivity of the contractile apparatus. Previous studies have demonstrated that an agonist-induced vascular tone is regulated by myosin light chain kinase, the activity of which is governed by a Ca²⁺-calmodulin-mediated phosphorylation (19). There is some evidence that [Ca²⁺]_i may also directly activate the Ca²⁺-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 [Ca²⁺]_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 K⁺_ATP channels open when the intracellular ATP concentration falls to <1 mM (21). Opening of the K⁺_ATP channels causes membrane hyperpolarization and relaxes the vascular smooth muscle by preventing Ca²⁺ entry via voltage-sensitive Ca²⁺ 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 Mg²⁺ (>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

There are a number of Ca²⁺-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 [Ca²⁺]_i in microvascular smooth muscle. Although fura 2 is useful for the evaluation of [Ca²⁺]_i, it must be noted that determination of the absolute value of [Ca²⁺]_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 [Ca²⁺]_i. For quantification of [Ca²⁺]_i, we used an in situ dual-wavelength calibration by obtaining R_max and R_min and thus estimated cytosolic [Ca²⁺]_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 [Ca²⁺]_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 [Mg²⁺] possibly prevents hyperkalemic cardioplegia-related intracellular Ca²⁺ overloading and preserves vascular function in coronary microvessels. To protect coronary microcirculation from Ca²⁺-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.