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J Thorac Cardiovasc Surg 1999;118:163-172
© 1999 Mosby, Inc.

CARDIOPULMONARY SUPPORT AND PHYSIOLOGY

INTRACELLULAR FREE CALCIUM ACCUMULATION IN FERRET VASCULAR SMOOTH MUSCLE DURING CRYSTALLOID AND BLOOD CARDIOPLEGIC INFUSIONS

From the Division of Cardiothoracic Surgery, Department of Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School,^a and Boston Biomedical Research Institute,^b Boston.

Supported by the National Institutes of Health grants HL46716 (F.W.S.) and HL31704 (K.G.M.).

Address for reprints: Frank W. Sellke, MD, Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, Dana 905, 330 Brookline Ave, Boston, MA 02215.

	Abstract

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Objective: The effects of magnesium- and potassium-based crystalloid and blood-containing cardioplegic solutions on coronary smooth muscle intracellular free calcium ([Ca²⁺]_i) accumulation and microvascular contractile function were examined.
Methods: Isolated ferret hearts were subjected to hyperkalemic (25 mmol/L K⁺) blood cardioplegic infusion, hypermagnesemic (25 mmol/L Mg²⁺, K⁺-free) crystalloid cardioplegic infusion, or hyperkalemic crystalloid cardioplegic infusion for 1 hour. Coronary arterioles were isolated, cannulated, and loaded with fura 2. Reactivity and [Ca²⁺]_i were assessed with videomicroscopy. [Ca²⁺]_i was measured at baseline and after application of 50 mmol/L KCl. In addition, [Ca²⁺]_i and vascular contraction were measured during exposure to Mg²⁺ and K⁺ cardioplegic solution at both 4°C and 37°C.
Results: From a baseline [Ca²⁺]_i of 177 ± 52 nmol/L, K⁺ cardioplegic infusion (302 ± 80 nmol/L potassium) markedly increased [Ca²⁺]_i, whereas blood cardioplegic infusion (214 ± 53 nmol/L) and Mg²⁺ cardioplegic infusion (180 ± 42 nmol/L) did not alter [Ca²⁺]_i. Although a difference between groups in percentage contraction after application of 50 mmol/L KCl was not observed, [Ca²⁺]_i increased significantly more in vessels in the control group (764 ± 327 nmol/L) and the K⁺ crystalloid cardioplegic infusion group (698 ± 215 nmol/L) than in vessels in the blood cardioplegic infusion group (402 ± 45 nmol/L) and the Mg²⁺ cardioplegic infusion group (389 ± 80 nmol/L). Mg²⁺ cardioplegic solution induced no microvascular contraction at either 4°C or 37°C, nor was an increase in [Ca²⁺]_i observed. K⁺ cardioplegic solution induced microvascular contraction at 37°C but not at 4°C; it increased [Ca²⁺]_i at both 4°C and 37°C.
Conclusion: An Mg²⁺-based cardioplegic solution, or appropriate Mg²⁺ or blood supplementation of a K⁺ crystalloid cardioplegic solution, may decrease the accumulation of [Ca²⁺]_i in the vascular smooth muscle during ischemic arrest.

	Introduction

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Intracellular calcium [Ca²⁺]_i accumulation in coronary smooth muscle cells is associated with derangements in coronary vasomotor tone and contractility after ischemic cardioplegic infusion. The altered myogenic contraction during and after potassium cardioplegic infusion is in part associated with opening of adenosine triphosphate–activated potassium channels (K+_ATP channels) and calcium accumulation in the coronary vascular smooth muscle. ^1,2 The inhibition of accumulation of [Ca²⁺]_i and maintenance of ATP tissue concentration with magnesium or other substances may theoretically favor a magnesium-based or blood-based cardioplegic solution. ³ Because the addition of blood to potassium cardioplegic solution ⁴ and magnesium cardioplegic solution ² have similar effects in preserving ß-adrenergic and endothelium-dependent relaxation and myogenic contraction, all of which are important in blood flow regulation, it was hypothesized that the addition of blood or Mg²⁺ to a hyperkalemic crystalloid cardioplegic solution could prevent functional damage to the vascular system caused by increased [Ca²⁺]_i accumulation in the coronary smooth muscle. In this study [Ca²⁺]_i accumulation in the coronary vascular smooth muscle and microvascular contraction were examined during ischemic arrest with a cold hyperkalemic blood cardioplegic solution, a hyperkalemic crystalloid cardioplegic solution, or a magnesium-based cardioplegic solution. Because the coronary microcirculation is the primary site of regulation of myocardial perfusion, microvascular tissue was examined.

	Materials and methods

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Male ferrets (8-12 weeks of age) were anesthetized with chloroform in a ventilation hood. The hearts were removed after injection of a small amount of heparin and were immediately immersed in a cold (4°C) saline solution. The following procedures were immediately undertaken.

Crystalloid cardioplegic infusion groups
After the cannulation of the ascending aortic root of isolated ferret hearts with a small catheter, 20 mL cold (4°C) Ca²⁺-free hypermagnesemic crystalloid cardioplegic solution (25 mmol/L Mg²⁺, n = 6) or cold Ca²⁺-free hyperkalemic crystalloid cardioplegic solution (25 mmol/L K⁺, n = 6) was infused into the coronary arteries through the aorta at a pressure of approximately 40 mm Hg. The composition of the Mg²⁺ cold crystalloid cardioplegic solution was as follows (in millimoles per liter): 121.0 NaCl, 25.0 MgCl₂, 4.7 KCl, 12.0 NaHCO₃, and 11.1 glucose in purified water. K⁺ cold crystalloid cardioplegic solution had the following composition (in millimoles per liter): 121.0 NaCl, 25.0 KCl, 12.0 NaHCO₃, and 11.1 mmol/L glucose in purified water. In both groups saline solution slush was placed on the surface of the heart to provide topical hypothermia during the ischemic period. Infusion of the cardioplegic solution (10 mL) in each group was repeated at 20-minute intervals for a total arrest time of 60 minutes (2 additional doses). The heart was then immediately placed in a cold Krebs buffer solution of the following composition (in millimoles per liter): 118.3 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 1.2 NaH₂PO₄, 25.0 NaHCO₃, and 11.1 glucose in purified water.

Blood cardioplegic infusion group
In 6 isolated ferret hearts, 20 mL hyperkalemic cold blood cardioplegic solution (4:1 mixture of oxygenated blood with high-potassium crystalloid cardioplegic solution resulting in 25 mmol/L KCl) was infused into the aortic root as described above previously, followed by intermittent infusion of 10 mL low-potassium cold blood cardioplegic solution (4:1 mixture of oxygenated blood with low-potassium crystalloid cardioplegic solution resulting in 12.5 mmol/L KCl) every 20 minutes (K⁺ blood cardioplegic infusion group). The compositions of the high- and low-potassium solutions used in this study to create the blood cardioplegic solutions contained 60 or 30 mmol/L KCl, respectively, 12.5 g mannitol, 50 mL citrate-phosphate-dextrose solution and 10 mEq tris(hydroxymethyl)aminomethane in 500 mL of 5% dextrose and 0.225% saline solution. In all groups, after 60 minutes of cardioplegic arrest the heart was immediately placed in a cold Krebs buffer solution.

Cold global ischemia group
In another 6 experiments isolated ferret hearts were flushed with cold Krebs buffer solution, placed in a cold saline solution for 60 minutes (cold global ischemia group) and then placed in a cold Krebs buffer solution.

Animal care
All animals received humane care in compliance with the Boston Biomedical Research Institute Committee on Animal Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institute of Health (NIH Publication No. 86-23, revised 1985).

Responses of nonischemic vessels
Microvessels were immediately obtained from nonischemic ferret hearts, cannulated as described previously, and then loaded with fura 2. Vessels were exposed to either Mg²⁺ cold crystalloid cardioplegic solution, Mg²⁺ cold crystalloid cardioplegic solution supplemented with 1.0 mmol/L Ca²⁺ cold crystalloid cardioplegic solution, K⁺ cold crystalloid cardioplegic solution supplemented with 1.0 mmol/L Ca²⁺, or K⁺ cold crystalloid cardioplegic solution supplemented with 1.0 mmol/L Mg²⁺ at either 4°C or 37°C (n = 4-5 hearts/group) for 5 to 10 minutes. The fluorescence and contraction responses were then measured.

Coronary microvessel preparations
After ischemic cardioplegic interventions, coronary arterioles (mean 133 ± 8 µm in internal diameter, ranging from 100 to 150 µm) were immediately dissected from the left anterior descending coronary artery–dependent subepicardial region of the free wall of the left ventricle with a x10 to x60 dissecting microscope (Olympus Optical Co, Ltd, Tokyo, Japan). The vessels were placed in a microvessel chamber (University of Iowa Medical Instrumentation, Iowa City, Iowa), cannulated with dual glass micropipettes measuring 40 to 80 µm in diameter, and secured with 10-0 nylon monofilament suture (Ethicon, Inc, Somerville, NJ). Oxygenated (95% oxygen and 5% carbon dioxide) Krebs buffer solution warmed to 37°C was continuously circulated through the microvessel chamber. Vessels were pressurized to 40 mm Hg in a no-flow state by means of a burette manometer filled with a Krebs buffer solution. With an inverted microscope (Zeiss IM 35, x40-200; Carl Zeiss, Inc, Thornwood, NY) connected to a camera (Hitachi model KP 115; Hitachi Denshi America, Ltd, Woodbury, NY), the microvessel image was projected onto a black and white television monitor. The microvessels were allowed to bathe in the chamber for at least 30 minutes before internal lumen diameter and [Ca²⁺]_i measurements were taken at baseline and under various conditions.

Coronary smooth muscle [Ca2+]_i handling and concentration
Vascular smooth muscle [Ca²⁺]_i was measured in isolated single coronary microvessels as previously reported by Meininger and colleagues. ⁵ In K⁺ blood cardioplegic infusion, Mg²⁺ cold crystalloid cardioplegic infusion, K⁺ cold crystalloid cardioplegic infusion, and cold global ischemia groups, after equilibration of the microvessels, fura 2 was loaded into the microvessels with a noncirculating Krebs solution containing 5 µmol/L fura 2 AM, which was dissolved in 0.05% dimethyl sulfoxide and 0.01% pluronic acid at room temperature (22°C-25°C), for 45 minutes. At the end of the loading period the extracellular fura 2 AM was washed out with Krebs solution and at least 30 minutes was allowed for de-esterification of intracellular fura 2 AM at 37°C. Fura 2 signals from the microvessels were recorded by positioning the pinhole on the lowest and most homogeneous location of the microvessel wall with a Nikon Fluor x40 (NA 0.8; Nikon Corporation, Tokyo, Japan) objective. To minimize bleaching and cell damage, the shutter was opened only when necessary for taking measurements. Excitation interference filters at 350 ± 5 nm and 390 ± 6 nm were used.

A computer-controlled filter wheel produced by Ludl switches filtered every 2 msec. Fluorescent signals were collected with a photomultiplier tube (Hamamatsu R928; Hamamatsu Photonics KK, Hananatsu City, Japan) after excitation at 350 nm and 390 nm. The signals at the 2 wavelengths from the photomultiplier tube were digitized by a Data Acquisition-EZ A/D Converter and processed using a DTVee program version 3.0 (Data Translation, Inc, Marlboro, Mass). The background signals at the 2 wavelengths were measured to subtract from subsequent fluorescent signal measurements. Baseline and KCl-induced (50 mmol/L) [Ca²⁺]_i changes in fluorescence at 350 and 390 nm were recorded at steady-state at the same time that the respective contraction responses were observed. A 50-mmol/L KCl concentration was chosen to induce an increase in [Ca²⁺]_i. Measurements were made and recorded 3 to 5 minutes after the drug administration, when the response had stabilized. The microvessels were washed with Krebs buffer solution at 37°C and allowed to equilibrate in a drug-free Krebs buffer solution for 20 minutes between interventions. Comparisons were made against the responses of microvessels and [Ca²⁺]_i from instrument-free control hearts.

Absolute ([Ca⁺²]_i) was calculated as follows:

[Ca⁺²]_i = K_d x [(R – R_min)/(R_max – R)] x S

where K_d was the dissociation constant; R was the ratio of the smooth muscle signal at 350 nm to that at 390 nm; Rmax was determined after treating microvessels with 50 µmol/L 4-bromo-A23187 containing 150 mmol/L KCl, 1 mmol/L Ca²⁺, and 5 mmol/L piperazine-N-Nī-bis (2-ethanesulfonic acid) (PIPES); Rmin was determined after treating microvessels with 4 mmol/L ethyleneglycol-bis-(ß-aminoethylether)-N,N,Nī,Nī-tetraacetic acid; and S was the ratio of the 390 nm signal in microvessels treated with ethyleneglycol-bis-(ß-aminoethylether)-N,N,Nī,Nī-tetraacetic acid to that in the presence of 4-bromo-A23187. A K_d value of 240 nmol/L at 37°C was used. The increase in absolute [Ca²⁺]_i was expressed as percentage change from baseline [Ca²⁺]_i (100% x given [Ca²⁺]_i/baseline [Ca²⁺]_I).

Drugs
Fura 2 AM was obtained from Molecular Probes (Molecular Probes, Inc, Eugene, Ore). The 4-bromo-A23187 and dimethyl sulfoxide were Calbiochem brand (CN Biosciences, Inc, San Diego, Calif). Ethyleneglycol-bis-(ß-aminoethylether)-N,N,Nī,Nī-tetraacetic acid, PIPES, magnesium chloride and pluronic acid were obtained from Sigma (Sigma, St Louis, Mo). KCl were obtained from Fisher Scientific (Fisher Scientific Worldwide, Laboratory Projects Division, Springfield, NJ). Pluronic acid was dissolved in dimethyl sulfoxide to make a stock solution that was stored at –20°C. Fura 2 AM solution was prepared on the day of the study. Other drugs were dissolved in ultrapure distilled water.

Statistical analysis
The contraction responses were expressed as the percentage contraction of the initial diameter of the microvessels. A negative value denoted relaxation response. All data were expressed as mean ± SD. Analysis of variance followed by the Fisher exact test was used to compare changes in percentage increase or absolute change in [Ca²⁺]_i between groups or to compare percentage vascular contraction between groups. Depending on the number of multiple comparisons performed, the Bonferroni correction was used to correct for multiple comparisons.

	Results

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Effects of ischemic cardioplegic infusion
Although exposure of microvessels to cold K⁺ crystalloid cardioplegic solution or cold global ischemia significantly increased [Ca²⁺]_i, exposure to cold K⁺ blood cardioplegic solution or cold Mg²⁺ crystalloid cardioplegic solution did not increase [Ca²⁺]_I (Fig. 1). These changes in [Ca²⁺]_i were not completely reversible after repetitive washing with Krebs buffer solution at 37°C (data not shown).

View larger version (27K): [in this window] [in a new window]   Fig 1. Coronary smooth muscle intracellular free calcium concentrations ([Ca2+]i) at baseline after 1 hour of cardioplegic infusion corresponding to ratios of fluorescence at 350 nm to that at 390 nm with fura2. K+-BCP, Cold blood cardioplegic infusion; K+-CCP, calcium-free hyperkalemic cardioplegic infusion; Mg2+-CCP, calcium free-hypermagnesemic cardioplegic infusion; CGI, cold global ischemia. Boxes represent mean; error bars indicate SD; n = 6 for each group.

View larger version (28K): [in this window] [in a new window]   Fig 2. Percentage contraction of initial internal diameter of microvessels in response to application of 50 mmol/L potassium chloride after cardioplegic infusion. K+-BCP, Cold blood cardioplegic infusion; K+-CCP, calcium-free hyperkalemic cardioplegic infusion; Mg2+-CCP, calcium-free hypermagnesemic cardioplegic infusion; CGI, cold global ischemia. Boxes represent mean; error bars indicate SD; n = 6 for each group.

View larger version (22K): [in this window] [in a new window]   Fig 3. Percentage increase from baseline of intracellular free calcium concentration ([Ca2+]i) in response to application of 50 mmol/L potassium chloride after cardioplegic infusion. K+-BCP, Cold blood cardioplegic infusion; K+-CCP, calcium-free hyperkalemic cardioplegic infusion; Mg2+-CCP, calcium free-hypermagnesemic cardioplegic infusion; CGI, cold global ischemia. Boxes represent mean; error bars indicate SD; n = 6 for each group.

View larger version (27K): [in this window] [in a new window]   Fig 4. Percentage contraction of initial internal diameter of nonischemic microvessels in response to application of hyperkalemic crystalloid cardioplegic solution and calcium-free hypermagnesemic crystalloid cardioplegic solution at 4°C and 37°C in nonischemic microvessels. K+-CCP, Calcium-free hyperkalemic cardioplegic solution; Mg2+-CCP, calcium free-hypermagnesemic cardioplegic solution. Boxes represent mean; error bars indicate SD; n = 4 to 5 for each group.

View larger version (19K): [in this window] [in a new window]   Fig 5. Increase from baseline in intracellular free calcium concentration ([Ca2+]i) in response to application of hyperkalemic crystalloid cardioplegic solution and hypermagnesemic crystalloid cardioplegic solution at 4°C and 37°C in nonischemic microvessels. K+-CCP, Calcium-free hyperkalemic cardioplegic solution; Mg2+-CCP, calcium-free hypermagnesemic cardioplegic solution. Boxes represent mean; error bars indicate SD; n = 4 to 5 for each group.

View larger version (37K): [in this window] [in a new window]   Fig 6. Increase from baseline in intracellular free calcium concentration ([Ca2+]i) after application of hyperkalemic crystalloid cardioplegic solution with 1.0 mmol/L calcium ion and hypermagnesemic crystalloid cardioplegic solution with 1.0 calcium ion at 4°C and 37°C in nonischemic microvessels. K+/1.0Ca2+-CCP, Hyperkalemic cardioplegic solution containing 1.0 mmol/L calcium ion; Mg2+/1.0Ca2+-CCP, hypermagnesemic cardioplegic solution containing 1.0 mmol/L calcium ion. Boxes represent mean; error bars indicate SD; n = 4 to 5 for each group.

View larger version (15K): [in this window] [in a new window]   Fig 7. Percentage contraction of initial internal diameter of nonischemic microvessels after application of hyperkalemic crystalloid cardioplegic solution with 1.0 mmol/L calcium ion and hypermagnesemic crystalloid cardioplegic solution with 1.0 mmol/L calcium ion at 4°C and 37°C in nonischemic microvessels. K+/1.0Ca2+-CCP, Hyperkalemic cardioplegic solution containing 1.0 mmol/L calcium ion; Mg2+/1.0Ca2+-CCP, hypermagnesemic cardioplegic solution containing 1.0 mmol/L calcium ion. Boxes represent mean; error bars indicate SD; n = 4 to 5 for each group.

View larger version (19K): [in this window] [in a new window]   Fig 8. Increase from baseline in coronary smooth muscle intracellular free calcium concentration ([Ca2+]i) in response to application of calcium-free hyperkalemic 1.0 mmol/L magnesium crystalloid cardioplegic solution (K+/1.0Mg2+-CCP) at 4°C and 37°C in nonischemic microvessels. Boxes represent mean; error bars indicate SD; n = 4 to 5 for each group.

	Discussion

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It is interesting to note that there was an apparent discrepancy between the baseline [Ca²⁺]_i and the observed KCl–induced increases in [Ca²⁺]_i and vascular contractions. In other words, baseline [Ca²⁺]_i was increased, but KCl-induced contraction and increase in [Ca²⁺]_i were similar in all experimental groups. Thus acute changes in baseline vascular smooth muscle [Ca²⁺]_i may not significantly affect vasomotor tone or contractile responses. However, only a purely calcium-dependent contractile agonist (KCl) was examined. Responses of microvessels to calcium-independent agonists may not be preserved, and calcium-dependent vasodilator mechanisms are known to be altered. ^2,4

Calcium accumulation in coronary smooth muscle cells
Depolarization-induced Ca²⁺ influx through voltage-operated Ca²⁺ channels, Ca²⁺-induced Ca²⁺ release from the sarcoplasmic reticulum, and Na⁺/Ca²⁺ exchange mechanisms are 3 major potential causes of Ca²⁺ accumulation during cardioplegic infusion. However, the exact cause of accumulation is controversial and dependent on the cell type, ^6,7 age, ⁸ species, and other factors. According to the Nernst equation, ^9,10 the open probability of voltage-operated Ca²⁺ channels is low during hyperkalemic (25 mmol/L K⁺) cardioplegic infusion, and maintained depolarization theoretically inhibits Ca²⁺ influx by inactivation of L-type Ca²⁺ channels. ^11,12 However, it has been reported that the increase in [Ca²⁺]_i during cardioplegic infusion may be inhibited by a calcium-channel blocker. ⁷ A contribution of Ca²⁺ in the sarcoplasmic reticulum to [Ca²⁺]_i accumulation is both probable and predicted, ¹³ but researchers have reported variable findings. ^6,7 Furthermore, increased intracellular free Na⁺ as a result of Na⁺/H⁺ exchange and depolarization of the membrane by hyperkalemic cardioplegic infusion can increase Ca²⁺ influx through the Na⁺/Ca²⁺ exchange system. ¹⁴ Decreased activity of the Na⁺/Ca²⁺ exchange mechanism during cardioplegic infusion may theoretically account for some of the increase in [Ca²⁺]_i. ¹⁵

In this study the cold blood cardioplegic solution used contained less than 1.0 mmol/L Ca²⁺ and 0.5 mmol/L Mg²⁺. This combination may cause a slight increase in [Ca²⁺]_i because it contains an inadequate concentration of the Mg²⁺ necessary to inhibit the increase in [Ca²⁺]_i (Fig. 8). Also, it is likely that the extracellular free Ca²⁺ concentration ([Ca²⁺]_ex) in part determines the degree of Ca²⁺ influx in response to a hyperkalemic environment. Changes in myocardial [Ca²⁺]_i during cardioplegic infusion have been reported to be minimal in the absence of extracellular Ca²⁺. ^8,9 In this study of vascular smooth muscle, however, we observed an increase in [Ca²⁺]_i during exposure of microvessels to a Ca²⁺-free hyperkalemic solution. These findings suggest a contribution of Ca²⁺ influx through release of Ca²⁺ from intracellular stores (sarcoplasmic reticulum) and Ca²⁺-induced Ca²⁺ release to add to the [Ca²⁺]_i during cardioplegic infusion. A significant defect in sarcoplasmic reticulum Ca²⁺ transport in the early phase of ischemia has been reported; this is characterized by a depression of Ca²⁺ uptake and Ca²⁺ channel ATPase activity. ¹⁶ The use of Mg²⁺ cold crystalloid cardioplegic solution completely abolished the increase in [Ca²⁺]_i, possibly as a result of Ca-Mg antagonism.

Hypothermia itself has been reported to augment the extent of depolarization, and this may lead to increased Ca²⁺ influx. ^17-19 Cold hyperkalemic cardioplegic infusion tends to induce more [Ca²⁺]_i overload than does infusion of a warm (37°C) crystalloid solution. ^20,21 In our study in nonischemic vessels, however, the difference in [Ca²⁺]_i accumulation between 4°C and 37°C during cardioplegic infusion was minimal but the increase in [Ca²⁺]_i remained elevated in the cytoplasm after washing with Krebs buffer solution, suggesting that impaired calcium mobilization may persist for a more prolonged time.

Although the movement of Mg²⁺ across the sarcolemmal membrane tends to be slow, intracellular free Mg²⁺ may double within 1 to 2 seconds after a sudden challenge with increased extracellular free Mg²⁺. ²² Increased extracellular Mg²⁺ has been reported to block Ca²⁺ channel currents ^23-25 and to act extracellularly by inhibiting Ca²⁺ influx through voltage-operated Ca²⁺ channels and intracellularly by blocking the movement of Ca²⁺ from the sarcoplasmic reticulum to the cytosol. ^13,25 Mg²⁺ may also increase sarcoplasmic and mitochondrial uptakes of calcium. ²⁶ In fact, both Mg²⁺ cardioplegic infusion and K⁺/Mg²⁺ cardioplegic infusion have been reported to significantly reduce [Ca²⁺]_i and provide enhanced myocardial functional recovery after ischemia. ^6,13,27-29

Limitations
The fact that fura 2 was loaded after rather than before ischemic cardioplegic infusion in isolated hearts may be a source of criticism. However, this was the only method available to examine the effects of blood on [Ca²⁺]_i during blood cardioplegic infusion. Because the data from K⁺– and Mg²⁺–cold crystalloid cardioplegic infusion in ischemic and nonischemic groups were very consistent and reproducible, we consider the data on blood cardioplegic infusion to be validated and credible. It should be noted that measurements of [Ca²⁺]_i and KCl-induced vascular contraction were obtained after a period of equilibration in normothermic Krebs buffer solution and loading of fura 2. Thus these values may be underestimates of the actual effect in vivo.

Hypothermia (4°C) in this study decreased the fluorescent ratio significantly (P = .002) by 7.3% ± 4.0% with respect to that seen under normothermic conditions (37°C), suggesting that hypothermia reduced [Ca²⁺]_i. In fact, calculation of the absolute [Ca²⁺]_i at baseline revealed that the [Ca²⁺]_i tended to be lower at 4°C. Although a K_d value at 4°C was not examined, the K_d value at 37°C according to the report by Pawlowski and associates ³⁰ would be expected and was therefore used in this study. R_max (0.983 ± 0.009 at 4°C and 1.015 ± 0.038 at 37°C) and R_min (0.412 ± 0.010 at 4°C and 0.417 ± 0.024 at 37°C) were not significantly different between 4°C and 37°C, but the S value at 4°C (1.11 ± 0.07) was significantly (P = .002) lower than that at 37°C (1.65 ± 0.19). This finding suggests that, contrary to the previous reports, ^17-19 hypothermia itself may decrease [Ca²⁺]_i. However, further examination is required to verify this speculation.

	Conclusions

Top Abstract Introduction Materials and methods Results Discussion Conclusions References

 
Cold hyperkalemic cardioplegic infusion led to an increase in [Ca²⁺]_i in the coronary vascular smooth muscle similar to that observed during cold unprotected ischemia. The addition of Mg²⁺ to the hyperkalemic solution and the use of a blood-based or Mg²⁺-based cardioplegic solution both markedly reduced this increase in [Ca²⁺]_i. Such solutions may better protect the coronary circulation during ischemic arrest.

	References

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